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Notice Medical knowledge is constantly changing. Standard safety precautions must be followed, but as new research and clinical experience broaden our knowledge, changes in treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current product information provided by the manufacturer of each drug to be administered to verify the recommended dose, the method and duration of administration, and contraindications. It is the responsibility of the practitioner, relying on experience and knowledge of the patient, to determine dosages and the best treatment for each individual patient. Neither the Publisher nor the author assume any liability for any injury and/or damage to persons or property arising from this publication. The Publisher
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To the memory of Ralph Wright, MA, MD, DPhil, FRCP. A leading first generation hepatologist, clinical scientist, teacher and father of one of the editors.
Contributors Paul C Adams MD Professor of Medicine Department of Medicine London Health Sciences Centre – University Campus London, ON, Canada Aijaz Ahmed MD Gastroenterology Fellow Hepatology Stanford University Medical Center Palo Alto, CA, USA Karl E Anderson MD Professor of Preventive Medicine and Community Health, Department of Preventive Medicine and Community Health University of Texas Medical Branch Galveston, TX, USA Miguel R Arguedas MD MPH Assistant Professor Gastroenterology – School of Medicine Birmingham, AL, USA Vicente Arroyo MD Professor of Medicine, Director Institute for Digestive Disease Hospital Clinic i Provincial Universitat de Barcelona Barcelona, Spain Veronica A Arteaga MD Doctor of Medicine Department of Surgery Cedars-Sinai Medical Center Los Angeles, CA; Clinical Researcher New England Hepatobiliary Disease Center Dartmouth-Hitchcock Medical Center Lebanon, NH, USA
Heike Bantel MD Department of Gastroenterology and Hepatology Hannover Medical School Hannover, Germany
Jordi Bruix MD Director Liver Unit IMD Hospital Clinic Provincial Catalonia, Spain
Angeline Bartholomeusz MD Research and Molecular Development Victorian Infectious Diseases Reference Laboratory North Melbourne, VIC, Australia
Kathleen M Campbell MD Assistant Professor Division of Gastroenterology, Hepatology and Nutrition Cincinnati Children’s Hospital Medical Center Cincinnati, OH, USA
Marina Berenguer MD Adjunct Professor Department of Digestive Medicine Hospital Universitari La Fe Valencia, Spain Annika Bergquist MD PhD Clinical Assistant Department of Gastroenterology and Hepatology Karolinska University Hospital Huddinge, Sweden Henri Bismuth MD Director Henri Bismuth Hepatobiliary Institute Villejuif, France Herbert L Bonkovsky MD Director of Clinical Research, the Lowell P. Weicker, Jr. General Clinical Research Center, and the Clinical Trials Unit Professor of Medicine and Molecular, Microbial, and Structural Biology Farmington, CT, USA Thomas D Boyer MD Director Arizona Liver Institute University of Arizona Tucson, AZ, USA
Rizwan Aslam MD ChB MRCP FRCR Assistant Clinical Professor Department of Radiology University of California, San Francisco San Francisco, CA, USA
Ulrika Broomé MD PhD Associate Professor of Medicine Department of Gastroenterology and Hepatology Karolinska University Hospital Huddinge, Sweden
Soon Koo Baik MD Associate Professor of Medicine Department of Medicine Yonsei University Wonju College of Medicine Wonju, Korea
Robert S Brown MD MPH Chief of Clinical Hepatology Medical Director Center for Columbia University College of Physicians and Surgeons New York, NY, USA
William F Balistreri MD Director Division of Paediatric Gastroenterology / Nutrition University of Cincinnati Children’s Hospital Medical Centre Cincinatti, OH, USA
Concepció Bru MD Senior Consultant, Associate Professor of Radiology BCLC Group Diagnosis Imaging Centre Barcelona, Spain
Martin Caselitz MD Consultant Medical Clinic II Klinikum Deggendorf Deggendorf, Germany John P Cello MD Professor of Medicine and Surgery Division of Gastroenterology, Hepatology and Clinical Nutrition San Francisco General Hospital San Francisco, CA, USA Naga Chalasani MD Associate Professor of Medicine Division of Gastroenterology/ Hepatology Indiana University School of Medicine Indianapolis, IN, USA Judy Chang BSc Department of Microbiology and Immunology The University of Melbourne Parkville, VIC, Australia Linda J Chen MD Clinical Instructor Division of Transplantation, Department of Surgery Stanford University School of Medicine Stanford, CA, USA Xin Chen PhD Assistant Professor Department of Biopharmaceutical Sciences University of California, San Francisco San Francisco, CA, USA Massimo Colombo MD Professor of Gastroenterology and Endocrinology Maggiore Hospital and University of Milan Milan, Italy Diane W Cox PhD CCMG FRSC Professor and Chair Department of Medical Genetics University of Alberta Edmonton, AB, Canada
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Contributors
Oscar W Cummings MD Associate Professor of Pathology Department of Pathology and Laboratory Medicine Indiana University School of Medicine Indianapolis, IN, USA John T Cunningham MD Professor of Clinical Medicine Section of Gastroenterology and Hepatology University of Arizona School of Medicine Tucson, AZ, USA Christopher P Day MD PhD FRCP Professor of Liver Medicine Department of Gastroenterology and Hepatology The Medical School University of Newcastle upon Tyne Newcastle upon Tyne, UK Laurie D DeLeve MD PhD Professor of Medicine Division of Gastrointestinal and Liver Diseases University of Southern California Keck School of Medicine Los Angeles, CA, USA R Brian Doctor PhD Associate Professor Division of Gastroenterology and Hepatology Department of Medicine University of Colorado Health Sciences Center Denver, CO, USA Scott A Elisofon MD Advanced Hepatology Fellow Division of Gastroenterology Hunnewell Ground Children’s Hospital Boston Boston, MA, USA Eric Esrailian MD MPH Clinical Instructor of Medicine Division of Digestive Diseases David Geffen School of Medicine at UCLA Los Angeles, CA, USA Carlos O Esquivel MD PhD The Arnold and Barbara Silverman Professor of Pediatric Transplantation Professor of Surgery and Chief, Division of Transplantation Stanford University School of Medicine Stanford, CA, USA Gregory T Everson MD Professor of Medicine; Director of Hepatology University of Colorado School of Medicine and Health Sciences Denver, CO, USA Michael B Fallon MD Associated Professor of Medicine Med – Gastroenterology University of Alabama at Birmingham Birmingham, AL, USA Diana M Flynn MB Bch Consultant in Paediatric Gastroenterology John Radcliffe Hospital Oxford, UK
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Scott L Friedman MD Professor of Medicine Division of Liver Diseases Mount Sinai School of Medicine New York, NY, USA Hans Fromm MD Professor of Medicine; Director Dartmouth-Hitchcock Hepatopancreaticobiliary Disease Center Lebanon, NH, USA Paul J Gaglio MD Associate Clinical Professor of Medicine (in Surgery) Center for Liver Disease and Transplantation Columbia University College of Physicians and Surgeons New York, NY, USA Guadalupe Garcia-Tsao MD Professor of Medicine Section of Digestive Diseases Yale University School of Medicine Yale, CT, USA Fayez K Ghishan MD Horace W. Steele Endowed Chair in Pediatric Research Professor and Head, Department of Pediatrics Director, Steele Memorial Children’s Research Center University of Arizona Health Sciences Center Tucson, AZ, USA Kathleen M Giacomini PhD Professor and Chair Department of Biopharmaceutical Sciences University of California, San Francisco San Francisco, CA, USA Pere Ginés MD Associate Professor of Medicine Liver Unit Institute for Liver Research Hospital Clinic i Provincial Universitat de Barcelona Barcelona, Spain
Mónica Guevara MD Associate Investigator Liver Unit Institute of Digestive Diseases Hospital Clinic Institut d’Investigacions Biomediques (IDIBAPS) Barcelona, Spain Françoise Imbert-Bismut MD Department of Biochemistry Hôpital de la Salpétrière Paris, France Sanjeev Gupta MD Professor of Medicine and Pathology Departments of Medicine and Pathology Albert Einstein College of Medicine Bronx, NY, USA Elizabeth J L Heathcote MD Professor of Medicine Toronto Hospital (Western) Toronto, ON, Canada Kristel Hunt MD Gastroenterology Fellow Division of Liver Diseases Mount Sinai School of Medicine New York, NY, USA John Hunter MD Professor and Chairman Department of Surgery, L223 Oregon Health Services University Portland, OR, USA Hartmut W Jaeschke PhD Professor of Pharmacology Associate Director Liver Research Institute University of Arizona College of Medicine Tucson, AZ, USA Peter L M Jansen MD Professor of Medicine Liver Center Division of Gastroenterology and Hepatology Academic Medical Center Amsterdam, The Netherlands Birgir Johannsson MD Infectious Disease Fellow Division of Infectious Diseases The University of Iowa Iowa City, IA, USA
Steven Goldschmid MD Associate Professor of Clinical Medicine Chief Section of Gastroenterology and Hepatology University of Arizona School of Medicine Tucson, AZ, USA
Maureen M Jonas MD Associate Professor of Pediatrics Division of Gastroenterology Harvard Medical School Children’s Hospital Boston, MA, USA
Gregory J Gores MD Professor of Medicine Mayo Medical School Rochester, MN, USA
Dean P Jones PhD Professor of Medicine Department of Biochemistry Emory University Atlanta, GA, USA
Albert K Groen PhD Associate Professor Department of Medical Biochemistry Academic Medical Center Amsterdam, The Netherlands
Emmet B Keeffe MD Professor of Medicine, Chief of Hepatology, Co-Director Stanford University Medical Center Palo Alto, CA, USA
Contributors
Deirdre A Kelly MD FRCP FRCPI FRCPH MB BA Professor of Paediatric Hepatology The Liver Unit Birmingham Children’s Hospital Birmingham, UK Percy A Knolle MD Professor of Molecular Medicine and Immunology Institute for Molecular Medicine and Experimental Immunology University of Bonn Bonn, Germany Jina Krissat MD Surgical Registrar Hepatobiliary and Pancreatic Surgery Unit The Royal London Hospital London, UK Manoj Kumar MD DM Senior Research Associate Department of Gastroenterology GB Pant Hospital New Delhi, India Douglas R LaBrecque MD Director, Liver Services Internal Medicine Liver Services University of Iowa Hospitals and Clinics Iowa City, IA, USA Konstantinos Lazaridis MD Assistant Professor of Medicine Center for Basic Research in Digestive Diseases Division of Gastroenterology and Hepatology Mayo Clinic College of Medicine Rochester, MN, USA Samuel S Lee MD Professor of Medicine Liver Unit Department of Medicine University of Calgary Calgary, AB, Canada Jay H Lefkowitch MD Professor of Clinical Pathology College of Surgeons and Physicians of Columbia University New York, NY, USA Riccardo Lencioni MD Associate Professor of Radiology Department of Oncology, Transplants and Advanced Technologies in Medicine Division of Diagnostic and Interventional Radiology University of Pisa Pisa, Italy Sharon Lewin MD Director, Infectious Diseases Unit; Professor, Department of Medicine Monash University The Alfred Hospital Melbourne, VIC, Australia
Keith D Lindor MD Professor of Medicine Division of Gastroenterology and Hepatology Mayo Medical School, Clinic and Foundation Rochester, MN, USA
Brent Neuschwander-Tetri MD Professor of Internal Medicine Division of Gastroenterology and Heptology Saint Louis University Liver Center St. Louis, MO, USA
Josep M Llovet, MD Senior Research Associate BCLC Group. Liver Unit, Digestive Disease Institute Mount Sinai School of Medicine Barcelona, Spain
Matthew Nichols MD Fellow in Gastroenterology and Hepatology University of Colorado Health Sciences Center Denver, CO, USA
Stephen Locarnini MD Divisional Head Research & Molecular Development Victorian Infectious Diseases Reference Laboratory North Melbourne, VIC, Australia Robert S McCuskey PhD Professor and Head of Cell Biology and Anatomy; Professor of Physiology; Professor of Pediatrics Department of Cell Biology and Anatomy College of Medicine, University of Arizona Tucson, AZ, USA Michael P Manns MD Professor of Medicine Head, Department of Gastroenterology and Hepatology Zentrum Innere Medizin and Dermatologie Medizinische Hochschule Hannover Hannover, Germany Enrique J Martinez MD FACP Associate Professor of Clinical Medicine Center for Liver Diseases University of Miami Miami, FL, USA Darius Moradpour MD Associate Professor of Medicine Division of Gastroenterology and Hepatology Centre Hospitalier Universitaire Vaudois Lausanne, Switzerland Kevin D Mullen MB FRCPI Professor of Medicine at Case Western Reserve University; Consultant of Gastroenterology Gastroenterology and Hepatology Division MetroHealth Medical Center Cleveland, OH, USA Satheesh Nair MD Medical Director of Liver Transplantation Ochsner Clinic Foundation New Orleans, LA, USA Russell Nash MD Assistant Professor Department of Pathology University of Colorado Health Sciences Center Denver, CO, USA James Neuberger DM FRCP Consultant Physician, Professor of Medicine Liver Unit Queen Elizabeth Hospital Birmingham, UK
David H Perlmutter MD Professor Pediatrics/Cell Biology Department of Pediatrics Washington University Pittsburgh, PA, USA Robert P Perrillo MD Director, Gastroenterology and Hepatology Section of Gastroenterology and Hepatology Ochsner Clinic Foundation New Orleans, LA, USA Thierry Poynard MD PhD Professor of Medicine Department of Hepato-Gastroenterology University of Paris VI Paris, France Jorge Rakela MD Professor of Medicine Department of Internal Medicine Mayo Clinic Scottsdale, AZ, USA Charles M Rice PhD Maurice R and Corinne P Greenberg Professor; Head, Laboratory of Virology and Infectious Disease; Scientific and Executive Director Center for the Study of Hepatitis C The Rockefeller University New York Presbyterian Hospital New York, NY, USA Mario Rizzetto MD Professor of Gastroenterology Department of Gastroenterology University of Torino – Molinette Torino, Italy Eve A Roberts MD FRCPC Professor of Paediatrics, Medicine and Pharmacology Division of Gastroenterology, Hepatology and Nutrition The Hospital for Sick Children Toronto, ON, Canada Don C Rockey MD Chief, Division of Digestive and Liver Diseases; Professor of Medicine University of Texas at Southwestern Medical Center Dallas, TX, USA Juan Rodés MD Professor of Medicine Hospital Clinic Barcelona, Spain
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Contributors
Hector Rodriguez-Luna MD Associate Consultant Division of Transplantation Medicine Mayo Clinic Phoenix, AZ, USA
Maria H Sjogren MD MPH Chief Department of Clinical Investigation Walter Reed Army Medical Center Washington, DC, USA
Sammy Saab MD MPH Associate Professor of Medicine and Surgery Division of Digestive Diseases David Geffen School of Medicine at UCLA Los Angeles, CA, USA
Jack T Stapleton MD Professor and Director Division of Infectious Diseases The University of Iowa and The Iowa City VA Medical Center Iowa City, IA, USA
Arun J Sanyal MBBS MD Charles Caravati Professor of Medicine Chairman: Division of Gastroenterology, Hepatology and Internal Medicine Medical College of Virginia Richmond, VA, USA S K Sarin MD DM FNA FNASc President, Asian Pacific Association Study of Liver; Adjunct Professor, Molecular Medicine, JNU; Professor and Head Department of Gastroenterology GB Pant Hospital New Delhi, India Thomas D Schiano MD Associate Professor of Medicine; Medical Director, Adult Liver Transplantation; Director of Clinical Hepatology Division of Liver Diseases Mount Sinai School of Medicine New York, NY, USA Leonard B Seeff MD Senior Scientist for Hepatitis Research Liver Disease Research Branch National Institute of Diabetic Digestive and Kidney Diseases (NIDDK) Bethesda, MD, USA Shobha Sharma MD Assistant Professor of Pathology Department of Pathology Emory University Hospital Atlanta, GA, USA Steven I Shedlofsky MD Marcos Lins Andrade Professor Division of Digestive Diseases and Nutrition University of Kentucky Lexington, KY, USA Oren Shibolet MD Lecturer in Medicine The Liver Unit Hadassah University Hospital Jerusalem, Israel Daniel Shouval MD Professor of Medicine; Director, Liver Unit Hadassah University Hospital Jerusalem, Israel
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Stephen F Stewart MBChB BSc PhD MRCP Consultant Hepatologist Department of Gastroenterology and Hepatology School of Clinical Medical Sciences The Medical School University of Newcastle upon Tyne Newcastle upon Tyne, UK Doris B Strader MD Associate Professor of Medicine Division of Gastroenterology/Hepatology Fletcher Allen Health Care Burlington, VT, USA
Dominique-Charles Valla MD Professor of Hepatology, University of Paris 7 Head, Federation Medico-Chirurgicale d’HepatoGastroentologie Hospital Beaujon Paris, France Rebecca W Van Dyke MD Professor of Medicine Gastroenterology Division University of Michigan School of Medicine Ann Arbor, MI, USA Hugo E Vargas MD Associate Professor of Medicine Mayo Clinic College of Medicine Phoenix, AZ, USA Siegfried Wagner MD Professor and Physician-in-Chief Medical Clinic II Klinikum Deggendorf Deggendorf, Germany
Christian P Strassburg MD Assistant Professor in Experimental Gastroenterology (Privatdozent) Department of Gastroenterology, Hepatology and Endocrinology Hannover Medical School Hannover, Germany
Jack R Wands MD Jeffrey and Kimberly Greenberg-Artemis and Martha Joukowsky Professor in Gastroenterology and the Professor of Medical Science Brown University; Director, Division of Gastroenterology and the Liver Research Center Rhode Island Hospital Providence, RI, USA
R Todd Stravitz MD Associate Professor of Medicine Section of Hepatology Virginia Commonwealth University Richmond, VA, USA
C Mel Wilcox MD Professor of Medicine Division of Gastroenterology and Hepatology University of Alabama at Birmingham Birmingham, AL, USA
Yee-Li Sun MD Resident Department of Radiology University of California, San Francisco San Francisco, CA, USA
Teresa L Wright MD Staff Physician Veterans’ Affairs Medical Center San Francisco, CA, USA
Jayant A Talwalkar MD MPH Assistant Professor of Medicine Mayo Clinic College of Medicine Rochester, MN, USA Ruedi Thoeni MD Chief GI Radiology University of California, San Francisco San Francisco, CA, USA Christian Trautwein MD Professor of Medicine Department of Gastroenterology, Hepatology and Endocrinology Zentrum Innere Medizin Hannover, Germany Daniel Tseng MD Minimally Invasive Surgery Fellow Oregon Health and Science University Portland, OR, USA
Judy Yee MD Associate Professor and Vice Chairman Department of Radiology University of California, San Francisco; Chief, Radiology Service Veterans’ Affairs Medical Center San Francisco, CA, USA Mahmoud M Yousfi MD Assistant Professor of Medicine Department of Internal Medicine Mayo Clinic Scottsdale, AZ, USA
Preface
The first edition of Hepatology appeared in 1982. The creation of the book came from a desire of Drs Zakim and Boyer to write a textbook on liver disease in which both pathophysiology and clinical material were presented in a manner that allowed the reader to understand current issues but, more importantly, to prepare the reader for new developments in the discipline of hepatology. David Zakim was the driving force behind the first edition and his expertise in understanding the basic science behind the clinical diseases was critical to the success of the book. With his leadership Hepatology met the goals set in the first and subsequent editions. David Zakim has now retired from academic medicine and for this book to continue to be one of the leading books in the field of liver disease it was essential that new editors be added. Drs Teresa Wright and Michael Manns are world leaders in hepatology with broad clinical expertise and outstanding research credentials. They continue the tradition established by Dr Zakim that a textbook should help in understanding a patient’s disease by providing fundamental knowledge of the pathophysiology of the disease process. Thus, their addition as editors allows Zakim and Boyer’s Hepatology to continue to evolve in this fifth edition. With the advent of the electronic age and ready availability of summaries of published works there has been a move away from reading source material and an increasing reliance on opinion articles frequently published with the support of the pharmaceutical industry. These ‘reviews’ are brief and cover the essentials but lack depth. In this environment books such as Zakim and Boyer’s Hepatology play an increasingly important role. The authoritative chapters in the book cover a subject in depth and give the reader both the basic and clinical information they need to grasp the area of interest. With a thorough understanding of an area gained by reading one or two chapters, the reader can then better understand new arti-
cles and judge the quality of ‘reviews’. Hepatology and the figures will be available on the Elsevier web site in a searchable and downloadable format to those who purchase the book. This edition continues the tradition of changing authors and adding new chapters to cover areas not previously discussed in detail in the previous edition. Fifty-one of the chapters have new or additional authors compared to the previous edition. We also have added 20 new chapters that cover subjects such as hepatotoxicity from herbal preparations, pediatric viral hepatitis and pharmacogenomics that were not in the previous edition. These new chapters reflect our commitment to keeping Zakim and Boyer’s Hepatology current and providing the reader with the latest advancements in liver disease as well as the influence of the new editors. Although we continue to have chapters on basic pathophysiology, we are focusing more and more on the clinical aspects of the discipline of hepatology. This evolution of the past 25 years reflects the advances in the field and the increasing number of treatment options available to the practicing physician for the management of hepatobiliary disorders. Lastly, the presentation of the book has been enhanced by drawing many of the figures in color, making the tables more pleasing to the eye and placing the color plates within the chapters rather than grouping them throughout the book. We believe these latter changes will make the book easier to use and more readable. The editors hope that this book will be of help to modern hepatologists and gastroenterologists around the world, as well as to physicians of other specialties and fellows in training, in order to increase their knowledge for the benefit of their patients. Thomas D. Boyer Teresa L. Wright Michael P. Manns
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Acknowledgements A book of this size involves numerous individuals in its creation. We are grateful to all of the contributors for their timely delivery of the manuscripts and the excellence of the chapters that they have written. This book would never have been published without the professional staff at Elsevier. Karen Bowler has been instrumental in the creation of this edition of Zakim and Boyer’s Hepatology. She was there at the first meetings with the new editors and has been there to solve problems and encourage all of us to make the best book possible. Claire Bonnett and Kathryn Mason have been of
equal importance during the production of Zakim and Boyer’s Hepatology. Their work has lead to a book with a new and exciting appearance, from the cover by illustrator Richard Tibbitts, to the tables, to the high quality color plates that are now distributed within the chapters. We realize that there are numerous other people at Elsevier who have contributed to Zakim and Boyer’s Hepatology and we are all grateful for their efforts as well. Lastly, we would like to thank our families who have supported us during the genesis of this book.
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Section I: Pathophysiology of the Liver
1
ANATOMY OF THE LIVER Robert S. McCuskey Abbreviations CGRP calcitonin gene-related peptide Da daltons DNA deoxyribonucleic acid GERL granules or secondary lysosomes HD high density HMS hepatic microvascular subunits ICAM intercellular adhesion molecule
IgA LAL LD LGL IL NK nNOS
immunoglobulin A liver-associated lymphocytes low density large granular lymphocytes interleukin natural killer neuronal nitric oxide
INTRODUCTION OVERVIEW OF THE STRUCTURE AND FUNCTION OF THE LIVER The liver is the largest organ in the body. In humans it is separated incompletely into lobes, covered on their external surfaces by a thin connective tissue capsule. The liver is composed of several cell types, which interact with each other but are adapted to performing specific functions. The principal cell type is the hepatic parenchymal cell, loosely referred to as the hepatocyte, which accounts for 60% of the total cell population and 80% of the volume of the organ. Hepatocytes are organized into plates or laminae that are interconnected to form a continuous three-dimensional lattice (Figure 1-1). Between the plates of hepatocytes are spaces occupied by hepatic sinusoids, the large-bore fenestrated capillaries of the liver that nourish each parenchymal cell on several sides (Figure 1-1). The sinusoidal space, and non-parenchymal cells associated with sinusoids, comprises the majority of the remaining liver volume. The non-parenchymal cells include sinusoidal endothelial cells, perisinusoidal stellate cells (fat-storing cells of Ito), and intraluminal Kupffer cells. An interconnecting network of minute intercellular channels form bile canaliculi, which course between adjacent hepatocytes (Figure 1-1A). These receive the bile secreted from hepatocytes and then drain through short bile ductules (cholangioles) partially lined by cuboidal epithelial cells to bile ducts. Hepatocytes carry out most of the functions generally associated with the liver. They extract and process nutrients and other materials from the blood, and they produce both exocrine and endocrine secretions, as follows:
Bile Synthesis and Secretion Hepatocytes synthesize bile acids from cholesterol; these function in the lumen of the small intestine to emulsify fats. Bilirubin, a toxic metabolite generated from the breakdown of hemoglobin, is excreted by hepatocytes as follows. Insoluble bilirubin is produced as a byproduct of red blood cell breakdown in the spleen; it circulates in the blood complex to albumin, and is taken up from the blood hepatocytes, conjugated to a soluble form, then secreted into bile canaliculi.
NPY RER SER SOM SP Tf VIP
neuropeptide Y rough endoplasmic reticulum smooth endoplasmic reticulum somatostatin substance P transferrin vasoactive intestinal peptide
Protein Synthesis Hepatocytes synthesize proteins for hepatic and non-hepatic use. Proteins for hepatic use include a wide variety of liver-specific enzymes that carry out the many synthetic and detoxifying functions of the liver. Proteins secreted by hepatocytes include all of the major plasma proteins except immunoglobulins (synthesized by plasma cells), e.g. albumin, transferrin, prothrombin, fibrinogen, lipoproteins and complement proteins.
Glucose Homeostasis Hepatocytes help to maintain blood glucose levels. In response to pancreatic islet hormones hepatocytes synthesize glycogen from glucose or break down glycogen and release glucose (glycogenolysis); hepatocytes can also synthesize glucose from other sugars (e.g. fructose) and from amino acids (gluconeogenesis).
Metabolism of Drugs and Toxins Hepatocyte enzymes metabolize drugs and toxins delivered to the liver from the gut via the portal circulation. The functions of the hepatic non-parenchymal cells are:
Kupffer Cells • Phagocytosis of bloodborne toxicants and particulates such as bacteria from the circulation. • Secretion of mediators (e.g. inflammatory mediators) that affect the function of adjacent cells and cells in distant sites. • Production of beneficial and toxic substances that contribute to host defense as well as liver injury.
Sinusoidal Endothelial Cells These form a leaky barrier between the parenchymal cells and the blood flowing in sinusoids. The endothelial cells are fenestrated and act as a sieve to prevent red blood cells and other cellular components from interacting with hepatocytes, while allowing rapid access to the other substances in the blood.
Stellate Cells • Storage of vitamin A and other fat-soluble vitamins. • Stellate cells when activated synthesize collagen, thus they are important in the development of cirrhosis.
3
Section I. Pathophysiology of the Liver
A
B
Figure 1-1. Laminae of hepatic parenchymal cells (H) interconnected to form a three-dimensional lattice containing a labyrinth of spaces occupied by sinusoids (S). BC, bile canaliculus; KC, Kupffer cell. (Modified from McCuskey RS. Functional morphology of the liver with emphasis on its microvasculature. In: Tavoloni N, Berk PD, eds. Hepatic transport and bile secretion. New York, Raven Press, 1993: 2, ©1993, with permission from Lippincott, Williams and Wilkins (http://lww.com).)
The structure of the liver at the tissue, cellular and molecular levels has evolved to subserve the above functions and is the subject of the remainder of this chapter.
GROSS ANATOMY The mature liver lies mainly in the right upper quadrant of the abdominal cavity, is attached to the diaphragm, and is protected by the ribcage. Its morphology has been extensively reviewed.1,2 Briefly, in adults the healthy liver weighs approximately 1500 g3 and extends along the midclavicular line from the right fifth intercostal space to just inferior to the costal margin. From there, the anterior border of the liver extends medially crossing the midline just inferior to the xiphoid process. A small portion of the organ projects across the midline and lies in the upper left abdominal quadrant. The liver has a dual blood supply which enters the liver at its hilus (porta hepatis) accompanied by the hepatic bile duct, lymphatics and nerves. Approximately 80% of the blood entering the liver is poorly oxygenated and is supplied by the hepatic portal vein. This is the venous blood flowing from the intestines, pancreas, spleen, and gallbladder. The remaining 20% of the blood supply is well oxygenated and delivered by the hepatic artery. Anatomically the liver is divided into right and left lobes by the falciform ligament, which is a peritoneal fold connecting the liver to the anterior abdominal wall and the diaphragm (Figure 1-2). The right lobe is further subdivided inferiorly and posteriorly into two smaller lobes – the caudate and quadrate lobes. The functional division, however, is a plane that passes through the gallbladder and
4
inferior vena cava which defines the halves of the liver supplied by the right and left branches of the portal vein and hepatic artery, together with biliary drainage into the right and left hepatic ducts.4 As a result, the quadrate lobe and a large portion of the caudate lobe belong functionally to the left hemiliver. Further functional subdivision of the liver into eight segments having independent vascular and biliary supplies has been reported (Figure 1-3) and is important when liver resection is required.1,2,5,6 The liver is encapsulated by a thin connective tissue layer (Glisson’s capsule) consisting mostly of regularly arranged type I collagen fibers, scattered type III fibers, fibroblasts, mast cells, and small blood vessels. On the surfaces facing the abdominal cavity this connective tissue layer is covered by the simple squamous mesothelial cells of the peritoneal lining. At the attachment of the falciform ligament to the liver, the two leaves of the ligament separate to form an area devoid of peritoneum, the ‘bare area’, on the superior surface of the liver. The right and left leaves of the falciform then merge with reflections of the peritoneum coming off the diaphragm, forming respectively the triangular and the coronary ligaments.
DEVELOPMENT OF THE LIVER The development of the liver has been extensively described1,2,7,8 and is illustrated in Figure 1-4. Briefly, the liver primordium appears in human embryos during the third week of gestation as an endodermal bud from the ventral foregut just cranial to the yolk sac. This bud becomes the hepatic diverticulum as it enlarges, elongates, and develops a cavity contiguous with the foregut. The hepatic
Chapter 1 ANATOMY OF THE LIVER
Diaphragmatic area Right lobe
Left triangular ligament
Coronary ligament
Lesser omentum
Right triangular Inferior vena cava Bare area ligament
Caudate lobe Left lobe
Renal area Portal vein
Porta hepatis
Hepatic duct Pyloric area
Cystic duct
Falciform ligament Colic areas Ligamentum teres
Falciform ligament Gallbladder Ligamentum teres
Gallbladder
Quadrate lobe
Figure 1-2. Lobes, surfaces, and ligaments of the liver viewed anteriorly (left) and from a posteroinferior perspective (right). (Modified from Moore KL, Dalley AF. Clinically oriented anatomy, 4th edn. Philadelphia: LWW, 1999: 264, ©1999, with permission from Lippincott, Williams and Wilkins (http://lww.com).)
Inferior vena cava Left and middle hepatic veins Right hepatic vein
Figure 1-3. Segmentation of the liver based on principal divisions of the portal vein and hepatic artery. (Modified from Moore KL, Dalley AF. Clinically oriented anatomy, 4th edn. Philadelphia: LWW, 1999: 268, © 1999, with permission from Lippincott, Williams and Wilkins (http://lww.com).)
II VII
VIII
I IV III
VI
Right and left branches of hepatic artery
V
Portal vein Gallbladder
Bile duct
Portal triad
Hepatic artery
diverticulum grows into the septum transversum, a plate of mesenchyme that incompletely separates the pericardial and peritoneal cavities, and separates into an hepatic portion that forms the hepatic parenchymal cells as well as the intrahepatic bile ducts, a cystic portion that forms the gall bladder, and a ventral portion that forms the head of the pancreas.
During the fourth week of development, buds of epithelial cells extending from the hepatic diverticulum into the mesenchyme of the septum transversum as thick, anastomosing cords several cells thick become interspersed within the developing anastomotic network of capillaries arising from the vitelline veins, thus beginning to establish the close relationship of hepatic parenchymal cells to
5
Section I. Pathophysiology of the Liver
A
Mesenchymal lining of coelomic tract
B Common cardinal vein
Sinus venosus
Left hepato-cardiac channel
Liver
Right hepato-cardiac channel
Left umbilical vein
Left umbilical vein
Septum transversum
Hepatic sinusoidal plexus Vitelline veins
C
Gut
D Inferior vena cava
Sinus venosus
Right umbilical vein
Right hepatic vein Hepatic sinusoidal plexus
Middle hepatic vein Left hepatic vein Ductus venosus (Dia. = 600μm)
Left umbilical vein Origin of hepatic bud Portal vein (Dia. = 100μm)
Left umbilical vein (Dia. = 600μm)
Right and left viteline veins Figure 1-4. Development of the liver. A Section through the region of the hepatic bud of a human embryo of 25 somites (26 days). B Vascular channels associated with the developing liver in a human embryo of 30 somites. C Vascular channels at a later stage showing development of the sinusoidal network. D Portal hepatic circulation in a human embryo of 17 mm (7 weeks). (Reproduced from MacSween RNM, Desmet VJ, Roskams T, Scothorne RJ. Functional morphology of the liver with emphasis on its microvasculature. In: MacSween NM, Burt AD, et al., eds. Pathology of the liver, 4th edn. London: Churchill Livingstone, 2002: 4, ©2002, with permission of Elsevier.)
the sinusoids.9 The anastomotic pattern of both multicellular cords of parenchymal cells and sinusoids persists until several years after birth, by which time cords two or more parenchymal cells thick bounded on several sides by sinusoids have become plates consisting of single parenchymal cells bounded on at least two sides by sinusoids, particularly in the centrilobular region.10,11 By 7 weeks the vitelline veins unite to form the portal vein. The hepatic artery is derived from the celiac axis and its ingrowth into the hepatic primordium closely follows that of the bile ducts.12–14 Between the sixth week and birth the fetal liver serves as a hematopoietic organ and as the primary site for fetal blood formation until the third trimester, when most hemopoietic sites disappear as the bone marrow develops.
6
MICROSCOPIC ANATOMY VASCULATURE, BILIARY SYSTEM, INNERVATION Vasculature Both the hepatic portal vein and the hepatic artery, together with afferent nerves, enter the liver at the hilus, where efferent bile ducts as well as lymphatics and nerves also exit the organ (see below). Branches of the hepatic artery, hepatic portal vein, main bile duct and main lymphatic vessel travel together in portal tracts through the liver parenchyma (Figure 1-5). Portal tracts are sometimes referred to as portal triads, because, of the five elements present, the lymphatic vessel is usually collapsed and inconspicuous, as are the autonomic nerves, resulting in only three elements being visible
Chapter 1 ANATOMY OF THE LIVER Figure 1-5. Hepatic microvasculature as determined by in vivo microscopic studies. PV, portal venule; HA, hepatic arteriole; L, lymphatic; BD, bile ductule; N, nerve; CV, central venule; SLV, sublobular hepatic vein. Arrows indicate direction of flow. (Modified from McCuskey RS. Functional morphology of the liver with emphasis on its microvasculature. In: Tavoloni N, Berk PD, eds. Hepatic transport and bile secretion. New York: Raven Press, 1993: 2, ©1993, with permission from Lippincott, Williams and Wilkins (http://lww.com).)
CV
Bile canaliculus
Sinusoid Perisinusoidal space
Inlet sphincter
Intersinusoidal sinusoid and sphincter Outlet sphincter
Arterio-sinus twig
Bile canaliculus Hepatic cell Sinusoid
Arterio-portal anastomoses N
PV
L BD
HA SLV
in sections through portal tracts. After repeated branching, terminal branches of the blood vessels (portal venules and hepatic arterioles) supply blood to the sinusoids (Figure 1-6). Branches of hepatic arterioles also supply the peribiliary plexus of capillaries nourishing the bile ducts, and then drain into sinusoids (via arteriosinus twigs) (Figure 1-7) or occasionally into portal venules (arterioportal anastomoses). Because all these vessels are independently contractile, the sinusoids receive a varying mixture of portal venous and hepatic arterial blood.15,16 After flowing through the sinusoids, blood is collected in small branches of hepatic veins termed central venules (central veins, terminal hepatic venules) (Figure 1-6). These course independently of the portal tracts and drain via hepatic veins, which leave the liver on the dorsal surface and join the inferior vena cava. Lymphatic vessels originate as blind-ending capillaries in the connective tissue spaces within the portal tracts.17 The fluid contained in these lymphatics flows toward the hepatic hilus and eventually into the cisternae chili and thoracic duct. The perisinusoidal space of Disse is thought by some to function as a lymphatic space that channels plasma to the true lymphatics coursing in the portal tract. However, anatomic connections between the space of Disse and the portal tract have not been identified.17,18 Lymph also leaves the liver in small lymphatics associated with the larger hepatic veins into lymphatics along the wall of the inferior vena cava.1 Lymphatics in the
Figure 1-6. Vascular cast of the hepatic microvasculature illustrating the tortuous anastomotic sinusoids adjacent to the portal venule (PV) and the more parallel and larger sinusoids near the central venule (CV). (Modified from McCuskey RS. The hepatic microvascular system. In: Tavoloni N, Berk PD, eds. Hepatic transport and bile secretion. New York: Raven Press, 1993: 4, ©1993, with permission from Lippincott, Williams and Wilkins (http://lww.com).)
7
Section I. Pathophysiology of the Liver
Figure 1-7. Terminal branches (arrowheads) from hepatic arteriole (HA) which frequently terminate in inlet venules or terminal portal venules where sinusoids originate. PV, portal venule; B, peribiliary plexus supplied by the adjacent hepatic arteriole. (Modified from McCuskey RS. Functional morphology of the liver with emphasis on its microvasculature. In: Arias IM, Boyer JL, et al., eds. The Liver: Biology and Pathobiology, 3rd edn. New York: Raven Press, 1994: 1095, ©1994, with permission from Lippincott, Williams and Wilkins (http://lww.com).)
Figure 1-9. Intrahepatic aminergic innervation in the dog. Brightly fluorescent nerve fibers are adjacent to the portal vein (PV), hepatic artery (HA), and bile duct (not visible in this section) and are also distributed intralobularly along the sinusoids (arrows).
of the portal vein and hepatic artery in portal tracts. Bile ducts drain through larger left and right hepatic ducts, which exit the liver at the hilus to form the common bile duct. These ducts are lined with simple columnar epithelial cells. Branches of the hepatic artery supply an extensive peribiliary plexus of capillaries (Figure 1-7).22
Innervation
Figure 1-8. Bile canalicular network filled with dye injected retrograde into the bile duct.
hepatic capsule drain to vessels either at the hilum or around the hepatic veins and inferior vena cava.1
Biliary System Bile canaliculi are spaces 1–2 mm wide formed between adjacent hepatocytes (Figure 1-1A).19–21 They are interconnected and form a network of minute intercellular channels (Figure 1-8) which receive the bile secreted from hepatocytes. These minute biliary channels are specialized regions of adjacent hepatic parenchymal cells and will be discussed in more detail together with the ultrastructure of these cells. The bile canaliculi drain through short bile ductules (cholangioles) partially lined by cuboidal epithelial cells to bile ducts, lined with simple cuboidal epithelium, which course along with branches
8
Aminergic, peptidergic, and cholinergic nerves are contained in the portal tracts and affect both intrahepatic blood flow and hepatic metabolism.23,24 The role of neural elements in regulating blood flow through the hepatic sinusoids, solute exchange, and parenchymal function is incompletely understood. This is due in part to limited investigation in only a few species, whose hepatic innervation may differ significantly from that of humans. For example, most experimental studies have used rats and mice, whose livers have little or no intralobular innervation. In contrast, most other mammals, including humans, have aminergic and peptidergic nerves extending from the perivascular plexus in the portal space into the lobule (Figure 1-9), where they course in the space of Disse in close relationship to stellate cells and hepatic parenchymal cells (Figure 1-10). Although these fibers extend throughout the lobule, they predominate in the periportal region. Cholinergic innervation, however, appears to be restricted to structures in the portal space and immediately adjacent hepatic parenchymal cells. Neuropeptides have been colocalized with neurotransmitters in both adrenergic and cholinergic nerves. Neuropeptide Y (NPY) has been colocalized in aminergic nerves supplying all segments of the hepatic–portal venous and the hepatic arterial and biliary systems. Nerve fibers immunoreactive for substance P (SP) and somatostatin (SOM) follow a similar distribution. Intralobular distribution of all of these nerve fibers is species dependent and similar to that reported for aminergic fibers. Vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) are reported to coexist in cholinergic and sensory afferent nerves innervating portal veins and hepatic
Chapter 1 ANATOMY OF THE LIVER
are mutually exclusive, have been proposed as follows and are illustrated in Figure 1-11. The classic hepatic lobule is a polygonal structure having as its central axis a central venule, with portal tracts distributed along its peripheral boundary.25 The peripheral boundaries of these lobules are poorly defined in most species, including man (Figure 1-12). In some species, e.g. pigs and seals, there is considerably more connective tissue in the liver and this is distributed along the peripheral boundary of classic lobules, thereby making them very distinct. Considerable sinusoidal anastomoses occur between adjacent lobules, and thus the blood collected by each central venule is supplied by several portal venules. The hepatic acinus26,27 is a unit having no distinct morphologic boundaries. Its axis is a portal tract and its peripheral boundary is circumscribed by an imaginary line connecting the neighboring terminal hepatic venules (central hepatic venules of the classic lobule), which collect blood from sinusoids. Contained within the acinus are three zones, each having different levels of oxygenation and metabolic function. In yet another model of lobular organization, the lobule is defined by bile drainage. So-called portal lobules28 have at their center a portal tract, with central veins present around the periphery of each lobule. Currently, the concept of subunits of the classic lobule forming functional units is the most consistent with existing evidence.29–32 In this model, each ‘classic’ lobule consists of several ‘primary lobules’. Each primary lobule is cone-shaped, having its convex surface at the periphery of the classic lobule supplied by terminal branches of portal venules and hepatic arterioles, and its apex at the center of the classic lobule drained by a central (terminal hepatic) venule.
arteries and their branches, but not the other vascular segments or the bile ducts. Nitrergic nerves immunoreactive for neuronal nitric oxide (nNOS) are located in the portal tract, where nNOS colocalizes with both NPY- and CGRP-containing fibers.
HEPATIC FUNCTIONAL UNITS The organization of each liver lobe into structural or functional units related to function and/or disease has been the subject of considerable debate during the past century. Several models, none of which
Figure 1-10. Nerve fiber (N) closely associated with a stellate cell (FSC) in the space of Disse in the dog. H, hepatic parenchymal cell; L, lipid droplet, C, collagen.
HA
Primary lobule
PV
Classic lobule A
B
C CV
HMS C B
Portal lobule
A
1
2
3
Acinus
Figure 1-11. Contiguous hepatic lobules illustrating the interconnecting network of sinusoids derived from two portal venules (PV). Note that the sinusoids become more parallel as they course toward the central venule (CV), which forms the axis of the classic lobule. Hepatic arterioles (HA) supply blood to sinusoids near the periphery of the lobule, usually by terminating in inlet venules or terminal portal venules. As a result, three zones (1, 2, 3) of differing oxygenation and metabolism have been postulated to compose a hepatic acinus, with its axis being the portal tract (lower left). Several acini would compose the portal lobule (lower right). Each classic lobule contains several cone-shaped subunits having convex surfaces fed by portal and arterial blood at the periphery and its apex at the central venule (upper left). A, B, and C represent hemodynamically equipotential lines in a ‘primary lobule.’ A recent modification further subdivides lobules into conical hepatic microcirculatory subunits (HMS), each being supplied by a single inlet venule. (Modified from McCuskey RS. Functional morphology of the liver with emphasis on its microvasculature. In: Tavoloni N, Berk PD, eds. Hepatic transport and bile secretion. New York: Raven Press, 1993: 4, ©1993, with permission from Lippincott, Williams and Wilkins (http://lww.com).)
9
Section I. Pathophysiology of the Liver
Figure 1-12. The liver is composed of lobules each having a central venule (CV) as its axis and peripheral boundaries which are poorly defined (arrows) but contain branches of the portal vein (PV), hepatic artery and bile duct.
These ‘primary lobules’ were renamed as ‘hepatic microvascular subunits (HMS)’ and were demonstrated to consist of a group of sinusoids supplied by a single inlet venule and its associated termination of a branch of the hepatic arteriole from the adjacent portal space (Figure 1-12). Further confirmation of this HMS concept was obtained by studying their development in neonatal livers.33 Accompanying the HMS are hepatic parenchymal cells and the associated cholangioles and canaliculi. Hepatocellular metabolic gradients also have been demonstrated to conform to this proposed functional-unit concept.34,35
HEPATIC PARENCHYMAL CELLS Hepatic parenchymal cells, commonly referred to as hepatocytes, are polyhedral cells about 20–30 mm in size, have a volume of approximately 5000 mm3 , and are organized into anastomotic sheets (Figure 1-1).36–38 They are epithelial cells, and like other polarized epithelial cells they have distinct apical, lateral and basal surfaces. The basal surfaces of hepatocytes face the sinusoidal endothelium. Their plasma membranes have microvilli which extend into the space of Disse (the space between hepatocytes and endothelial cells) to increase surface area for the exchange of materials between hepatocytes and blood plasma. The apical surfaces of hepatocytes face adjacent hepatocytes and enclose the bile canaliculi, minute spaces forming a network of channels that carry the bile secretion (exocrine secretion) of hepatocytes (Figures 1-13 and 1-14). The apical surfaces also form microvilli to increase the surface area for secretion. This is also referred to as the canalicular domain of the plasma membrane. The lateral membranes of hepatocytes extend from the bile canaliculi to the space of Disse and form cell–cell junctions, including gap junctions which facilitate communication between hepatocytes, and tight junctions which seal the bile canalicular lumen from the interstitial space (Figures 1-13 and 1-14). These tight junctions are critical in that they prevent leakage of plasma into bile as well as backflow of bile from canaliculi into the blood. Functionally, the basal and lateral membranes are frequently considered a unit, the basolateral membrane.
10
Figure 1-13. Portions of three hepatic parenchymal cells having bile canaliculi (BC) located between adjacent cells. RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum; M, mitochondria; L, lysosome; S, sinusoid; D, space of Disse; E, endothelial cell. Inset is a higher magnification of a tight junction (arrowhead) between adjacent parenchymal cells. DS, desmosome. (Reproduced from Jones AL. Anatomy of the normal liver. In: Zakim D, Boyer TD, eds. Hepatology: A Textbook of Liver Disease, 3rd edn. Philadelphia: WB Saunders, 1996: 22, ©1996, with permission of Elsevier.)
Hepatocytes have one, or frequently two, nuclei and their cytoplasm contains numerous mitochondria as well as a prominent Golgi apparatus located between the nucleus and the bile canaliculi, rough endoplasmic reticulum, and smooth endoplasmic reticulum, with associated rosettes of glycogen particles.38 They also contain numerous endosomes, lysosomes, and peroxisomes. Fat droplets also may be present.
PLASMA MEMBRANE The plasma membrane is a dynamic structure38–41 that has a variety of regions having specific functions and characteristics. The basal plasma membrane of each hepatocyte faces one or more sinusoids, where its surface area is greatly increased by microvilli that extend into the space of Disse (Figure 1-13) to facilitate the uptake of bloodborne substances into hepatocytes and the secretion of constitutively produced substances into the blood. This exchange of products across the plasma membrane in the space of Disse is further facilitated by the absence of a typical epithelial basal lamina; the sinusoidal endothelium also has a greatly reduced or absent basal
Chapter 1 ANATOMY OF THE LIVER
Figure 1-14. Two adjacent hepatic parenchymal cells and enclosed bile canaliculus (BC) and associated organelles. L, lysosome; G, Golgi; SER, smooth endoplasmic reticulum; Mb, microbody (peroxisome); M, mitochondria, g, glycogen; N, nucleus; arrowheads, tight junctions. (Reproduced from Jones AL. Anatomy of the normal liver. In: Zakim D, Boyer TD, eds. Hepatology: A Textbook of Liver Disease, 3rd edn. Philadelphia: WB Saunders, 1996: 22, ©1996, with permission of Elsevier.)
lamina. The apical surface of the plasma membrane is limited to the bile canaliculi, which are channels formed by tight junctions between adjacent hepatocytes (Figures 1-13 and 1-14). Microvilli extend into the bile canaliculi, expanding the surface area of the apical plasma membrane for secretion of bile. Communication between hepatocytes is provided by gap junctions, which are an assemblage of many connexons, membrane pores formed by the circular arrangement of six transmembrane proteins called connexins. Connexons in apposing plasma membranes are directly aligned with each other and form aqueous channels that allow the passage of ions and small molecules. Cellular metabolic products, as well as chemical and electrical signals, can pass from cell to cell. Hepatocytes express specific genes for their unique connexin proteins. Desmosomes, as well as ‘knob and groove’ or interdigitating undulations of adjacent plasma membranes, attach cells together in addition to the tight junctions forming bile canaliculi. The molecular structure of the hepatocyte plasma membrane includes specializations such as membrane proteins that are receptors for hormones, for example insulin and glucagon; and receptors that bind other substances, such as circulating immunoglobulin A (IgA), and also contribute the secretory component required for IgA function. Assorted carrier and channel protein membrane components regulate/facilitate the great variety of substances that enter and leave hepatocytes by ways other than receptors, endocytosis, and exocytosis. Hepatocyte uptake and the release of glucose affects the regulation of blood glucose levels, and also accounts for the variable intracellular glycogen deposits that have been characterized in a variety of physiological conditions.
NUCLEUS Hepatocytes have one or two spherical nuclei containing one or more prominent nucleoli (Figures 1-13 and 1-14).1,2,8,38 Some of the nuclei are polyploid and their number increases with age.42 Polyploid nuclei are characterized by their greater size, which is proportional
to their ploidy. Multinucleated hepatocytes and polyploidy are consistent with high cellular function and demands, and are mechanisms by which both nuclear and cytosomal ‘machinery’ are increased to meet these functional demands. The high level of hepatocellular activity is also reflected in the high percentage of nuclei that are euchromatic, which indicates that transcription of most of the genome is occurring continuously; thus, almost all of the deoxyribonucleic acid (DNA) is in the extended configuration, and little heterochromatin is observed. Hepatocytes engaged in the synthesis of many proteins have a large nucleolus (sometimes several) that can be recognized by light microscopy, and this characteristic is typical of hepatocytes. Electron microscopy reveals the nucleolus to consist of pale-staining areas of nucleolar organizer DNA, an electron-dense granular portion of ribonucleoprotein particles forming ribosomal subunits, and a fibrillar region of transcripts of rRNA. Heterochromatic nucleolar-associated chromatin is found at the nucleolus periphery. Nucleoli are the sites of the translation of rRNA into protein-rich ribosomal subunits that exit the nucleus through pores in the double membrane nuclear envelope.
ENDOPLASMIC RETICULUM, RIBOSOMES, AND GOLGI APPARATUS Rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), and Golgi complex are abundant in mammalian hepatocytes (Figures 1-13 and 1-14).1,2,38,43,44 Their functions are related mainly to the synthesis and conjugation of proteins, metabolism of lipids and steroids, detoxification and metabolism of drugs, and breakdown of glycogen. The endoplasmic reticulum forms a continuous three-dimensional network of tubules, vesicles, and lamellae.43,45 About 60% of the endoplasmic reticulum has ribosomes attached to its cytoplasmic surface and is known as the RER. The remaining 40% constitutes the SER, which lacks a coating of ribosomes. The membranes of the endoplasmic reticulum are 5–8 nm thick. The lumen of the RER is about 20–30 nm in width, whereas that of the SER is larger (30–60 nm). The morphological characteristics and amount of the endoplasmic reticulum may vary in the different zones of the liver lobule. RER is arranged in aggregates of flat cisternae that may be found in the whole cytoplasm. It is more frequently distributed in the perinuclear, pericanalicular, and vascular regions of hepatocytes, and it is more abundant in periportal cells than in centrilobular cells.46,47 The numerous attached membrane-bound ribosomes consist of a large and a small subunit; the former is the part found attached to the RER. Free ribosomes and polyribosomes are also present within the hepatocyte cytoplasm. Ribosomes contain RNA and ribosomal proteins and play a key role in the synthesis of proteins. SER is less common and has a more complex arrangement than RER.38 It is usually much more abundant in centrilobular than in periportal hepatocytes.46–48 The cytoplasm within the SER tubules is usually slightly more electron dense than the surrounding cytoplasm. SER membranes are irregular in size and present a tortuous course. They may be tubular or vesicular in type, with a width of 20–40 nm. SER is mainly distributed near the periphery of the cell. It is often in close relation to RER and Golgi membranes, as well as to glycogen inclusions.49 The ER is not the only site for the protein synthesis in hepatocytes. Abundant free ribosomes in the cytoplasm participate in the
11
Section I. Pathophysiology of the Liver
synthesis of some proteins that will be secreted, but especially of all structural proteins for the hepatocyte. Messages encoding proteins that are to remain within the cytoplasm or are destined to enter the nucleus, peroxisomes, or mitochondria are completely synthesized by free ribosomes. The Golgi complex is a three-dimensional structure in hepatocytes consisting of numerous membranes and vacuoles.8,38,43,44 Multiple Golgi complexes exist in each hepatic parenchymal cell. Whether or not these complexes are connected with each other (functionally forming a single large organelle) is uncertain. The Golgi generally is distributed near the bile canaliculus or nucleus. The Golgi apparatus presents a characteristic heterogeneity. It is usually formed by a stack of four to six parallel cisternae, often with dilated bulbous ends containing electron-dense material. The cisternae may show a size up to 1 mm in diameter with a lumen 30 nm wide. This structure shows a convex or proximal part facing the nucleus and the endoplasmic reticulum (cis Golgi) where small vesicles transfer proteins from the endoplasmic reticulum, and a concave part (trans Golgi) where vesicles and vacuoles (secretory granules) originate to transport the contained secretory proteins to the plasma membrane for discharge into the space of Disse. Cis and trans Golgi are connected by means of the medial Golgi. The latter is the intermediate station between endoplasmic reticulum and Golgi products, such as secretory granules or secondary lysosomes (GERL). This arrangement of Golgi stacks corresponds to its morphofunctional polarization related to the pathway of protein passage through this structure. Proteins in fact enter via the cis Golgi, pass through the medial Golgi and leave this structure via the exit pole (trans Golgi). Two main types of secretory vesicle can be considered within the Golgi apparatus: smaller presecretory granules of 50 nm in diameter and larger secretory granules of 400–600 nm in diameter containing proteins such as very low-density lipoproteins.50
MITOCHONDRIA Mitochondria are large organelles and are very numerous in hepatocytes (1–2000/cell) (Figures 1-13 and 1-14), making up about 18–20% of the cell volume.51 They play a role in the oxidative phosphorylation and oxidation of fatty acids and in all metabolic processes of the hepatocyte.38 Although the mitochondria are dispersed ubiquitously within hepatocytes, they are more concentrated near sites of ATP utilization52 and are often associated with the RER.53 Such a relationship seems to be important during the formation of cytoplasmic membranes (SER) and cytochromes.53 Mitochondria in hepatocytes may be round or elongated, have a width of 0.4–0.6 mm and a length of 0.7–1.0 mm. Longer (up to 4 mm) and larger (up to 1.5 mm in diameter) mitochondria are more numerous in periportal hepatocytes.46,51 Mitochondria are bounded by an outer and an inner membrane, each 5–7 nm thick. The outer membrane possesses special small pores, which allow the passage of molecules smaller than about 2000 daltons (Da). The inner membrane’s surface area is greatly increased by the presence of numerous cristae, which fold within the mitochondrial matrix. The space between inner and outer membranes presents a low-density matrix and ranges from about 7 to 10 nm in thickness. Mitochondria show a relatively low-density matrix in which lamellar or tubular cristae and a variable amount of small dense granules can be observed. The dense granules have a diameter of 20–50 nm. In addition, filaments
12
of the circular mitochondrial DNA about 3–5 nm in width and granules containing mitochondrial RNA of about 12 nm in diameter are also present. The DNA codes for some of the mitochondrial proteins that are synthesized in ribosomes within the organelle, but most of the mitochondrial protein is encoded by nuclear DNA. Mitochondria are self-replicating and have a half-life of approximately 10 days.
LYSOSOMES Lysosomes in hepatocytes (Figures 1-13 and 1-14) consist of a heterogeneous population of organelles containing hydrolytic enzymes that are morphologically and functionally interrelated.38,54,55 These organelles form rounded single-membrane-bound dense bodies, autophagic vacuoles, multivesicular bodies, coated vesicles, and the GERL. The latter is like a cytoplasmic pool of structures located proximal to the Golgi apparatus (but is not part of it), consisting of smooth-surfaced membranes (like a specialized area of smooth ER) with the same hydrolase activity of the lysosome (but without the typical morphology of spherical organelles) that probably have a major role in the formation of lysosomes and hepatocyte lipoprotein metabolism. Several classes of lysosome can be identified within the hepatocyte cytoplasm: (1) the primary lysosomes, small in size, which are considered from a functional point of view to be in a resting phase; (2) the secondary lysosomes, which are functionally activated; (3) the autophagic vacuoles, containing parts of the degrading cytoplasmic organelles, and which often are delimited by a double membrane; and, finally (4) the residual bodies, which are larger than primary and secondary lysosomes and are usually more frequent in older organisms. The residual bodies contain the residues of non-digested material or pigments such as lipofuscins (which are considered undigestible permanent residues). Lipofuscin granules are the most numerous lysosomal bodies present in human hepatocytes.47 Lysosomes are frequently found near the plasma membrane proximal to the bile canaliculus, forming the so-called ‘peribiliary dense bodies’ of early histological descriptions. The lysosomes in periportal hepatocytes are often larger and more positive for acid phosphatase than those in centrilobular hepatocytes.47,48
PEROXISOMES (MICROBODIES) Peroxisomes are subcellular organelles that are usually rounded or slightly oval in shape, surrounded by a single membrane (Figure 1-14), and participate mainly in oxidative processes.38,56,57 Each hepatocyte may contain 300–600 peroxisomes. These organelles are characteristically more numerous and larger in hepatocytes than in other mammalian cells.47 They contain a fine granular matrix in which in some species (but not in humans) a denser paracrystalline structure may be present. The peroxisome size ranges between 0.2 and 1.0 mm. They are often found grouped in clusters near the endoplasmic reticulum. However, the presence of direct connections (the so-called ‘tails’) with endoplasmic reticulum or other peroxisomes (peroxisomal reticulum) is still under investigation. Peroxisomes may be more numerous in pericentral hepatocytes, but they are generally homogeneously distributed within the hepatic lobule.47,48 The origin and formation of peroxisomes is still under debate. Nevertheless, they have been said to originate as a focal protrusion of the RER.
Chapter 1 ANATOMY OF THE LIVER
CYTOPLASMIC INCLUSIONS The hepatocyte is extremely rich in cytoplasmic inclusions. These are functionally related to the enhanced metabolic activity of the liver cells. The more frequently observed cytoplasmic inclusions are glycogen granules, lipid droplets, and pigments of various natures.38 Glycogen granules are the most abundant inclusions in normal hepatocytes (Figures 1-13 and 1-14).38,47 At the electron microscopy level they are stained by lead salts, and may occur either in the monoparticulate form (b particles, 15–30 nm in size) or, more frequently, as aggregates of smaller particles arranged to form ‘rosettes’ (a particles). Glycogen granules are dispersed in the cytoplasm, but are often associated with the SER. Glycogen is depleted during fasting, disappearing first from periportal hepatocytes and then from centrilobular cells. Upon refeeding, the sequence reverses. Lipid inclusions appear as empty vacuoles or osmiophilic droplets usually not surrounded by membranes. Fat droplets may vary in size and quantity, and correspond mainly to triglyceride levels in the hepatocyte.48 A variable amount of iron-containing granules may often be present within the hepatocyte cytoplasm. These are related to the apoferritin–ferritin system (the so-called ‘hepatic iron buffer’). Liver iron metabolism occurs in hepatocytes; nevertheless, the pathway of iron transport from the blood to the hepatocytes has not yet been fully elucidated. In addition to hepatocytes, liver endothelial cells and Kupffer cells58,59 also possess receptors for transferrin, a glycoprotein implicated in cellular iron uptake, thus suggesting that iron transport involves a transendothelial (transcytosis) mechanism. Hepatocytes contain iron in the form of ferritin particles, i.e. an iron-containing protein ultrastructurally characterized by a roughly spherical shape and comprising a protein shell (apoferritin) 11 nm in diameter and a central core of about 5 nm in diameter containing iron. Hepatocyte iron deposits may also occur as single-membrane-bound lysosomal bodies (residual bodies) forming aggregates of iron-containing electron-dense particles (siderosomes–hemosiderin granules).
CYTOSKELETON AND CYTOMATRIX The cytoskeleton is a structure that is thought to regulate the shape, subcellular organization and movements of the cells. In the hepatocyte, the cytoskeletal organization50,60,61 is dependent on the arrangement of the three main components of this structure: the microfilaments, the intermediate filaments, and the microtubules. These filament types are regularly distributed in the cytoplasm and characterize the cytomatrix, which together with other finer filaments (microtrabeculae) is thought to give the ‘gel’ consistency to the cell cytoplasm. Microfilaments, made of actin, and microtubules, consisting of tubulin, are both related to intracellular motility. Microtubules are thought to be involved in determining cell shape, in mitosis, and in regulating the intracellular transport of vesicles.62 Especially in the liver, these structures assume a relevant role in the secretion of lipoproteins, albumin, and the release of lipids into bile. Microfilaments are more directly related to bile secretion. In fact, they are normally found around the bile canaliculi (pericanalicular web). Many experimental studies have shown that microfilaments play an active role in the dilatation and contraction of bile canaliculi.63–65 Thus, they may control the bile canalicular
caliber and bile flow. Intermediate filaments show a more complex architecture. They correspond to the epithelial cell ‘tonofilaments’ of the old nomenclature. In the liver they show a relationship with the Mallory bodies (the structural marker of human alcoholic liver disease).66 They are located around the nucleus, near the cell border, in the cytoplasmic network and around the bile canaliculi. There is very little information on the presence of microtubules or microfilaments in differentiating hepatocytes. In mice, these structures have been recognized as dense bundles occurring near the nucleus and the plasma membrane in late developmental stages.67 Their presence could have some importance in bile canaliculus and desmosome differentiation.
NON-PARENCHYMAL CELLS The hepatic sinusoid is an unique, dynamic microvascular structure which serves as the principal site of exchange between the blood and the perisinusoidal space (of Disse), into which project the microvilli of the hepatic parenchymal cells that form the external lining of this space.15 The sinusoid is composed of non-parenchymal cells, of which there are four recognized types (Figures 1-15 and 1-16).15,68 These are fenestrated endothelial cells and phagocytic Kupffer cells, which form the sinusoid lining that is in contact with the blood; extraluminal fat-storing cells (of Ito), also referred to as stellate cells, lipocytes, or perisinusoidal cells, which serve as specialized pericytes extending processes throughout the space of Disse; and pit cells, which are immunoreactive natural killer (NK) cells that are attached to the luminal surface of the sinusoid and are part of a population of liver-associated lymphocytes (LAL).69 Additional cells and cell processes may be present in the perisinusoidal space (of Disse) of some species, most notably mast cells in the dog70 and adrenergic and peptinergic nerves in most mammalian species except mouse and rat.23 The perisinusoidal space is thought by some to function as a lymphatic space that channels plasma to the true lymphatics coursing in the portal tract. Although this hypothesis would help to explain the large efflux of lymph from the liver, it may not be valid as anatomic connections between the space of Disse and the portal tract have not been identified.17,18 For a review of intrahepatic lymphatics see Trutmann and Sasse.17 The majority of the non-parenchymal cells have been studied both in situ and in vitro. Together, sinusoidal cells represent about 6% of the total liver volume, but account for 30–35% of the total number of liver cells.71,72 The purpose of this chapter is to present an overview of the structural and functional features of these sinusoidal cells, which together provide a physical and selective barrier between the blood and the parenchyma that is dynamic and responsive to a wide variety of physical and chemical stimuli. Whereas sinusoidal lining cells have the capacity to divide and proliferate, especially when stimulated by immune system modifiers,73 sinusoidal macrophages and NK cells may also be increased in numbers by the respective recruitment and subsequent modification of monocytes and lymphocytes, principally of bone marrow origin.74
SINUSOIDAL ENDOTHELIAL CELLS Like endothelial cells in capillaries elsewhere in the body, contiguous sinusoidal endothelial cells in the liver form the basic tubular
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Section I. Pathophysiology of the Liver
KC
E
SP
SD
SC
BC
HC
Figure 1-15. Sinusoid wall and contiguous hepatic parenchymal cells (HC). E, endothelium; KC, Kupffer cell; SD, space of Disse; SC, stellate cell; SP, sieve plate of fenestrae; BC, bile canaliculus. (Modified from McCuskey RS. In: Tavoloni N, Berk PD, eds. Hepatic transport and bile secretion. New York: Raven Press, 1993: 6, ©1993, with permission from Lippincott, Williams and Wilkins (http://lww.com).)
14
Figure 1-17. Sinusoidal endothelial cell with limited perinuclear cytoplasm, containing a few organelles, such as mitochondria, a lysosome, and a few cisternae of endoplasmic reticulum. The endothelial cell rests on the microvilli filling the space of Disse. L, sinusoidal lumen; N, nucleus. (Modified from Wisse E, Braet F, et al. Structure and function of sinusoidal lining cells in the liver. Toxicol Pathol 1996;24:100–111, with permission.)
Figure 1-16. Sinusoid (S) lined by endothelial cells (SEC) having attenuated cytoplasm with Kupffer cell (KC) attached to the luminal surface and a stellate cell (SC) lying externally in the space of Disse. (Modified from McCuskey RS. Functional morphology of the liver with emphasis on its microvasculature. In: Tavoloni N, Berk PD, eds. Hepatic transport and bile secretion. New York: Raven Press, 1993: 6, ©1993, with permission from Lippincott, Williams and Wilkins (http://lww.com).)
Figure 1-18. Sinusoid illustrating fenestrae organized in clusters as ‘sieve’ plates (arrowheads). SD, space of Disse; H, hepatic parenchymal cell. (Reproduced from McCuskey RS. Functional morphology of the liver with emphasis on its microvasculature. In: Tavoloni N, Berk PD, eds. Hepatic transport and bile secretion. New York: Raven Press, 1993: 7, ©1993, with permission from Lippincott, Williams and Wilkins (http://lww.com).)
vessel for transvascular exchange between the blood and the surrounding tissue (Figure 1-15) and represent approximately 50% of the numbers and volume of sinusoidal cells.71,72 The morphology of hepatic sinusoidal endothelial cells has been reviewed by several authors.68,75 These cells are unique to the liver in that their extensive, attenuated cytoplasm contains numerous fenestrae, approximately 170 nm in diameter, which lack diaphragms and which are
clustered together in groups known as ‘sieve plates’76 (Figures 1-16–1-18). In addition, this specialized endothelium generally lacks a basal lamina during health, so that solutes and small particles have direct access to the perisinusoidal space containing processes of fat-storing cells and the microvilli of hepatic parenchymal cells. The endothelium of the sinusoids exhibits heterogeneity. The fenestrae are not uniform in size or distribution throughout the length
Chapter 1 ANATOMY OF THE LIVER
of the sinusoid, from its origin at the portal venule to its termination in the central venule. At the periportal end of the sinusoid the fenestrae are somewhat larger than those located centrilobularly, but their numbers are fewer which, when combined with the sinusoid having a smaller diameter at the periportal rather than the centrilobular end, results in a higher centrilobular endothelial porosity.76–78 The functional significance of these regional differences is unclear, but it is tempting to relate them to the functional metabolic heterogeneity that has been demonstrated for hepatocytes in different regions of the lobule,35,79–81 as well as the portal-to-central intralobular oxygen gradient.82 The fenestrae constitute only 6–8% of the surface area of the endothelial lining. They form a selective barrier between the blood and parenchyma that acts as a dynamic, selective sieve for particulates such as chylomicron remnants.76,83 Transport of particulates somewhat larger than the size of the fenestrae is postulated to be accomplished by the ‘forced sieving’ and ‘endothelial massage’ concomitant with the passage of blood cells, particularly leukocytes, through the sinusoids and the resulting interaction of these cells with the endothelial wall.76 The endothelial fenestrae are dynamic structures whose diameters are affected by luminal blood pressure, vasoactive substances, drugs, and toxins.76,83–85 The mechanism for active control of the diameters of these fenestrae appears to reside in actin-containing components of the cytoskeleton.86–89 Additional cytoskeletal components form rings that delineate both the fenestrae and the sieve plates.87,90 As a result, the fenestrae are thought to regulate the passage of large substances such as chylomicron remnants through the endothelium while allowing free exchange of plasma and large proteins between the blood and the space of Disse. Thus, the sinusoidal endothelial filter influences the fat balance between the liver and other organs, the cholesterol level in the plasma, and the delivery of retinoids to parenchymal and fat-storing cells. There is a reduction of the numbers of fenestrae with age.91–93 The surfaces of the sinusoidal endothelial cells are relatively smooth compared to that of Kupffer cells and are generally lacking in filopodia or lamellopodia (Figures 1-16–1-18). The perikaryon contains mitochondria, some scattered components of both smooth and rough endoplasmic reticulum, and a well developed Golgi apparatus. Throughout the cytoplasm are located numerous vacuoles and organelles associated with the uptake, transport and degradation of material. These include bristle-coated pits, which are invaginations from the cell membrane, bristle-coated micropinocytotic vesicles, endosomes, transfer tubules, and lysosomes.68,94 The fact that these endothelial cells contain 45% by volume of the pinocytotic vesicles in the liver as well as 14% of the lysosomes71,72 indicates the high degree of endocytotic activity present in these cells. The variety of substances known to be endocytosed by sinusoidal endothelial cells includes proteins, glycoproteins, lipoproteins, glycosaminoglycans95,96 and, under certain conditions, larger particulates which are phagocytosed in the absence of functional Kupffer cells.97 A number of receptors to accomplish this have been identified on the cell surface, including Fc receptors for immune complexes, transferrin (Tf) receptors, scavenger receptors, mannose, galactose, apo E and C-III receptors. Of these, the scavenger and apo-E receptors are particularly abundant on endothelial cells compared to Kupffer cells, as are mannose/N-acetyl glucosamine recep-
tors. The former indicate the important role played by the sinusoidal endothelial cells in the processing and metabolism of lipoproteins. Recently, they have been demonstrated to play a significant role in the removal of AGE molecules.93 The endothelial cells also are secretory and release interleukin (IL)-1, IL-6, and interferon.68,95 In addition, these cells produce eicosanoids, particularly PGI2 , PGE2 , and TXA2 , as well as endothelin and nitric oxide.68 Thus, along with Kupffer cells, the endothelium participates in host defense mechanisms and regulation of sinusoidal blood flow in the liver. Finally, sinusoidal endothelial cells constitutively express the intercellular adhesion molecule ICAM-1, which along with VCAM-1 is up-regulated by inflammatory stimuli either directly or by mediators released from stimulated Kupffer cells, resulting in increased adhesion of leukocytes to the endothelial surfaces.98
KUPFFER CELLS Kupffer cells constitute the largest population of fixed macrophages in most vertebrates. They are components of the walls of hepatic sinusoids and play a significant role in the removal by endocytosis of particulates and cells from the portal blood, as well as toxic, infective and foreign substances, particularly those of intestinal origin.96 Kupffer cells also are the source of a variety of beneficial, vasoactive, and toxic mediators which are thought to be involved in host defense mechanisms, as well as some disease processes in the liver.96,99 Included among the substances released are eicosanoids, free radicals, cytokines, interferon, platelet-activating factor, and lysosomal enzymes. The morphology of mammalian Kupffer cells, including those in humans, has been described and extensively reviewed.96,100 Kupffer cells are macrophages that constitute one of the cellular components of hepatic sinusoids (Figures 1-15, 1-16, 1-19, 1-20). In this site they are anchored to the luminal surface of the sinusoidal endothelium and thus are exposed to the bloodstream. Occasionally, Kupffer cells also are interdigitated between endothelial cells. However, Kupffer cells are unevenly distributed within hepatic lobules, with
Figure 1-19. Kupffer cell (KC) attached to luminal surface of sinusoidal endothelium by processes that penetrate fenestrae. (Modified from McCuskey RS. Functional morphology of the liver with emphasis on its microvasculature. In: Tavoloni N, Berk PD, eds. Hepatic transport and bile secretion. New York: Raven Press, 1993: 7, ©1993, with permission from Lippincott, Williams and Wilkins (http://lww.com).)
15
Section I. Pathophysiology of the Liver
Figure 1-20. Kupffer cell, having lysosomes with varying density and diameter, vacuoles, and a nucleus (N). Kupffer cells are sometimes seen in direct contact with the microvilli of the parenchymal cells (arrowhead). L, sinusoidal lumen; f, fenestrae; SD, space of Disse. (Modified from Wisse E, Braet F, et al. Structure and function of sinusoidal lining cells in the liver. Toxicol Pathol 1996;24:100–111, with permission.)
the majority being found in the periportal region, where they are larger and have greater phagocytic activity than Kupffer cells located in the centrilobular region of the lobule.73,101–103 In addition, Kupffer cells are often located at the junctions of sinusoids. As a result, the majority of Kupffer cells are strategically located to remove foreign materials as they enter the liver lobule. Kupffer cells often present a large irregular surface, caused by numerous microvilli, filopodia and lamellopodia extending from the cellular surface (Figures 1-16 and 1-19).100 Attachment to the endothelium appears to be by cytoplasmic processes which often penetrate the endothelial fenestrae to enter the space of Disse, where they may come in contact with fat-storing cells and, occasionally, parenchymal cells. Other processes frequently extend across the lumen to anchor in the opposite wall of the sinusoid. As a result, Kupffer cells often have a branched or ‘stellate’ appearance. Whereas Kupffer cells frequently contact other sinusoidal cell types, no organized junctions have been visualized between Kupffer cells and these contiguous cells. The surface of Kupffer cells is covered with a fuzzy coat of unknown composition which normally is not preserved by perfusion fixation with glutaraldehyde.104,105 It can, however, be seen coating the inner surface of the membranes of large pinocytotic vacuoles, and as a dense midline within membranous invaginations known as ‘worm-like’ bodies or vermiform processes.106 These structures are thought to be unique to Kupffer cells, as are annulate lamellae.106 The latter are sometimes found connected to the rough endoplasmic reticulum (RER) and are thought to represent a particular arrangement of the RER. These latter two structures, along with the nuclear membrane, stain positive for endogenous peroxidase. Although this is a specific marker for Kupffer cells in the rat liver105,107,108 it is not as useful in other species, because of a similar positivity in large numbers of endothe-
16
lial cells. More recently, monoclonal antibodies also have been used to identify macrophages and Kupffer cells.109–111 In addition to the above structures, the cytoplasm of Kupffer cells contains bristle-coated micropinocytotic vesicles and a number of clear vacuoles and dense bodies (lysosomes) which, along with the vermiform processes and fuzzy-coated vacuoles, are involved in the high level of endocytotic and digestive activity attributed to these cells.96,106 Additionally, the usual set of cellular organelles is also present in the cytoplasm, including mitochondria, RER, free ribosomes, Golgi apparatuses, microtubules, microfilaments, intermediate filaments, centrioles and a nucleolus.96,100,106 However, fat droplets, autophagic vacuoles, multivesicular bodies, peroxisomes, and smooth endoplasmic reticulum have not been reported in Kupffer cells in situ. The endocytotic mechanisms of Kupffer cells have been studied both in situ and, in greater detail, in isolated cultured cells. Four morphologically recognizable endocytotic mechanisms for Kupffer cells fixed in situ by perfusion have been described: bristle-coated micropinocytosis; pinocytosis veriformis; pinocytosis (fuzzy-coated vacuole); and phagocytosis.96,106 Of these, the principal endocytotic mechanisms, both in vivo and in vitro, are thought to be phagocytosis and bristle-coated micropinocytosis. Phagocytosis of particulates larger than 0.3–0.5 mm (e.g. latex, bacteria, etc.) is performed by hyaloplasmic pseudopodia, which extend from the cell surface to engulf the particulate. Phagocytosis of particulates >0.5 mm, e.g. latex, has been used as a marker to distinguish Kupffer cells from other sinusoidal lining cells under normal conditions.108 However, as noted previously the sinusoidal endothelium is also capable of phagocytosing latex particles if Kupffer cells are injured.97 Bristlecoated micropinocytosis is thought to be responsible for both receptor-mediated and non-receptor mediated fluid-phase endocytosis. Several receptors have been demonstrated on Kupffer cells, including Fc and C3 receptors, N-acetyl-D-galactosamine receptors, and N-acetyl-glucosamine/mannose receptors. The origin and cell kinetics of Kupffer cells continues to be debated between those who are proponents of a monocytic origin and those favoring self-replication.73,112–114 Taken together, the data seem to support both points of view. Kupffer cells during health have long residence times and slow rates of self-replication, augmented by some recruitment and transformation of monocytes. Monocyte recruitment becomes more important during stimulation of Kupffer cell function (e.g. zymozan, BCG, etc.).115–118
STELLATE CELLS External to the endothelium, perisinusoidal cells known as stellate cells (previously known as fat-storing cells, Ito cells or lipocytes) are located in the space of Disse (Figures 1-15, 1-16, 1-21), with a higher frequency in the periportal area.119–121 These cells contain fat droplets and are the major storage site of retinoids, including vitamin A, which emits a characteristic, rapidly quenched autofluorescence when excited with ultraviolet light at 328 nm. Two types of fat droplet are recognized, one with and one without a limiting membrane.120 The nuclear area of the stellate cell is frequently located in recesses between hepatic parenchymal cells, whereas the thin, multiple cytoplasmic processes of these cells course though the perisinusoidal space and extensively embrace the abluminal surfaces of
Chapter 1 ANATOMY OF THE LIVER
Figure 1-21. Stellate cell lying within the space of Disse, covered by the endothelial lining. Fat droplets (*) and cisternae of the endoplasmic reticulum are located in the cytoplasm. A small bundle of collagen fibers (arrow) is associated with the cell. L, sinusoidal lumen; f, fenestrae; N, nucleus; SD, space of Disse. (Modified from Wisse E, Braet F, et al. Structure and function of sinusoidal lining cells in the liver. Toxicol Pathol 1996;24:100–111, with permission.)
the endothelium that surrounds the sinusoid like a cylindrical basket.122 This close relationship of the processes of the stellate cell to the sinusoid wall, the presence of large numbers of cytoplasmic microtubules and microfilaments, the positive immunostaining of desmin and a-smooth muscle actin, and the close association of nerve fibers (Figure 1-10), coupled with the demonstration of contractile activity in these cells both in vivo and in vitro, strongly suggests that stellate cells play a role in the local regulation of blood flow through the hepatic sinusoids.123–126 In health, little or no basal lamina and collagen is associated with the sinusoidal endothelium. As a result, the sinusoid wall is a highly permeable structure that permits continuity of plasma between the blood and the hepatocyte. However, during certain types of liver injury, e.g. cirrhosis, basement membrane material and collagen fibrils accumulate in the perisinusoidal space, resulting in ‘capillarization’ of the sinusoid and impaired transvascular exchange.127 The perisinusoidal stellate cells are thought to be responsible for the synthesis of this material, following their transformation into myofibroblast-like cells having reduced numbers of fat droplets and vitamin A, as well as an increased capacity to secrete extracellular matrix materials, including collagen types I and III–VI, fibronectin, laminin, tenascin, undulin, hyaluronic acid, biglycan, decorin, syndecan-containing chondroitin sulfate, heparan, and dermatan sulfate.128
LIVER-ASSOCIATED LYMPHOCYTES 129
Pit cells are derived from circulating large granular lymphocytes (LGL)130 that become attached to the sinusoidal wall (Figure 1-22) and which possess natural killer (NK) activity and are part of a population of liver-associated lymphocytes (LAL).69,131,132 Pit cells contain azurophilic granules which stain for acid phosphatase, sug-
Figure 1-22. Pit cell with typical dense granules. This pit cell is in close contact with the endothelial lining and is seen to contact microvilli of the parenchymal cells (arrowhead). Ec, endothelial cell; f, fenestrae; L, sinusoidal lumen; N, nucleus; SD, space of Disse. (Modified from Wisse E, Braet F, et al. Structure and function of sinusoidal lining cells in the liver. Toxicol Pathol 1996;24:100–111, with permission.)
gesting that they are lysosomal in nature.133,134 In addition, the cytoplasm of these cells contains characteristic rod-cored vesicles as well as multivesicular bodies, a Golgi apparatus, and mitochondria, all of which exhibit polarity toward one side of an eccentric, indented nucleus. Although the majority of attachments to the sinusoidal wall are to endothelial cells, adhesion to Kupffer cells is not uncommon. Pit cells have been shown to spontaneously kill tumor cells as well as produce a cytolytic factor which is up-regulated by biological response modifiers such as zymosan, as well as by interleukin-2.131 These substances also induce proliferation of pit cells, as does partial hepatectomy, perhaps through the activation of Kupffer cells. Finally, two types of pit cell have been recognized: high density (HD) and low density (LD). The LD pit cells have a larger number of granules, which are also smaller than those in HD cells; in addition, LD cells exhibit more cytotoxicity.135
HETEROGENEITY Within the hepatic lobules, the parenchyma exhibits considerable heterogeneity along the portal venous–central venous axis, both ultrastructurally and in various enzyme activities. This results in an intralobular metabolic zonation, with different cellular functions represented in different zones within each lobule.79,80 For example, the key enzymes involved in glucose uptake and release and in the
17
Section I. Pathophysiology of the Liver
formation of urea and glutamine are reciprocally located with glucogenic and urea cycle enzymes, principally in the periportal zone, and glycolytic and glutaminogenic enzymes in the centrilobular zone. Mixed-function oxidation and glucuronidation are mainly centrilobular functions, whereas sulfation is principally a periportal function. This zonation of enzymatic functions also is reflected ultrastructurally in differences in mitochondria and smooth endoplasmic reticulum between different zones. As a result of this zonation, as well as the portal–central oxygen gradient, most toxicologic and pathologic events in the liver show a considerable degree of zonal preference. An example of toxicants eliciting periportal injury is allyl alcohol; carbon tetrachloride and acetaminophen elicit centrilobular injury. The sinusoids are composed of specialized non-parenchymal cells and also exhibit structural and functional heterogeneity.15,16 Near their origins from portal venules and hepatic arterioles, sinusoids are slightly narrower as well as being tortuous and anastomotic, forming interconnecting polygonal networks; farther away from the portal venules the sinusoids become organized as parallel vessels that terminate in central venules (terminal hepatic venules). Short intersinusoidal sinusoids connect adjacent parallel sinusoids. The volume of liver occupied by sinusoids in the periportal area is also greater than that surrounding central venules. However, because of the smaller size and the anastomotic nature of the periportal sinusoids, the surface available for exchange in this area (surface/volume ratio) is greater than in centrilobular sinusoids. The size and pattern of distribution of endothelial fenestrae differs along the length of the sinusoid. At the portal end, the fenestrae are larger but comprise less of the endothelial surface area than they do in the pericentral region. The functional significance of these regional differences is unclear, but relates to the functional metabolic heterogeneity that has been demonstrated for hepatocytes in different regions of the lobule. This, in turn, may depend on the recognized portal–central intralobular oxygen gradient.
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54. Novikoff AB, Novikoff PM. Lysosomes. In: Arias IM, Jakoby WB, Popper H, et al., eds. The liver: biology and pathobiology. New York: Raven Press, 1988: 227–239. 55. Sahagian GG, Novikoff PM. Lysosomes. In: Arias IM, Boyer JL, Fausto N, et al., eds. The liver: biology and pathobiology. New York: Raven Press, 1994: 275–291. 56. Lazarow PB. Peroxisomes. In: Arias IM, Boyer JL, Fausto N, et al., eds. The liver: biology and pathobiology, 3rd edn. New York: Raven Press, 1994: 293–307. 57. Sotto U, Rapp S, Gorgas K, Just WW. Peroxisomes and lysosomes. In: LeBouton AV, ed. Molecular and cell biology of the liver. Boca Raton: CRC Press, 1994:; 181–262. 58. Kishimoto T, Tavassoli M. Transendothelial transport (transcytosis) of iron-transferrin complex in the rat liver. Am J Anat 1987;178:241–249. 59. Soda R, Tavassoli M. Blood 1984;63:270–276. 60. Feldmann G. The cytoskeleton of the hepatocyte. Structure and functions. J Hepatol 1989;8:380–386. 61. Philips MJ, Satir P. The cytoskeleton of the hepatocyte: organisation, relationships and pathology. In: Arias IM, Jakoby WB, Popper H, et al., eds. The liver: biology and pathobiology. New York: Raven Press, 1988: 11–27. 62. Crawford J. The role of vesicle-mediated transport pathways in hepatocellular bile secretion. Semin Liver Dis 1996;16:169–189. 63. Philips MJ, Oshio C, Miyami M, et al. A study of bile canalicular contractions in isolated hepatocytes. Hepatology 1982;2:763–768. 64. Kawahara H, French SW. Role of cytoskeleton in canalicular contraction in cultured differentiated hepatocytes. Am J Pathol 1990;136:521–532. 65. Watanabe N, Tsukada N, Smith CR, Phillips MJ. Motility of bile canaliculi in the living animal: implications for bile flow. J Cell Biol 1991;113:1069–1080. 66. Phillips MJ. A study of bile canalicular contractions in isolated hepatocytes. Lab Invest 982;47:311–313. 67. Sugisaki T, Sagakuchi T. Intracytoplasmic tonofilaments: a desmosome-like structure in the mouse fetal liver cell. J Ultrastruct Res 1977;59:178–184. 68. Wisse E, Braet F, Luo DZ, et al. Structure and function of sinusoidal lining cells in the liver. Toxicol Pathol 1996;24:100–111. 69. Winnock M, Barcina MG, Lukomska B, et al. Liver-associated lymphocytes: Role in tumor defense. Semin Liver Dis 1993;13:81–92. 70. McCuskey PA. Electron and fluorescence microscopic study of mast cells and adrenergic innervation in Beagle dog liver. In: Wisse E, Knook DL, Decker K, eds. Cells of the hepatic sinusoids. Vol. 2. Leiden: Kupffer Cell Foundation, 1989: 260–265. 71. Blouin A. Morphometry of the liver sinusoidal cells. In: Wisse E, Knook DL, eds. Kupffer cells and other liver sinusoidal cells. Amsterdam: Elsevier Biomedical, 1977: 61–71. 72. Blouin A, Bolender RP, Weibel ER. Distribution of organelles and membranes between hepatocytes and non hepatocytes in the rat liver parenchyma. A stereological study. J Cell Biol 1977;72:441–455. 73. Bouwens L, Baekeland M, Zanger RD, Wisse E. Quantitation, tissue distribution and proliferation kinetics of Kupffer cells in normal rat liver. Hepatology 1986;6:718–722. 74. Bouwens L, Knook DL, Kuppen PJK, et al. Electron microscopic observations on the accumulation of large granular lymphocytes (pit cells) and Kupffer cells in the liver of rats treated with continuous infusion of interleukin-2. Hepatology 1990;12:1365– 1370. 75. Brouwer A, Knook DL, Wisse E. Sinusoidal endothelial cells and perisinusoidal fat-storing cells. In: Arias IM, Jakoby WB, Popper H, et al., eds. Liver: biology and pathobiology. New York: Raven Press, 1988: 665–682. 76. Wisse E, DeZanger RB, Jacobs R, et al. The liver sieve: consideration concerning the structure and function of
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endothelial fenestrae, the sinusoid wall and the space of Disse. Hepatology 1985;5:683–692. Horn T, Henriksen JH, Christoffersen P. The sinusoidal lining cells in ‘normal’ human liver. A scanning electron microscopic investigation. Liver 1986;6:98–110. Vidal-Vanaclocha F, Barbera-Guillem E. Fenestration patterns in endothelial cells of rat liver sinusoids. J Ultrastruct Res 1985;90:115–123. Gumucio JJ, Bilir BM, Moseley RH, Berkowitz CM. The biology of the liver cell plate. In: Arias I, Boyer JL, Fausto N, et al., eds. The liver: biology and pathobiology, 3rd edn. New York: Raven Press, 1994: 1143–1163. Jungermann K. Metabolic zonation of liver parenchyma. Semin Liver Dis 1988;8:329–341. Teutsch HF. Regionality of glucose-6-phosphate hydrolysis in the liver lobule of the rat: Metabolic heterogeneity of ‘portal’ and ‘septal’ sinusoids. Hepatology 1988;8:311–317. Lemasters JJ, Ji S, Thurman RG. Centrilobular injury following hypoxia in isolated, perfused rat liver. Science 1981; 213:661–663. Fraser R, Day WA, Fernando NS. Review: The liver sinusoidal cells. Their role in disorders of the liver, lipoprotein metabolism and atherogenesis. Pathology 1986;18:5–11. Fraser R, Dobbs BR, Rogers GWT. Lipoproteins and the liver sieve: The role of the fenestrated sinusoidal endothelium in lipoprotein metabolism, atherosclerosis, and cirrhosis. Hepatology 1995;21:863–874. Oda M, Azuma T, Watanabe N, et al. Regulatory mechanism of hepatic microcirculation: Involvement of contraction and dilatation of sinusoids and sinusoidal endothelial fenestrae. In: Hammersen MK, ed. Progress in applied microcirculation. Vol. 17. Basel: Karger, 1990: 103–128. Arias IM. The biology of hepatic endothelial fenestrae. In: Schaffner F, Popper H, eds. Progress in liver diseases. Vol. IX. Philadelphia: WB Saunders, 1990: 11–26. Braet F, DeZanger R, Baekeland M, et al. Structure and dynamics of the fenestrae-associated cytoskeleton of rat sinusoidal endothelial cells. Hepatology 1995;21:180–189. Oda M, Han JY, Yokomori H. Local regulators of hepatic sinusoidal microcirculation: recent advances. Clin Hemorheol Microcirc 2000;23:85–94. Oda M, Yokomori H, Han JY, et al. Hepatic sinusoidal endothelial fenestrae are a stationary type of fused and interconnected caveolae. In: Wisse E, Knook DL, DeZanger R, et al., eds. Cells of the hepatic sinusoid. Vol. 8. Leiden: Kupffer Cell Foundation, 2001: 94–98. Braet F, Spector I, Zanger RD, Wisse E. A novel structure involved in the formation of liver endothelial cell fenestrae revealed by using the actin inhibitor misakinolide. Proc Natl Acad Sci USA 1998;95:13635–13640. Cogger VC, Warren A, Fraser R, et al. Hepatic sinusoidal pseudocapillarization with aging in the non-human primate. Exp Gerontol 2003;38:1101–1107. LeCouteur DG, Fraser R, Cogger VC, McLean AJ. Hepatic pseudocapillarisation and atherosclerosis in ageing. Lancet 2002;359:1612–1615. Smedsrod B, Melkko J, Araki N, et al. Advanced glycation end products are eliminated by scavenger-receptor-mediated endocytosis in hepatic sinusoidal Kupffer and endothelial cells. Biochem J 1997;322:567–573. Wisse E. An electron microscopic study of the fenestrated endothelial lining of rat liver sinusoids. J Ultrastruct Res 1970;31:125–150. Smedsrod B, DeBleser PJ, Braet F, et al. Cell biology of liver endothelial and Kupffer cells. Gut 1994;35:1509–1516. Wisse E, Braet F, Luo D, et al. Structure and function of sinusoidal lining cells in the liver. Toxicol Pathol 1996;24:100–111.
97. Steffan A-M, Gendrault J-L, McCuskey RS, et al. Phagocytosis, an unrecognized property of murine endothelial liver cells. Hepatology 1986;6:830–836. 98. VanOosten M, Bilt Evd, Vries HEd, et al. Vascular adhesion molecule-1 and intercellular adhesion molecule-1 expression on rat liver cells after lipopolysaccharide administration in vivo. Hepatology 1995;22:1538–1546. 99. Decker K. Biologically active products of stimulated liver macrophages. Eur J Biochem 1990;192:245–261. 100. McCuskey RS, McCuskey PA. The fine structure and function of Kupffer cells. J Electron Microsc Tech 1990;14:237–246. 101. McCuskey PA, McCuskey RS. In vivo and electron microscopic study of the development diabetic cerebral microangiopathy. Microcirc Endothel Lymphatics 1984;1:221–224. 102. McCuskey RS, McCuskey PA, Urbaschek R, Urbaschek B. Species differences in Kupffer cells and endotoxin sensitivity. Infection and Immunity 1984;45:278–280. 103. Sleyster ECH, Knook DL. Relation between localization and function of rat liver Kupffer cells. Lab Invest 1982;47: 484–490. 104. Emeis JJ. Morphologic and cytochemical heterogeneity of the cell coat of rat liver Kupffer cells. J Reticuloendothelial Soc 1976;20:31–50. 105. Wisse E. Observations on the fine structure and peroxidase cytochemistry of normal rat liver Kupffer cells. J Ultrastruct Res 1974;46:393–426. 106. Wisse E, Knook DL. The investigation of sinusoidal cells: a new approach to the study of liver function. In: Popper H, Schaffner F, eds. Progress in liver diseases. Vol. VI. New York: Grune & Stratton, 1979: 153–171. 107. Fahimi HD. The fine structural localization of endogenous and exogenous peroxidase activity in Kupffer cells of rat liver. J Cell Biol 1970;47:247–262. 108. Widmann JJ, Cotran RS, Fahimi HD. Mononuclear phagocytes (Kupffer cells) and endothelial cells. Identification of two functional cell types in rat liver sinusoids by endogenous peroxidase activity. J Cell Biol 1972;52:159–170. 109. Bodenheimer HC, Faris RA, Charland C, Hixson DC. Characterization of a new monoclonal antibody to rat macrophages and Kupffer cells. Hepatology 1988;8:1667–1672. 110. Malorny U, Michels E, Sorg C. A monoclonal antibody against an antigen present on mouse macrophages and absent from monocytes. Cell Tissue Res 1986;243:421–428. 111. Ramadori G, Dienes H, Burger R, et al. Expression of Iaantigens on guinea pig Kupffer cells. Studies with monoclonal antibodies. J Hepatol 1986;2:208–217. 112. Bouwens L, Baekeland M, Wisse E. Cytokinetic analysis of the expanding Kupffer-cell population in rat liver. Cell Tissue Kinet 1986;19:217–226. 113. Crofton RW, Dulk MMCD-D, Furth RV. The origin, kinetics, and characteristics of the Kupffer cells in the normal steady state. J Exp Med 1978;148:1–17. 114. Volkman A. Disparity in origin of mononuclear phagocyte populations. J Reticuloendothelial Soc 1976;19:249–268. 115. Bouwens L, Baekeland M, Wisse E. Importance of local proliferation in the expanding Kupffer cell population of rat liver after zymosan stimulation and partial hepatectomy. Hepatology 1984;4:213–229. 116. Bouwens L, Knook DL, Wisse E. Local proliferation and extrahepatic recruitment of liver macrophages (Kupffer cells) in partial-body irradiated rats. J Leukocyte Biol 1986;39:687–697. 117. Bouwens L, Wisse E. Proliferation, kinetics, and fate of monocytes in rat liver during a zymosan-induced inflammation. J Leukocyte Biol 1985;37:531–543. 118. Deimann W, Fahimi H. The appearance of transition forms between monocytes and Kupffer cells in the liver of rats treated with glucan. J Exp Med 1979;149:883.
Chapter 1 ANATOMY OF THE LIVER
119. Wake K. Sternzellen in the liver: Perisinusoidal cells with special reference to storage of vitamin A. Am J Anat 1971;132:429–461. 120. Wake K. Development of vitamin A-rich lipid droplets in multivesicular bodies of rat liver stellate cells. J Cell Biol 1974;63:683–691. 121. Wake K. Perisinusoidal stellate cells (fat-storing cells, interstitial cells, lipocytes), their related structure in and around liver sinusoids, and vitamin A-storing cells in extrahepatic organs. Int Rev Cytol 1980;66:303–353. 122. Wake K, Motomatsu K, Dan C, Kaneda K. Three-dimensional structure of endothelial cells in hepatic sinusoids of the rat as revealed by the Golgi method. Cell Tissue Res 1988;253:563–571. 123. Kawada N, Tran-Thi TA, Klein H, Decker K. The contraction of hepatic stellate (Ito) cells stimulated with vasoactive substances. Possible involvement of endothelin 1 and nitric oxide in the regulation of the sinusoidal tonus. Eur J Biochem 1993;213:815–822. 124. Pinznai M, Faili P, Ruocco C, et al. Fat-storing cells as liverspecific pericytes: Spatial dynamics of agonist-stimulated intracellular calcium transients. J Clin Invest 1992;90:642–646. 125. Zhang JX, Pegoli W, Clemens MG. Endothelin-1 induces direct constriction of hepatic sinusoids. Am J Physiol 1994;266:G624–G632. 126. Rockey DC. Hepatic blood flow regulation by stellate cells in normal and injured liver. Semin Liver Dis 2001;21:337–349.
127. LeBail B, Bioulac-Sage P, Senuita R, et al. Fine structure of hepatic sinusoids and sinusoidal cells in disease. J Electron Microsc Tech 1990;14:257–282. 128. Gressner AM. Perisinusoidal lipocytes and fibrogenesis. Gut 1994;35:1331–1333. 129. Wisse E, Noordende JMVt, Meulen JVD, Daems WT. The pit cell: Description of a new type of cell occurring in rat liver and peripheral blood. Cell Tissue Res 1976;173: 423–435. 130. Vanderkerken K, Bouwens L, Neve WD, et al. Origin and differentiation of hepatic natural killer cells (pit cells). Hepatology 1993;18:919–925. 131. Bouwens L, Wisse E. Pit cells in the liver. Liver 1992;12:3–9. 132. Kaneda K, Dan C, Wake K. Pit cells as natural killer cells. Biomed Res 1983;4:567–576. 133. Bouwens L, Remels L, Baekeland M, et al. Large granular lymphocytes or pit cells from rat liver: Isolation, ultrastructural characterization and natural killer cell activity. Eur J Immunol 1987;17:37–42. 134. Kaneda K, Wake K. Distribution and morphological characteristics of the pit cells in the liver of the rat. Cell Tissue Res 1983;233:485–505. 135. Vanderkerken K, Bouwens L, Wisse E. Characterization of a phenotypically and functionally distinct subset of large granular lymphocytes (pit cells) in rat liver sinusoids. Hepatology 1990;12:70–75.
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2
LIVER REGENERATION Christian Trautwein Abbreviations APC adenomatous polyposis coli APR acute-phase response CCL4 carbontetrachloride C/EBPs CCAAT/enhancer-binding proteins CDK cyclin-dependent kinases CKIS cyclin-dependent kinase inhibitors CNTF ciliary neurotropic factor CT-1 cardiotropin 1 DSH dishevelled EGF epidermal growth factor EGFR/ epidermal growth factor receptor Erbb1 ERK/ extracellular signal-regulated ECM extracellular matrix FADD Fas-associated death domain FOXM1B forkhead box M1B transcription factor Gab1 growth-factor-receptor-bound protein 2 (Grb 2)-associated binder 1
GSK glycogen synthase-3b HB-EGF heparin-binding EGF HGF hepatocyte growth factor IL interleukin IKK I-kB kinase JAKs Janus kinases JNK Jun terminal kinase LIF leukemia inhibitory factor LRP lipoprotein receptor-related protein MAPkinase kinase/mitogen-activated protein kinase MAPKKK mitogen-activated protein kinase kinase kinase NEMO NF-kB Essential Modulator NF-kB nuclear factor-kB NK natural killer OSM oncostatin M Pak p21-activated protein kinase Pl3K phosphotidylinositol3-kinase
INTRODUCTION The liver stands out for its unique ability to regenerate and thereby restore its original mass after tissue loss. Major progress has been achieved during the last 50 years in understanding the mechanisms involved in controlling this process. However, this phenomenon was already known in Greek mythology: the Titan Prometheus stole the fire from Zeus and took it to mankind. Zeus punished Prometheus by chaining him to a rock in the Caucasus mountains. Every day an eagle came and ate from his liver, which regenerated overnight. The liver has a large metabolic task to perform, and normally hepatocyte proliferation in the liver is a rare event. However, liver regeneration is induced following different mechanisms of injury. In humans, examples are acute liver damage after virus infection or liver resection. Moreover, in recent years liver transplantation, and especially split liver transplantation, has become of major importance owing to organ shortage. Therefore, models to study liver regeneration are of direct relevance to a better understanding of the mechanisms that are important also in patients. Additionally, the direct clinical application of split liver transplantation allows further proof of the concepts developed in animal models. Different animal models have been established to study liver regeneration, mainly in rats and mice. More recently, mouse models have been favored because genetic manipulation in this species allows us to directly address the function of specific genes in hepatocyte proliferation after injury. The best-studied model to investigate the mechanisms involved in liver regeneration is that of partial
PKC PKB PPAR RIP Shp2 SODD SOS Stat TGF TNF TRADD TRAF2 TR u-PA
proteinkinase C protein kinase B peroxisome proliferator-activated receptor receptor-interacting protein SH2-domain containing protein tyrosine phosphatase 2 silencer of death domains son-of-sevenless signal transducer and activator of transcription transforming growth factor tumor necrosis factor TNF receptor-associated death domain TNF-R-associated factor 2 thyroid hormone receptor urokinase-type plasminogen activator
hepatectomy. In mice or rats at least 60% of the liver is surgically removed and the impact on cell cycle progression of parenchymal and non-parenchymal liver cells can be investigated. In this classic model, a first wave of hepatocyte proliferation is found in rats after 24 hours and in mice after 40 hours. Non-parenchymal cells follow hepatocyte proliferation several hours later (Figure 2-1).1 In this simple model of partial hepatectomy, basic mechanisms have become evident in the control of hepatocyte proliferation. The correct liver/body-weight ratio can be restored very rapidly, which takes between 7 and 10 days in rodents. This ratio is relatively constant and reflects the balance between liver function and the body’s demands. After resection the removed liver lobes are not replaced, but hepatocytes in the remaining lobes proliferate. Therefore liver regeneration can also be described as compensatory hyperplasia. After liver mass is restored, hepatocytes receive signals that lead to a halt in proliferation. Thus liver regeneration is a tightly regulated process where hepatocytes enter the cell cycle and become quiescent again. Besides the proliferative response after resection or injury, the liver also has the capacity to proliferate without loss of tissue, and this is known as direct hyperplasia. Different agents, e.g. nuclear receptors, have been characterized that trigger direct hyperplasia.2 However, this chapter will focus on the mechanism of liver regeneration known as compensatory hyperplasia. In the last 50 years different aspects of liver regeneration have been covered. In the beginning, the model of partial hepatectomy was established and morphological and metabolic changes during hepatocyte proliferation were studied. Subsequently it became obvious that growth factors are involved in controlling the exact
23
Section I. Pathophysiology of the Liver
created. Before mitosis of a somatic cell can occur an interphase has to be established where certain cellular processes are performed in order to allow cell division. The interphase can be divided into a Gap1 (G1) phase, a DNA-synthesis (S) phase and a Gap2 (G2) phase (Figure 2-2). The G1 and G2 phases are characterized by increasing cell volume and processes that are essential to prepare the cell for chromosomal doubling in S phase or chromosomal segregation during mitosis. Challenging events during cell cycle progression, such as incomplete DNA replication, DNA damage, and depletion of growth factors or mismatching of metaphase chromosomes, result in blocking of cell cycle progression at so-called checkpoints. These are found at late G1 phase (restriction checkpoint), at the end of G2 (G2/M checkpoint) and during mitosis (spindle checkpoint). These checkpoints are crucial in order to stop cell cycle progression until defects have been repaired or, alternatively, cells activate a suicide program leading to the elimination of cells with a dysregulated cell cycle in order to avoid uncontrolled growth. In the adult liver hepatocytes are in a resting state, which is also called G0 (Figure 2-2). The different phases of cell cycle progression are coordinated by proteins called cyclins. Accordingly different members of this family are expressed in a defined manner throughout the cell cycle. Cyclins are the regulatory subunits of cyclin-dependent kinases (CDK) and activate kinases in order to specifically phosphorylate substrates that are crucial in regulating the different phases of cell cycle progression. Members of the cyclin D family (D1, D2, and D3) are expressed in early and middle G1 phase, where they bind to CDK4 and CDK6. Expression of D-cyclins is dependent on stimulation by growth factors. Therefore D-cyclins represent sensors that are needed for the interaction of the cell with the extracellular environment. During this period after partial hepatectomy the induction of cyclin D1 in particular plays a pivotal role in triggering the proliferation of hepatocytes.3,4 As a consequence c-myc expression is induced, which is important to trigger hepatocyte proliferation. Additionally, there is evidence that overexpression of c-myc alone is sufficient to stim-
timing of cell cycle progression of hepatocytes. In further studies the intracellular events, especially in the nucleus, were investigated, which resulted in an analysis of changes in the expression and activity of transcription factors. Meanwhile, through the powerful tools offered by genetically manipulated mice, the complex interacting pathways of liver regeneration have been investigated, and these tools help us discover the essential mechanisms required to restore liver mass after injury.
MECHANISMS OF CELL CYCLE REGULATION Eukaryotic cells have the capacity to divide. This process is called mitosis, and during the event two identical daughter cells are
30 0 Percent labelled cells
Hepatocytes Biliary ductular cells Kupffer and lto cells Sinusoidal endothelial cells
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Days after partial hepatectomy Figure 2-1. Time kinetics of DNA synthesis in different liver cell types during liver regeneration after partial hepatectomy. The four major types of liver cell undergo DNA synthesis at different time points after partial hepatectomy. In rats, hepatocyte DNA synthesis peaks at 24 hours, whereas the other cell types proliferate later. Regenerating hepatocytes produce growth factors that can function as mitogens for these cells. This has suggested that hepatocytes stimulate proliferation of the other cells via a paracrine mechanism. (Reprinted from Science 1977; 276:60–66; Michalopoulos GK, DeFrances MC. Liver regeneration.)
G0
P18ink4C P21WAF1 P27KIP1
P21WAF1 P27KIP1
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G2/Mcheck point Spindelcheck point
Figure 2-2. Cell cycle progression. Cell cycle progression is dependent on the orchestrated expression and activation of specific catalytic enzymes (CDKS) with their regulatory units (cyclins). The resting hepatocyte (G0) is activated by different stimuli to enter the cell cycle. During G1 phase cyclin D–CDK4/6 complexes become activated, followed by cyclin E–CDK2/3 at G1/S phase. During S phase the cyclin A–CDK1/2, and during G2/M cyclin A/B–CDK1, are required. Additionally, cell cycle progression is controlled through a complex network of proteins where the cyclin-dependent kinase inhibitors (CKIs) play an important role. They interact with the specific cyclin–Cdk complexes and thus can manipulate their activity. Using knockout animals in hepatocytes, so far a role especially for p21, p27 and p18I has been shown in manipulating cell cycle progression during liver regeneration.
Chapter 2 LIVER REGENERATION
ulate proliferation and oncogenic transformation of hepatocytes in vivo, whereas blocking its expression reverses this effect.5 At late G1 phase control of cell cycle progression by the cyclin D family becomes less relevant and is followed by the expression of cyclin E (E1, E2). During the transition, the strength and timing of DNA synthesis after partial hepatectomy is strongly controlled by the expression and cooperation of CDK inhibitors, which are involved in controlling CDK activity. In particular, p18, p21 and p27 have been shown to play an important role during this period.6–8 After passing the restriction point cell cycle progression will become independent of growth factors, meaning that after this point the depletion of growth factors will have no impact on cell cycle progression, as these cells will complete mitosis. After the restriction point, expression of E-cyclins will increase until G1-/S-phase transition, whereas at later time points the protein will become degraded. Cyclins E1 and E2 build complexes with CDK2 and CDK3, respectively. It has long been thought that the activity of these complexes is necessary to coordinate the mechanisms essential for G1-/S-phase progression. The fundamental understanding has recently been questioned by the work of different groups. Experiments using knockout animals for CDK2 or double-knockout mice for cyclins E1 and E2 indicated that the cyclin E/CDK2 complex might be less essential than at first thought for G1-/ S-phase transition, and as originally described in Drosophilz melanogaster.9–12 However, at present it is not clear whether these findings are also important for liver regeneration, as experiments that address the question whether a resting cell in G0 phase may need the complex in order to go into mitosis are lacking. In S phase CDK2 and CDK1 interact with cyclin A, whereas in G2 complexes consisting of cyclins A and B with CDK1 become relevant to drive cell cycle progression. After reaching the G2-/Mphase passage members of the cyclin B family alone take over to control mitosis. In recent years Costa’s group used the partial hepatectomy model to further characterize the role of the forkhead box M1B transcription factor (FoxM1B) – a winged helix transcription factor – for cell cycle progression. This transcription factor is expressed in all replicating cells and plays a role especially after the G1-/S-phase checkpoint, although it is not expressed in terminally differentiated cells.13 The group demonstrated that hepatocyte-specific deletion of FoxM1B results in strong reduction of DNA synthesis. Lack of FoxM1B expression is associated with increased protein expression of the CDK inhibitor p21 and reduced Cdc25A phosphatase levels, which leads to decreased CDK2 activation and progression into S phase.14 In addition, FoxM1B has been shown to correlate with the regenerative capacity of the aging liver. Re-establishing FoxM1B expression in the aging liver reverses proliferative capacity after partial hepatectomy, indicating that FoxM1B might be an important limiting factor for hepatocyte proliferation in the aging liver.15 The links of FoxM1B to cell cycle progression are CDKspecific phosphorylation sites in the transactivation domain of FoxM1B. Phosphorylation of these sites results in target gene transcription of genes involved in S- or M-phase progression.16 The results as found for FoxM1B demonstrate that the partial hepatectomy model is also useful to study general cell cycle-dependent mechanisms in vivo.
MECHANISMS CONTROLLING TRANSITION OF RESTING HEPATOCYTES INTO MITOSIS Mitosis in an uninjured liver is a rare event. After loss of tissue mass different growth factors, cytokines and molecular events were characterized for their role to mediate transition of the resting hepatocytes into mitosis. As outlined above, cells are sensitive to extracellular stimuli until they pass the G1 restriction point. In order to better define the events that drive hepatocytes during liver regeneration from G0 until the G1 restriction point, two time periods have been defined: the priming and the transition phase. The priming phase reflects the first hours after it becomes evident that the liver needs to restore its mass. This is the period that triggers hepatocytes to leave G0 and enter G1 phase. The transition phase describes the period after entry into G1 until the G1 restriction time point. In these two phases different growth factors and intracellular events become relevant, and so these will be outlined in more detail. The time period of the two phases may vary between mouse and rat and their respective strains. However, the first priming period is short and the events take place in the first 1–3 hours after injury. In the priming phase the extracellular environment and, as a consequence, also the intracellular pathways that become activated, changes dramatically. Among the mechanisms that are relevant to prime the hepatocytes are changes in the extracellular matrix, and activation of growth factors, cytokines and chemokines. These different components interact with each other. Interestingly, lack of expression of one of the genes frequently has a rather mild phenotype, as a redundancy of the systems appears to exist so that pathways can at least in part compensate for each other. Additionally, in nature the artificial differentiation between the priming and the transition periods is less strict, as during liver regeneration many steps occur in parallel.
CHANGES IN THE EXTRACELLULAR MATRIX Besides parenchymal and non-parenchymal cells, the extracellular matrix is important for liver homeostasis and plays an important role in initiating liver regeneration. In order to allow cell proliferation the extracellular matrix (ECM) has to be modified. Changes in the extracellular matrix have different effects. They allow the hepatocyte to be less attached to surrounding cells, and thus give the cells space for mitosis. Additionally, these events trigger intracellular pathways, which are important for G0/G1 transition of hepatocytes. One of the first ECM components to become activated is the urokinase-type plasminogen activator (u-PA). One minute after partial hepatectomy higher u-PA activity can already be detected, and continuously increases during the first 60 minutes.17 u-PA is one of the major initiators of the metalloproteinase cascade leading to ECM degradation and proteolysis, and in addition is an activator of plasminogen and hepatocyte growth factor (HGF). Consecutively a
25
Section I. Pathophysiology of the Liver
higher conversion rate of plasminogen to plasmin and increased fibrinogen breakdown can be detected in the first minutes after partial hepatectomy.17 Matrix degradation results in the release of matrix-bound factors such as HGF. Furthermore, the event is also involved in activating HGF: the inactive single chain of HGF is transformed into the mitotically active two-chain form, which is important to trigger the intracellular pathways required for hepatocyte proliferation.18–21 An important finding is the observation that disruption of the interaction between the cell adhesion molecules E-cadherin and bcatenin is an early step during liver regeneration. In the first 5 minutes after partial hepatectomy nuclear translocation of b-catenin increases, which results in target gene transcription.22 These are genes involved in mediating cell proliferation, such as cyclin D1 and c-myc. E-cadherin shows an opposite regulation to b-catenin. Together these results indicate that modulation of cell adhesion molecules at the outer surface of hepatocytes activates b-catenin, which is involved in contributing to prime hepatocytes. Besides the interaction with cell adhesion molecules, growth factors also have an impact on disruption of the interaction between b-catenin and E-cadherin (Figure 2-3). These are, for example, the HGF/C-metand the EGF/EGF-receptor systems.23
Wnt Cadherin LRP
Frizzled
The pathways interacting with b-catenin are even more complex, as this system also allows interaction with growth factors. Besides binding to the intracellular part of E-cadherin, b-catenin is also present in the cytoplasm, where it directly interacts with other proteins that direct the molecule to the ubiquitin degradation system. After binding of Wnt to its receptor, frizzled degradation of bcatenin is inhibited and as a result the protein translocates to the nucleus, where it activates target gene transcription. Therefore, bcatenin can be activated via two independent mechanisms – Ecadherin and Wnt – which also interact with each other through a complex network.23
LIGAND–RECEPTOR INTERACTION ACTIVATED DURING LIVER REGENERATION Several growth factors, cytokines and chemokines have been shown to interact with receptors on the cell membrane, activating the intracellular signaling pathways involved in target gene activation in hepatocytes. The growth factors were characterized by their mitogenic capacity. Therefore, at present HGF, EGF and transforming growth factor (TGF)-a can be differentiated as mitogens, whereas insulin and epinephrine are classified as co-mitogens. Additionally, growth inhibitory factors – TGF-b and activin A – have been described that are downregulated during the early phase of liver regeneration, but are important to inhibit cell proliferation after liver mass has been restored.
b-catenin a-catenin Dsh b-catenin
Axin GSK
APC b-catenin
b-catenin
TCF
Figure 2-3. Schematic of b-catenin activation via Wnt and cadherin. Binding of Wnt to its receptors Frizzled and LRP (lipoprotein receptor-related protein) results in Dsh activation and the accumulation of b-catenin and interaction with TCF that regulates target gene transcription. In unstimulated cells the level of b-catenin is kept low through degradation by the proteosome system involving Axin, adenomatous polyposis coli (APC) and glycogen synthase-3b (GSK). Dsh (dishevelled) uncouples b-catenin from this protein complex. Additionally, the cytoplasmic domains of type I cadherin binds b-catenin and thus links the protein via a-catenin to the actin cytoskeleton. The interaction of these molecules is controlled by phosphorylation. In general activation of tyrosine kinases, e.g. by growth factors, results in loss of cadherin-mediated cell–cell adhesion and thus increases b-catenin expression and gene transcription. Both possibilities result in the activation of processes involved in cell adhesion and cell migration that play a role during liver regeneration.
26
MITOGENIC GROWTH FACTORS HEPATOCYTE GROWTH FACTOR (HGF) HGF is the best-characterized mitogenic growth factor involved in stimulating liver regeneration and was first isolated and purified from the serum of a patient with fulminant hepatic failure and from rats after partial hepatectomy.24,25 Meanwhile, it has become evident that HGF and the scatter factor, or HGF, are the same molecules,26 and thus the protein also has tasks in cell tissues besides the liver. Activation of intracellular pathways via HGF occurs after binding to its receptor c-met (Figure 2-427). Crucial for the downstream activation of c-met-dependent pathways is the phosphorylation of two tyrosine residues at its intracellular domain. Tyrosine phosphorylation creates docking sites for substrates. The most relevant partner is Gab1. Through a specific Met-binding-site, Gab1 interacts with c-met and becomes phosphorylated. Phosphorylated Gab1 binds signal molecules such as the SH2 domain-containing protein tyrosine phosphatase 2 (Shp2), PI3K, phospholipase C and CRK. One of the major downstream pathways to become activated through c-met/Gab1/Shp2, is the ERK/MAPK pathway that triggers transcription factors such as ETS/AP1 and adhesion molecules. This mechanism is directly involved in mediating cell proliferation, whereas PI3K, via Akt/protein kinase B, confers cell survival. Besides these main signaling pathways c-met also activates Jun terminal kinase (JNK), signal transducer and activator of transcription (Stat) 3, nuclear factor-kB (NF-kB) and b-catenin (for review, see 27).
Chapter 2 LIVER REGENERATION
HGF a b c-met p p
Grb2 Sos
Rac1
Cdc42
Ras
Gab1
Crk
Shp2
Raf
PI3K
C3G Akt/PKB
Pak
Rap1
ERK/MAPK
Cadherins
pRB Cdk6 p27
Cell polarity Motility
Proliferation Cell-cycle
uPA MMPs Fibronectin
FAK Integrins
Migration Cell junction
c-Met can also interact with other membrane receptors on the cell surface, e.g. E-cadherin, b4-integrin or Fas. This receptor cross-talk may also have a direct effect on the cellular response of the cell (for review, see 27). Therefore, HGF/c-met can interact on different levels with cell cycle progression during liver regeneration. HGF serum concentrations are elevated after partial hepatectomy, and in particular the active two-chain form can be detected.28 Activation of HGF/c-met-dependent signaling is found directly after partial hepatectomy, but also at later time points. Phosphorylation of c-met was detected within the first 5 minutes after surgery.29 The role of c-met in cell cycle progression during liver regeneration has recently been further clarified by two independent groups using conditional c-met knockout mice.30,31 These experiments provided evidence that after partial hepatectomy ERK activation is selectively mediated via the HGF/c-met system, which is associated with a reduction in DNA synthesis.30 Also after carbontetrachloride (CCL4) injury impaired regeneration was found and inflammatory changes in the livers of these animals were more prolonged. Additionally, the animals, which lacked c-met expression, showed higher sensitivity versus Fas-induced apoptosis.31 Therefore, using conditional ablation of the c-met receptor in hepatocytes shows that the signaling pathway is essential for providing proliferative and protective signals during liver regeneration.
EPIDERMAL GROWTH FACTOR (EGF) AND TRANSFORMING GROWTH FACTOR-a (TGF-a) The epidermal growth factor receptor (EGFR/Erbb1) belongs to a family of structurally related tyrosine kinase receptors, including
Figure 2-4. HGF/c-met-dependent signaling. After ligand binding tyrosines at the intracellular part of the c-met receptor become phosphorylated and serve as docking sites for different adapter molecules, e.g. Gab1, Grb2, phosphotidylinositol 3kinase and others. As a consequence, specific signaling molecules such as Ras, Shp2 and Crk become activated that trigger downstream pathways. These cascades are essential in stimulating gene expression and/or functions involved in, for example, proliferation, migration or survival. Some of the prominent pathways that are activated are the Ras/Raf/ERK/MAPkinase or PI3K/Akt/PKB pathways. Abbreviations: ERK/MAPkinase, extracellular signal-regulated kinase/mitogen-activated protein kinase; Gab1, growth-factor-receptor-bound protein 2 (Grb2)-associated binder 1; Pak, p21-activated protein kinase; PI3K, phosphotidylinositol3kinase; PKB, protein kinase B; Shp2, SH2-domain containing protein tyrosine phosphatase 2; Sos, son-of-sevenless; met a and b receptor subunit (modified from 27).
Bad Caspase-9
Survival
Erbb2/neu, Erbb3 and Erbb4. After ligand binding the receptor can bind homo- or heterodimers. Several growth factors, such as EGF, TGF-a, amphiregulin, heparin-binding EGF (HB-EGF), b-cellulin and epiregulin, can bind EGFR and induce receptor dimerization. Consecutive activation of the intrinsic tyrosine kinase induces complex downstream pathways (Figure 2-5).32–34 The mitogenic role of the EGFR in liver regeneration is thought to be mediated by stimulating MAP kinase activity. Studies in hepatocytes revealed that only EGFR and Erbb3 are expressed in the adult liver. No expression of Erbb2 and Erbb4 can be detected. Additionally, the expression of EGFR and Erbb3 does not change in hepatocytes during liver regeneration.35 For two ligands – TGF-a and EGF – a potential role during liver regeneration has been suggested. The two molecules show around 35% homology. Good evidence for the important role of EGF during liver regeneration first came from experiments in sialoadenectomized rats. In these animals DNA synthesis is reduced after 48 hours and the application of EGF can rescue the phenotype. An effect on DNA synthesis was evident when sialoadenectomy was performed 3 hours after partial hepatectomy, indicating that EGF is not required during the priming phase.36 Additionally, recent experiments using a transgenic mouse that overexpressed heparin-binding (HB)-EGF-like growth factors showed that after partial hepatectomy a stronger impact on DNA synthesis could be found. As HB-EGF also signals via EGFR, these results further provide evidence that factors binding to EGF receptors are able to stimulate DNA synthesis after partial hepatectomy.37 However, there is also evidence that tyrosine phosphorylation of EGFR in the liver is constitutive and the status
27
Section I. Pathophysiology of the Liver
NOTCH/JAGGED SIGNALING
EGF-R1 (Erbb1) Erbb2/Erbb3/Erbb4 EGF TGF-a
PTK P
P
SATs
Ras PKC
PI3K
Raf
AKT
MAPK
PTEN
FKHR
c-myc c-fos c-jun
GSK-3b
Cyclin D1 Cyclin E
Figure 2-5. Signaling via the epidermal growth factor (EGF) receptor. Binding of EGF or TGF-a to the EGF receptor (EGF-R/Erbb1) or other members of the family (Erbb2/neu, Erbb3, and Erbb4) results in intracellular receptor homo- or heterodimerization. This event stimulates intrinsic tyrosine kinases and phosphorylation of the intracellular receptor domains. As a consequence, the Ras/Raf/MAPkinase cascade, but also other signaling pathways, e.g. PI3K, proteinkinase c (PKC) and Stat proteins, are activated which translate the different EGF-dependent functions at the cellular level.
The Notch/Jagged signaling pathway is relevant in different systems for cell growth and differentiation. Notch genes encode for a family of transmembrane receptors. After ligand binding of Jagged, proteolytic cleavage of intracellular domains occurs. One part of the cytoplasmic domain (NICD) translocates to the nucleus, where NICD binds to the transcription factor CBF1/RBP-Jk, which turns the complex from a repressor into a transactivator protein. Recent evidence demonstrates that NCID nuclear translocation is an early event that occurs in the first 15 minutes after partial hepatectomy.48 When Notch or Jagged expression is blocked before partial hepatectomy via siRNA, this results in a reduction of DNA synthesis in the animals. Thus the Notch/Jagged system seems to be directly involved in triggering cell proliferation during liver regeneration in the priming phase.
ROLE OF CYTOKINES DURING LIVER REGENERATION Besides the direct role of growth factors, it also became evident that cytokines such as TNF-a and interleukin (IL)-6 are involved in priming hepatocytes towards cell cycle progression. Initially it was found that there is a cascade of events leading first to elevated TNFa, and subsequently to increased IL-6 serum levels. Additionally, experiments indicated that anti-TNF antibodies inhibit the proliferation of hepatocytes, again indicating that the molecule might be involved in controlling cell cycle progression.
TUMOR NECROSIS FACTOR-a (TNF-a)
does not change during liver regeneration, indicating that there is no further activation of the receptor after partial hepatectomy.29 Serum levels of TGF-a increase after partial hepatectomy,38 although this increase is modest (twofold).39 TGF-a levels in the serum directly correlate with hepatocyte proliferation after partial hepatectomy.40 TNF has been discovered to be a potential regulator of TGF-a expression after partial hepatectomy.41 During liver regeneration TNF stimulates the metalloproteinase TACE, also known as ADAM 17. TACE cleaves TGF-a at the surface and increases its serum expression. Transgenic animals overexpressing TGF-a in the liver show an increased liver/body-weight ratio.42 However, TGF-a -/- animals have no defect in liver regeneration, indicating that TGF-a in physiological doses is dispensable for liver regeneration.43 Knockout mice constitutive for Erbb2, Erbb3 and Erbb4 die during mid gestation.44–46 Knockout animals for EGFR die at the latest 20 days after birth.47 However, so far no liver phenotype has been reported for any of these mice. As only EGFR and Erbb3 are expressed in hepatocytes, conditional knockout experiments will ultimately resolve whether the pathway is required under normal condition to restore liver mass.
28
TNF signals through two distinct cell surface receptors, TNF-R1 and TNF-R2, of which TNF-R1 initiates the majority of TNF’s biological activities in hepatocytes. Binding of TNF to its receptor leads to the release of the inhibitory protein silencer of death domains (SODD) from TNF-R1’s intracellular domain. This leads to the recognition of the intracellular TNF-R1 domain by the adapter protein TNF receptor-associated death domain (TRADD), which recruits additional adapter proteins: receptor-interacting protein (RIP), TNF-R-associated factor 2 (TRAF2), and Fas-associated death domain (FADD). These proteins then activate distinct signaling cascades (Figure 2-6). FADD recruits caspase 8, which can trigger apoptosis. TRAF2 is upstream of several cascades. It activates cIAP-1 and -2, a mitogenactivated protein kinase kinase kinase (MAPKKK) which ultimately activates c-Jun NH2-terminal kinase (JNK). Additionally, TRAF2 is involved in NF-kB activation. Here also RIP is required, but it does not need its enzymatic activity (for review, see 49). Especially for NF-kB, but also for JNK, a role in liver regeneration has been reported. Activation of NF-kB by TNF requires a complex network of kinases. First, the IKK complex interacts with TRAF2 and RIP. Upon activation the IKK kinase phosphorylates I-kB, which results in its degradation, and as a consequence NF-kB is released to the nucleus, where target gene transcription starts.
Chapter 2 LIVER REGENERATION
TNFa TNF-R1 FADD
Pro-Caspase 8
TRADD
RIP
IKK1 HSP90
CDC3
Caspase 8
cIAP
TRAF2
Anti-apoptosis IKK2
Figure 2-6. TNF-dependent signal transduction. Binding of TNF to its cognate receptor TNF-R1 results in the release of SODD and the formation of a receptor-proximal complex containing the important adapter proteins TRADD, TRAF2, RIP and FADD. These adapter proteins in turn recruit additional key pathway-specific enzymes (for example caspase-8 and IKK2) to the TNF-R1 complex, where they become activated and initiate downstream events leading to apoptosis via caspase 8, NF-B activation involving the IKK-complex, and Jun kinase (JNK) activation.
Mitochondria
NEMO ASK1
RelA
p50
Caspase 9
PP IKBa
IKBa
RelA
RelA
p50
RelA
p50
MKK4/6
Cytochrome C Apaf-1
p50
Proteosome
JNK
c-jun, ATF-2, Elk-1 other substrates
Caspase 3 Cytoplasmic targets
Nuclear targets
Inflammatory/antiapoptotic genes
The high molecular weight IKK complex that mediates the phosphorylation of I-kB has been purified and characterized. This complex consists of three tightly associated I-kB kinase (IKK) polypeptides: IKK1 (also called IKK-a) and IKK2 (IKK-b) are the catalytic subunits of the kinase complex and have very similar primary structures with 52% overall similarity.50–52 Moreover, it contains a regulatory subunit called NEMO (NF-kB Essential Modulator), IKK-g or IKKAP-1.53,54 In vitro, IKK1 and IKK2 can form homoand heterodimers.55 Both IKK1 and IKK2 are able to phosphorylate I-kB in vitro, but IKK2 has a higher kinase activity in vitro than IKK1.56–58 The IKK complex phosphorylates I-kBs at the N-terminal domain at two conserved serines (S32 and S36 in human I-kBa). After phosphorylation, the I-kBs undergo a second post-translational modification: polyubiquitination by a cascade of enzymatic reactions, mediated by the b-TrCP–SCF complex (or the E3IkB ubiquitin ligase complex). This process is followed by the degradation of I-kB proteins by the proteasome, thus releasing NF-kB from its inhibitory IkB-binding partner, so it can translocate to the nucleus and activate transcription of NF-kB-dependent target genes.51,59 Because the enzymes that catalyze the ubiquitination of I-kB are constitutively active, the only regulated step in NF-kB activation appears in most cases to be the phosphorylation of I-kB molecules. NF-kB was first identified in the liver as a factor that is rapidly activated within 30 minutes after partial hepatectomy.60 The importance of NF-kB and TNF signaling was further confirmed by the
fact that liver regeneration is defective in TNF-receptor1 knockout mice that do not show hepatic NF-kB activation after partial hepatectomy.61 The question remained as to whether NF-kB is able to directly promote hepatocyte proliferation in this model. NF-kB has been shown to be capable of directly stimulating the transcription of genes that encode G1-phase cyclins, and a kB-site is present within the cyclin D1 promoter.62,63 Additionally, experiments using an adenovirus of non-degradable I-kBa super-repressor, which blocks NF-kB activation, indicated that NF-kB activation after partial hepatectomy is required for liver regeneration. Animals treated with the virus showed a lack of hepatocyte proliferation and increased apoptosis.64 In contrast, Chaisson et al. used transgenic mice that expressed the non-degradable I-kBa super-repressor specifically in hepatocytes, but only 60% of the hepatocytes expressed the transgene. These mice – in contrast to the adenovirus experiments – showed normal hepatocyte proliferation after partial hepatectomy.65 However, both systems, which were used to block NF-kB activation, have some experimental problems. Therefore, at present it is unclear which level of NF-kB activation is required to allow normal liver regeneration after partial hepatectomy. TNF also triggers Jun kinase (JNK) activity and c-Jun activation during liver regeneration.66,67 Both factors are essential for cell cycle progression after partial hepatectomy. Inhibition of JNK activity results in reduced hepatocyte proliferation and G0/G1 transition of
29
Section I. Pathophysiology of the Liver
hepatocytes. However, no impact on apoptosis was observed.68 Conditional knockout mice for c-Jun have a severe phenotype after partial hepatectomy as half of the mice died, or showed impaired regeneration, increased cell death and lipid accumulation in hepatocytes.69 Together these results demonstrate that JNK/c-Jun activation is crucial to stimulate liver regeneration after partial hepatectomy. Via FADD, TNF can trigger apoptosis via caspase 8 activation. Fas can use the same pathway. However, unlike TNF, hepatocytes are more sensitive to Fas-induced apoptosis as the counterbalancing effect of NF-kB activation is missing.70 During liver regeneration after partial hepatectomy, hepatocytes are less sensitive to Fasinduced apoptosis. Additionally, Fas stimulation enhances hepatocyte proliferation, indicating that the FADD/caspase 8 pathway during liver regeneration induces pro-proliferative effects.71
INTERLEUKIN-6 Interleukin-6 (IL-6) belongs to a family comprising IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotropic factor (CNTF) and cardiotropin 1 (CT-1), all of which need the gp130 molecule for signal transduction.72,73 Cytokines of the IL6 family interact with a receptor complex on the cell surface in which gp130 is the central molecule, as it is used by several family members for signal transduction. IL-6 first binds the IL-6 receptor (gp80) and then interacts with gp130. Subsequently, dimerization of two gp130 molecules activates Janus kinases (Jaks), which phosphorylate specific tyrosine residues of gp130 and thus activate the SHP2/ERK/MAP pathways or the transcription factors STAT1 and STAT3 (Figure 2-7).72,73
IL-6
JAKs (Jak1/2/Tyk2) Y2 Shp2 Y3 Y4 Y5 Y6 Y1
IL-6R/ gp80 gp130
STAT3 activation
STAT1 Ras/Mapactivation pathway
Figure 2-7. Interleukin-6-dependent signaling. On the cell surface interleukin6 (IL-6) first interacts with the IL-6 receptor (IL-6R)/gp80. This complex interacts with gp130 molecules and in turn triggers intracellular dimerization. Receptorbound Janus kinases (JAKs: Jak1/2/Tyk2) became activated and phosphorylate tyrosines as the intracellular part of gp130. The phosphorylated tyrosines are essential to activate downstream pathways. Although phosphorylation of the second tyrosine is important to trigger the Ras/Map pathway via SH2-domaincontaining protein tyrosine phosphatase 2 (Shp2), the four distal tyrosines are essential to activate Stat transcription factors.
30
Shortly after the STAT transcription factors were identified74 it became evident that there is transient IL-6-dependent STAT3 activation after partial hepatectomy, which is restricted to the first hours as in turn its inhibitor SOCS3 is immediately induced and thus limits its activity.75–77 The ultimate proof for the relevance of IL-6 for liver regeneration came from experiments with IL-6-/- mice. First experiments published by Taub’s group demonstrated that these animals had a defect in hepatocyte proliferation after partial hepatectomy. Significantly more of the IL-6-/- animals died than did the wt control mice.78 The relevance of these findings was further emphasized as the defect in liver regeneration found in TNFR-1-/- mice could be reversed by IL-6 injection.61 Through these two findings the hypothesis was raised that IL-6 is an essential factor in driving the resting hepatocyte into the cell cycle. Further experiments were aimed at better defining the pathways activated by IL-6 that are essential for liver regeneration. The most prominent factor activated by IL-6 in hepatocytes is STAT3. Treatment of IL-6-/- mice after partial hepatectomy with stem cell factor restored Stat3 activation and DNA synthesis.79 As Stat3 knockout mice are embryonal lethal,80 conditional knockout mice with a hepatocyte-specific knockout for STAT3 were used to study the role of IL-6/gp130-dependent STAT3 activation during liver regeneration. These animals also showed impairment in liver regeneration, resembling the results of IL-6-/- animals.81 Therefore these results suggested that the STAT3 pathway in particular seems necessary for liver regeneration following partial hepatectomy. However, in these animals there was strong STAT1 activation, which is normally not found after partial hepatectomy. STAT1 is known to mediate effects opposite to those of STAT3. Therefore this experimental setting has major problems to solve the role of STAT3 during liver regeneration. Blindenbacher et al. performed a careful study in IL-6-/- mice to better define the role of IL-6 during liver regeneration.82 They tested to see whether IL-6 has a direct impact on hepatocyte proliferation or body homeostasis. Using intravenous or subcutaneous IL-6 injection the authors found that the role of IL-6 seems not to be directly involved in stimulating hepatocyte proliferation, but in maintaining body homeostasis in order to allow normal liver regeneration. These results were further confirmed in conditional knockout animals for gp130. These animals showed normal liver regeneration compared to wt animals.83 However, after LPSinjection – mimicking bacterial infection – more of the gp130-/animals died than did controls, and showed impaired hepatocyte proliferation. Taken together, the work of these groups indicates that IL-6/gp130 is involved in contributing to liver regeneration through mechanisms that are not directly related to cell cycle control. At present the pathways relevant to mediate this effect are not completely understood. However, in recent years several reports have demonstrated that IL-6 activates antiapoptotic pathways also in hepatocytes. Earlier experiments by Kovalovich et al. demonstrated that IL-6 can activate BcL-xL expression, and a role for activating Akt has also been suggested.84,85 Therefore these results indicate that IL-6/gp130 might be relevant to directly protect hepatocytes during cell cycle progression. Additionally, IL-6 induces pathways involved in mediating immune-dependent mechanisms. IL-6 via STAT3 is the major cytokine to induce the acute-phase response (APR) in the liver. The
Chapter 2 LIVER REGENERATION
APR is a first line of defense in the body, but is also involved in the regulation of other pathophysiologic mechanisms, e.g. macrophage activation and interaction with the complement system.86 Besides controlling APR expression, IL-6 contributes to the regulation of the Th1/Th2 response.87 Therefore, these IL-6-dependent tasks could also be relevant in contributing to body homeostasis after partial hepatectomy.
INTERFERON-g Intracellular pathways activated via interferon-g comprise distinct pathways. However, the most prominent one is STAT1. STAT1 has been shown to directly activate the cyclin-dependent kinase inhibitors p21 and IRF-1. For both factors a direct role in inhibiting cell cycle progression has been demonstrated.88,89 After partial hepatectomy overexpression of p21 in hepatocytes results directly in inhibition of liver regeneration.90 Also for IRF-1 there is in vitro evidence in hepatocytes that IFN-g-induced expression results in cell cycle arrest.91 During liver regeneration after partial hepatectomy natural killer (NK) cells are activated, which results in secretion of interferon-g. Induction of NK cell activity, for example by pIpC or murine CMV infection, results in a strong release of IFN-g by NK cells, which is associated with inhibition and retardation of hepatocyte proliferation. Additionally, stronger DNA synthesis after partial hepatectomy was found in interferon-g-/- animals.92 Therefore, interferon-g is a physiological inhibitor of liver regeneration, and a strong immunological response, e.g. during immune-mediated liver disease, might be associated with a lack of liver regeneration.
CHEMOKINES TNF is able to induce multiple intracellular functions. Among these is the induction of several members of the CXC chemokine family. These are heparin-binding proteins with four conserved cysteine amino acids, where the first two cysteines are separated by one nonconserved amino acid.93 Members of this family are IL-8, MIP-2, ENA-78, IP-10 and others. They are best known because of their chemotactic properties. ENA-78 and MIP-2 in particular can be produced after TNF stimulation also by hepatocytes, and therefore the question emerged as to whether these chemokines could be responsible for some of the TNF-dependent effects during liver regeneration.94,95 Hepatic ENA-78 and EPA levels are increased after partial hepatectomy,96 and manipulating both chemokine-dependent effects has a major impact on liver regeneration. Blockage of either of the two chemokines impairs liver regeneration, whereas stimulation experiments with MIP-2 results in enhanced hepatocyte proliferation after partial hepatectomy.95 As shown for IL-6, MIP-2 via its receptor CXCR2 also activates STAT3,96,97 suggesting that this could be one of the mechanisms by which MIP-2 enhances cell cycle progression of hepatocytes. A positive effect on liver regeneration has also been demonstrated for IP-10. IP-10 activates intracellular signals as shown for MIP-2 via the CXC-R2 receptor.98 Therefore it is likely that all members of the CXC chemokine family are able to stimulate hepatocyte proliferation, at least to a certain extent, using the CXC-R2 receptor.
ENERGY METABOLISM DURING LIVER REGENERATION In the early phase of liver regeneration after partial hepatectomy the liver accumulates large amounts of triglycerides.99 Fat accumulation is reduced at the start of DNA synthesis. Therefore fat accumulation in hepatocytes seems important during the process of liver regeneration. In order to better characterize the relevance of this observation, Shteyer et al. blocked fat accumulation after partial hepatectomy by two different approaches and in both cases liver regeneration was impaired. These results demonstrate that the tight regulation of fat accumulation is essential to allow proliferation of hepatocytes.100 The molecular mechanisms behind these observations are at present not completely understood. However, in recent years mainly two families of transcription factors – CCAAT/enhancer-binding proteins (C/EBPs) and PPAR – that are important in regulating genes involved in lipid metabolism have been studied during liver regeneration. The C/CBP family consists of several members and a role for hepatocyte proliferation both in vivo and in vitro has been demonstrated.101,102 After partial hepatectomy an up-regulation of C/EBPb and a down-regulation of C/EBPa can be observed which triggers changes in target gene expression.103 These changes occur before the start of DNA synthesis. Lack of C/EBPb expression in knockout animals results in impaired DNA synthesis and is associated with hypoglycemia and a lack of immediate early gene expression.104 Additionally, lack of expression of insulin-like growth factor-binding protein also blocks C/EBPb induction after partial hepatectomy and a phenotype related to the phenotype found in C/EBPb-/- mice.105 Therefore, direct cross-talk between metabolic functions and C/EBPb expression is obviously important during liver regeneration, and these changes are also essential regulators during adipocyte differentiation.106 Thus changes in C/EBP activity may also determine adipogenic changes found during liver regeneration. PPARa (peroxisome proliferator-activated receptor a) belongs to the family of nuclear receptors and there is emerging evidence that it might be a critical modulator controlling the energy flux important for repair of liver damage. PPARa especially controls the constitutive expression of genes involved in mitochondrial fatty acid b-oxidation, including carnitine palmitoyltransferase-1.107,108 Mitochondrial b-oxidation of fatty acids has been shown critical for energy metabolism during liver regeneration.109 Therefore, partial hepatectomy experiments in PPARa-/- mice were interesting as they showed delayed and reduced DNA synthesis. These findings were associated with a lack of cyclin D1 and c-myc expression, indicating that energy metabolism is critical in the early priming phase, allowing hepatocytes to leave the resting G0 state.110 Besides its role in energy metabolism there is also evidence that PPARa directly induces cyclin D1 expression. Experiments with nafenopin, a peroxisome proliferator, and a PPARa ligand show that via this mechanism this drug can induce hyperplasia of the liver.111 In recent years a second class of nuclear receptor thyroid hormone receptor (TR) has been shown to stimulate liver regeneration after partial hepatectomy via its ligand thyroid hormone (T3), apart from its effect on direct hyperplasia. As shown for PPARs, T3 directly induces cyclin D1 expression112 and thus enhances liver regenera-
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tion after partial hepatectomy, especially during the first 24 hours after surgery.113 At present there are no data concerning whether T3 may also have an effect on the energy metabolism of hepatocytes during liver regeneration. Together these results indicate that hepatocytes require a tightly regulated network controlling energy metabolism in order to allow cell cycle progression. Further work in this area might lead to the possibility of stimulating the regenerative capacity of hepatocytes in diseased livers.
INHIBITION OF HEPATOCYTE PROLIFERATION DURING LIVER REGENERATION Several factors have been defined that are involved in mediating hepatocyte proliferation. In contrast, the mechanisms leading to cessation of liver growth are incompletely understood. The most prominent factor that seems important is transforming growth factor-b (TGF-b). TGF-b is a multifunctional cytokine involved in different mechanisms, e.g. growth and development. Three forms of TGF-b (TGFb 1–3) are known in mammals, which have 80% identity on the amino acid level. All TGF-b forms bind directly or via co-receptors to the TGF-b type II receptor. In turn they recruit, bind and transphosphorylate type I receptors, thereby stimulating their
protein kinase activity. The activated type I receptors phosphorylate Smad2 or Smad3, which then bind to Smad4. The resulting Smad complex translocates to the nucleus in order to interact in a cellspecific manner with various transcription factors to regulate target gene transcription (Figure 2-8) (for review see 114). In many cells TGF-b inhibits cell proliferation in G1 as it stimulates cyclin-dependent protein kinase inhibitor p15 and blocks the function or production of essential cell cycle regulators, e.g. cyclin-dependent protein kinases 2 and 4 and cyclins D1 and D4.115 A role for TGF-b in hepatocytes was first detected in vitro, where it has strong antiproliferative activity.116 After partial hepatectomy TGF-b mRNA increases immediately117, and infusion of TGF-b after partial hepatectomy transiently delays the start of DNA synthesis.118 Additionally, during liver regeneration hepatocytes acquire a transient resistance against TGF-b by down-regulating TGF-b receptors119 or up-regulating inhibitors of the TGF-b signaling pathway.120 Therefore, these results suggest that TGF-b-dependent signaling is directly involved in controlling liver regeneration at different stages. At the beginning the pathway is down-regulated in order to allow hepatocytes to enter the cell cycle, whereas after DNA synthesis TGF-b sensitivity is restored so as to limit hepatocyte proliferation and terminate liver regeneration.121 The concept that TGF-b-dependent signaling is especially involved in the early phase of liver regeneration has been further confirmed in hepatocyte-specific knockout mice for TGF-b receptor II (TGF-RII). These animals show an earlier and increased DNA
Figure 2-8. TGF-b-dependent signaling. Binding of TGF-b induces phosphorylation and activation of TGF-b-receptor1 (TGFbR1) by the TGF-b-receptor2 (TGFbR2). TGFbR1 phosphorylates Smad2 and Smad3. Both factors interact with Smad4 in the cytoplasm or nucleus and regulate gene transcription in several ways. This includes binding and interaction with other transcription factors, interacting with co-repressors, and binding to factors such as CBP and p300 involved in mediating gene transcription. Smad7 represses signaling by other Smads in order to down-regulate the cascades. Besides activating Smads TGF-b also induces the ERK/MAPkinase cascade that is involved in modulating/inhibiting Smad proteins.
TGFb TGFbR2
TGFbR1 PP Smad3
P
PP Smad2
Smad7
P P Smad2 Smad3
Smad4
Smad4
PP Smad2
PP Smad4
PP Smad2
PP Smad2 PP
Smad2
PP Smad3
Smad2 Smad2
PP
PP
Smad3
PP Smad3
Smad4
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synthesis. Additionally, the weight of the regenerating liver is increased after partial hepatectomy. However, there was no major difference in cessation of DNA synthesis between TGF-RII-/animals and controls, indicating that additional pathways are involved in blocking DNA synthesis after partial hepatectomy.122 A second member of the TGF-b superfamily, activin A, has also been suggested for blocking hepatocyte proliferation in vitro and after partial hepatectomy.123 Activin A induces intracellular Smad activation through its type II receptor, comparable to TGF-b. Therefore similar roles as for TGF-b in terminating liver regeneration have been suggested. Kogure and co-workers infused follistatin – an activin A receptor antagonist – during liver regeneration and demonstrated that hepatocyte proliferation and liver weight are induced.124 In the TGF-RII-/- animals activin A expression was increased compared to controls. Additionally, after follistatin infusion in TGFRII-/- hepatocyte proliferation was increased after 120 hours, again indicating that the activin A-induced pathway is involved in terminating liver regeneration.122 However, as activin A and TGF-b activate very similar intracellular pathways there is a high chance that the two pathways are able to compensate for each other in both ways. Besides TGF-b and activin A, alternative signaling cascades have been discussed that might be involved in terminating liver regeneration. An attractive candidate is sphingosine 1-phosphate (S1P), as interaction with the G-protein-coupled endothelial differentiation gene Edg-5 activates Rho activity in hepatocytes, which is growth inhibitory. As shown for TGF-b, Edg-5 also increases 24–72 hours after partial hepatectomy, and administration of S1P during liver regeneration increases Rho activity and inhibits DNA synthesis.125 Together the results of the inhibitor pathways of hepatocyte proliferation demonstrate that different pathways do exist which are involved in inhibiting hepatocyte proliferation. Further work in this area will probably help to develop new treatment options to limit the uncontrolled growth of hepatocytes.
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114. Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 2003;113:685–700. 115. Alexandrow MG, Moses HL. Transforming growth factor beta 1 inhibits mouse keratinocytes late in G1 independent of effects on gene transcription. Cancer Res 1995;55:3928–3932. 116. Nakamura T, Tomita Y, Hirai R, et al. Inhibitory effect of transforming growth factor-beta on DNA synthesis of adult rat hepatocytes in primary culture. Biochem Biophys Res Commun 1985;133:1042–1050. 117. Braun L, Mead JE, Panzica M, et al. Transforming growth factor beta mRNA increases during liver regeneration: a possible paracrine mechanism of growth regulation. Proc Natl Acad Sci USA 1988;85:1539–1543. 118. Russell WE, Coffey RJ Jr, Ouellette AJ, et al. Type beta transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat. Proc Natl Acad Sci USA 1988;85:5126–5130. 119. Chari RS, Price DT, Sue SR, et al. Down-regulation of transforming growth factor beta receptor type I, II, and III during liver regeneration. Am J Surg 1995;169:126–131. 120. Macias-Silva M, Li W, Leu JI, et al. Up-regulated transcriptional repressors SnoN and Ski bind Smad proteins to antagonize
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3
MECHANISMS OF LIVER CELL DESTRUCTION Hartmut Jaeschke Abbreviations AIF apoptosis-inducing factor AMAP 3¢-hydroxyacetanilide A-SMase acidic sphingomyelinase Apaf-1 apoptosis protease-activating factor-1 APAP acetaminophen CAD caspase-dependent DNase CXC cys-X-cys c-FLIP cellular FLICE-inhibitory protein DD death domain DED death effector domain DIABLO direct inhibitor of apoptosis-binding protein with low PI DISC death-inducing signaling complex FADD Fas-associated death domain
FAN FasL IAPs ICAD IL-6 IKK iNOS KC MAT1 MCP-1 MIP-2 MPT mtDNA NAPQI
factor associated with N-SMase Fas ligand inhibitors of apoptosis proteins inhibitor of caspase-dependent DNase interleukin-6 inhibitor of NF-kB kinase inducible nitric oxide synthase keratinocyte-derived chemokine methionine adenosyltransferase 1A monocyte chemoattractant protein-1 macrophage inflammatory protein-2 membrane permeability transition pores mitochondrial DNA N-acetyl benzoquinone imine
INTRODUCTION Most acute and chronic liver diseases are characterized by an excessive rate of death of hepatocytes and other types of liver cell. Therefore, investigating the mechanisms of how liver cells die under various pathophysiological conditions is critically important for the development of therapeutic intervention strategies. In recent years much progress has been made in understanding the intracellular signaling mechanisms that cause cell death. This led to the elucidation of signaling pathways of ‘self-destruction’ (apoptosis) and to increased insight into mechanisms of ‘forced destruction’ (oncotic necrosis). In addition, it is recognized that there is a balance between liver cell death and regeneration. This chapter will focus on recent advances in our understanding of apoptotic and necrotic signaling mechanisms in liver cells.
APOPTOSIS OR ONCOTIC NECROSIS Until about 10 years ago almost all research on mechanisms of cell death focused on necrosis or oncotic necrosis. However, more recently, apoptosis signaling pathways became the center of attention. As a result, many previous conclusions regarding necrotic cell death were challenged,1,2 and today there is a controversy over which is the predominant pathway in most forms of liver disease.3–5 Given the overlap in the signaling pathways between the two modes of cell death, it appears less relevant to argue about the label but preferable instead to focus on the intracellular signaling events. Nevertheless, the term apoptosis should not be used unless the critical
NK NKT cells NO N-SMase PARP-1 RIP1 Smac tBid TGF-a TRADD TRAF2 TUNEL
natural killer NK cells with T-cell receptors nitric oxide neutral sphingomyelinase poly-(ADP-ribose)-polymerase-1 TNF-receptor-interacting protein 1 second mitochondria-derived activator of caspases truncated Bid transforming growth factor-a TNFR1-associated death domain protein TNF-receptor associated factor 2 transferase-mediated deoxyuridine triphosphate nick-end labeling
morphological characteristics of apoptotic cell death are fulfilled, i.e. cell shrinkage, chromatin condensation, and margination and formation of apoptotic bodies (Table 3-1).3 Apoptosis is generally a single cell event and does not per se cause an inflammatory response. Apoptotic bodies are removed without a trace by neighboring cells or tissue macrophages. On the other hand, if the cell swells, shows evidence of vacuolation, karyolysis and karyorrhexis, together with the release of cell contents, it is said to undergo oncotic necrosis. In general, oncotic necrosis affects large numbers of cells clustered together. In addition, oncotic necrosis triggers a significant inflammatory response. Although most of these cell death characteristics can be identified in a tissue (Table 3-1), there are a number of important issues to consider.
SEVERITY OF INSULT DETERMINES MODE OF CELL DEATH Not every type of cellular stress triggers a uniform response with a predetermined mode of cell death. In fact, a very mild insult affecting only a few cellular organelles, e.g. mitochondria, may not cause cell death but instead lead to removal of the damaged mitochondria in the surviving cell by autophagy.6 If the insult is more severe, more mitochondria are involved and the cell undergoes apoptosis. A moderate insult may lead to the release of enough cytochrome c and other mediators into the cytosol to propagate the cell death signal and, on the other hand, leave enough mitochondria intact to maintain cellular ATP levels. However, if the insult is too severe and most of the mitochondria are damaged, cellular ATP levels drop and the cell undergoes oncotic necrosis.3,7 This concept was recently supported in an in vivo model. Short periods of hemorrhage, which
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Section I. Pathophysiology of the Liver
Table 3-1. Morphological Characteristics and Biochemical Assays for Apoptosis and Oncotic Necrosis
Morphological characteristics Cell morphology
Nuclear morphology (H&E, DAPI) Mitochondrial morphology Inflammation (mainly neutrophil accumulation) Biochemical parameters TUNEL Assay (DNA strand breaks in nucleus and detection of large DNA fragments in cytosol) Antihistone ELISA (detection of small DNA fragments in cytosol) Internucleosomal DNA damage (DNA ladder on agarose gels) Caspase enzyme activities (fluorescence assays are most sensitive)
APOPTOSIS
ONCOTIC NECROSIS
Cell shrinkage, eventually break-up into apoptotic bodies; single cell event even if many cells are affected Chromatin condensation and margination No relevant changes initially; later: swelling and rupture of outer membrane Negative; can be positive during excessive apoptotic cell death and secondary necrosis
Cell swelling, eventually cell contents release; affects large numbers of clustered cells
Nuclear and cytoplasmic staining when nucleus is degraded
>> 10-fold increase over baseline
3–10-fold increase over baseline during karyolysis Positive during karyolysis
Positive Strongly positive (up to >100-fold increase over baseline) Strongly positive; shows decline of proenzymes and the appearance of active fragments
Caspase processing, e.g. active caspase-3 (immunohistochemistry) Cleavage of caspase substrates, e.g. PARP-1 (Western blot) Release of mitochondrial proteins, e.g. cytochrome c, Smac/DIABLO, endonuclease G, AIF Translocation of proteins to mitochondria, e.g. Bax, Bid, tBid.
Strongly positive
Cell contents release, e.g. ALT, AST, caspases, etc. Nuclear staining with propidium iodide, trypan blue etc. Annexin V staining (phosphatidyl serine externalization) Mitochondrial depolarization (detected by membrane potential-indicating probes, e.g. rhodamine 123, JC-1 and others)
Strong inflammatory response can aggravate the existing injury
Nuclear staining
Caspase processing (Western blot)
Death receptor and death ligand expression
Karyolysis, karyorrhexis Swelling and rupture of outer membrane
Strongly positive; shows decline of intact protein and the appearance of fragment Positive; best detected as increase of proteins levels in the cytosol; decline of protein levels in mitochondria is less sensitive Positive, increase of protein levels in mitochondria is more sensitive to detect than reduction of concentration in cytosol Death receptors are constitutively expressed on hepatocytes and nonparenchymal cells; increased expression is no evidence for apoptosis Initially negative; increase in liver enzyme activities in plasma is observed during secondary necrosis Initially negative; will become positive during secondary necrosis when cell membrane permeability is increased Positive; early event in apoptosis Positive; initially affects a small number of mitochondria; progresses during secondary necrosis
Negative or minor increase (1 mmol/l) without any increase in biliary bile acid secretion.157
BENIGN RECURRENT INTRAHEPATIC CHOLESTASIS TYPE 2 Not all patients with BRIC have mutations of ATP8B1. In a subset of patients with episodic cholestasis, mutations of BSEP were found. The disease was called BRIC type 2. It appears that these patients are particularly prone to the development of cholelithiasis and less to pancreatitis. This distinguishes them from patients with BRIC type 1. Serum GGT levels are low in both diseases. Patients with BRIC type 1 can be completely asymptomatic between attacks of cholestasis, but whether this is also true for patients with BRIC type 2 needs to be studied.158
FAMILIAL HYPERCHOLANEMIA Familial hypercholanemia is characterized by elevated serum bile salt levels, severe pruritus and fat malabsorption.159 So far this
disease has been identified among Amish individuals. It was originally thought to result from a sinusoidal uptake defect. Recently it has been reported to be caused by mutations of one of two genes, one that encodes tight junction protein 2 (ZO-2) and BAAT, which encodes bile acid coenzyme A: amino acid N-acyltransferase. In these latter patients glycine and taurine bile acid conjugates cannot be formed.160
BILE ACID SYNTHESIS DEFECTS Defects of bile acid synthesis resemble PFIC type 2. Clayton et al.161 described a defect of 3b-D5-C27-hydroxysteroid oxidoreductase as a cause of giant cell hepatitis. Deficiency of D4-3-oxosteroid-5b reductase and 3b-hydroxy C27steroid dehydrogenase/isomerase and mutations of the oxysterol 7a-hydroxylase gene may also be causes of neonatal hepatitis and cholestasis.162–164 In these diseases toxic intermediates are formed which cause cholestasis by interaction with the hepatic bile acid transporter.165 Bile acid synthesis defects are called PFIC type 4 by some authors.
PROGRESSIVE FAMILIAL INTRAHEPATIC CHOLESTASIS TYPE 3 The third PFIC subtype, PFIC type 3, is quite different from the others. The serum GGT activity is markedly elevated in these patients and liver histology shows extensive bile duct proliferation, and portal and periportal fibrosis.64,166,167 Phenotypically PFIC type 3 resembles the Mdr2(-/-) mice. In humans with PFIC type 3, mutations of the ABCB4 (MDR3) gene are the underlying cause.129,167,168 Phosphatidylcholine, the main phospholipid in bile, is washed down from the canalicular membrane by bile acids (Figure 5-4A). In contrast to PFIC type 2, in PFIC type 3 bile acid transport proceeds unimpaired, but this occurs without phospholipids because of the MDR3 deficiency. This has major pathophysiologic consequences. In normal bile the inherent toxicity of bile acids is quenched by phosphatidylcholine. In the bile of PFIC 3 patients bile acid toxicity is unantagonized by phospholipids, and this causes damage to bile duct epithelium, with periportal inflammation and fibrosis and bile duct proliferation as a result (Figure 5-4B). In humans this is even more extreme than in Mdr2(-/-) mice, as human bile acids (e.g. chenodeoxycholic acid) are more toxic than those of the mouse – mainly the very hydrophilic muricholate. In patients with PFIC type 3 symptoms present somewhat later in life than in PFIC types 1 and 2, and liver failure also occurs at a later age. Jaundice may be less apparent, but pruritus is usually severe. Patients with a partial ABCB4 (MDR3) defect respond to ursodeoxycholic acid therapy.147 The majority of patients, however, have to be transplanted. Mutations of the ABCB4 gene on chromosome 7q21 are the underlying cause of the disease. Although PFIC3 is discussed as a cholestatic disease, in a strictly physiologic sense there is no cholestasis, as the bile flow is not impaired.58
INTRAHEPATIC CHOLESTASIS OF PREGNANCY Jacquemin et al.168 reported a high incidence of intrahepatic cholestasis of pregnancy (ICP) in families with PFIC type 3. This suggests that in persons carrying one mutated ABCB4 gene, cholestasis may occur during pregnancy. Mutations leading to single
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Cholesterol BSEP
ABC G5/G8
Bile acids
Bilirubin
MRP2 MDR3
Phosphatidyl choline
related to ATP8B1 serum GGT is not elevated. Ursodeoxycholic acid has been shown to be of benefit in patients with ICP, with a reduction in fetal loss.173,174
OTHER FORMS OF INTRAHEPATIC CHOLESTASIS More forms of intrahepatic cholestasis exist. Aageneas syndrome is a combination of severe progressive lymphedema and episodic intrahepatic cholestasis.175 The locus for this disease has been mapped to chromosome 15q.176
DUBIN–JOHNSON SYNDROME GGT
GGT GGT
GGT A
Cholesterol BSEP
Bilirubin
ABC G5/G8
BA
BA BA BA
Bile acids
Phosphatidyl choline MDR3
MRP2
GGT GGT GGT
GGT
REFERENCES
BA BA BA BA BA BA BA
GGT GGT GGT
GGT GGT
GGT
B Figure 5-4. Hepatocanalicular secretion of bile salts, phosphatidylcholine and cholesterol. A The toxicity of bile salts is neutralized in mixed micelles by the presence of phosphatidylcholine and cholesterol. B In progressive familial cholestasis type 3 bile salt secretion is unimpaired but the secretion of phosphatidylcholine is strongly reduced. Thus, bile salt toxicity is unantagonized by the lack of phosphatidylcholine, and this affects the cell integrity of the biliary epithelium. Bile duct epithelial cell proliferation, periportal inflammation and fibrosis and elevation of g-glutamyltransferase is a result of this.
amino acid substitutions of the MDR3 protein may cause intracellular traffic mutants, that is, the protein is synthesized but does not reach its destination in the canalicular membrane.169 One can hypothesize that in patients carrying these mutations the hormones in the third trimester of pregnancy impair the intracellular targeting that causes the disease to become clinically manifest. ICP has also been described in families with PFIC type 1.170 BSEP (ABCB11) polymorphisms seem to be of less importance for ICP.171 ICP not related to MDR3 or ATP8B1 has been reported in a Finnish group of patients.172 In contrast to ICP related to ABCB4, in ICP
80
Dubin–Johnson syndrome is described here not because it is an important cholestatic disease, but because it is caused by a mutation of ABCC2 encoding MRP2 (Table 5-2).133,177 Dubin–Johnson syndrome is characterized by conjugated hyperbilirubinemia without other serum enzyme abnormalities. Patients have a normal lifespan. A black or brownish lysosomal pigment in the hepatocytes is a characteristic histological feature. The excretion of urinary coproporphyrin isomer I is elevated in these patients. TR- and EHBR rats are animal models for this disease. These animals have a decreased hepatobiliary secretion of organic anions because of a mutation of the Abcc2 gene.81,178 Patients with Dubin–Johnson syndrome are homozygous carriers of ABCC2 gene mutations. Rapid degradation of mutated ABCC2 mRNA, or impaired MRP2 protein maturation and trafficking, may be the underlying cause of the disease.179 Dubin–Johnson syndrome has no influence on longevity. The disease needs no treatment.
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6
HEPATIC FIBROSIS AND CIRRHOSIS Don C. Rockey and Scott L. Friedman Abbreviations ALT alanine aminotransferase AST aspartate aminotransferase AUROC area under the receiver operator characteristic BMI body mass index CINC cytokine-induced neutrophil chemoattractant CTGF connective tissue growth factor DDR discoidin domain receptors ECM extracellular matrix EGF epidermal growth factor ELF European liver fibrosis ET-1 endothelin-1 FGF fibroblast growth factor FPI fibrosis probability index GGT g-glutamyl transferase
GnT-III HA HBV HCV HGF HIV HOMA-IR IL-10 JI LPS MCP-1 MEGX MELD MMPs MMP-2
N-acetylglucosaminyl transferase III hyaluronic acid hepatitis B virus hepatitis C hepatocyte growth factor human immunodeficiency virus insulin resistance by the homeostasis model assessment interleukin-10 jejuno-ileal lipopolysaccharide monocyte chemotactic protein-1 monoethylglycinexylidide model for end-stage liver disease matrix metalloproteinases matrix metalloproteinase 2
INTRODUCTION Hepatic fibrosis has emerged as a highly relevant aspect of liver biology because of the significant progress in uncovering its mechanisms, combined with a growing realization that effective antifibrotic therapies may soon alter the natural history of chronic liver disease. Thus, liver fibrosis can now be viewed as a clinical problem whose diagnosis and treatment will soon have rational, evidence-based approaches. This progress is very timely, as the continued ‘aging’ of the HCV-infected cohort and the growing prevalence of obesityrelated liver diseases are leading to precipitous increases in the prevalence of advanced liver disease.1 With these issues in mind, this chapter will review clinical aspects of hepatic fibrosis, including natural history, pathophysiologic mechanisms, current and future tools for diagnosis, and emerging antifibrotic strategies. In addition, several recent reviews highlight many of these aspects in greater detail.2–6 Hepatic fibrosis is the accumulation of extracellular matrix, or scar, in response to acute or chronic liver injury. Fibrogenesis represents a wound healing response to injury (Figure 6-1), and ultimately leads to cirrhosis. Cirrhosis is the end-stage consequence of fibrosis of the hepatic parenchyma, resulting in nodule formation that may lead to altered hepatic function and blood flow. Both fibrosis and cirrhosis are the consequences of a sustained wound-healing response to chronic liver injury from a range of causes, including viral, autoimmune, drug induced, cholestatic and metabolic diseases. The clinical manifestations of cirrhosis vary widely, from no symp-
MMP-9 NASH NGFR NO PDGF PELD PIIINP PPAR QTL ROC TGF-b1 TIMPs ULN VEGF
matrix metalloproteinase 9 non-alcoholic steatohepatitis nerve growth factor receptor nitric oxide platelet-derived growth factor pediatric end-stage liver disease propeptide of type III collagen peroxisomal proliferator-activated receptor quantitative trait loci receiver operating characteristic transforming growth factor beta 1 tissue inhibitors of metalloproteinases upper limit of normal vascular endothelial growth factor
toms at all to liver failure, and are determined by both the nature and severity of the underlying liver disease as well as the extent of hepatic fibrosis. Up to 40% of patients with cirrhosis are asymptomatic and may remain so for long periods, but progressive deterioration leading to death or liver transplantation is typical once complications (such as ascites, variceal hemorrhage or encephalopathy) develop. In such patients there is a 50% 5-year mortality, with approximately 70% of these deaths directly attributable to liver disease.7 In asymptomatic individuals cirrhosis may be first suggested during routine examination, although histologic analysis may be required to establish the diagnosis. Cirrhosis affects hundreds of millions of patients worldwide. The overall burden of liver disease in the United States – the vast majority of which is due to chronic disease with fibrosis – continues to expand, exacting an increasing economic and social cost.1 Indeed, in the US cirrhosis is the most common non-neoplastic cause of death among hepatobiliary and digestive diseases, accounting for approximately 30 000 deaths per year. In addition, 10 000 deaths are due to liver cancer, the majority of which arise in cirrhotic livers, consistent with a steadily rising mortality rate from hepatic cancer.8 Notably, hepatocellular carcinoma is the most rapidly increasing neoplasm in the US and western Europe.9 Initial studies of hepatic fibrosis focused on the composition of extracellular matrix in liver, and continued incremental progress in this area is still anticipated. However, attention has gradually shifted towards exploring the cellular basis of fibrosis and the cellular mediators that drive fibrosis progression and regression (see Pathophysiology, below). In general, the molecular composition of the scar
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Normal liver
Liver injury
Hepatocytes
Loss of Hepatocyte microvilli
Space of Disse
Quiescent stellate cell Kupffer cell
Activated stellate cell Endothelial cell
Hepatic sinusoid
Deposition of scar matrix
Loss of fenestrae
Kupffer cell activation
Figure 6-1. Hepatic liver cells and the hepatic sinusoid in normal and injured liver. On the left panel is shown the multiple key liver-specific cellular elements in the normal liver, including hepatocytes, endothelial cells, Kupffer cells, and stellate cells. Stellate cells are located within the subendothelial space of Disse (i.e. between the sinusoidal endothelium and hepatocytes). The figure emphasizes the close physical relationships between the various cellular elements in the liver. After liver injury, changes in numerous cells occur; for example, stellate and Kupffer cells become activated (see Figure 6-3), hepatocytes lose their microvilli, and endothelial cells lose their characteristic fenestrae. All of these features contribute to continued cell activation and injury, as well as dysfunction at the whole organ level.
tissue in cirrhosis is similar regardless of etiology, and resembles that of other parenchymal scarring (e.g. kidney), consisting of the extracellular matrix constituents, collagen types I and III (i.e. ‘fibrillar’ collagens), sulfated proteoglycans, and glycoproteins.10 However, some isoforms of extracellular matrix constituents, for example fibronectin11 and proteoglycans,12 may be relatively enriched during progressive injury. These scar constituents accumulate from a net increase in their deposition in liver and not simply from the collapse of existing stroma.
CLINICAL ASPECTS OF HEPATIC FIBROSIS NATURAL HISTORY AND RISK FACTORS Fibrosis leading to cirrhosis can accompany virtually any chronic liver disease that is characterized by the presence of architectural disruption and/or inflammation. The vast majority of patients with liver disease worldwide have chronic viral hepatitis, or steatohepatitis associated with either alcohol or obesity; other etiologies of liver disease include parasitic infestation (e.g. schistosomiasis), autoimmune attack on hepatocytes or biliary epithelium, neonatal liver disease, metabolic disorders including Wilson’s disease,
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hemochromatosis and a variety of storage diseases, chronic inflammatory conditions (e.g. sarcoidosis), drug toxicity (e.g. methotrexate or hypervitaminosis A), and vascular derangements, either congenital or acquired. Of the many causes of chronic liver disease, our understanding of natural history of fibrosis is most complete in HCV, with some information about HBV and steatohepatitic diseases, including alcoholic liver disease and NASH. Information about fibrosis progression in other diseases is largely anecdotal, but the development of cirrhosis typically requires many years to decades. There are, however, some notable exceptions in which the development of cirrhosis can be greatly accelerated, possibly occurring within months rather than years: (1) neonatal liver disease – infants with biliary atresia may present at birth with severe fibrosis and marked parenchymal distortion; (2) HCV-infected patients after liver transplantation – a subset of patients who undergo liver transplantation for HCV cirrhosis may develop rapidly progressive cholestasis and recurrent cirrhosis within months, requiring retransplantation;13 (3) patients with HIV/HCV co-infection – these patients have relatively rapid fibrosis compared to those with HCV alone,14 especially if the HIV is untreated (see below); (4) severe delta hepatitis;15 and (5) some cases of drug-induced liver disease. These examples of ‘fulminant fibrosis’ probably reflect dysregulation of several pathways, including defective immunity, massive inflammation and necrosis, and/or
Chapter 6 HEPATIC FIBROSIS AND CIRRHOSIS
altered matrix resorption. Together, they highlight the highly dynamic nature of scar accumulation and degradation. Moreover, when matrix accumulation is unopposed because degradation is ineffective, more rapid fibrosis may ensue. Once cirrhosis and its complications develop the prognosis is predicted by widely used systems, including Child–Pugh, PELD16 and MELD,17 which are predictive independent of the etiology of liver disease.
Hepatitis C Virus The risk and natural history of fibrosis associated with HCV have been greatly clarified as a result of several large clinical studies incorporating standardized assessments of fibrosis that combine detailed historical and clinical information.18 The disease can run a remarkably variable course, from decades of viremia with little fibrosis to a rapid onset of cirrhosis within 10–15 years. It appears to be host factors rather than viral factors that correlate with fibrosis progression in HCV. The data supporting this conclusion include the following: (1) there is no relationship between viral load or genotype and severity of fibrosis even though these former factors affect the response to antiviral therapy; (2) human promoter polymorphisms (e.g. TGF-b1 and angiotensin) appear to correlate with fibrosis risk,19 with large-scale efforts currently under way to identify additional genetic markers of fibrosis risk;20 (3) host immune phenotype may be critical, as there is more rapid progression in immunosuppressed patients, whether due to HIV or to immunosuppressive drugs.14 In mice, a Th2 phenotype strongly correlates with fibrogenic potential,21 which has led to successful efforts to use quantitative trait loci (QTL) mapping to identify specific fibrosis risk genes in these animals.22 Other identified host risk factors for more rapid progression of HCV include: (1) older age at the time of infection; (2) concurrent liver disease due to HBV or alcohol (>50 g/day); it is uncertain, however, whether lesser amounts of alcohol intake are additive towards fibrosis progression: recent studies suggest that less than 50 g/day of alcohol results in a neglible increased risk of hepatic fibrosis;23 (3) male gender; (4) increased body mass index (BMI), associated with hepatic steatosis;24 (5) HIV infection or immunosuppression following liver transplantation. Because standard clinical indices cannot distinguish between minimal and even advanced fibrosis, knowledge about these risk factors and duration of infection can greatly inform clinical management. Thus, for chronic HCV, if the time of infection is known and a biopsy obtained at any time thereafter, the rate of progression per year based on either Ishak or METAVIR scoring can be estimated.25 Although initial analyses of this type suggested that fibrosis progression is truly linear, it is now increasingly clear that the progression rate accelerates as the disease advances,26 such that it takes less time to progress between Metavir stages 3 and 4, than from stage 1 to 2, for example. Assessment of fibrosis stage and rate of fibrosis progression can be valuable for at least three reasons: (1) the actual stage of fibrosis will indicate the likelihood of response to a-interferon or ainterferon/ribavirin, as the advanced stages of fibrosis (F3 or F4) generally have a lower response rate to antiviral therapy;27,28 (2) if little fibrosis progression has occurred over a long interval, then treatment with antiviral therapy may be deemed to be less urgent
and it may be safe to await more effective and/or better-tolerated therapy; (3) the approximate time to the development of cirrhosis can be estimated. This would not, however, indicate if or when clinical liver failure would occur, as the complications of liver disease may be delayed for up to a decade or more after the establishment of cirrhosis. As genetic risk markers that predict a rapid fibrosis progression rate are developed, this information, combined with the absolute stage of fibrosis, may enable more accurate identification of patients at risk for disease and thus in need of antifibrotic therapy.
Hepatitis B Virus Very few studies have assessed the progression rate of fibrosis in chronic HBV infection. In general, inflammatory activity, as influenced by viral factors, including e Ag status, that indicate active viral replication, correlates with fibrosis.29,30 Fibrosis progression has been correlated with HBV genotype in at least one study.31 In a subset of patients a rapidly progressive ‘fibrosing cholestatic hepatitis’ may occur,32 but there are neither definitive risk factors for this condition nor unique etiologic, cellular or molecular determinants identified. In addition, delta hepatitis superinfection or co-infection may greatly accelerate the risk of advanced fibrosis and cirrhosis.15 What is striking, however, is that virologic suppression in response to potent antiviral regimens can effect remarkable improvement not only in serum alanine aminotransferase (ALT) levels and histologic inflammation, but also in fibrosis.15,33–35 Indeed, dramatic resolution of cirrhosis in a 10-year follow-up has been reported in patients with delta hepatitis who were successfully treated with a-interferon.15
Alcoholic Liver Disease The clearest clinical determinant of fibrosis is continued alcohol abuse: patients with fibrosis who continue to drink are virtually assured of progression. In addition, two clinical features commonly seen in steatohepatitis, elevated BMI and serum glucose, also confer an increased risk of fibrosis in alcoholic liver disease.36 Pathologically, the presence of pericentral fibrosis (central hyaline sclerosis) carries a high risk of eventual panlobular cirrhosis, which is almost certain if alcohol intake continues.
Non-Alcoholic Steatohepatitis There is a critical need for better data about natural history, risk factors for fibrosis, and rate of fibrosis progression in NASH, issues now being addressed in several multicenter studies. Patients with only steatosis and no inflammation appear to have a benign course when followed for up to 19 years;37 however, it is unclear whether this lesion is completely distinct from steatohepatitis, or instead represents a precursor of NASH. It is instructive to remember that HCV fibrosis progression rates were underestimated shortly after the virus was first identified, as many patients had a relatively early fibrosis stage. With continued infection, however, a sizeable fraction eventually have progressed to more advanced stages. In a parallel situation, the obesity epidemic in the US and the developed world is only now being fully appreciated, and a threshold level of obesity may have only begun to confer a risk of liver disease that will become clinically significant in the next decade. In patients with sustained
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NASH spontaneous histologic improvement is very uncommon, but better longitudinal data are needed to understand the natural history of this disease; for example, data examining the evolution of NASH in patients undergoing bariatric surgery who have extensive weight loss and improvement in their metabolic syndrome are awaited. In three combined studies of 26 patients followed with sequential biopsies for up to 9 years, 27% had progression of fibrosis and 19% advanced to cirrhosis, but none had reversal of fibrosis.37 Of interest is the recurrence of NASH following liver transplantation in some patients with cryptogenic cirrhosis, implicating an underlying metabolic defect that may account for liver disease in both the native and the transplanted organs. Risk of fibrosis and rate of progression are critical issues that will influence risk stratification and patient selection for clinical trials, as progression to cirrhosis is the most important clinical consequence of NASH. Recently developed systems to grade and stage liver disease in NASH38 should allow for improved, prospective collection of standardized data that can further address these vital questions. In general, increasing obesity (BMI) >28 kg/m2) correlates with severity of fibrosis and risk of cirrhosis. Other risk factors include necroinflammatory activity with ALT >2¥ normal and/or AST/ALT >1, age, elevated triglycerides, insulin resistance and/or diabetes mellitus, and systemic hypertension.39 It is uncertain whether these features are comparable across the spectrum of disorders associated with NASH, including obesity with insulin-resistance, JI (jejuno-ileal) bypass, total parenteral nutrition and rapid weight loss, among others. Whether these factors represent surrogates for other risk factors (i.e. reduced antioxidant levels in older patients, increased renin–angiotensin activity in hypertensives) is unknown. Ratziu and colleagues40 have reported a clinicobio-logical score that combines age, BMI, triglycerides and ALT and which reportedly has 100% negative predictive value for excluding significant fibrosis.
REVERSIBILITY OF FIBROSIS AND CIRRHOSIS There is now clear evidence that fibrosis and even cirrhosis can be reversible. The feature common to all cases of cirrhosis improvement is the elimination of the underlying cause of liver disease, whether due to eradication of HBV,41 delta hepatitis15 or HCV,42 decompression of biliary obstruction in chronic pancreatitis,43 or to immunosuppressive treatment of autoimmune liver disease.44 Moreover, there is ample evidence of reversibility in animal models, which provide vital clues to underlying mechanisms.45 Earlier studies demonstrated that fibrosis improves with treatment of HCV,46 and even cirrhosis can regress following HCV eradication with a-interferon/ribavirin.42 Among a large cohort of patients successfully treated with this combination there were 150 with cirrhosis, half of whom had a reduction in their fibrosis score according to METAVIR staging, with several regressing by two or more stages. Because fibrosis in HCV typically progresses over three decades, one might anticipate an equally slow but steady regression of fibrosis following viral clearance.
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It remains unclear what distinguishes those patients whose cirrhosis is reversible from those in whom it is fixed. Potential factors influencing reversibility probably include: (1) a prolonged period of established cirrhosis, which could reflect a longer period of crosslinking of collagen, rendering this collagen less sensitive to degradation by enzymes over time. Animal studies now clearly support this possibility;47 (2) total content of collagen and other scar molecules, which might lead to a large mass of scar that is physically inaccessible to degradative enzymes; (3) reduced expression of enzymes that degrade matrix, or sustained elevation of proteins that inhibit the function of these degradative enzymes, in particular elevated levels of tissue inhibitors of metalloproteinases (TIMPs), which block matrix proteases and also prevent apoptosis of activated stellate cells.48,49 All three scenarios highlight the dynamic process of collagen deposition and degradation.
PATHOPHYSIOLOGY OF HEPATIC FIBROSIS AND CIRRHOSIS EXTRACELLULAR MATRIX (ECM) IN THE NORMAL AND THE FIBROTIC LIVER Extracellular matrix refers to the array of macromolecules that comprise the scaffolding of normal and fibrotic liver. These macromolecules consist of three main families: collagens, glycoproteins and proteoglycans (see 10 for review). The number of collagens identified in liver is rapidly growing, and includes collagen XVIII, which is a precursor to the molecule angiostatin. Glycoproteins include fibronectin, laminin, merosin, tenascin, nidogen, and hyaluronic acid, among others. Proteoglycans include heparan, dermatan sulfates, chondroitin sulfates, perlecan dystroglycan syndecan, biglycan and decorin. There is tremendous heterogeneity of these matrix macromolecules with respect to their different isoforms, variable combinations within different tissue regions, and changes related to age. In normal liver the subendothelial space of Disse separates the epithelium (hepatocytes) from the sinusoidal endothelium. This space contains a basement membrane-like matrix which, unlike the typical basement membrane, is not electron dense. The hepatic basement membrane is composed of non-fibril-forming collagens, including types IV, VI and XIV, glycoproteins and proteoglycans. This normal subendothelial ECM is critical for maintaining the differentiated functions of resident liver cells, including hepatocytes, stellate cells and sinusoidal endothelium. In contrast to basement membrane-type matrix, in normal liver the so-called interstitial ECM is largely confined to the capsule, around large vessels, and in the portal areas. It is composed of fibrilforming collagens (e.g. types I and III) together with cellular (EDA) fibronectin, undulin, and other glycoconjugates. As the liver becomes fibrotic, the total content of collagens and non-collagenous components increases three- to fivefold, accompanied by a shift in the type of ECM in the subendothelial space from the normal low-density basement membrane-like matrix to interstitial-type matrix (see 10 for review). This ‘capillarization’ leads to the loss of hepatocyte microvilli and the disappearance of endothelial fenestration (Figure 6-1).
Chapter 6 HEPATIC FIBROSIS AND CIRRHOSIS
The outcome of fibrogenesis is the conversion of normal lowdensity basement membrane-like matrix to high-density interstitialtype matrix. A number of components are responsible for ECM remodeling (see 49,50 for reviews) (Figure 6.2). These include a family of zinc-dependent enzymes matrix metalloproteinases (MMPs),51 their inhibitors (tissue inhibitor of metalloproteinases, TIMP), and several converting enzymes (MT1-MMP, and stromelysin, for example). In human liver diseases there is down-regulation of MMP1 (interstitial collagenase, collagenase I) and up-regulation of MMP2 (gelatinase A) and MMP9 (gelatinase B). Based on the differing substrate specificities of these enzymes, the result is increased degradation of basement membrane collagen and decreased degradation of interstitial collagens. These activated MMPs are regulated in part by their tissue inhibitors, the so-called TIMPs. TIMP1 and TIMP2 are upregulated relative to MMP1 in progressive experimental liver fibrosis, which may explain the decreased degradation of interstitial-type matrix observed in experimental and human liver injury. In contrast, during the resolution of experimental liver injury TIMP-1 and TIMP-2 expression is decreased whereas collagenase expression is unchanged, resulting in a net increase in collagenase activity and increased resorption of scar matrix. Stellate cells are a key source of MMP-2 and stromelysin. They also express TIMP-1 and TIMP-2 mRNAs and produce TIMP-1 and MT1-MMP MMP-9, which is a type IV collagenase locally secreted by Kupffer cells, and may also be produced by stellate cells in
response to interleukin-1.52 The source of MMP-1, which plays a crucial role in degrading the excess interstitial matrix in advanced liver disease, is still uncertain.53 However, interstitial collagenase activity in liver may be attributable to either MT1-MMP or even MMP-2, although further studies are required.
ECM–CELL INTERACTIONS Changes in the microenvironment of the space of Disse result in phenotypic changes in all resident liver cells. Hepatic stellate cells are activated by the surrounding increase in interstitial matrix.54 Sinusoidal endothelial cells produce cellular fibronectin in very early liver injury, which also contributes to stellate cell activation. In addition, endothelial cells produce type IV collagen, proteoglycans and factors (e.g. urokinase-type plasminogen activator) that participate in the activation of latent cytokines such as TGF-b1. Activated Kupffer cells release cytokines and reactive oxygen intermediates that may stimulate stellate cells in a paracrine manner.55 Platelets are also an abundant source of cytokines upon injury, producing a rich array of important growth factors. Hepatocytes, the most abundant cells in the liver, generate lipid peroxides following injury that lead to stellate cell activation, a prerequisite for fibrogenesis (see below). The dynamic interactions between fibrogenic cells in liver and the ECM is an important determinant of fibrogenesis. The ECM is a reservoir for growth factors, for example platelet-derived growth factor (PDGF).10 Like all cytokines, PDGF signals by binding to
Activated stellate cell Early pathological degradation
Regression Apoptotic stellate cell Normal ECM
MT1-MMP MMP-2 TIMP-2
Kupffer cell MMP-1 MMP-13 Other MMPs Progression
TIMP-1 TIMP-2
Figure 6-2. Emerging Mechanisms of Early Pathologic Matrix Degradation, Fibrosis Progression & Fibrosis Resolution in Chronic Liver Disease. Activation of stellate cells (top left panel) is a key event in hepatic fibrosis, and is associated with pathologic matrix degradation due to increased production of membrane type matrix metalloproteinase 1 (MT1-MMP), matrix metalloproteinase-2 (MMP-2), and tissue inhibitors of metalloproteinases (TIMPs), leading to replacement by interstitial collagen or scar matrix. As fibrosis progresses (middle panel), sustained expression of TIMPs prevents matrix degradation and apoptosis of activated stellate cells. Regression of fibrosis (upper right panel) is associated with increased apoptosis of activated stellate cells. Apoptosis requires decreased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), yielding a net increase in protease activity. These events may occur coincident with production of matrix metalloproteinases, which could include MMP-1 (in humans) and/or MMP-13 (in rodents), although cellular sources of these enzymes (possibly including Kupffer cells), and clear evidence of their induction in vivo are still lacking. Validation of these events and further elucidation of mechanisms underlying fibrosis regression represent key challenges for future studies.147a
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membrane receptors. The PDGF receptor belongs to a receptor family known as receptor tyrosine kinases, which collectively are key transducers for many important cytokines, including hepatocyte growth factor (HGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Interestingly, a new subclass of receptor tyrosine kinases, socalled discoidin domain receptors (DDR), has been identified; this group of receptors signal in response to fibrillar collagens rather than peptide ligands.56 Indeed, stellate cell activation is accompanied by up-regulation of DDR2 receptors, and increased signaling is associated with altered MMP-2 expression.57,58 Intracellular signaling cascades downstream of receptor tyrosine kinases and other receptors are pervasive (see 59 for review). Integrins are another type of membrane receptor that transduce extracellular signals in liver. These are heterodimeric transmembrane proteins composed of an a and a b subunit whose ligands are matrix molecules rather than cytokines. Several integrins and their downstream effectors have been identified in stellate cells, including a1b1, a2b1, a5b1, avb1, avb3 and a6b4.6,60,61 Integrins may also complex with other receptor families in mediating cell motility and fibrogenesis, for example the tetraspanin family of receptors.62
HEPATIC STELLATE CELL ACTIVATION – THE COMMON PATHWAY LEADING TO HEPATIC FIBROSIS The identification of stellate cells as the key cellular source of extracellular matrix in liver has been a major advance. This distinct cell population, located in subendothelial space of Disse between hepatocytes and sinusoidal endothelial cells (Figure 6-1), represents onethird of the non-parenchymal population or about 15% of the total number of resident cells in normal liver.63 In normal liver they are the principal storage site for retinoids (vitamin A metabolites),
Inciting injury Recruitment of inflammatory cells T-cell NK cell Hepatocyte Stellate cell Kupffer cell
Expression of cytokines
Activated stellate cell
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Stellate cell activation
which accounts for 40–70% of retinoids in the body. Most of the retinoids are in the form of retinyl esters and are confined to cytoplasmic droplets. Preferential expression of ECM genes in stellate cells has been confirmed in mechanistically distinct experiment models of injury. Recent studies have emphasized the heterogeneity of mesenchymal populations in the liver, with variable expression of neural,64 angiogenic,65 contractile,66 and even bone marrow-derived67 markers. Moreover, experimental genetic ‘marking’ of stellate cells by the expression of fluorescent proteins downstream of either fibrogenic or contractile gene promoters illustrates the plasticity of fibrogenic populations in vivo.68 In view of this capacity for ‘transdifferentiation’ between different mesenchymal cell lineages, and possibly even from epithelium,69 the key issue is whether fibrogenic cells express target molecules such as receptors or cytokines in sufficient concentrations in vivo to merit their targeting by diagnostic agents or antifibrotic compounds. Following liver injury of any etiology, stellate cells undergo a process known as ‘activation’, which is characterized by the transition of quiescent vitamin A-rich cells into proliferative, fibrogenic, and contractile myofibroblasts.54 Stellate cell activation is typically a result of complex interplay among ECM (Figure 6.2) and cellular (Figure 6.3) elements found in the local environment. It should be noted that activation most often occurs in the setting of hepatocellular injury and subsequent inflammation. Activation can be conceptually viewed as a two-stage process: initiation (also referred to as ‘preinflammatory’) and perpetuation54 (Figure 6-4). Initiation refers to early changes in gene expression and phenotype that render the cells responsive to other cytokines and stimuli, whereas perpetuation results from the effects of these stimuli on maintaining the activated phenotype and generating fibrosis. Initiation is largely due to paracrine stimulation, whereas perpetuation involves autocrine as well as paracrine loops.
Figure 6-3. Cellular response to wound healing. Most forms of liver injury result in hepatocyte injury followed by inflammation, which in turn leads to activation of hepatic stellate cells. Inflammatory effectors are multiple and include T cells, NK and NKT cells as well as Kupffer cells. These cells produce growth factors, cytokines, and chemokines that play an important role in stellate cell activation. Additionally, injury leads to disruption of the normal cellular environment, and also to stellate cell activation (right upper panel). Once activated, stellate cells themselves produce a variety of compounds, including growth factors, cytokines, chemokines, and vasoactive peptides. These substances have pleotropic effects in the local environment, including many which have autocrine effects on stellate cells themselves. One of the major results of stellate cell activation is extracellular matrix synthesis, as well as the production of matrix degrading enzymes.
Chapter 6 HEPATIC FIBROSIS AND CIRRHOSIS
PERPETUATION PROLIFERATION
CONTRACTILITY
INITIATION
ET-1 PDGF
FIBROGENESIS TGF-β1
RESOLUTION MMP-2 MATRIX DEGRADATION PDGF MCP-1 REVERSION?
PDGF MCP-1
CHEMOTAXIS APOPTOSIS LEUKOCYTE CHEMOTAXIS
RETINOID LOSS
Figure 6-4. Stellate cell activation. Stellate cell activation is a key pathogenic feature underlying liver fibrosis and cirrhosis. Multiple and varied stimuli contribute to the induction and maintenance of activation, including (but not limited to) cytokines, peptides, and the extracellular matrix itself. Key phenotypic features of activation include the production of extracellular matrix, loss of retinoids, proliferation, of up-regulation of smooth muscle proteins, secretion of peptides and cytokines (which have autocrine effects), and up-regulation of various cytokine and peptide receptors. Reprinted with permission from ref 54a.
Initiation Oxidant stress may be an early determinant of stellate cell activation. In hepatic injury, whether subclinical or overt, there is a perturbation of normal liver homeostasis, with extracellular release of either free radicals (i.e. ‘oxidant stress’), intracellular constituents, and/or cytokines and signaling molecules. Sources of these mediators may be circulating (i.e. endocrine), paracrine or autocrine. In particular, oxidant stress-mediated necrosis leading to stellate cell activation may underlie a variety of liver diseases, including hemachromatosis, alcoholic liver disease, viral hepatitis and nonalcoholic steatohepatitis (NASH).55,70,71 Liver injury is typically associated with infiltration of inflammatory cells, but even in their absence the liver contains sufficient resident macrophages (Kupffer cells) and natural killer cells (pit cells) to initiate local inflammation prior to the arrival of extrahepatic cells. In addition to oxidant stress, following early injury endothelial cells produce a splice variant of cellular fibronectin that is able to stimulate stellate activation.
Endothelial cells in early injury may also participate in the conversion of latent TGF-b1 to its active, profibrogenic form through the activation of plasmin. Whereas necrosis is considered a classic inflammatory and fibrogenic stimulus, recent findings also suggest that apoptosis may provoke a fibrogenic response in stellate cells. Apoptotic fragments released from hepatocytes are fibrogenic towards cultured stellate cells,72 and Fas-mediated hepatocyte apoptosis in vivo in experimental animals is also fibrogenic.73 Platelets in injured liver are a potent source of paracrine stimuli by generating multiple potentially important mediators, including PDGF, TGF-b1, and epidermal growth factor (EGF). Additionally, activated stellate cells have also been observed in primary and metastatic human tumors, as well as a murine model of metastatic melanoma to liver.74 In recent years, increasing interest has been focused on the molecular regulation of gene expression during early stellate cell activa-
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tion. There have been many advances in dissecting pathways of membrane and intracellular signaling and transcriptional gene regulation in activated hepatic stellate cells that are too numerous to detail here.75 A growing number of transcription factors may regulate stellate cell behavior, including peroxisomal proliferator-activated receptors (PPAR) a, b and g,76 retinoid receptors,77 NF-kB,78,79 Jun D,75 Krüppel-like factor 6 (previously called ‘Zf9’),80 Foxf1,81 and CRP282 among others.
Perpetuation After initiation, activated stellate cells undergo a series of phenotypic changes that collectively lead to the accumulation of ECM. These include proliferation; contractility; fibrogenesis; chemotaxis; matrix degradation; retinoid loss; and proinflammatory responses and cytokine release. The following sections detail the mechanisms underlying each of these events.
Proliferation An increase in the number of stellate cells has been documented after both human and experimental liver injury, in large part due to local proliferation. Following liver injury, many mitogenic factors as well as their cognate tyrosine kinase receptors are unregulated, primariliy through receptor tyrosine kinases.59 PDGF is the bestcharacterized and most potent mitogen towards stellate cells. Upregulation of PDGF receptor following liver injury enhances the responsiveness to autocrine PDGF, whose expression is also increased. The downstream signaling pathways involve ERK/MAP kinase, phosphoinositol 3 kinase (PI 3-kinase) and STAT-1 (signal transducers and activators of transcription) (see 3 for review). PDGF-induced proliferation correlates with increased intracellular Ca2+ and pH, raising the possibility that calcium channel blockers might modulate stellate cell mitogenesis or activation. Other stellate cell mitogens include endothelin-1 (ET-1),83,84 thrombin,85 FGF,86 and IGF,87,88 among others (see 89,90 for reviews). A recent study has documented increased sensitivity to ET-1 during activation,91 suggesting potentiation of autocrine/paracrine stimulation.
Contractility Contraction by stellate cells may be a major determinant of early and late increases in portal resistance during liver fibrosis. Activated stellate cells impede hepatic blood flow both by constricting individual sinusoids and by contracting the cirrhotic liver, as the collagenous bands typical of end-stage cirrhosis contain large numbers of activated stellate cells (see 66 for review). A key contractile stimulus towards stellate cells is ET-1.66 Other contractile agonists include arginine vasopressin, adrenomedullin, and eicosanoids.66 The regulation of stellate cell contraction is complex. The endothelium-derived relaxing factor nitric oxide (NO) appears to be an important relaxing factor in the sinusoid (although other factors, such as carbon monoxide, also play a role). The net contractile activity of stellate cells in vivo therefore reflects the relative strength of each of these opposing activities. Current evidence suggests that intrahepatic portal hypertension probably results from diminished NO (and/or other vasodilators) activity as well as increased stimulation by ET-1 (or other constrictors).66
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The expression of smooth muscle a actin is increased during stellate cell activation. ET-1 and other vasoactive mediators increase their expression.83 Thus studies of contractile proteins in stellate cells may yield a therapeutic target for the treatment of intrahepatic portal hypertension.
Fibrogenesis Fibrogenesis is perhaps the key component of the stellate cell’s contribution to hepatic fibrosis. TGF-b1 is the most potent fibrogenic factor, with some fibrogenic activity documented for interleukin-1b, TNF, lipid peroxides, acetaldehyde, and others (see 2,6 for reviews). Because of its importance, TGF-b1 regulation has received considerable attention. TGF-b1 is up-regulated in experimental and human hepatic fibrosis. Although sources of this cytokine are many, autocrine expression is among the most important (see 2 for review). Several mechanisms underlie the increase in TGF-b1 expression by stellate cells during liver injury, including TGF-b transcriptional upregulation, activation of latent TGF-b1, increased TGF-b receptor expression, and up-regulation of TGF-b signaling components.92–96
Chemotaxis Stellate cells may accumulate both through proliferation and via directed migration into regions of injury, or chemotaxis. PDGF, the leukocyte chemoattractant MCP-1, and a growing family of chemokines have been identified as key stellate cell chemoattractants.97 In addition to tyrosine kinase receptors, new agents have been implicated in stellate cell migration, in particular tetraspanin receptors.3,62
Matrix Degradation A greater understanding of matrix degradation in liver is emerging. Quantitative and qualitative changes in the activity of MMPs and their inhibitors play a vital role in extracellular matrix remodeling in liver fibrogenesis (see ECM in the normal and fibrotic liver and Figure 6.2 above). As noted above, the net effect of changes in matrix degradation is the conversion of the low-density subendothelial matrix to one rich in interstitial collagens.
Retinoid Loss Stellate cell activation is accompanied by loss of their characteristic perinuclear retinoid (vitamin A) droplets. Although the intracellular form is largely retinyl esters, when retinol is exported from the cell during activation it is primarily as retinol, suggesting the possibility of intracellular hydrolysis of esters before being exported. Several nuclear retinoid receptors that bind intracellular retinoid ligands have been identified and their effects characterized in stellate cells.77,98
Proinflammatory Responses and Cytokine Release Hepatic stellate cells and sinusoidal endothelial cells have emerged as inflammatory effectors. Sinusoidal endothelial cells, normally fenestrated to allow rapid bidirectional transport of solutes between sinusoidal blood and parenchymal cells, may rapidly lose their fenestrations upon injury and express proinflammatory molecules, including ICAM-1, VEGF and adhesion molecules.74,99 Together with stellate cells, they activate angiogenic pathways in response to hypoxia associated with local injury or malignancy.74,97,100,101
Chapter 6 HEPATIC FIBROSIS AND CIRRHOSIS
Key inflammatory pathways converge on stellate cells, leading to fibrosis (see Figure 6-3). Thus, the cell type is a central mediator in inflammation, rather than just a passive target. Upon activation, they release chemokines97,102 and other leukocyte chemoattractants, proteinase-activated receptors,103 and up-regulate expression of key inflammatory receptors, including ICAM-1,104 chemokine receptors,105 and those mediating lipopolysaccharide (LPS) signaling, including Toll-like receptor 4.78 Stellate cells may also contribute to intrahepatic apoptosis of T lymphocytes.106 Remarkably, little attention has focused on the contribution of different lymphocyte subsets to hepatic fibrogenesis. Interest has increased recently, in part because of the observation that patients with HCV who are co-infected with HIV, as well as those who are immunosuppressed following liver transplantation, have accelerated fibrosis rates, implicating the immune system as a determinant of fibrogenesis. These observations have been supported by animal studies demonstrating that the immune phenotype regulates fibrogenesis independent of effects on injury, which in turn have led to efforts to map the genetic loci accounting for these differences.22 Most recently, CD8 lymphocytes have emerged as potential profibrogenic cells, based on their ability to induce early fibrogenesis following adoptive transfer to naïve SCID mice from animals with liver injury.107 Autocrine cytokines play vital roles in regulating the activation process of stellate cells. These cytokines include TGF-b1, PDGF, FGF, HGF, PAF, stem cell factor and ET-1, among others.59,90,97,108 Furthermore, stellate cells release neutrophil and monocyte chemoattractants, which can amplify inflammation in liver injury. These chemokines include colony-stimulating factor, monocyte chemotactic protein-1 (MCP-1), and cytokine-induced neutrophil chemoattractant (CINC).97 Anti-inflammatory cytokines produced by stellate cells have also been identified. Up-regulation of interleukin-10 (IL-10) occurs in early stellate cell activation. The anti-inflammatory effects of this cytokine are demonstrated by its ability to down-regulate TNF-a production from macrophages. Knockout mice lacking IL-10 have more severe hepatic fibrosis following CCl4 administration, and transgenic mice expressing IL-10 in liver have reduced fibrosis.107 Based on the consistent antifibrotic effect of IL-10 in experimental liver disease, a clinical trial was undertaken which failed to show an antifibrotic effect in patients with HCV infection, possibly because of marked increases in HCV replication109 (see Therapy of hepatic fibrosis, below).
DISEASE-SPECIFIC MECHANISMS REGULATING HEPATIC FIBROSIS – HCV AND NASH In addition to generic mechanisms of fibrogenesis common to all experimental and human liver disease, there has been progress in elucidating disease-specific mechanisms, in particular in hepatitis C (HCV) and NASH (non-alcoholic steatohepatitis). In HCV, stellate cells might be infectable by the virus because they express putative HCV receptors.104,110 Moreover, adenoviral transduction of HCV non-structural and core proteins induces stellate cell proliferation and the release of inflammatory signals.104 In HCV-infected liver chemokines and their receptors are up-regulated, stimulating lymphocyte recruitment.111 HCV proteins may also interact directly with sinusoidal endothelium.112
The increasing prevalence of obesity in the US and western Europe is associated with an alarming increase in NASH,39 leading to advanced fibrosis and cirrhosis. Leptin, a circulating adipogenic hormone that is proportionate to adipose mass in circulating blood, has been clearly linked to stellate cell fibrogenesis.113–115 Sources are likely to be both endocrine and autocrine, associated with enhanced signaling through the leptin receptor, which is up-regulated during stellate cell activation.113 Concurrently, down-regulation of adiponectin, a counterregulatory hormone, in obesity may amplify the fibrogenic activity of leptin. This possibility is supported by findings in mice lacking adiponectin, which have enhanced fibrosis following toxic liver injury.116
RESOLUTION OF LIVER FIBROSIS AND THE FATE OF ACTIVATED STELLATE CELLS During recovery from acute human and experimental liver injury the number of activated stellate cells decreases as tissue integrity is restored. Either reversion of stellate cell activation, or selective clearance of activated stellate cells by apoptosis, may explain the loss of activated cells in resolving liver injury. To date, evidence is strongest for stellate cell apoptosis in this setting. Apoptosis of stellate cells probably accounts for the decrease of activated stellate cells during resolution of hepatic fibrosis.49 Following injury, apoptosis may be inhibited by soluble factors and matrix components that are present during injury, whereas an apoptotic pathway otherwise represents a ‘default’ mode. Furthermore, cell death ligands, including TRAIL and fas, are expressed in liver injury, and activated stellate cells are more susceptible to TRAILmediated apoptosis.73,117,118 Another death receptor, nerve growth factor receptor (NGFR), is also expressed by activated stellate cells, and its stimulation with ligand drives apoptosis.119 Survival factors also regulate the net activity of stellate cell apoptosis. IGF-I promotes stellate cell survival via the PI3-K/c-Akt pathway and TNF-a has the same effect, but utilizes the NF-kB pathway.120,121 Molecules regulating matrix degradation appear closely linked to survival and apoptosis. Active MMP2 correlates closely with apoptosis, and in fact may be stimulated by it.122 Inhibition of MMP2 activity by TIMP-1 blocks apoptosis in response to a number of apoptotic stimuli.123 Interactions between stellate cells and the surrounding matrix also influence their propensity towards apoptosis, and this might partly explain the antiapoptotic activity of TIMP-1. Moreover, the fibrotic matrix may provide important survival signals to activated stellate cells. For example, animals expressing a mutant collagen I resistant to degradation have more sustained fibrosis and less stellate cell apoptosis following liver injury,48 and transgenic animals expressing TIMP-1 in liver have delayed resolution of fibrosis.124 Studies using gliotoxin,125 a fungal toxin that induces apoptosis in stellate cells, emphasize the role of this pathway in stellate cell removal during resolution of liver fibrosis. It is unknown whether an activated stellate cell can revert to a quiescent state in vivo, although it has been observed in culture. When stellate cells are grown on a basement membrane substratum (Matrigel) they remain quiescent, and plating of highly activated cells on this substratum down-regulates stellate cell activation.58,126
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METHODS TO MEASURE FIBROSIS OVERVIEW Measurement of fibrosis not only helps to stage the severity of disease, it allows serial determination of disease progression. The level of fibrosis may play an important role in clinical management and determine patients’ prognosis. For example, aggressive therapy is more appropriate in HCV-infected patients with advanced fibrosis. Further, the fibrosis progression rate is an important predictor of the time to develop cirrhosis.18 It is essential to measure fibrosis accurately, given the growing prospect of antifibrotic therapies and the need to track their efficacy. Moreover, with growing evidence that fibrosis is reversible, methods will need to assess both progression and regression accurately. For example, specific therapy leads to a reduction in fibrosis in a number of diseases, including autoimmune liver disease, hepatitis C, hepatitis B, and others.34,35,42,44,127–129 Percutaneous liver biopsy has traditionally been considered to be the gold standard test to assess liver fibrosis. However, a variety of non-invasive tests have been advanced as potential alternatives to biopsy. These include clinical signs, routine laboratory tests, quantitative assays of liver function, markers of extracellular matrix synthesis and/or degradation, and radiologic imaging studies. In addition to individual indicators of fibrosis, combination tests, and a number of models for predicting liver fibrosis have been developed. Individual and combination tests are discussed below. The ideal method to measure fibrosis would be simple, noninvasive, reproducible, inexpensive, accurate, and readily available. Unfortunately, none of the currently available approaches fulfills all of these criteria.
BEDSIDE DIAGNOSTIC TOOLS Clinical signs and symptoms of liver disease are frequently highlighted in assessing patients with liver disease, but these are of little value in detecting early, precirrhotic stages of liver fibrosis. In contrast, a number of clinical features can be utilized to assess whether cirrhosis with portal hypertension may be present. Signs of cirrhosis include spider angiomata, distension of abdominal wall veins, ascites, splenomegaly, muscle wasting, Dupuytren’s contractures (especially with ethanol-associated cirrhosis), gynecomastia and testicular atrophy in males, and palmar erythema. However, it is important to emphasize that even in patients with histologic cirrhosis, and in those with portal hypertension, these physical signs may not be present.
NON-INVASIVE MARKERS OF FIBROSIS Blood-Based Markers – Overview A wide variety of blood, serum, or plasma ‘markers’ for fibrosis have been proposed. There are several categories of marker or test. For example, some detect abnormalities in serum chemistries. Included in these types of test are aspartate aminotransferase (AST), alanine aminotransferase (ALT), g-glutamyl transferase (GGT), bilirubin, albumin, and a2-macroglobulin, among others. Moreover, some of these individual tests have been incorporated into simple and/or complex mathematical models or algorithms (see below).
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Another major category of test includes those that are based specifically on the pathogenesis of fibrosis (see above). For example, proteins that are produced as a result of the fibrogenic process itself that have been studied as markers of fibrosis include procollagen I, fibronectin, tenascin, laminin, hyaluronic acid and others. Other markers have included cytokines (i.e. TGF-b1), connective tissue growth factor (CTGF), PDGF and others, matrix degrading enzymes (i.e. TIMP1), and others (Table 6-1). Finally, groups of tests, including those that utilize markers of fibrosis in combination with each other or in combination with other types of test, have been advanced in an attempt to detect and measure fibrosis. Ideally, a blood-based test should have both high sensitivity and high specificity. Many of the available tests have a high specificity (>95%) for advanced fibrosis. However, few (including algorithms) have great sensitivity to detect moderate levels of fibrosis. Moreover, a serum-based assay ideally should be linear over the full range of fibrosis, follow the natural history, and accurately reflect the effect of treatment.
Routine Laboratory Tests A number of studies have used routine laboratory tests in an attempt to determine whether a patient may have advanced liver disease, in particular to exclude or confirm portal hypertension and/or esophageal varices.130,131 Although tests such as the prothrombin time, albumin level, and portal vein diameter (measured by ultrasound) have all been associated with varices, studies have been remarkably consistent in their identification of the platelet count as
Table 6-1. Cytokines, Growth Factors, Peptides, Proteases, and other Components Important in Hepatic Fibrogenesis Cytokines
Growth factors
Peptides
Transforming growth factor-b Transforming growth factor-a Interleukin-1 Interleukin-4 *Interleukin-6 Interleukin-10 Interleukin-13 *Monocyte chemotactic factor
Transforming growth factor-b
Endothelin-1
Transforming growth factor-a
Norepinephrine
*Insulin-like growth factor (I, II) *Platelet-derived growth factor *Fibroblast growth factor Vascular endothelial growth factor Hepatocyte growth factor Connective tissue growth factor
Angiotensin II
Proteases and their inhibitors Matrix-metalloproteinase-1 (interstitial collagenase) Matrix-metalloproteinase-2 (gelatinase A) Matrix-metalloproteinase-3 (stromelysin-1) Matrix-metalloproteinase-7 (matrilysin) Matrix-metalloproteinase-8 Matrix-metalloproteinase-9 (gelatinase B) Matrix-metalloproteinase-10 (stromelysin-2) Tissue inhibitor of metalloproteinase-1
Miscellaneous Thrombospondin (1,2) Leptin Activin A *Thrombin Osteopontin
Agents may have direct effects on hepatic stellate cells, or indirect effects in the wounding environment. *Compounds whose effect is largely via stimulation of proliferation.
Chapter 6 HEPATIC FIBROSIS AND CIRRHOSIS
the best single predictor of esophageal varices. For example, in one study, cirrhotics without splenomegaly on physical examination and with a platelet count >88 000/mm3 had a risk of large esophageal varices of 7.2%, whereas the risk was 28% if the platelet count was less than 88 000/mm3.130 An AST/ALT ratio >1 has been proposed to indicate the presence of cirrhosis.132 In one study of patients with HCV, a ratio >1 had 100% specificity and positive predictive value for distinguishing cirrhotic from non-cirrhotic patients, with a 53.2% sensitivity and 80.7% negative predictive value.133 In addition, the ratio correlated positively with the stage of fibrosis, but not with the grade of activity or other biochemical indices. Of cirrhotic patients, 17% had no clinical or biochemical evidence of chronic liver disease except for an elevated AST/ALT ratio. In another study, the AST/ALT ratio had 81.3% sensitivity and 55.3% specificity in identifying cirrhotic patients who died within 1 year of follow-up.132,134 In a further attempt to develop non-invasive tools for the measurement of liver fibrosis, Forns and coworkers developed a model using data from HCV patients that included age, GGT, cholesterol, and platelet count.135 This model was developed with the intention to differentiate patients with significant fibrosis from those without. The sensitivity for detecting METAVIR F2–F4 fibrosis was 94%, and the presence of significant F2–F4 fibrosis could be excluded with high accuracy (negative predictive value of 96%).135 Likewise, Wai et al.136 constructed a simple model utilizing routine laboratory data (Table 6-2). The authors devised a novel index, termed the AST to platelet ratio index, or APRI, which is the AST level/upper limit of normal (ULN) divided by the platelet count (109/l) multiplied by 100. The sensitivity and specificity for fibrosis of the APRI value depended on the cut-offs used. Using an APRI value of 1.50, the positive and negative predictive values for significant fibrosis (Ishak score = 3) were 91% and 65%, respectively, whereas for cirrhosis and an APRI of 2.00, the positive and negative predictive values were 65% and 95%, respectively. Thus for a hypothetical patient, if the AST was 90 IU/l (and the ULN 45) and platelet count was 100 ¥ 109/l, then the APRI would be 2.00. This means that the patient has essentially a 90% chance of having significant fibrosis, and somewhat less likelihood of having cirrhosis. However, cirrhosis could not be excluded with certainty. Although the APRI is attractive because of its simplicity, it can neither definitively diagnose nor exclude cirrhosis, and it will not identify patients with early fibrosis. Other simple quantitative systems based on routine laboratory values have been developed. One early example was the ‘PGA index’, which combined prothrombin time, GGT and apolipoprotein A1 (Table 6-2); this test was examined in patients with alcoholic cirrhosis.137 The diagnostic accuracy of this index was later improved by the addition of a2-macroglobulin (and hence termed the ‘PGAA index’).138 The test characteristics of many of these indirect assays have been derived from datasets, but have not been validated on independent datasets. More complicated algorithms based on commonly available laboratory tests include the ‘Fibrotest,’ reported by the French MULTIVIRC group.25 This group used mathematical modeling to develop an algorithm including five different markers to predict fibrosis (the markers selected were a2-macroglobulin, haptoglobin, GGT, apolipoprotein A1, and total bilirubin). This index predicted a spe-
Table 6-2. Combined Panels of Blood Markers used to Detect Liver Fibrosis Panel
Components
References
AST/ALT § Forns APRI PGA index Fibrotest
AST/ALT Platelets, GGT, cholesterol AST, Platelets Platelets, GGT, apolipoprotein A GGT, haptoglobin, bilirubin, apolipoprotein A, a2-macroglobulin Hyaluronic acid, TIMP-1, a2-macroglobulin ECM proteins AST, cholesterol, HOMA-IR
132–134 135 136 137–138
Fibrospect *ELF FPI
25, 139–142 146 147
ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, g-glutamyl transpeptidase; APRI, AST to platelet ratio index; TIMP-1, tissue inhibitor of metalloproteinase 1; ECM, extracellular matrix; ELF, European liver fibrosis; FPI, fibrosis probability index; HOMA-IR, insulin resistance by the homeostasis model assessment. § Also includes age in the panel. *Components tested include collagen IV, collagen VI, amino terminal propeptide of type III collagen (PIIINP), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), tenascin, laminin, and hyaluronic acid (HA).
cific biopsy category in 46% of patients139 and has been validated in a number of hepatitis C patient cohorts, having been found to have an area under the receiver operator characteristic (AUROC) curve of 0.73–0.87.140 The addition of ALT to the marker panel allows for prediction of METAVIR necroinflammatory activity.140 The panel has also been examined in other liver disease cohorts.141,142 Limitations of this panel in fibrosis include false positive results due to increases in bilirubin or decreases in haptoglobin, for example from hemolysis secondary to ribavirin therapy. Likewise, false positive results may also occur in situations where there is hyperbilirubinemia, such as Gilbert’s disease and cholestasis. Acute inflammation may also affect the results of the test owing to changes in a2macroglobulin or increases in haptoglobin. Currently, it is unclear whether the ‘fibrotest’ assay meets sufficiently rigorous criteria, given a predictive value of only 46%, for routine clinical use.
Tests Using Extracellular Matrix/Fibrosis Markers Analyses of serum markers of extracellular matrix/fibrosis include many proteins important in fibrogenesis, ECM constituents (i.e. fibronectin, collagen I, collagen IV, collagen VI, amino terminal propeptide of type III collagen (PIIINP), tenascin, and hyaluronic acid, metalloproteinases (including many of those listed in Table 6-1), inhibitors of matrix metalloproteinases (i.e. TIMP-1, TIMP-2), and other proteins, peptides, and cytokines, as highlighted in Table 6-1. Although many tests have been studied individually, they are generally not sensitive for detection of fibrosis143,144 (see 145 for review).
Tests Using Combinations of Extracellular Matrix and/or Routine Markers
A combination test including hyaluronic acid, TIMP1, and a2macroglobulin was examined in a cohort of 294 patients with HCV infection and subsequently validated in a second cohort of 402 patients146 (‘Fibrospect’, Table 6-2). This had a combined AUROC of 0.831 for METAVIR F2–F4 fibrosis. The positive and negative predictive values were 74.3% and 75.8%, respectively, with an accuracy of 75%. This three-marker panel thus may help differentiate
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patients with HCV infection with moderate/severe fibrosis from those with no/mild fibrosis, although it was not possible to differentiate specific stages accurately. Another combination test was developed by the European Liver Fibrosis (ELF) Study Group.147 This group examined collagen IV, collagen VI, PIIINP, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), TIMP-1, tenascin, laminin, and hyaluronic acid (HA). The study was unique in that it examined patients with a wide variety of liver diseases, including those with chronic hepatitis C virus infection (n = 496), alcoholic liver disease (n = 64), non-alcoholic fatty liver disease (n = 61), chronic hepatitis B virus infection (n = 61), primary biliary cirrhosis or primary sclerosing cholangitis (n = 53), recurrent liver disease post orthotopic liver transplantation (n = 48), autoimmune hepatitis (n = 45), hemochromatosis (n = 32), cryptogenic cirrhosis (n = 19), both hepatitis B and C (n = 4), and other or no known diagnosis (n = 138); the cohort also had a wide distribution of fibrosis stages (Scheuer fibrosis stages were as follows: stage 0 = 24.6%; stage 1 = 35.5%; stage 2 = 13.4%; stage 3 = 14.9%; and stage 4 = 11.8%). An algorithm was developed that detected the upper third of fibrosis groups (Scheuer stages 2, 3, and 4) with a sensitivity of 90% and accurately detected the absence of fibrosis (Scheuer stages 0, 1), with a negative predictive value for this level of fibrosis of 92%. The AUC of a receiver operating characteristic (ROC) plot was 0.804. Interestingly, the addition of clinical chemistry tests including liver function tests, or hematological indices including platelet count and prothrombin time, did not improve test performance. The test appeared to be best in patients with hepatitis C, non-alcoholic fatty liver disease and alcoholic liver disease. The inclusion of patients with multiple etiologies of liver disease, although appealing, has the potential to limit the accuracy of these and other panels, as the characteristics of specific assays may be disease specific. Another model, including AST, cholesterol, and insulin resistance (as well as age and an estimate of past alcohol intake) in patients with HCV147a found that the sensitivity for detection of advanced fibrosis depended on the index value used. At a low probability index, the sensitivity for predicting significant fibrosis was high, but specificity was low, while at a high probability index, sensitivity for significant fibrosis was low, but specificity was high.
Proteomics With the recent explosion in proteomics, proteomic approaches have attempted to identify unique protein fingerprints in patients with liver disease. Various platforms are available, including those that measure protein expression, protein–protein interactions, or even enzymatic activity. The majority of approaches have used highthroughput technologies to identify novel protein expression patterns. For example, a recent study in 46 patients with chronic hepatitis B identified 30 proteomic features predictive of significant fibrosis (Ishak stage = 3) and cirrhosis. The AUROC for this analysis was 0.906 and 0.921, for advanced fibrosis and cirrhosis, respectively.148 Another study in 193 patients with chronic hepatitis C identified eight peaks that differentiated METAVIR fibrosis stages with an AUROC of 0.88; this was compared to an AUROC 0.81 for the Fibrotest.149 Another report in patients with HCV fibrosis identified several serum proteins to be differentially regulated.150 In
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this study, patients with advanced fibrosis had elevated levels of a2macroglobulin, haptoglobin, and albumin, but apolipoprotein AI, apolipoprotein A-IV, complement C4, and serum retinol-binding protein were reduced. Another approach has included measurement of labeled Nglycans found in serum.151 The technique exploits the ability to analyze the desialylated total serum N-glycome on a DNA analyzer. The authors focused on cirrhosis (primarily ethanol induced), demonstrating unique patterns of serum N-glycans in those with cirrhosis compared to those with chronic liver disease alone. It was postulated that in cirrhotic livers characteristic N-glycans with a bisecting GlcNAc residue were prominent. In normal liver, the enzyme responsible for this modification, N-acetylglucosaminyl transferase III (GnT-III), is found only in non-parenchymal cells, but in regenerating liver (two-thirds partial hepatectomy) this enzyme is produced in hepatocytes. Thus, GnT-III expression is presumably a manifestation of hepatocellular regeneration, reflected by regenerative nodules. This approach was most sensitive for the detection of cirrhosis and was also able to exclude cirrhosis with great accuracy. When combined with the commercially available Fibrotest this test had 100% specificity and 75% sensitivity for diagnosing compensated cirrhosis.151
Summary of Blood-based Markers A key advantage of serum markers to detect fibrosis is their noninvasiveness. Additionally, it has been argued that serum markers overcome sampling problems associated with liver biopsy. However, these approaches have several drawbacks. First, most of the studies examining serum markers have been performed in cohorts of patients that have been biased toward advanced fibrosis/cirrhosis. A further problem is that the currently proposed serum marker algorithms use dichotomous rather than continuous scales. The dichotomous nature of these variables would be less problematic if there were clear clinical associations, for example if prognosis or treatment response were highly linked to stage 0–1 versus stages 2–4. In the absence of clinical correlates between dichotomous variables and outcomes, it remains important to diagnose the different stages of fibrosis accurately (0–4). Unfortunately, current tests and algorithms are unable to do this, and perhaps most importantly, the tests do not differentiate between intermediate levels of fibrosis. Thus, although assessments of fibrosis with approaches that use serum markers have great appeal, and indeed, in some areas the tests have begun to replace liver biopsy. Further investigation is required to optimize these tests.
Imaging Tests A wide variety of radiographic tests have been used to image patients with fibrosis/cirrhosis. Included in this group are ultrasound, CT, and MRI. In general, these tests are capable of detecting evidence of portal hypertension, thus they have the ability to detect advanced disease. As currently used in clinical practice, however, they are insensitive for the detection of moderate degrees of fibrosis. Transient elastography, which uses pulse-echo ultrasound acquisitions to measure liver stiffness and predict fibrosis stage, has gained interest as a method to quantify fibrosis as it appears that liver ‘stiffness’ may accompany the fibrogenic response.152 In a prospective multicenter study of 327 chronic HCV patients, the AUROCs for
Chapter 6 HEPATIC FIBROSIS AND CIRRHOSIS
METAVIR stage F2–F4 and cirrhosis were 0.79 and 0.97, respectively.153 In a separate study of 183 chronic HCV patients, transient elastography compared favorably with the Fibrotest and APRI (AUROC for F2–F4 = 0.83, 0.85 and 0.78, for transient transient elastography, Fibrotest and APRI, respectively).154 When transient elastography was combined with the Fibrotest, the predictive value for fibrosis stage F2–F4 was improved, with an AUROC of 0.88.154 Transient elastography (Fibroscan) reportedly offers good reproducibility with low inter- and intraobserver variability. The procedure is performed by obtaining multiple validated measurements in each patient, further reducing the potential for sampling errors. The depth of measurement from the skin surface is between 25 and 65 mm, raising the possibility that this technique may be difficult to use in obese patients or those with ascites. However, newer probes are being developed for obese patients, and further investigation is expected. Finally, it would theoretically be desirable to utilize advances in the molecular understanding of liver fibrosis to image the liver. For example, the number of activated stellate cells, which reflect fibrogenic activity, might be identified by tagging them with cell-specific markers.155 Alternatively, matrix or matrix turnover could be labeled using molecular tools. Although such approaches are appealing, they remain experimental at present.
in the other. Finally, in 10% of subjects, stage 0–2 disease was identified in one lobe and stage 3–4 fibrosis was found in the other. Similar variability was reported in another study in patients with fatty liver disease.161 There are several other limitations of liver biopsy. Quantification of fibrosis in biopsies is subject to significant interobserver variation. In chronic hepatitis C, for example, standardized grading systems, including Knodell, METAVIR, Scheuer or Ishak, are concordant in only 70–80% of samples. Specimen quality is very important, with smaller samples leading to an underestimation of disease severity.162 A recent study created digitized virtual image biopsy specimens of varying length from large liver sections, and revealed that 75% of 25-mm biopsy specimens were correctly classified using the METAVIR staging system, compared to only 65% for biopsies 15 mm long.163 Interestingly, a recent study noted that the experience of the pathologist may have more influence on interobserver agreement than specimen length.164 Another major problem with using liver biopsy or serum markers to quantify fibrosis is that all of the currently utilized grading systems use a simple linear numerical scoring approach, implying that they represent linear changes in fibrosis content. Such an inference is highly inaccurate, as METAVIR stage 4 fibrosis does not represent twice as much fibrosis as stage 2, but rather a 5–20-fold difference.
Tests of Liver Function A variety of bona fide liver function tests have been used to assess liver fibrosis and cirrhosis. Such tests generally measure advanced disease and several depend on perfusion, such as indocyanine green, sorbitol and galactose clearance tests, or tests such as the 13C– galactose breath test and the 13C–aminopyrine breath test that depend on the functional capacity of the liver.156–158 Another test, the MEGX test, which measures monoethylglycinexylidide (MEGX) formation after the administration of lidocaine, depends upon the activity hepatic cytochrome P450 3A4 isoenzyme (which catalyzes oxidative N-de-ethylation of lidocaine to MEGX.159 The MEGX test has a sensitivity and specificity in the 80% range for distinguishing chronic hepatitis from cirrhosis in comparison to standard liver tests.159 Unfortunately, although the MEGX test and other function tests may predict prognosis in cirrhotic patients, they are insensitive for quantifying fibrosis in patients with less advanced disease.156–158
Liver Biopsy Percutaneous liver biopsy has traditionally been considered to be the gold standard test to measure fibrosis. Although there is great experience with liver biopsy, this procedure is time consuming, inconvenient, uncomfortable, invasive, and makes both patients and physicians anxious. Further, liver biopsy can be associated with substantial sampling-error (see Chapter 12 for further details about liver biopsy). In a recent study in which 124 patients with chronic HCV infection underwent laparoscopy-guided biopsy of each the right and left hepatic lobes, 33.1% had a difference of at least one histologic stage (modified Scheuer system) between the two lobes.160 Furthermore, in 18 study subjects a stage consistent with cirrhosis was found in one lobe, whereas stage 3 fibrosis was reported
TREATMENT OF FIBROSIS Specific therapy for the treatment of liver fibrosis is attractive because the scarring response leads to many if not all of the complications of chronic liver disease, in particular impaired synthetic function, liver failure, and perhaps hepatocellular cancer. Fibrosis, particularly in its advanced stages, may also contribute to portal hypertension, by preventing blood flow through fibrotic nodules. Although attempts have been made previously to treat specifically the ‘fibrosis’ component of liver disease, these approaches have generally been unsuccessful. Thus, there remains a major unmet need for novel and effective antifibrotic therapy. Advances in elucidating the pathogenesis of fibrosis have led to renewed efforts in this area. Additionally, data indicating that fibrosis is reversible have helped fuel this effort (Figure 6.5). Although fibrosis is commonly accepted as the precursor to cirrhosis, it is not clear that mortality risk increases directly with the stage of fibrosis, until the patient actually becomes cirrhotic. Even with established cirrhosis, in a cohort of patients with chronic HCV infection Fattovich and colleagues demonstrated that complications of cirrhosis developed over prolonged periods, and only when complications occurred was mortality increased.7 The most effective ‘antifibrotic’ therapies are currently those that treat or remove the underlying stimulus to fibrogenesis (Table 6-3). In addition, preclinical and human clinical studies have highlighted a number of therapies that may abrogate fibrogenesis without affecting the underlying disease, by targeting specific steps in the fibrogenic response. Anti-inflammatory therapies have been based on the knowledge that inflammation drives the fibrogenic cascade. Other treatments have attempted to inhibit cellular injury or focused on stellate cell activation, whereas others have targeted collagen syn-
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Section I. Pathophysiology of the Liver
Figure 6-5. Reversal of fibrosis. An example of reversal of advanced fibrosis (cirrhosis in this situation) is depicted. A liver biopsy prior to lamivudine treatment is shown (upper panel and left panel). After treatment with lamivudine, liver biopsy was repeated and reveals almost complete dissolution of fibrosis. Data similar to these have been published in autoimmune liver disease, hepatitis C, alcoholic hepatitis, hepatitis B, and others. (Reprinted with permission, Wanless, et al: Arch Pathol Lab Med 2001;124:1599–1607.)
Inset in A
B
A
C
Table 6-4. Diseases and Therapies in which there is Strong Evidence that Treatment Reduces Liver Fibrosis
Table 6-3. Approaches to Treat Liver Fibrosis Approach
Example
Disease
Therapy
References
Remove injurious agent Anti-inflammatory agents Antioxidants Cytoprotective agents Inhibit stellate cell activation Inhibit stellate cell activation phenotypes (fibrogenesis)
Eradication of HBV Corticosteroids in AIH PPC in alcoholic hepatitis Ursodeoxycholic acid Interferon-g Colchicine
Hepatitis B Hepatitis C Bile duct obstruction Autoimmune hepatitis Hemochromatosis Alcoholic hepatitis
Lamivudine Interferon-a* Surgical decompression Corticosteroids Iron depletion Corticosteroids
33–35,129 42 43 44 165,166 168,169
Note: some approaches have not been demonstrated to be successful. AIH, autoimmune hepatitis; PPC, polyenylphosphatidylcholine.
thesis and matrix deposition. The following section highlights human studies in these areas.
THERAPIES DIRECTED AT THE UNDERLYING DISEASE In many forms of liver disease treatment of the underlying inciting lesion leads to an improvement in fibrosis (Table 6-4). For example, eradication or inhibition of HBV33,34,129 or HCV replication42 leads to reversion of fibrosis, even in patients with histological cirrhosis. Fibrosis reverts in patients with hemochromatosis during iron depletion,165,166 after corticosteroid therapy in autoimmune hepatitis,165,166 and in patients with secondary biliary cirrhosis after decompression of bile duct obstruction.43 In a preliminary report in patients with non-alcoholic steatohepatitis (NASH) treated with the peroxisomal proliferator active receptor (PPAR)-g agonist rosiglitazone both steatosis and fibrosis were reduced.167
ANTI-INFLAMMATORY COMPOUNDS Many liver diseases, such as HCV disease, have an important inflammatory component. Inflammation in these disorders typically drives stellate cell activation and fibrogenesis, and it is these diseases in particular that have been studied in order to evaluate the efficacy of anti-inflammatory drugs.
Corticosteroids Classic examples of the benefits of steroids include autoimmune hepatitis and alcoholic hepatitis. In patients with autoimmune hep-
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*or PEG-interferon-a, with or without ribavirin. MTX, methotrexate; PPAR, peroxisomal proliferator-activated receptor.
atitis who respond to medical treatment (prednisone or equivalent) advanced fibrosis and cirrhosis are reversible.44 Fibrosis may improve in patients with alcoholic liver disease who respond to corticosteroids.168,169 Thus, corticosteroids appear to have antifibrotic effects in patients with certain liver disorders.
Interleukin-10 (IL-10) Interleukin (IL)-10 has both anti-inflammatory and immunosuppressive effects. IL-10 has been shown to reduce the production of proinflammatory cytokines, such as TNF-a, IL-1, interferon-g, and IL-2 from T cells. These cytokines belong to the Th1 family. Endogenous IL-10 reduces the intrahepatic inflammatory response, shifts the cytokine milieu towards a Th2 predominance, and reduces fibrosis in several in vivo models of liver injury.170 It was hypothesized that in vivo administration of IL-10 in patients with hepatitis C virus infection may have an anti-inflammatory and hence an antifibrotic effect.109 Therefore, 30 patients with advanced HCV-mediated fibrosis who had failed standard interferon-a-based antiviral therapy were enrolled in a 12-month treatment trial of IL-10 given daily or thrice weekly subcutaneously. In 13 of 28 of these patients the hepatic inflammation score decreased by at least two points (Ishak score) and 11 of 28 had a reduction in fibrosis score (mean change from 5.0 ± 0.2 to 4.5 ± 0.3, p < 0.05). However, serum HCV RNA levels increased during therapy (mean HCV RNA at day 0: 12.3 ± 3.0 mEq/ml; and at 12 months: 38 mEq/ml; p < 0.05). Changes in liver histology and HCV RNA levels were accompanied by an apparent shift in toward a Th2-predominant lymphocyte phenotype, as had been originally hypothesized. Long-term therapy with
Chapter 6 HEPATIC FIBROSIS AND CIRRHOSIS
IL-10 decreased hepatic inflammatory activity and appeared to have an inhibitory overall effect on fibrosis. Thus, although IL-10 appears to reduce inflammation and fibrosis, it has not been pursued as an antifibrotic compound because of putative detrimental virologic effects.
Miscellaneous Anti-inflammatory Drugs A number of other anti-inflammatory approaches have gained attention as therapies for fibrosis. Because TNF-a drives inflammation in many diseases, and because TNF-a is up-regulated in liver diseases (such as alcoholic hepatitis), an anti-TNF-a compound should theoretically reduce inflammation and hence the stimulus for fibrosis.171–174 Preliminary analyses from a study of anti-TNF-a therapy suggest an improvement in inflammation which presumably precedes fibrosis in patients with alcoholic hepatitis,173 although there was little effect of anti-TNF-a on hepatic fibrosis. Such data, in addition to the favorable effects of the TNF-a inhibitor pentoxifylline on mortality, provide the rationale for future study in patients with alcoholic hepatitis. These approaches and others that broadly inhibit inflammation must be considered cautiously because of the concern about disruption of the immune system, with increased risk of infection. Penicillamine is a heavy metal chelating compound that has been proposed to have anti-inflammatory and thus antifibrogenic effects.175 However, this compound had no effect on fibrogenesis in patients with primary biliary cirrhosis.176,177 Metrothrexate is thought to have anti-inflammatory properties, but interestingly has typically been considered to be profibrogenic in the liver for patients receiving methotrexate for treatment of rheumatologic diseases178 (although it is noteworthy that the risk of fibrosis progression may be less prominent than typically believed178,179). Metrothrexate has been studied in patients with primary biliary cirrhosis. Although some investigators have reported highly favorable effects in this disease, including improvement of the disease and reversion of fibrosis,180 the majority of the data on methotrexate have either been negative181,182 or show that the drug’s effects have been marginal, either alone181 or in combination with colchicine.183 It is important to emphasize that if methotrexate is used to treat patients with primary biliary cirrhosis, this must be undertaken by an experienced hepatologist.
ANTIOXIDANT AGENTS Oxidative stress is thought to play an important role in injury, stellate cell activation, and the stimulation of extracellular matrix production, as discussed above. Thus, a wide variety of antioxidants have received attention as potential antifibrotics.
Polyenylphosphatidylcholine Polyenylphosphatidylcholine is a mixture of polyunsaturated phosphatidylcholines, extracted from soybeans. This compound has antioxidant properties and oxidant stress (see above) is thought to be important in the inflammatory and fibrogenic response to injury, particularly in alcoholic liver disease. As oxidative stress leads to lipid peroxidation, and lipid peroxidation is injurious at the level of the cell membrane, phosphatidylcholine has been proposed to be protective against injury to cell membranes, resulting in reduced cellular injury and fibrogenesis.184
A VA cooperative multicenter clinical trial examined the effect of polyenylphosphatidylcholine in 789 patients with alcoholic hepatitis who had a very high daily average alcohol intake (16 drinks/day).185 Subjects were randomized to either polyenylphosphatidylcholine or placebo for 2 years. The long period of treatment is noteworthy as it is likely that long periods of treatment will be required to effect changes in the liver fibrosis. Many subjects substantially reduced their ethanol consumption during the trial, which probably accounted for improvement in fibrosis in the control group, making it difficult to demonstrate an improvement in fibrosis in the polyenylphosphatidylcholine group. Thus, overall, polyenylphosphatidylcholine failed to lead to significant improvement in fibrosis.
Silymarin Silymarin is derived from the milk thistle Silybum marianum. This extract has been shown to reduce lipid peroxidation and inhibit fibrogenesis in rodent animal models,186,187 as well as in baboons.188 It has been tested in several carefully performed human clinical trials, although fibrosis was not used as an endpoint. The compound has been found to be safe, but reportedly has mixed effects.189,190 In one study examining silymarin in alcoholics189 mortality was reduced; in addition, patients with early stages of cirrhosis also appeared to benefit. However, in another study in alcoholics no survival benefit could be identified.190 In both of these trials, silymarin appeared to be safe. Thus, although the agent is safe and is commonly used by patients with fibrosing liver disease, there is limited evidence of its efficacy.
Other Antioxidants Antioxidants such as vitamin E have been examined in animal models191 as well as in humans.192–195 A vitamin E precursor, D-atocopherol (1200 IU/day for 8 weeks), was studied in six patients with hepatitis C virus infection who failed to respond to interferon therapy,192 and was found to inhibit stellate cell activation but did not affect fibrosis. A randomized controlled trial examined vitamin E in patients with mild to moderate alcoholic hepatitis and found that vitamin E reduced serum hyaluronic acid, but did not lead to a change in type III collagen.194 Combined antioxidant therapy, including vitamin E, had no effect on outcome in patients with severe alcoholic hepatitis, although fibrosis was not specifically addressed.195 Malotilate is another potential cytoprotective agent, perhaps acting via inhibition of cytochrome P450 2E1; in addition, this compound may have anti-inflammatory properties. In patients with primary biliary cirrhosis, although it was found to diminish plasma cell and lymphocytic infiltrate and piecemeal necrosis, it had no significant effect on fibrogenesis.196 Another agent used to antagonize oxidative stress is S-adenosylmethionine; this compound is important in the synthesis of the antioxidant glutathione. The enzyme (methionine adenosyltransferase) responsible for its synthesis is reduced in the injured liver;197 thus it has been hypothesized that if S-adenosylmethionine were replaced, then injury and fibrogenesis might be slowed. S-adenosylmethionine has been tested in a large randomized trial in patients with alcoholic cirrhosis.198 There was an improvement in overall mortality/need for liver transplantation in the treatment arm, espe-
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cially in patients with Child’s A/B cirrhosis, although histologic assessment of fibrosis was not specifically assessed.198 Propylthiouracil is an antithyroid drug that reacts with some of the oxidizing species derived from the respiratory burst and may thus be protective in alcoholic liver disease, a disease in which an increase in hepatic oxygen consumption may predispose the liver to ischemic injury. Thus, propylthiouracil has been tested in a number of randomized clinical trials in patients with alcoholic liver disease. Unfortunately, a systematic review and meta-analysis found that propylthiouracil had no benefit in fibrosis, or in any other outcome measured.199
CYTOPROTECTIVE AGENTS Ursodeoxycholic Acid Ursodeoxycholic acid binds to hepatocyte membranes, where it presumably stabilizes them and is thus cytoprotective. This cytoprotective action in turn theoretically reduces inflammation and may in turn have a beneficial effect on fibrogenesis.200 Neither experimental data nor human studies indicate a primary antifibrotic effect of ursodeoxycholic acid in the liver, but the compound has been examined extensively.201–209 Ursodeoxycholic acid has been studied in patients with cystic fibrosis, primary biliary injury (primary biliary cirrhosis, primary sclerosing cholangitis and progressive familial intrahepatic cholestasis), and miscellaneous liver diseases. Results with ursodeoxycholic acid in these conditions have been mixed. Both symptomatic and biochemical improvement has been observed in these diseases, in particular the biliary diseases, but data on histologic improvement (and survival) have not been consistent. For example, in a randomized controlled trial in patients with primary biliary cirrhosis, ursodeoxycholic acid led to reduced fibrosis in those with mild disease but had no effect on those with severe disease.202 In another study, survival was improved in patients treated with ursodeoxycholic acid but fibrosis was not improved.206 Further, in a histopathologic study of 54 patients with primary biliary cirrhosis and paired liver biopsies, 4 years of ursodeoxycholic acid therapy was associated with a significant decrease in the prevalence of florid interlobular bile duct lesions, lobular inflammation, and necrosis. Worsening of fibrosis was observed in 14 patients (the majority had only a onegrade progression in fibrosis score), whereas stabilization was noted in the 40 remaining patients.207 A recent combined analysis of the histologic effect of ursodeoxycholic acid on paired liver biopsies including a total of 367 patients (200 ursodeoxycholic acid and 167 placebo) revealed that subpopulations of patients with initial earlystage disease may benefit from therapy.208 Results of meta-analyses examining ursodeoxycholic acid have been mixed, and have largely reported that ursodeoxycholic acid is not effective in primary biliary cirrhosis.205 The aggregate data suggest that ursodeoxycholic acid may impede the progression of fibrosis in primary biliary cirrhosis via effects on (bile duct) inflammation, particularly if given early in the disease course. It should be emphasized that so far as we know, ursodeoxycholic acid is extremely safe. Thus, although it is also expensive, the available data justify its use as an antifibrotic in patients with primary biliary cirrhosis. Ursodeoxycholic acid has also been studied in children with progressive familial intrahepatic cholestasis,203 where it appeared to
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improve fibrogenesis. Additionally, a small series indicated that seven of 10 patients with cystic fibrosis treated with ursodeoxycholic acid had a reduction in liver fibrosis.204 Although these effects are promising, it should be emphasized that the numbers of patients studied has been small. Finally, in a large randomized controlled trial of ursodeoxycholic acid in patients with non-alcoholic steatohepatitis over 2 years, including 107 subjects who had paired biopsy data, there was no improvement in fibrosis.209
MISCELLANEOUS Anabolic–androgenic steroids such as oxandrolone have been examined in randomized trials including patients with alcoholic liver disease, but have not been found to have significant effects on fibrosis (or other outcomes).210
Stellate Cell-Specific Compounds Interferon-g A wealth of data supports the antifibrotic potential of interferon-g. The interferons consist of a family of three major isoforms, a, b and g. These isoforms are unique, not only in structure but also in their biologic actions. Interferons-a and -b bind to the same receptor, whereas interferon-g binds to a different receptor. Interferon-a has more potent antiviral effects than does interferon-g, and interferong has been shown to specifically inhibit extracellular matrix synthesis in isolated cells, including stellate cells.211,212 Interferon-g potently inhibits multiple aspects of stellate cell activation,211,212 and appears to have antifibrotic effects in patients with pulmonary fibrosis.213 Such data have generated considerable enthusiasm about the use of interferon-g in patients with hepatic fibrosis, although there is theoretical concern about its use because it is proinflammatory, and moreover its overexpression in the liver leads to chronic hepatitis.214 None the less, it has now been tested in humans with fibrosing liver disease, and appears to be safe.215 Although this pilot study provides a firm foundation highlighting the potential use of interferon-g in patients, larger studies are needed to prove a therapeutic benefit.
COMPOUNDS THAT INHIBIT FIBROGENESIS Colchicine Colchicine is a plant alkaloid that inhibits polymerization of microtubules, a process that in turn is believed to be required for collagen secretion. Based on this concept, colchicine has been advanced as an antifibrotic agent. A sizeable body of literature indicates that colchicine has antifibrotic properties in experimental animal models.216 This work has led to a number of human clinical trials.217–220 A wide variety of liver diseases has been studied, including primary biliary cirrhosis, alcoholic cirrhosis, cryptogenic cirrhosis, and miscellaneous other liver diseases. In a double-blind randomized controlled trial examining colchicine in primary biliary cirrhosis improvements were noted in a number of biochemical markers, but the drug failed to reduce fibrosis.217 In an often-cited popularized double-blind randomized controlled trial of colchicine versus placebo in patients with various liver diseases, colchicine led to improved fibrosis as well as a dramatic improvement in survival.218 However, this study has been extended to clinical practice with great caution because of a variety of methodological concerns. First,
Chapter 6 HEPATIC FIBROSIS AND CIRRHOSIS
Table 6-5. New Potential Antifibrotic Targets in Humans Agent
Comments
Anti-TGF-b Anti-PDGF Interferon-g PPAR ligands
Blocks stellate cell fibrogenesis Blocks stellate cell proliferation Inhibits multiple features of stellate cell activation ? Stellate cell-specific effects
many patients were lost to follow-up, and in addition there was substantial unexplained excess mortality in the control group (unrelated to liver disease). In a recent large VA cooperative multicenter study involving 549 patients comparing colchicine (0.6 mg orally b.i.d.) to placebo in patients with alcoholic liver disease, there was no apparent effect of active treatment on survival. Histologic data that might have provided information on the anti-inflammatory effects of colchicine were not obtained.219 A meta-analysis including 1138 subjects found that colchicine had no effect on hepatic fibrosis or mortality.220 In summary, the data surrounding colchicine suggest that this compound is safe but likely to be ineffective.
FUTURE ANTIFIBROTICS Given the major effort in understanding the biology of hepatic fibrogenesis, it is not surprising that numerous pathways have been targeted as having therapeutic potential. Many compounds have been studied in experimental models and have been shown to have antifibrotic properties, including several with great potential in human liver disease (Table 6-5). Several important pathways merit discussion. One of the most important examples is the TGF-b pathway, as it plays a central role in the fibrogenic cascade. Several approaches to inhibit the action of TGF-g have been proposed and include the use of molecules such as decorin, the protein core component of proteoglycan, which binds and inactivates TGF-b,221 antibodies directed against TGF-b1, and soluble receptors which typically encode for sequences that bind active TGF-b and prevent it from binding to its cognate receptors. The concept has been well established experimentally; indeed, the effect of inhibition of TGF-b in animal models of liver injury and fibrogenesis has been striking.222,223 A limitation of approaches that target TGF-b is that the cytokine potently inhibits cellular proliferation, and inhibition of its effects in vivo could predispose to malignant transformation. Another critical pathway involves PDGF. PDGF is the most potent stellate cell mitogen known,3,224 and in addition stimulates stellate cell migration.225 A number of approaches have been used to inhibit the effect of PDGF. For example, kinase inhibitors that specifically inhibit PDGF signaling might be useful,226 as could those with more general effects on tyrosine kinase receptors. Additionally, stellate cells express angiotensin and endothelin receptors and their cognate ligands appear to be overproduced in the liver; further, stimulation of stellate cells with their respective ligands leads to stellate cell activation.66 Thus, inhibition of their binding may be clinically beneficial. Among others agents, compounds such as pirfenidone,227 peroxisomal proliferator-activated receptor (PPAR)-g ligands,76,228 and halofuginone229 appear to have direct effects on stellate cells and
thus could evolve into effective antifibrotic compounds. Many others have been highlighted (see 5 for review).
SUMMARY AND FUTURE DIRECTIONS FOR ANTIFIBROTIC THERAPY The explosion of information about the pathogenesis of fibrogenesis has spawned a field dedicated to antifibrotics focused on the activation of hepatic stellate cells. Stellate cell activation is characterized by a number of important features, including enhanced matrix synthesis and a prominent contractile phenotype, processes that each contribute to the dysfunction of the liver typically found in advanced disease. It should be emphasized that the control of activation is multifactorial, and thus several potential therapeutic interventions are possible. A further critical concept is that fibrosis, in particular the ECM component of fibrosis, is dynamic, and the accumulation of fibrosis may be inhibited. It is likely that fibrosis, including even advanced fibrosis, may be reversible under the appropriate conditions. Currently, effective therapy for hepatic fibrogenesis exists for several diseases in which the cause of the underlying disease is removed. In contrast, specific therapy directed only at the fibrotic lesion is not currently available; the most effective therapies will most likely be directed at the stellate cell. Additionally, approaches that regulate matrix remodeling (i.e. by enhancing matrix degradation or inhibiting factors that prevent matrix breakdown) will be attractive. Thus, multiple potential targets have been identified, and it is highly likely that candidates will emerge. The ideal antifibrotic compound will be specific, effective, safe, and inexpensive.
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REPLICATION OF HEPATITIS B VIRUS AND PATHOGENESIS OF DISEASE Angeline Bartholomeusz, Judy Chang, Stephen Locarnini, and Sharon Lewin Abbreviations ALT alanine aminotransaminase APC antigen-presenting cells BCP basal core promoter ccc covalently closed circular DC dendritic cells DHBV Duck hepatitis B virus EnhI enhancer I EnhII enhancer II ER endoplasmic reticulum FasL Fas ligand HBcAg hepatitis B core protein or antigen HBeAg hepatitis B e antigen HBsAg hepatitis B surface antigen HBSP hepatitis B splice protein HBx hepatitis Bx protein HBV hepatitis B virus HCC hepatocellular carcinoma
HLA HSC GAPD IFN LCMV LHBs lxRNA MHBs MHR MIP-1a NK NKT NLS nt PAMP pg pHSA
human leukocyte antigen hepatic stellate cells glyceraldehyde-3-phosphate dehydrogenase
interferon lymphocyte choriomeningitis virus large hepatitis B surface protein long-X RNA medium hepatitis B surface protein major hydrophilic region macrophage inflammatory protein 1a natural killer natural killer T nuclear localization signal nucleotide pathogen-associated molecular patterns pregenomic polymerized human serum albumin
INTRODUCTION Hepatitis B virus (HBV) is the prototype member of the family Hepadnaviridae, which also includes viruses that can infect higher primates such as chimpanzees and lower primates such as tupaia (tree shrews).1 Other hepadnaviruses can infect a range of other mammals, including woodchucks and ground-squirrels, and birds such as herons and ducks. HBV is an enveloped virus and contains a circular, partially double-stranded DNA genome. There are three types of virus-associated particle: the virion, and spherical and filamentous particles found in serum. Only the HBV virion is infectious, as the spherical particles and filaments do not contain the HBV genome and represent excess production of the viral envelope. The HBV virion is 42 nm in diameter and comprises an outer envelope that contains the three envelope proteins, all of which express the hepatitis B surface antigen (HBsAg). This envelope surrounds an inner nucleocapsid made up of the hepatitis B core protein or antigen (HBcAg) that packages the viral genome and associated polymerase. The spherical particles are 17–25 nm in diameter and can occur in large numbers up to 1013/ml. The filaments or tubular structures are approximately 20–22 nm in diameter. In spite of its small genome size, HBV is a complex virus. The HBV genome is only 3.2 kb in length, and so to compensate for this
PKR POL RANTES RNase H rt SHBs Th1 Th2 TLR TLR-2 TNF TNF-R1 TRAIL Tyr WMHBV
protein kinase activity polymerase regulated on activation, normal T expressed and secreted ribonuclease H reverse transcriptase small hepatitis B surface protein T-helper cell type 1 T-helper cell type 2 toll-like receptors Toll-like receptor 2 tumor necrosis factor TNF receptor 1 TNF-related apoptosis-inducing ligand tyrosine Woolly Monkey hepatitis B virus
limited coding potential HBV DNA is organized into a series of overlapping reading frames and co-terminal reading frames that encode for proteins which are either multifunctional or have very different functions despite sharing similar amino acid residues (Figure 7-1). Furthermore, HBV has also evolved unique strategies for genomic replication (Figure 7-2). It is the intricacies of this replication strategy and its impact on the hepatocyte and the host’s immune response that this chapter will unravel, as well as new systems that have been developed to facilitate research into HBV replication and pathogenesis.
MOLECULAR VIROLOGY NEW CELL LINES AND MODEL SYSTEMS THAT HAVE BEEN DEVELOPED TO INVESTIGATE HBV REPLICATION AND PATHOGENESIS A number of new cell lines and cloning strategies have been developed to deliver HBV into cells and to investigate HBV replication, virus–host interactions, pathogenesis and antiviral sensitivities. The major problem of studying HBV replication is the lack of a suitable cell-culture system for infecting cells in vitro. One of the recent advances has been the development of a cell line, HepaRG, by
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Pre-S2/S promoter
2.1 4 2.
kb
A RN
kb R
NA
Pre-S 2
S1 ePr
–S
d
S F-
n tra
OR
Pre-S1/S promoter
Figure 7-1. Diagrammatic representation of the HBV genome. The inner circle represents the virion genome DNA that is packaged within the viral particles in the cytoplasm of infected cells, and the dashes represent the region of the positive-sense DNA which is incompletely synthesized. The middle circle represents the four open reading frames corresponding to precore, core, HBx, polymerase, and the envelope proteins. The outer circle represents the HBV RNAs. The promoter and enhancer regions are indicated.
+ nd ra
DR
1
DR2 5'
5' AA A AA A AA A AA A
Basal core promoter
F
F-C OR
3.5 kb RNA
St
OR LPO
O RF -X Enhancer 1/X promoter
NA bR 0.7 k Enhancer 11
Gripon et al.2 that can be infected with HBV. The wider availability of this cell line should facilitate research into the early events of HBV replication, including virus entry and the hunt for the elusive cellular receptor for HBV. A number of delivery systems have been developed to transport HBV into cells. This includes the recombinant adenovirus3 and the recombinant baculovirus system.4 To study HBV replication and antiviral sensitivities from patient samples a number of groups have developed strategies for the PCR amplification of full genome length (or near full genome length) HBV from the patient and directly cloning the amplified product into vectors.5 These new patientspecific clones will enable mutants to be studied in the context of the entire authentic genetic framework, and thus is extremely useful for determining phenotypically the antiviral resistance profile for a patient at a given time. Other groups have developed particular plasmid vectors that facilitate the cloning and expression of fulllength HBV genomes amplified from the sera of patients6 that can then be used to enable efficient phenotypic analysis of patientderived virus in cell culture.7 These vectors will be useful in future studies of drug resistance, including surveillance and cross-resistance testing.8
HEPATITIS B VIRAL GENOME: MAJOR TRANSCRIPTS Transcription of the HBV genome results in the formation of the pregenomic (pg) RNA and the precore mRNA, which are approxi-
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mately 3.5 kb and three subgenomic RNAs.9 The polymerase gene is the largest open reading frame and encodes for the multifunctional polymerase (POL) protein. The polymerase gene overlaps all six other genes, including the core gene that encodes for HBcAg and the precore gene that encodes for the hepatitis B e antigen (HBeAg), the three envelope genes PreS1, PreS2 and S that encode for the large, middle and small envelope proteins, respectively (LHBs, MHBs and SHBs), and the X gene encoding for the multifunctional X protein.
EARLY EVENTS: ATTACHMENT, PENETRATION AND ENTRY Current studies have demonstrated that LHBs are involved in virus entry and binding to hepatocytes.10 Monoclonal antibodies to the PreS1 region of LHBs (amino acids 20–23; Asp- Pro-Ala- Phe) prevented virus attachment.10 Interestingly, antisera to a conformational epitope within the S coding region also prevented attachment, whereas monoclonal antibodies directed to the PreS2 coding region did not totally prevent virus attachment. Earlier studies had determined that the N terminus region of the LHBs protein (codons 21–30) is critical for species specificity, as all hepadnaviruses are highly species and cell-type specific.11 However, using the HDVHBV system with Woolly Monkey hepatitis B virus (WMHBV), Barrera et al.12 found that replacement with codons 1–40 of HBV in the HDV-WMHBV was insufficient to enable infectivity of human
Chapter 7 REPLICATION OF HEPATTIS B VIRUS AND PATHOGENESIS OF DISEASE
Infectious virion S particles
Virus attachment Uncoating Nuclear transport 3'
5' 5'
2.1kb
Synthesis of ccc DNA
AAA
3' AAA
2.4kb
ccc DNA
S HBsAg
Generation of minichromosome
Pre-S1 Pre-S2
5'
3' 0.7kb
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AAA
HBx 5'
Precore mRNA 3.5kb
mRNA Transcription 3'
AAA 5' 3.5kb
Precore Protein synthesis Intracellular conversion pathway
Capsid protein (Core)
Pregenomic mRNA 3' AAA
Figure 7-2. Life cycle of HBV. After attachment, penetration and uncoating of the infectious virion, the viral nucleocapsid is released into the cytosol and transported to the nuclear pore. The viral genome is delivered into the nucleus, where it is converted into cccDNA and the viral minichromosome is generated. The minichromosome is the major transcriptional template from which all RNA transcripts are generated. The pregenomic RNA is translated in the cytosol to produce both the core and the polymerase protein. The polymerase and the pregenomic RNA are encapsidated. HBV DNA synthesis occurs within the nucleocapsid. The nucleocapsids can then either be transported back to the nucleus via the intracellular conversion pathway or be enveloped and secreted into the extracellular space as virions. The envelope proteins are translated at the rough endoplasmic reticulum, and in addition to the virions are also secreted as small particles and tubules containing only envelope proteins. The precore protein is synthesized from the precore mRNA, which is slightly larger than the pregenomic RNA. The precore protein is processed through the Golgi apparatus, where it undergoes multiple cleavages at both its N and its C terminus and is secreted as HBeAg.
Encapsidation with polymerase Capsid protein (Core)
POL
Hepadnavirus DNA synthesis
RNA (+)
Golgi apparatus Envelope and secretion
Mature genome
DNA (+)
DNA (–)
Free virions Secreted HBeAg
hepatocytes, suggesting that other regions of the PreS1 protein may be required for species specificity. The search for the HBV receptor using LHBs has uncovered a large number of potential candidates, including the receptors for immunoglobulin A, interleukin-6, transferrin and asialoglycoprotein, glyceraldehyde-3-phosphate dehydrogenase (GAPD), apolipoprotein H, and human liver annexin V. Unfortunately, none has been unequivocally identified as the major receptor for the specific binding of HBV.13 A number of cofactors as well as a primary receptor may be required for HBV attachment and penetration. MHBs are not necessary for infectivity, yet this region does interact with cellular proteins, which may possibly enhance infection uptake and uncoating.14 The PreS2 encoded region contains a binding site for polymerized human serum albumin (pHSA) and also the transferrin receptor that may facilitate attachment and penetration of HBV to target cells.14
S particles
TRANSPORT OF VIRAL GENOME TO THE NUCLEUS, UNCOATING AND FORMATION OF THE MAJOR TRANSCRIPTIONAL TEMPLATE The HBc protein of HBV encodes a nuclear localization signal (NLS) for the transport of the mature capsids containing the HBV genomes into the nucleus. The nuclear transport is mediated by the imporin pathway using nuclear transport receptors Impb/Impa. The phosphorylation of the C-terminal sequences on the HBc protein is linked with capsid maturation and exposure of the NLS signal. Rabe et al.15 have demonstrated that it is only these mature capsids that are able to move the capsid protein from the collection of nuclear proteins referred to as the ‘nuclear basket’ into the karyoplasms and uncoat their HBV DNA into the nucleus. Hepadnaviruses have an unusual process, referred to as the ‘intracellular conversion pathway’, in which newly synthesized viral
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nucleocapsids can be directed back into the nucleus to increase the pool of the transcriptional template, the covalently closed circular (ccc) DNA. In DHBV the PreS region is involved in the regulation of this intracellular conversion pathway. However, studies examining HBV envelope proteins have not demonstrated the same effect on regulation of the capsids into the nucleus, nor increased cccDNA pools.16 The next stage in HBV viral replication is the conversion of the viral genome into the minichromosome. The HBV viral genome is a partially double-stranded molecule. The HBV viral polymerase may mediate the repair of the ‘gap’ and, in association with host cellular DNA, repair enzymes that facilitate the conversion of viral genome into cccDNA. This conversion also requires the removal of the HBV polymerase protein and oligoribonucleotide and ligation of DNA. Kock et al.17 have demonstrated that the nucleos(t)ide analogs adefovir and lamivudine can inhibit the initial DNA repair process.17 The HBV cccDNA is chromatinized by cellular histone and nonhistone protein and converted into a minichromosome.18 The cccDNA is the major transcriptional template, thus the chromatinization of the HBV genome will affect the binding of transcription factors, thereby regulating transcription. The HBV core protein may influence the spacing of the nucleosome complex on the HBV DNA and hence also the binding of transcription factors.19
TRANSCRIPTION AND TRANSLATION OF THE HBV VIRAL PROTEINS Five promoters control the synthesis of the six viral transcripts of HBV. The HBV genome contains two enhancers, designated enhancer I (EnhI) and enhancer II (EnhII), both of which exhibit greater activity in cell lines of hepatic origin. Although the enhancers are located upstream of specific promoters, EnhI regulates all viral promoters and EnhII regulates the basal core promoter (BCP) as well as the transcription of the PreS2/S promoters. Doitsh et al.9 have proposed that HBV may have both early and late transcriptional events in which EnhI may regulate the expression of the early transcripts of X, and a long X-related transcript of 3.9 kb known as long-X RNA (lxRNA), whereas EnhII appears to regulate late gene transcription events. All RNA molecules are transcribed by the host cell RNA polymerase II using the cccDNA template, are capped and are polyadenylated. The pgRNA and the precore mRNA are longer than genomic length, and transcription is controlled by the BCP. The bifunctional pregenomic mRNA is utilized as the genomic template for reverse transcription of the viral negative-sense DNA and for translation of HBc, Ag and POL proteins, whereas the slightly longer precore mRNA encodes for only the precore protein, which is subsequently processed and secreted as HBeAg.
The Hepatitis B Core Protein (HBcAg) The HBc protein is translated in the cytosol, where it initially forms dimers, followed by multimerization of the dimers to form the nucleocapsid. The HBc protein has been crystallized and the nucleocapsid protein has been studied using cryoelectron microscopy.20 The multimerization of the HBc protein can occur independently of the encapsidization of the pgRNA–POL complex. The HBc protein possesses two distinct domains:14 the N-terminal domain (amino acid residues 1–144), which is involved in dimerization and multi-
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merization of the nucleocapsid; and the C terminal domain, which includes a potential nuclear localization sequence and is arginine rich. This region is required for nucleic acid binding and in viral encapsidation. The negatively charged codons 113 and 117 were determined to be essential for pgRNA encapsidation.21 When expressed in bacteria the core protein can self-assemble into capsids. Two icosahedral shells of different sizes are observed. Particles with a T = 3 symmetry containing 90 homodimers of 32 nm, and particles with a T = 4 symmetry consisting of 120 homodimers of 36 nm.22 Deletion of an arginine-rich domain at the C terminus allows efficient expression of the protein in bacteria and favors the formation of T = 4 over T = 3 capsids. To date, the preference of T4 over T3 in infected patients has not been determined. Mutational analysis of core has been used to investigate potential interactions between the capsid and the viral envelope.23 The mutations that affected envelopment clustered around the base of the spike and to a small area at the capsid surface close to the pores in the capsid shell.
Hepatitis Be Antigen The precore mRNA is not used for reverse transcription and functions only in translation of the secreted HBeAg, which is one of two accessory proteins of the virus. The first 19 amino acids of the precore protein comprise a secretion signal that allows for the translocation of the precore protein into the lumen of the endoplasmic reticulum (ER). The signal sequence is cleaved off by a host cell signal peptidase and the protein is secreted through the ER and Golgi apparatus. A further modification of the C terminus results in the secretion of a heterogeneous population of 14–17 kDa proteins serologically defined as HBeAg. Alternatively, the precore protein also expresses a signal for transport into the nucleus. The role of HBeAg appears to be for the establishment of persistent infection in vivo,24 to serve an immunoregulatory role in natural infection, and to activate or tolerize T cells.25
Hepatitis B Polymerase (POL) The POL protein is translated from pregenomic RNA. POL contains four functional regions: (1) terminal protein (tp) used in priming HBV DNA synthesis; (2) the spacer region; (3) the reverse transcriptase (rt) that has RNA- and DNA-dependent DNA polymerase activities; and (4) the ribonuclease H (RNase H) that cleaves the RNA in the RNA–DNA hybrids during reverse transcription. The POL protein is covalently attached to the genome and is packaged within the nucleocapsid. The rt region encodes for seven domains G, F, and A to E, which are conserved in other polymerase proteins.26 The HBV POL has not been crystallized, but various homology models have been developed based on the structure of HIV.26 The antiviral nucleos(t)ide analogs that have been developed for the treatment of HBV are all targeted to the rt function of the POL. Resistance to lamivudine,27 adefovir28 and entecavir29 has now been detected. HBV DNA synthesis requires the encapsidation of the HBV POL protein (see below). However, in Duck hepatitis B virus (DHBV), and now recently in HBV-infected cells, a cytoplasmic form of HBV POL has been detected.30 This was detected at relatively lower amounts than DHBV POL, but had a shorter half-life. DHBV POL
Chapter 7 REPLICATION OF HEPATTIS B VIRUS AND PATHOGENESIS OF DISEASE
translation was detected at the same time or earlier than core, even though it is of the same template, and the ratio of POL relative to core dropped with time. Investigations of the translation of DHBV POL have determined that a shunting mechanism is used, and ribosomes are transferred from a donor region near the 5¢ end of the pgRNA to an acceptor site at or near the start AUG of POL. This shunting mechanism may be involved in the regulation of the amount of POL translated. Based on current understanding of HBV DNA replication, the cytoplasmic POL is unlikely to play a role in DNA replication. Mizukoshi et al.31 have identified 10 CD4 T cell epitopes in HBV POL. Cytoplasmic HBV POL may have a role in the adaptive or the innate immune regulation of HBV.
Envelope Proteins: Large (LHBs), Medium (MHBs) and Small Hepatitis B Surface Protein (SHBs) The 2.6 and 2.1 kb RNA transcripts encode the envelope proteins LHBs, MHBs and SHBs. The viral envelope, the small particles and filaments are synthesized and assembled at the ER membranes and then bud into its lumen. The SHBs is 226 amino acids long and is the most abundant protein in all three HBV-associated particles. The SHBs encodes a glycosylation site at codon 146 and both glycosylated and non-glycosylated forms are produced. SHBs contains a high number of cysteine residues that are cross-linked with each other, forming the major hydrophilic region (MHR) that is the major antigenic determinant of HBsAg, described as the ‘a’ determinant. The most-characterized vaccine escape mutant sG145R is located within this region. The MHBs containing the PreS2 domain is a minor component of the virion or HBs particle and consists of the S plus a 55 amino acid N-terminal extension. The MHBs protein is not required for infectivity or virus assembly; however, the glycosylated MHBs is required for virus secretion.32 An N-linked carbohydrate is also attached to Asn-4 of the PreS2 domain of the MHBs protein, which contains an additional modification of an O-linked glycosylation at Thr-37 in the PreS2 domain. The MHBs is considerably more immunogenic than SHBs, and PreS2-containing HBs particles generated from animal cell lines have been used in some countries as a prophylactic vaccine.33 LHBs is more prevalent than MHBs in virions and filaments, but less prevalent in the HBs spherical particles. LHBs contains a further 108 or 119 amino acids (depending on the subtype/genotype) compared to MHBs. LHBs is glycosylated and is modified at Gly-2 of the PreS1 domain by myristylation. The myristylation is essential for viral infection as well as assembly and release.
Hepatitis Bx Protein (HBx) The 1.1 kb transcript encodes for the X protein (HBx), which is 154 amino acids in length and is the second accessory protein of HBV. HBx is not required for in vitro virus replication but is required for the establishment of hepadnaviral infection in woodchucks.34 HBx is located in both the cytoplasm and the nucleus of the cell. The level of HBx expression can influence its cellular localization. It is predominantly nuclear when expressed in cells at very low levels, but becomes largely cytoplasmic as its expression level increases. A number of conflicting studies have been reported for the HBx
protein that are probably related to the expression levels of HBx, its ability to both be a substrate and an inhibitor of the proteasome complex, and its effect in the modulation of cytosolic calcium, which activates various signaling pathways involving Src kinases.1,35,36 HBx is a multifunctional viral protein. In the nucleus, HBx is a modest promiscuous trans-activator that can regulate transcription via direct interaction with different transcription factors. This regulation of viral and cellular genes affects viral replication and viral proliferation, directly or indirectly, and so, not surprisingly, HBV can influence apoptosis and cell cycle regulatory pathways. However, the trans-activation function of X was found to be reduced when proteasome inhibitors were investigated. This interaction of HBx with the 26S proteasome complex may also affect immune evasion by suppressing viral antigen presentation.37 HBx affect on cytosolic calcium may involve an interaction with or action upon the mitochondrial voltage-dependent anion channel,36,38 thereby functioning in the cytoplasm to activate various signaling pathways. In the nucleus, HBx can regulate transcription through a direct interaction with different transcription factors, and in some cases enhance their binding to specific transcriptional elements.39 Overall, whether the HBx activation of transcription or its effect on the cytoplasmic signaling pathways plays a significant role during natural infection with HBV remains an open question.
GENOMIC REPLICATION The process of HBV DNA synthesis is complex and involves not only reverse transcription but also a complicated process of three translocations of the polymerase and primer to complete the doublestranded genome synthesis. The major features of this complex synthesis are: (1) POL protein binds to the encapsidation signal (epsilon, or e) at the 5¢ end of the pgRNA; (2) using part of the bulge region of e as template, the POL protein synthesizes a complementary 3–4 nucleotide (nt) DNA primer that is covalently linked to a tyrosine (Tyr) residue of POL; (3) the covalent complex translocates to a complementary region in the 3¢-proximal DR-1; (4) the primer is extended to a complete negative (-)-strand DNA with concomitant degradation of the RNA template, except for some 15–18 nt at its 5¢ end; (5) this RNA, containing the 5¢ DR-1, is transferred to the complementary DR-2 on the newly made (-)-strand DNA, serving there as primer for the (+)-strand DNA; (6) (+)strand DNA is extended to the physical end of (-)-DNA; (7) using the short terminal redundancy (r), a template switch from the 5¢ to the 3¢ end of (-)-strand DNA occurs and the (+) strand is extended to form the typical relaxed circular (RC) DNA.40 HBV and the other members of the Hepadnaviridae replicate their DNA genome by reverse transcription of a pgRNA template within the subviral core particle. Mature nucleocapids and virions contain the HBV 3.2 kb RC DNA genome with the POL covalently attached to the 5¢ end of the (-)-strand DNA.
ASSEMBLY AND RELEASE OF HBV VIRION The assembly of nucleocapsids containing RC DNA occurs in the cytosol, and these are then selectively enveloped prior to exiting the cell. The LHBs, MHBs and SHBs have different but interrelated functions during viral assembly, related to their transmembrane topologies.41
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The topologies of the SHBs and MHBs are similar and are determined by at least two signal sequences, resulting in a short luminal exposed N-terminal sequence, two transmembrane regions separated by a 55 amino acid cytosolic loop, and a luminal 70 amino acid domain containing the major epitope of the protein and a glycosylation site. The PreS2 region of MHBs is located in the ER lumen. LHBs protein biosynthesis is different, with the entire PreS domain initially remaining in the cytosol following translation. The PreS1 region of LHBs within the virion has a dual topology in which half are on the exterior surface of the virion and half are on the internal surface.41 A specific region within PreS1 at amino acids 70–94 binds to a heat-shock protein that controls the topology of this protein.42 Deletion of this translocation control region results in PreS with a uniform topology. The dual topology of the PreS1 region is required for virus infection and interaction with cellular receptors, as well for as the envelopment of the replicating core particles. The envelope proteins can assemble into either spherical or filamentous subviral lipoprotein particles as well as being incorporated into the outer shell of virions. The L and S proteins are essential for virion formation.43 Studies to determine which regions of the envelope proteins are important for virion formation versus subparticle formation have been performed.44,45 One region was located in LHBs between amino acids 103 and 124. The other region was located in SHBs between amino acids 35 and 46. Although the latter sequence is also present in the C-terminal part of L, the mutations affected virion morphogenesis mainly in the context of S. The MHBs protein is not required for assembly.
DEFECTIVE HBV PARTICLES: ROLE OF SPLICING In addition to the unspliced major HBV RNA transcripts described above, single- or double-spliced 2.2 kb RNAs have been detected in HBV-DNA-transfected hepatoma cells46 and in infected human livers.47 Sequencing of the single-spliced 2.2 kb HBV RNAs typically reveals a deletion from the last codon of the core gene to the middle of the S gene, that creates a new open reading frame, known as the hepatitis B splice protein (HBSP), which includes truncated S and POL proteins.48 The in vivo expression of HBSP is associated with viral replication and, more importantly, liver fibrosis.49 As well as pathogenesis, this alternative replicative strategy may be a mechanism of viral persistence,50 and further studies are clearly indicated to determine its role in viral replication and its effect on the host.
IMMUNOPATHOGENESIS OF HBV HBV infection results in an initial hepatitis that may or may not be symptomatic. Viral clearance depends on the age and immune status of the individual. Most infections in immunocompetent adults are self-limiting. Persistent or chronic infection is more likely to occur following perinatal transmission (from mother to child) or after horizontal transmission to children or immunocompromised adults. The immune determinants of successful clearance of HBV are not fully understood, but depend on both the cellular and the humoral immune responses. At the same time, however, liver inflammation and disease are also believed to be largely immune mediated. Therefore, a complex interaction exists between HBV and the host in both
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the initial clearance of HBV and the long-term pathogenesis of HBV disease. As discussed above, the study of the replication and pathogenesis of HBV has been limited owing to the lack of available animal models and in vitro cell lines that support HBV infection. HBV can infect chimpanzees, who only acquire a self-limiting acute hepatitis. Other animal models include infection of ducks with Duck hepatitis B and woodchucks with Woodchuck hepatitis B virus. More recent developments in transgenic mouse models have allowed a better understanding of the relative contributions of the different arms of the immune system to HBV, in particular the contribution of the early innate immune response.
ACUTE HBV INFECTION From studies of acute HBV infection in chimpanzees it is clear that HBV DNA peaks and declines prior to the onset of symptoms of acute hepatitis or the induction of a T cell-mediated response. Although no immunological data are available about early intrahepatic events in human infection, animal data support the concept that clearance of HBV DNA is largely mediated by antiviral cytokines produced by cells of the innate and adaptive immune response. In particular, interferon (IFN)-g, tumor necrosis factor (TNF)-a and IFN-a/b are believed to trigger several pathways leading to the inhibition of viral replication without the direct destruction of infected cells. Clinical hepatitis is observed following the decline of HBV DNA levels and is associated with the influx of inflammatory cells, including both HBV-specific and non-specific T cells.51,52 In particular, the appearance of CD8+ T cells that mediate cytolytic activity against HBV-infected cells coincides with an increase in alanine aminotransaminase (ALT) detected in serum.51 Following clearance of the virus and a reduction in ALT, HBV surface antibodies are detected. HBV-specific antibodies, together with HBVspecific memory T cells, incur protective immunity against future infections.
The Innate Immune Response The initial response to HBV is believed to be mediated by nonspecific mechanisms that can be activated in a very short time, ranging from minutes to hours. Among these mechanisms, killing of virus-infected cells without human leukocyte antigen (HLA) restriction or apparent specificity of viral antigens is believed to occur via natural killer (NK) cells, natural killer T (NKT) cells and Kupffer cells (liver-residing macrophages).52–54 NKT cells depend on nonclassic major histocompatibility complex class I-like CD1 molecules for their development, and mainly recognize glycolipids presented by CD1 (recently reviewed in 55). Following infection with HBV, it is believed that hepatocytes, which have low expression of human leukocyte antigen (HLA) class I, release IFN-a and IFN-b. Initial recognition of HBV infection may be mediated by toll-like receptors (TLR) following the detection of pathogen-associated molecular patterns (PAMP).56 These PAMPs probably include viral envelope glycoproteins, double-stranded or single-stranded RNA, and viral accessory proteins such as HBeAg and HBx. Recent studies have shown that several TLRs can recognize viral components and be important mediators of innate immune responses to various viral infections.57 In hepatitis C, the core and
Chapter 7 REPLICATION OF HEPATTIS B VIRUS AND PATHOGENESIS OF DISEASE
NS-3 proteins trigger Toll-like receptor 2 (TLR-2)-mediated pathways and inflammatory activation in vitro.58 In the peripheral blood monocytes of individuals chronically infected with HBV there is a substantial down-regulation of the TLR-2 receptor which is reversed during effective antiviral therapy.56 This latter finding indicates that viruses such as HBV have evolved powerful and effective mechanisms to suppress or block the innate immune response in order to establish productive replication and/or persistence. Further studies in this exciting area are clearly indicated. In HBV transgenic mice the production of IFN-a/b is associated with a 10-fold reduction of viral capsids containing HBV pregenomic RNA and the activation of double-stranded dependent protein kinase activity (PKR), which inhibits HBV protein synthesis.59 In addition to this, IFN-a/b recruits and mediates the activities of antigen-presenting cells (APC), in particular Kupffer cells and dendritic cells (DC). These APCs in turn produce interleukin-18 (IL18) and chemokine CCL3, which induces NK and NKT cell activity (Figure 7-3).60 NKT cells in HBV transgenic mice directly inhibit HBV replication via IFN-g.53,54,60 Injection of a-galactosylceramide, a ligand of CD1d which is used to stimulate NKT cells, resulted in the secretion of cytokines IFN-g and IFN-a/b and subsequent control and inhibition of HBV replication.53 Suppression of HBV was still detected even in T cell-depleted mice, suggesting that IFN-g production is not dependent on CD4+ and CD8+ T cell activity.53 The activity of NK and NKT cells is likely to be an important anti-HBV response that precedes the up-regulation of HLA class I expression on hepatocytes. Up-regulation of HLA class I expression is critical for the presentation and recognition of foreign antigen by T cells in the adaptive arm of the immune response.54 Therefore, the innate immune system probably controls a substantial burden of HBV replication in the early stages of infection prior to the detection of any hepatic inflammatory cell infiltrates or associated liver damage. Kupffer cells play a major role in mediating both early innate and adaptive immune responses. The activation of Kupffer cells via other viral infections, such as malaria, can also lead to enough cytokine production to effectively control and clear HBV. Following infection
of HBV transgenic mice with a liver-specific malaria strain, Kupffer cells produced cytokines that led to the reduction of both the malaria infection and the chronic HBV infection.61 Furthermore, Kupffer cells coordinate the recruitment and maturation of HBVspecific T cells via synthesis of several cytokines and chemokines, including IFN-g, CXCL9, and CXCL10.62 However, complete eradication and control of viral infection cannot be accomplished by the innate immune system alone: the adaptive immune response is needed for total clearance and protection from further HBV infection.
The Adaptive Immune Response Antigen-Presenting Cells (APC) APCs, namely Kupffer cells and in particular DCs, are important for the presentation and maturation of HBV-specific T cells, the main effectors of HBV clearance. APCs present foreign antigen to CD4+ and CD8+ T cells and produce cytokines, IL-12 and TNF-a, which induce IFN-g production and proliferation of CD8+ T cells. IL-12 also induces CD4+ T-cell differentiation into the T-helper cell type 1 (Th1) subset (Figure 7-4).60
CD4+ T Cells
Activated HBV-specific Th1 CD4+ T cells are multispecific, although strong responses against peptides c50–69, found in both HBcAg and HBeAg, are observed following resolution of acute HBV infection, regardless of the HLA phenotype of the infected individual. In acute HBV infection HBV-specific CD4+ T cells can be detected at the time of elevated HBV DNA (before the peak of liver damage) and persist long after recovery. Following the maturation of CD4+ T cells, communication between these Th1 cells and CD8+ T cells is needed for the activation of CD8+ T cell activity. This communication is also mediated by DCs, which are stimulated by the antigenspecific Th1 cells. The altered DCs can then present foreign antigens
Antigen presentation to both CD4+ and CD8+ T-cells DC
HBV
Hepatocyte
CD8+ T-cell
Kupffer cells
Cytolytic activity and production of IFN- and TFN-
IFN-
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NK cells
IL-18 CCL3
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Antigen presentation
CD4 differentiates into two subsets CD4+ T-cell
IL-12
IL-2 IFN- TNF- IFN- Th1 TNF- CD4
Hepatocyte
IL-10 IL-4 Adaptive immune responses
Cytokine and chemokine production Figure 7-3. Innate immune response to HBV. A number of these steps are hypothetical and/or derived from animal model studies, with confirmation required from clinical investigation.
Th2 CD4
IL-10 IL-4
B cell
HBeAb HBcAb HBsAb
Figure 7-4. The adaptive immune response to HBV. The complexity of the dynamic cellular interactions is explained in detail in the text.
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to the CD8+ T cells and also induce CD8+ T cell maturation into HBV-specific cytolytic T cells.
CD8+ T Cells
Mature CD8+ T cells are the main effector cells involved in HBV clearance. This was clearly shown by depletion of CD8+ T cells following acute HBV infection in chimpanzees.63 Depletion of CD8+ T cells led to persistence of HBV infection and demonstrated the importance of both cytolytic and non-cytolytic activity of HBVspecific CD8+ T cells.63 In humans, not all those who recover from acute HBV infection have elevated ALT levels or clinical symptoms, suggesting that non-lytic mechanisms such as those induced by IFNg and TNF-a are used to clear acute HBV infection.51 IFN-g is produced mainly by HBV-specific CD8+ T cells, but can also be produced by NK, NKT cells and HBV-specific Th1 CD4+ T cells.53,64 TNF-a and IFN-g clear HBV via several mechanisms, including destabilization of the viral capsid via the NF-kB pathway, degradation of viral proteins via nitric oxide and proteosome activity, and post-transcriptional degradation of HBV RNA.65–68 The importance of these cytokines was confirmed in studies of HBV transgenic mice, where administration of anti-IFN-g and anti-TNF-a antibodies obliterated the ability of CD8+ T cells to clear HBV RNA intermediates and nucleocapsid protein (HBcAg). In acute HBV infection the HBV-specific CD8+ T-cell response is polyclonal and multispecific to most HBV proteins, meaning that there are several receptors present for each epitope and that several epitopes are recognized by a single CD8+ T cell. These two factors respectively increase recognition of the target epitope and reduce viral ‘escape’ via mutation. Therefore, the presence of functional HBV-specific CD8+ T cells, which are maintained by Th1 CD4+ T cells and IL-12, may be more important than the quantity.69 These HBV-specific CD8+ responses have been assessed mainly in HLAA2-positive individuals in whom CD8+ T-cell responses most frequently target core (c18–27), envelope/surface (s183–191, s250–258, s335–343) and polymerase (p455–463, p575–583) epitopes with increasing frequency.70
B Cells The humoral response is also critical to long-term clearance of HBV and protection from infection with HBV. In individuals who recover from acute HBV infection, activated T-helper cell type 2 (Th2) CD4+ T cells induce B-cell production of anti-HBs, anti-HBc and anti-HBe. Anti-HBs antibodies are synthesized early in infection but are not detectable because they are complexed with the excess of envelope antigens produced during virus replication. Anti-HBs is important in providing protective immunity against subsequent HBV infections, and is the basis of protection in vaccinated individuals. The pathogenetic role of antibody to non-envelope protein remains controversial. It is generally accepted that anti-HBc does not have virus-neutralizing activity, although protection of chimpanzees against HBV infection by passive administration of antiHBc/anti-HBe antibodies has been observed, suggesting a possible but undefined role for anti-HBc.
OCCULT HBV INFECTION Both HBV-specific humoral and cellular responses persist after recovery from acute HBV infection. However, in some individuals
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who recover from acute HBV infection the long-lasting HBVspecific CD8+ T cell response is believed to be a combination of persistence of HBV-specific memory T cells as well as constant restimulation of CD8+ and CD4+ T cells by low amounts of HBV DNA. Persistent HBV at low levels may be considered an occult infection. This long-lasting presence of HBV DNA may be due to the stable nature of cccDNA. Evidence has also shown that HBV infection of immunologically privileged sites, although rare, does occur and may also contribute to occult HBV infection. Therefore, in the setting of immunodeficiency, such as advanced HIV infection or chemotherapy, relapse of HBV infection may occur even if the individual has previously cleared the virus and is anti-HBs positive.
CHRONIC HBV INFECTION The mechanism of HBV persistence is not fully understood but is probably multifactorial, including HBV-specific immune suppression, persistence of stable forms of HBV such as cccDNA, and/or infection of immunologically privileged sites.
HBV-Specific Immune Suppression In individuals with chronic HBV infection the HBV-specific CD4+ and CD8+ T-cell response is significantly diminished.70,71 In particular, in HBeAg-positive chronic carriers the core epitope (in region c18–27) specific CD8+ T cells are almost undetectable and have diminished ability to produce IFN-g. Some HBV-specific CD80+ T cells in persistent HBV infection have been described as ‘partially tolerant’, being unable to bind specific tetramers, to produce IFNg, to lyze, and to expand following stimulation.72 Circulating HBVspecific CD8+ T cells from individuals with chronic HBV infection and a high ALT and high HBV viral load also have decreased proliferative capacity compared to circulating HBV-specific CD8+ T cells from individuals with low ALT and viral loads.70 The reduction in HBV-specific T cells and reduction in IFN-g production is consistent with the responses observed in animal models of other persistent viral infections, such as lymphocyte choriomeningitis virus (LCMV), where there is consecutive elimination of TNF-a and IFNg-producing CD8+ T cells.73 HBV-specific CD8+ T cells are, however, found in the liver, where they may cause an inflammatory response but are ineffective in clearing HBV infection.70 Using HLA-A2-restricted epitopes and tetramers to evaluate HBV-specific T-cell responses, a comparison of CD8+ T-cell responses between HBV chronic carriers with and without an abnormal ALT showed no difference in the absolute quantity of HBV-specific CD8+ T cells in the liver. Individuals with chronic HBV infection and an elevated serum ALT had a larger infiltrate of non-specific CD8+ T cells than patients with a normal ALT.70 Therefore, control of HBV replication may occur by HBV-specific cytotoxic T lymphocytes without causing hepatocyte destruction. Indeed, the presence of these cells is associated with inhibition of viral replication in the absence of liver damage. A generalized CD4+ T-cell hyporesponsiveness in individuals with chronic HBV infection has also been demonstrated. This may be a consequence of impaired function of HBV-infected DCs, which have reduced IFN-g, TNF-a and IL-12 production.74 A reduced overall responsiveness of CD4+ T cells may also contribute to the
Chapter 7 REPLICATION OF HEPATTIS B VIRUS AND PATHOGENESIS OF DISEASE
lack of production of neutralizing antibodies to HBV in chronically infected individuals, as demonstrated in other chronic viral infections, such as that of LCMV.75 The effect of HBV infection on DC function is not well understood, but recent in vitro analysis of monocyte-derived DCs from individuals chronically infected with HBV showed that there is a reduction in the ability of DCs to prime T cells.74 Although there is no difference in absolute numbers of myeloid or plasmacytoid dendritic cells in individuals with HBV infection compared to uninfected controls, significant functional defects were recently demonstrated in both subsets of DCs.76 The mechanism of how HBV impairs dendritic cell function is not currently understood.
Viral Factors Leading to HBV Persistence HBx Protein HBx can modify several cellular pathways, including NF-kB, and this may subsequently affect immune response and antigen presentation.77 HBx protein trans-activates HLA I molecules on hepatocytes, which are normally expressed at low levels. Although an increase in HLA I expression on hepatocytes may assist recognition of foreign antigen by T cells, in the case of HBV it could also recruit T cells to the liver and hence lead to liver damage. Furthermore, in chronic HBV carriers CD8+ T cells have a reduced capacity to downregulate HBx post-transcriptional mRNAs. Therefore, HBx may contribute to the ongoing expression of HLA I on hepatocytes and the recruitment to the liver of inflammatory cells that lack effective antiviral activity.
HBe Antigen (HBeAg) HBeAg acts both as an immunogen and a tolerogen. HBeAg (precore protein) and HBcAg (core protein) share an overlapping reading frame. HBeAg has a leading peptide sequence and a different conformational structure.25 Although HBcAg is needed for the formation of viral capsid and replication, HBeAg is not necessary for infection or replication of HBV.25 However, HBeAg has been conserved in all hepadnaviruses and is believed to be important in the persistence of HBV infection. HBeAg may play a role in immune regulation by depletion of HBeAg- and HBcAg-specific Th1 CD4+ T cells via Fas-mediated apoptosis. This is thought to occur in the setting of perinatal transmission, where HBeAg crosses the placenta from the infected mother and establishes tolerance to HBV in vitro. The imbalance of Th1/Th2 responses leads to the production of anti-inflammatory cytokines such as IL-4 and IL-10, and promotes suppression of HBeAg/HBcAg-specific CD8+ T cell responses and Th1 effector cells. This ‘suppressive’ cytokine profile is reversed when patients chronically infected with HBV seroconvert from HBeAg to anti-HBe, leading to an increase in IL-12 and IFN-g (a Th1 cytokine profile) that may enhance CD8+ T cell function. In keeping with this hypothesis, perinatal transmission from a mother who has persistent HBV infection with an HBeAg-negative mutant (such as HBV with a mutation in the basal core promoter or precore stop codon) is more likely to lead to an acute rather than a persistent HBV infection in the infant. Although infection with an HBeAgnegative mutant is associated with a reduced likelihood of chronic HBV infection, there are conflicting reports demonstrating that HBeAg-negative HBV chronic infection can lead to worse liver
injury and a poorer long-term prognosis,78 but this is always in the setting of previous HBeAg-positive chronic hepatitis B.24 Infection with HBeAg-negative strains has been associated with cases of severe acute hepatitis or fulminant hepatitis.79 The increase in liver injury may be due to the lack of the Th2 ‘skew’ observed in HBeAgpositive HBV chronic infections, which would lead to increased Th1 CD8+ T cell activation.
Other Mechanisms Other possible mechanisms leading to HBV persistence include the replication of HBV in ‘privileged’ locations, such as extrahepatic sites, but why this should occur in some individuals and not others is unclear. Alternatively, induction of Fas-L on hepatocytes could delete HBV-specific CTL more efficiently. Virus-specific CTL might be inactivated if antigen is presented in the absence of co-stimulatory signals in the liver, a mechanism that would specifically allow for T-cell tolerance.80 Finally, viral mutations that abrogate or antagonize antigen recognition of HBV have been reported. Therefore, selection for CTL escape may occur, either during acute infection or once persistent infection is already established.
MECHANISMS OF HBV DISEASE Abnormal Liver Function or ‘Flares’ Levels of HBV viremia in persistent infection are substantially lower than in primary infection, although they vary from person to person. Higher titers of HBV DNA are often indicated by the continued presence of HBeAg. With the passage of time there is a decrease in HBV DNA titer and a tendency for HBeAg to disappear along with the development of anti-HBe antibodies. Seroconversion from HBeAg to anti HBe occurs at a rate of 5–10% per year.81 Often, the disappearance of HBeAg is preceded or accompanied by a transient rise in ALT, known as a flare. A significant reduction of HBV DNA may accompany seroconversion to anti-HBe antibodies.82 These acute exacerbations can be mild with no detectable symptoms, or lead to severe disease, including hepatic decompensation and failure. It is believed that hepatic flares are an attempt by the immune response to clear the virus, and flares occur more commonly in HBeAg-positive individuals.25 However, hepatic flares are also common in the phase of HBeAg-negative chronic HBV.24 In HBeAgpositive HBV, it has been suggested that these responses are triggered by the increasing concentrations of serum HBeAg and intracellular HBcAg. High antigen concentrations would be required for the induction of a T-cell response because of the low avidity of HBcAg and HBeAg-specific T cells in patients with chronic HBV infection.83 Hepatitis flares can also be followed by a significant rise in IL-12 levels that can precede or occur simultaneously with HBeAg seroconversion. The immunopathogenesis of hepatic flares in HBeAg-negative chronic HBV is unknown. Liver biopsies taken during a hepatic flare show infiltration of the lobules with both HBV-specific and non-specific T cells. The recruitment of non-specific inflammatory cells, including macrophages, T cells and neutrophils, may allow for control of HBV viral load before an increase in ALT is detected.51,52 It is believed that IFN-g, needed for recruitment of HBV-specific T cells and also for non-cytopathic clearance of HBV, is also responsible for increasing the susceptiblity of hepatocytes to TNF-a-induced apoptosis and mediating
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macrophage recruitment of more necroinflammatory cells. IFN-ginducible chemokines, including macrophage inflammatory protein 1a (MIP-1a), MIP-1b and RANTES (Regulated on Activation, Normal T Expressed and Secreted), are up-regulated and together with CXCL9 and CXCL10 bind chemokine co-receptor CCR5, which activates lymphocytes regardless of their specificity. Interestingly, in transgenic mice the administration of antibodies against CXCL9 and CXCL10 can lead to reduced intrahepatic inflammation without a reduction in the non-cytopathic effects of IFN-g.62
Chronic Liver Disease, Cirrhosis and Fibrosis Repeated flares or continuous recruitment of inflammatory cells to the liver ultimately results in fibrosis, cirrhosis, and eventually hepatocellular carcinoma (HCC).84,85 Inflammatory cells recruited to the liver cause cell death and hence liver injury through several different mechanisms. Apoptosis of hepatocytes is largely induced by CD8+ T cells via several pathways, including TNF-a.86 Although TNF-a has a non-cytopathic antiviral effect, it can also cause apoptosis via TNF receptor 1 (TNF-R1) and TNF-related apoptosisinducing ligand (TRAIL). In vitro transfection of Hep G2 and Chang cell lines leads to an increase in the susceptibility of hepatocyte cell lines to apoptosis via TRAIL. HBx protein mediates TNF-a-induced apoptosis by increasing the expression of TRAIL receptors on hepatocytes by up to fourfold.87 During HBV infection Fas expression is also up-regulated on hepatocytes.88 Interaction between Fas ligand (FasL; expressed on CD8+ T cells) and Fas (expressed on hepatocytes) also leads to cell apoptosis. Fas-mediated cell death is believed to also play an important role in the severe acute hepatitis observed in fulminant hepatitis where expression of FasL is elevated.89 Finally, the perforin/granzyme pathway has been shown to also mediate hepatocyte apoptosis in HBV infection. Measurement of perforin and granzyme mRNA shows a correlation with histologic activity index and an increase in serum ALT.88 The combined effects of these various apoptosis-inducing mechanisms results in long-term liver damage. Hepatic stellate cells (HSC) are perisinusoidal mesenchymal cells that are activated during liver injury through controlled pathways.84 Activated HSC release extracellular matrix that contains mainly type I collagen, leading to fibrosis. Following liver injury HSC can also phagocytose residual dead cells, and this further induces the release of type I collagen.85 HSCs are themselves protected against apoptosis. The establishment of fibrosis in individuals chronically infected with HBV is largely non-reversible. Individuals with chronic HBV infection have a 200-fold increased risk in HCC compared to HBV-uninfected individuals. HBx protein is thought to have an important role in the pathogenesis of HCC. HBx protein has a high frequency of overexpression in HCC tissue.90,91 HBx protein alters signal transduction pathways and is therefore thought to induce HCC by altering oncogene expression, or by sensitizing HBV-infected cells to other carcinogens.92 Analysis of HBx sequences in HCC show an association with HCC and truncation of the C-terminus of the HBx protein.93 This truncated HBx protein is less effective in inhibiting cellular proliferation, and at the same time suppresses full-length HBx protein activity, allowing for increased proliferation of cells and the development of HCC.94 Likewise, mutations in envelope proteins of HBV have also been shown to be overexpressed in tissue from patients with HCC. Deletions in
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the PreS1 and PreS2 regions of surface protein lead to the accumulation of truncated surface antigens in the endoplasmic reticulum. Long-term accumulation of the truncated surface antigens leads to oxidative stress, which can cause mutations in hepatocyte DNA and promote HCC formation.95
ANTI-HBV THERAPY AND VIRAL CLEARANCE A reduction in HBV viral load in natural infection appears to precede the detection of HBV-specific T-cell responses in both self-limiting acute infections and flares associated with HBeAg seroconversion in chronic HBV infection. Therefore, a reduction of HBV viral load by antiviral chemotherapy could potentially reduce ‘anergy’ associated with persistent HBV infection. Treatment of HBV-infected patients includes the use of IFN-a and reverse transcriptase inhibitors such as nucleoside analogs (lamivudine; 2¢-deoxy-3¢-thiacytidine; 3TC), and nucleotide analogs (tenofovir [9-(R)-(2-phosphonylmethoxypropyl)adenine] and adefovir [9-(2-phosphonylmethoxyethyl)adenine]). Interferon-a was the first drug to be licensed to treat chronic HBV infection and induce HBeAg and HBsAg seroconversion. Following interferon-a treatment, roughly 35–40% of individuals have a reduction in HBV viral load, and in 8–10% of cases HBsAg seroconversion and viral clearance occurs. Unlike reverse transcriptase inhibitors, IFN-a is capable of mediating immunomodulatory effects that lead to HBV suppression by the immune response. Examination of IFN-a efficacy in perinatally acquired HBV infection has demonstrated mixed results, with some studies showing reduced efficacy and others showing similar seroconversion rates as in Caucasian patients who did not acquire the disease through perinatal transmission. More recently the use of pegylated forms of interferon have shown promising results, with HBeAg seroconversion rates of ~29–37% following 48 weeks of therapy.96 In HBeAg-negative chronic HBV the use of peginterferon-a2a, either alone or in combination with lamivudine, leads to better virological and immunological outcome than the use of lamivudine alone.97 A number of nucleoside analogs have been developed for the treatment of HBV. Lamivudine is most commonly used and has been associated with an increase in HBV-specific CD4+ and CD8+ T-cell responses in both HBeAg-positive and -negative individuals.31,98,99 Lamivudine can successfully suppress HBV viral load, and an increase in functional HBV-specific CD4+ and CD8+ T cells has been detected within the first few months of treatment.99 These HBVspecific T cells can proliferate and produce IFN-g in response to HBV antigen stimulation. Detection of HBV-specific T cells was not associated with HBeAg seroconversion or viral clearance, and appears to be transient.99 Long-term lamivudine therapy (more than 100 weeks) results in HBeAg seroconversion in 27% of individuals. Withdrawal of lamivudine, even in individuals who have HBeAg seroconverted, can lead to a relapse of HBV DNA replication. Interestingly, in individuals infected with both HIV and HBV who are receiving HBV-active therapy, such as lamivudine or tenofovir, HBVspecific CD8+ T cells are detected, but HBV-specific CD4+ T cells are not reconstituted.71 Therefore, it currently appears that antiviral therapy such as lamivudine or adefovir will not cure HBV infection but will provide long-term suppression of HBV replication until drug resistance emerges. The role of a therapeutic vaccine for the treatment of HBV infection remains an area to be further explored.
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41. Bruss V, Thomssen R. Mapping a region of the large envelope protein required for hepatitis B virion maturation. J Virol 1994;68:1643–1650. 42. Loffler-Mary H, Werr M, Prange R. Sequence-specific repression of cotranslational translocation of the hepatitis B virus envelope proteins coincides with binding of heat shock protein Hsc70. Virology 1997;235:144–152. 43. Bruss V, Ganem D. The role of envelope proteins in hepatitis B virus assembly. Proc Natl Acad Sci USA 1991;88:1059–1063. 44. Bruss V. A short linear sequence in the pre-S domain of the large hepatitis B virus envelope protein required for virion formation. J Virol 1997;71:9350–9357. 45. Loffler-Mary H, Dumortier J, Klentsch-Zimmer C, Prange R. Hepatitis B virus assembly is sensitive to changes in the cytosolic S loop of the envelope proteins. Virology 2000;270: 358–367. 46. Chen PJ, Chen CR, Sung JL, Chen DS. Identification of a doubly spliced viral transcript joining the separated domains for putative protease and reverse transcriptase of hepatitis B virus. J Virol 1989;63:4165–4171. 47. Su TS, Lui WY, Lin LH, et al. Analysis of hepatitis B virus transcripts in infected human livers. Hepatology 1989;9:180–185. 48. Soussan P, Garreau F, Zylberberg H, et al. In vivo expression of a new hepatitis B virus protein encoded by a spliced RNA. J Clin Invest 2000;105:55–60. 49. Soussan P, Tuveri R, Nalpas B, et al. The expression of hepatitis B spliced protein (HBSP) encoded by a spliced hepatitis B virus RNA is associated with viral replication and liver fibrosis. J Hepatol 2003;38:343–348. 50. Rosmorduc O, Petit MA, Pol S, et al. In vivo and in vitro expression of defective hepatitis B virus particles generated by spliced hepatitis B virus RNA. Hepatology 1995;22:10–19. 51. Webster GJ, Reignat S, Maini MK, et al. Incubation phase of acute hepatitis B in man: dynamic of cellular immune mechanisms. Hepatology 2000;32:1117–1124. 52. Tang TJ, Kwekkeboom J, Laman JD, et al. The role of intrahepatic immune effector cells in inflammatory liver injury and viral control during chronic hepatitis B infection. J Viral Hepatol 2003;10:159–167. 53. Kakimi K, Guidotti LG, Koezuka Y, Chisari FV. Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J Exp Med 2000;192:921–930. 54. Kimura K, Kakimi K, Wieland S, et al. Interleukin-18 inhibits hepatitis B virus replication in the livers of transgenic mice. J Virol 2002;76:10702–10707. 55. Raulet DH. Interplay of natural killer cells and their receptors with the adaptive immune response. Nature Immunol 2004;5:996–1002. 56. Visvanathan K, Skinner N, Locarnini S, et al. Impaired toll-like receptor expression in chronic hepatitis B. Gut 2003;52:130. 57. Boehme KW, Compton T. Innate sensing of viruses by toll-like receptors. J Virol 2004;78:7867–7873. 58. Dolganiuc A, Oak S, Kodys K, et al. Hepatitis C core and nonstructural 3 proteins trigger toll-like receptor 2-mediated pathways and inflammatory activation. Gastroenterology 2004;127:1513–1524. 59. Wieland SF, Guidotti LG, Chisari FV. Intrahepatic induction of alpha/beta interferon eliminates viral RNA-containing capsids in hepatitis B virus transgenic mice. J Virol 2000;74:4165–4173. 60. Kimura K, Kakimi K, Wieland S, et al. Activated intrahepatic antigen-presenting cells inhibit hepatitis B virus replication in the liver of transgenic mice. J Immunol 2002;169:5188–5195. 61. Pasquetto V, Guidotti LG, Kakimi K, et al. Host–virus interactions during malaria infection in hepatitis B virus transgenic mice. J Exp Med 2000;192:529–536. 62. Kakimi K, Lane TE, Chisari FV, Guidotti LG. Cutting edge: Inhibition of hepatitis B virus replication by activated NK T cells does not require inflammatory cell recruitment to the liver. J Immunol 2001;167:6701–6705.
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63. Thimme R, Wieland S, Steiger C, et al. CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection. J Virol 2003;77:68–76. 64. Szkaradkiewicz A, Jopek A, Wysocki J, et al. HBcAg-specific cytokine production by CD4 T lymphocytes of children with acute and chronic hepatitis B. Virus Res 2003;97:127–133. 65. Biermer M, Puro R, Schneider RJ. Tumor necrosis factor alpha inhibition of hepatitis B virus replication involves disruption of capsid integrity through activation of NF-kappaB. J Virol 2003;77:4033–4042. 66. Guidotti LG, McClary H, Loudis JM, Chisari FV. Nitric oxide inhibits hepatitis B virus replication in the livers of transgenic mice. J Exp Med 2000;191:1247–1252. 67. Robek MD, Wieland SF, Chisari FV. Inhibition of hepatitis B virus replication by interferon requires proteasome activity. J Virol 2002;76:3570–3574. 68. Guidotti LG, Morris A, Mendez H, et al. Interferon-regulated pathways that control hepatitis B virus replication in transgenic mice. J Virol 2002;76:2617–2621. 69. Maini MK, Reignat S, Boni C, et al. T cell receptor usage of virus-specific CD8 cells and recognition of viral mutations during acute and persistent hepatitis B virus infection. Eur J Immunol 2000;30:3067–3078. 70. Maini M, Boni C, Lee C, et al. The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection. J Exp Med 2000;191:1269. 71. Chang J, Wightman F, Bartholomeusz A, et al. Reduced hepatitis B virus (UBV)-specific CD4+ T cell responses in human immunodeficiency virus type 1-HBV coinfected individuals receiving HBV-active antiretroviral therapy. J Virol 2005;79:3038–3051. 72. Reignat S, Webster GJ, Brown D, et al. Escaping high viral load exhaustion: CD8 cells with altered tetramer binding in chronic hepatitis B virus infection. J Exp Med 2002;195:1089–1101. 73. Wherry EJ, Blattman JN, Murali-Krishna K, et al. Viral persistence alters CD8 T cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J Virol 2003;77:4911–4927. 74. Beckebaum S, Cicinnati V, Zhang X, et al. Hepatitis B virusinduced defect of monocyte-derived dendritic cells leads to impaired T helper type 1 response in vitro: mechanisms for viral immune escape. Immunology 2003;109:487–495. 75. Ciurea A, Hunziker L, Klenerman P, et al. Impairment of CD4(+) T cell responses during chronic virus infection prevents neutralizing antibody responses against virus escape mutants. J Exp Med 2001;193:297–305. 76. van der Molen RG, Sprengers D, Binda RS, et al. Functional impairment of myeloid and plasmacytoid dendritic cells of patients with chronic hepatitis B. Hepatology 2004;40:738–746. 77. Murakami S. Hepatitis B virus X protein: a multifunctional viral regulator. J Gastroenterol 2001;36:651–660. 78. Funk ML, Rosenberg DM, Lok AS. World-wide epidemiology of HBeAg-negative chronic hepatitis B and associated precore and core promoter variants. J Viral Hepatol 2002;9:52–61. 79. Bartholomeusz A, Locarnini S. Hepatitis B virus mutants and fulminant hepatitis B: fitness plus phenotype. Hepatology 2001;34:432–435. 80. Limmer A, Ohl J, Kurts C, et al. Efficient presentation of exogenous antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T cell tolerance. Nature Med 2000;6:1348–1354. 81. Ribeiro RM, Lo A, Perelson AS. Dynamics of hepatitis B virus infection. Microbes Infect 2002;4:829–835. 82. Tedder RS, Ijaz S, Gilbert N, et al. Evidence for a dynamic host–parasite relationship in e-negative hepatitis B carriers. J Med Virol 2002;68:505–512. 83. Chen M, Sallberg M, Thung SN, et al. Nondeletional T cell receptor transgenic mice: model for the CD4(+) T cell
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84.
85.
86.
87.
88.
89.
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repertoire in chronic hepatitis B virus infection. J Virol 2000;74:7587–7599. Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem 2000;275:2247–2250. Canbay A, Taimr P, Torok N, et al. Apoptotic body engulfment by a human stellate cell line is profibrogenic. Lab Invest 2003;83:655–663. Jo M, Kim TH, Seol DW, et al. Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosisinducing ligand. Nature Med 2000;6:564–567. Janssen HL, Higuchi H, Abdulkarim A, Gores GJ. Hepatitis B virus enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity by increasing TRAIL-R1/death receptor 4 expression. J Hepatol 2003;39:414–420. Lee JY, Chae DW, Kim SM, et al. Expression of FasL and perforin/granzyme B mRNA in chronic hepatitis B virus infection. J Viral Hepatol 2004;11:130–135. Rivero M, Crespo J, Fabrega E, et al. Apoptosis mediated by the Fas system in fulminant hepatitis by hepatitis B virus. J Viral Hepatol 2002;9:107–113. Wang XZ, Chen XC, Chen YX, et al. Overexpression of HBxAg in hepatocellular carcinoma and its relationship with Fas/FasL system. World J Gastroenterol 2003;9:2671–2675. Hwang GY, Lin CY, Huang LM, et al. Detection of the hepatitis B virus X protein (HBx) antigen and anti-HBx antibodies in cases of human hepatocellular carcinoma. J Clin Microbiol 2003;41:5598–5603.
92. Tralhao JG, Roudier J, Morosan S, et al. Paracrine in vivo inhibitory effects of hepatitis B virus X protein (HBx) on liver cell proliferation: an alternative mechanism of HBx-related pathogenesis. Proc Natl Acad Sci USA 2002;99:6991–6996. 93. Iavarone M, Trabut JB, Delpuech O, et al. Characterisation of hepatitis B virus X protein mutants in tumour and non-tumour liver cells using laser capture microdissection. J Hepatol 2003;39:253–261. 94. Tu H, Bonura C, Giannini C, et al. Biological impact of natural COOH-terminal deletions of hepatitis B virus X protein in hepatocellular carcinoma tissues. Cancer Res 2001;61:7803–7810. 95. Hsieh YH, Su IJ, Wang HC, et al. Pre-S mutant surface antigens in chronic hepatitis B virus infection induce oxidative stress and DNA damage. Carcinogenesis 2004;25:2023–2032. 96. Cooksley WG, Piratvisuth T, Lee SD, et al. Peginterferon alpha-2a (40 kDa): an advance in the treatment of hepatitis B e antigen-positive chronic hepatitis B. J Viral Hepatol 2003;10:298–305. 97. Marcellin P, Lau GK, Bonino F, et al. Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B. N Engl J Med 2004;351:1206–1217. 98. Malacarne F, Webster GJ, Reignat S, et al. Tracking the source of the hepatitis B virus-specific CD8 T cells during lamivudine treatment. J Infect Dis 2003;187:679–682. 99. Boni C, Penna A, Bertoletti A, et al. Transient restoration of antiviral T cell responses induced by lamivudine therapy in chronic hepatitis B. J Hepatol 2003;39:595–605.
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REPLICATION AND PATHOGENESIS OF HEPATITIS C VIRUS
8
Darius Moradpour and Charles M. Rice Abbreviations ARF alternative reading frame ARFP alternative reading frame protein BVDV bovine viral diarrhea virus cDNA complementary DNA CRE cis-acting replication element CsCl cesium chloride DC-SIGN dendritic cell-specific intercellular adhesion molecule-3-grabbing integrin ER endoplasmic reticulum F frameshift protein GBV GB virus HCC hepatocellular carcinoma
HCV HCVDB HGV hVAP-A HVR IRES ISDR LDL LDLR L-SIGN
hepatitis C virus Hepatitis C Virus DataBase hepatitis G virus human vesicle-associated membrane protein-associated protein A hypervariable region internal ribosome entry site interferon sensitivity-determining region low-density lipoprotein low-density lipoprotein receptor liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin
NCR NK NS PEG-IFN-a RdRp SR-BI TBE VLDL VSV YFV
non-coding region natural killer non-structural pegylated interferon-a RNA-dependent RNA polymerase scavenger receptor class B type I tickborne encephalitis very low-density lipoprotein vesicular stomatitis virus yellow fever virus
INTRODUCTION
TAXONOMY
Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) worldwide.1 A protective vaccine is not available and therapeutic options are limited. Current standard therapy, pegylated interferon-a (PEGIFN-a) combined with ribavirin, results in a sustained virologic response in 20–80% of patients, depending on the HCV genotype and other factors.2–5 However, in clinical practice many patients do not qualify for or do not tolerate IFN-based therapy.6 As a consequence, the number of patients presenting with long-term sequelae of chronic hepatitis C, including HCC, is expected to further increase over the next 20–30 years.7 Thus, there is an urgent need to develop more effective and better-tolerated therapies for chronic hepatitis C. A detailed understanding of the molecular virology of hepatitis C underpins these efforts. HCV was identified in 1989 as the most common etiologic agent of post-transfusion and sporadic non-A, non-B hepatitis by the use of recombinant DNA technology.8 Investigation of the viral life cycle has been limited by the low viral titers found in the sera and livers of infected individuals and the lack of efficient cell culture systems or small animal models permissive for HCV. Nevertheless, considerable progress has been made using heterologous expression systems,9,10 functional cDNA clones,11 the replicon system,12,13 functional HCV pseudoparticles,14,15 and most recently, recombinant infections HCV produced in vitro181–183 (see 16–19 for recent reviews). These and other milestones in HCV research are listed in Table 8-1.
HCV has been classified in the Hepacivirus genus within the family Flaviviridae, which includes the classic flaviviruses, such as yellow fever (YFV) and dengue viruses, the animal pestiviruses, such as bovine viral diarrhea virus (BVDV), and the as yet unassigned GB viruses A (GBV-A), GBV-B and GBV-C20 (Figure 8-1). GBV-C was also designated hepatitis G virus (HGV). However, it was subsequently found that GBV-C/HGV is not a common agent of viral hepatitis and its pathogenic relevance, if any, remains unknown. An important feature of HCV is its high genetic variability.21 HCV isolates fall into three major categories, depending on the degree of sequence divergence: genotypes, subtypes, and isolates. There are six major genotypes (also called ‘clades’) that differ in their nucleotide sequence by 30–35%. Within an HCV genotype, several subtypes (designated a, b, c etc.) can be defined that differ in their nucleotide sequence by 20–25%. The term quasispecies refers to the genetic heterogeneity of the population of HCV genomes coexisting in an infected individual. The genetic variability of HCV may have important implications for the pathogenesis, natural course and prevention of hepatitis C. The E1 and E2 glycoprotein regions are particularly variable, whereas the core and some of the non-structural protein sequences are more conserved. The highest degree of sequence conservation is found in the 5¢ and 3¢ non-coding regions (NCR). In the United States and western Europe, genotypes 1a and 1b are the most frequent, followed by genotypes 2 and 3. In Europe, genotype 3 is distributed widely among injection drug users. In
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Pestivirus BVDV
CSFV
GBV-B GBV-C Flavivirus
3a
YFV HCV
1b 2a 1a
JEV
DENV
Hepacivirus
Identification of HCV Polyprotein processing Three-dimensional structure of the NS3 serine protease Infectious clone of HCV Replicon system HCV pseudoparticles Proof-of-concept clinical studies of an HCV protease inhibitor Recombinant infectious HCV
southern and eastern Europe, genotype 1b is most frequent. In Japan, China, and Taiwan genotypes 1b and 2 are predominant. Genotype 4 is found primarily in Egypt, North and Central Africa, and the Middle East. It is typically associated with past medical treatment, e.g. parenteral treatment for schistosomiasis. Genotype 5 is found commonly only in South Africa and genotype 6 is found among intravenous drug users in Hong Kong, Vietnam and, more recently, Australia. Patients infected with genotype 1 have a poorer response to IFNa therapy than those infected with genotype 2 or 3. However, the clinical significance of HCV genotypes with respect to the natural history of hepatitis C is controversial. Close to 20 000 HCV sequences, including nearly 200 full-length genomes, have so far been deposited in generic databanks such as GenBank, the EMBL Nucleotide Sequence Database or the DNA Data Bank of Japan (DDBJ). A number of sequence databases are dedicated specifically to HCV, including the Hepatitis C Virus DataBase (HCVDB) of the French Réseau National Hépatites (http://hepatitis.ibcp.fr), the Los Alamos Hepatitis C Virus Databases (http://hcv.lanl.gov), and the Japanese Hepatitis Virus Database (http://s2as02.genes.nig.ac.jp). These offer a number of specialized features as well as useful links for HCV sequence analysis, structure predictions, CD4+ and CD8+ T-cell epitope compilations etc.
GENETIC ORGANIZATION HCV contains a 9.6 kb positive-strand RNA genome composed of a 5¢ NCR, a long open reading frame encoding a polyprotein precursor of about 3000 amino acids, and a 3¢ NCR (Figure 8-2).
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GBV-A
2b
Table 8-1. Milestones in HCV Research 1989 1993 1996 1997 1999 2003 2003 2005
Figure 8-1. Simplified phylogenetic tree of the Flaviviridae family. The Flaviviridae comprise the genera Flavivirus, Pestivirus and Hepacivirus as well as the as yet unassigned GB viruses A (GBV-A), GBVB and GBV-C. Only few examples of flavi- and pestiviruses, as well as few HCV genotypes and subtypes, are shown. YFV, yellow fever virus; JEV, Japanese encephalitis virus; DENV, Dengue virus; BVDV, bovine viral diarrhea virus; CSFV, classical swine fever virus.
It took 8 years from the discovery of HCV to establish the first infectious cDNA clone,11 because in the absence of a robust tissue culture system the only read-out for infectivity was the direct inoculation of in vitro transcribed, synthetic RNA into the liver of a chimpanzee. In addition, owing to the variation present in the quasispecies and errors introduced by PCR (polymerase chain reaction) amplification, construction of infectious cDNA clones required the preparation of a consensus sequence from a number of clones. Functional cDNA clones now exist for genotypes 1a,11,22–24 1b,25 and 2a.26 Genetic studies using infectious clones have shown the essential nature of the HCV enzymes, the conserved elements of the 3¢ NCR and the difficult-to-study proteins such as p7.27,28
THE 5¢ AND 3¢ NON-CODING REGIONS The 5¢ NCR is highly conserved among different HCV isolates and contains an internal ribosome entry site (IRES) essential for capindependent translation of the viral RNA.29,30 Because the vast majority of cellular mRNAs are translated by a cap-dependent mechanism, the HCV IRES represents an attractive antiviral target. The 5¢ NCR contains four highly ordered domains, numbered I–IV. Domain I is not required for IRES activity, but is essential for HCV RNA replication.31 Domains II and III include two large stemloops. Subdomain IIIf forms a pseudoknot with domain IV, which contains the translation initiation codon. Domains II, III and IV, together with the first 24–40 nucleotides of the core coding region, constitute the IRES. The key element is domain III, which permits direct binding of the 40 S ribosomal subunit to subdomains IIIa, IIIc, IIId and IIIe, as well as of eukaryotic translation initiation factor 3 (eIF3) to subdomain IIIb. The three-dimensional structure of the HCV IRES bound to the 40 S ribosomal subunit was resolved at 20 Å resolution by cryoelectron microscopy.32 Strikingly, it was found that IRES binding induces a significant conformational change in the 40 S subunit, indicating that the HCV IRES dynamically modulates the host translational machinery. In addition, high-resolution structural information is now available for critical elements of the IRES, including stem loops II, IIIb, IIId and IIIe, as well as the IIIabc four-way junction, facilitating the design of small molecule inhibitors of HCV translation initiation.33–36 A current model of HCV translation initiation includes the formation of a binary complex between the IRES and the 40 S ribosomal subunit, followed by assembly of a 48 S-like complex at the
Chapter 8 REPLICATION AND PATHOGENESIS OF HEPATITIS C VIRUS
5´ NCR
5B-SL3 5B-SL3.2
III
b II a
c e
d
(U)n
9.6 kb
f
I
3´ NCR
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IRES-mediated translation
C
E1
E2
NS2
NS3
A NS4 B
A
NS5
B
Polyprotein processing 192
1 C Core
E1
384
747 810 E2
* * * * * * * * Envelope glycoproteins
p7
NS2 Protease
1658 1712
1027 NS3
A
NS4B
Serine Helicase Serine Membr. web protease protease cofactor
3011
2421
1973 NS5A
NS5B
?
RNA-dependent RNA polymerase
Figure 8-2. Genetic organization and polyprotein processing of HCV. The 9.6 kb positive-strand RNA genome is schematically depicted at the top. Simplified RNA secondary structures in the 5¢ and 3¢ non-coding regions (NCRs) as well as in the NS5B stem-loop 3 cis-acting replication element (5B-SL3) are shown. Internal ribosome entry site (IRES)-mediated translation yields a polyprotein precursor of about 3000 amino acids that is processed into the mature structural and non-structural proteins. Amino acid positions are shown above each protein (HCV H strain; genotype 1a; GenBank accession number AF009606). Solid diamonds denote cleavage sites of the HCV polyprotein precursor by the endoplasmic reticulum signal peptidase. The open diamond indicates further C-terminal processing of the core protein by signal peptide peptidase. Arrows indicate cleavages by the HCV NS2-3 and NS3 proteases. Asterisks in the E1 and E2 region indicate glycosylation of the envelope proteins. Note that polyprotein processing, illustrated here as a separate step for simplicity, occurs both co- and posttranslationally.
AUG initiation codon upon association of eIF3 and ternary complex (eIF2•Met-tRNAiMet•GTP) and, in a rate limiting step, GTPdependent association of the 60 S subunit to form the 80 S complex.37 The 3¢ NCR is composed of a short variable region, a poly(U/UC) tract with an average length of 80 nucleotides, and an almost invariant 98 nucleotide RNA element, designated the X-tail.27,38–43
CIS-ACTING REPLICATION ELEMENTS Promoter elements regulating the replication of positive-strand viral RNAs, called cis-acting replication elements (CRE), are often found at or near the 5¢ and 3¢ termini of genome RNA (or its complement). Although only the 5¢-terminal 125 bases including domain I are absolutely required, downstream sequences that include the entire 5¢ NCR dramatically enhance the efficiency of HCV RNA replication.31,44 The conserved elements in the 3¢ NCR, including a minimal poly(U) tract of about 25 bases, are also essential for replication both in cell culture41–43 and in vivo.27,40 Besides the 5¢ and 3¢ NCRs, conserved RNA structures have been predicted within the HCV open reading frame.45,46 A new CRE was recently confirmed in the sequence encoding the C-terminal domain of non-structural protein 5B (NS5B).47 An essential stem-loop, designated 5B-SL3.2, was identified within a larger cruciform RNA
element designated 5B-SL3 (Figure 8-2). More recently, it was shown that the upper loop of 5B-SL3.2 is engaged in a kissing interaction with a stem-loop in the X-tail, suggesting that a pseudoknot structure is formed at the 3¢ end of the HCV genome that is essential for RNA replication.48
POLYPROTEIN PROCESSING IRES-mediated translation of the HCV open reading frame yields a polyprotein precursor that is co- and post-translationally processed by cellular and viral proteases into the mature structural and nonstructural proteins (Figure 8-2). The structural proteins include the core protein and the envelope glycoproteins E1 and E2. These are released from the polyprotein precursor by the endoplasmic reticulum (ER) signal peptidase. The structural proteins are separated from the non-structural proteins by the p7 polypeptide. The nonstructural proteins include the NS2-3 protease and the NS3 serine protease, an RNA helicase/NTPase located in the C-terminal twothirds of NS3, the NS4A polypeptide, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase (RdRp). The NS2-3 protease cleaves at the NS2/NS3 site, whereas the NS3 serine protease is responsible for processing of the downstream nonstructural proteins (Figure 8-2). These are cleaved in a preferential
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order, as shown in heterologous expression systems and in HuH-7 cells harboring HCV replicons (see below).10,49 The first cleavage occurs co-translationally and liberates NS3 from the remainder of the polyprotein. Subsequent processing events can be mediated in trans, with rapid processing at the NS5A/NS5B site. The resulting NS4A-5A precursor is cleaved first between NS4A and NS4B, resulting in a relatively stable NS4B-5A intermediate, and subsequently between NS4B and NS5A.
STRUCTURAL PROTEINS CORE The first structural protein encoded by the HCV open reading frame is the core protein, which presumably forms the viral nucleocapsid. During translation of the HCV polyprotein, the nascent polypeptide is targeted to the ER membrane for translocation of the E1 ectodomain into the ER lumen, a process mediated by an internal signal sequence located between the core and E1 sequences. Cleavage of the signal sequence by signal peptidase yields an immature 191-amino-acid core protein, which contains the E1 signal peptide at its C terminus. This signal peptide is further processed by signal peptide peptidase (SPP), yielding the mature 21 kDa core protein of approximately 179 amino acids.50 The N-terminal hydrophilic domain of core contains a high proportion of basic amino acid residues and has been implicated both in RNA binding and homo-oligomerization. When expressed in mammalian cells, core is found on membranes of the ER, in seemingly ER-derived membranous webs (see below), and on the surface of lipid droplets.51–54 It is at present unclear whether the association with lipid droplets, which is mediated by the central, relatively hydrophobic domain of core and was detected in different heterologous expression systems, in transgenic mice and in liver specimens from HCV-infected chimpanzees, plays a role during viral replication or virion morphogenesis. It has been speculated that the interaction of core with lipid droplets may affect lipid metabolism, which in turn may contribute to the development of liver steatosis. The observation that certain HCV core-transgenic mice develop steatosis and HCC has lent further support to this hypothesis.55,56 A small proportion of the core protein may also be found in the nucleus. Little is known about the assembly of core into nucleocapsids. In vitro studies with recombinant HCV core proteins demonstrated that the N-terminal 124 amino acid residues are sufficient for the assembly of nucleocapsid-like structures, and that the presence of structured RNA is required for this process.57 However, under these experimental conditions RNA encapsidation is not specific, and the signals and processes that mediate RNA packaging and nucleocapsid assembly during HCV replication are unknown. More recently, assembly of nucleocapsid-like particles has been observed in cellfree translation systems.58 These capsids sediment at 100S and have a buoyant density of 1.28 g/ml on CsCl gradients. Intriguingly, the core protein has been reported to interact with a variety of cellular proteins and to influence numerous host cell functions, including apoptosis, cell cycle control, gene expression and many others.59,60 However, the relevance of these observations, derived mainly from heterologous overexpression experiments, for the natural course and pathogenesis of hepatitis C is currently unknown.
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ARFP/F PROTEIN An alternative reading frame (ARF) was recently identified in the HCV core region which, as a result of a -2/+1 ribosomal frameshift, has the potential to encode a protein of up to 160 amino acids, designated ARFP (alternative reading frame protein) or F (frameshift) protein46,61,62 (Figure 8-3). Expression of the ARFP/F protein of HCV genotype 1a in vitro or in mammalian cells yields a 17 kDa protein. Amino acid sequencing indicated that the frameshift probably occurs at or near codon 11 of the core protein sequence.61 However, multiple frameshifting events have recently been reported in this region, and a 1.5 kDa protein could also be produced by -1/+2 frameshifting.63 In addition, the frameshift position seems to be genotype dependent, as a +1 frameshift at codon 42 was recently reported for genotype 1b.64 Detection of antibodies65 and T cells66 specific for the ARFP/F protein in patients with hepatitis C suggests that this protein is expressed during HCV infection. However, given that the ARF is not present in subgenomic HCV replicons, the ARFP/F protein is not required for HCV RNA replication in vitro. Recent studies incorporating multiple stop codons into the ARF have shown that ARFP/F protein expression is not absolutely required for replication in vivo.67 Rather, these results suggest that the ARF may harbor additional conserved RNA elements that are required for translation and/or replication of full-length HCV genome RNAs. Thus, the functions, if any, of the ARFP/F protein in the life cycle and pathogenesis of HCV remain to be elucidated.68
ENVELOPE GLYCOPROTEINS The envelope proteins E1 and E2 are extensively glycosylated and have an apparent molecular weight of 30–35 and 70–72 kDa, respectively. They form a non-covalent complex, which is believed to represent the building block for the viral envelope.69 E2 is believed to make contact with the cellular receptor(s) for HCV, whereas E1 has been predicted to possess fusion activity.
5´ NCR
C
E1
ARF
ARFP/F Figure 8-3. The alternative reading frame (ARF) in the HCV core coding region and the ARF protein (ARFP)/F protein. The 5¢ non-coding region (5¢ NCR) of the HCV genome contains extensive secondary structures forming an internal ribosome entry site. The main open reading frame of the HCV genome with the core protein (C) and the N-terminal portion of envelope glycoprotein 1 (E1) are depicted in gray. The ARF is illustrated in red. The putative ARFP/F protein is shown at the bottom. The frameshift from the core reading frame into the ARF occurs at or near codon 11 of the core coding sequence.
Chapter 8 REPLICATION AND PATHOGENESIS OF HEPATITIS C VIRUS
E1 and E2 are type I transmembrane glycoproteins. Interestingly, the transmembrane domains, located at their C termini, are involved in heterodimerization and have ER retention properties. Each of these transmembrane domains is composed of two stretches of hydrophobic amino acids separated by a short polar segment. The second hydrophobic stretch acts as an internal signal peptide for the downstream protein. Before signal sequence cleavage, the E1 and E2 transmembrane domains have been proposed to adopt a hairpin structure at the translocon. After cleavage, the signal sequence is reoriented towards the cytosol, resulting in a single transmembrane passage.70 The ectodomains of E1 and E2 contain numerous highly conserved cysteine residues that may form four and nine intramolecular disulfide bonds, respectively. In addition, E1 and E2 contain up to five and 11 glycosylation sites, respectively. Thus, HCV glycoprotein maturation and folding is a complex process that involves the ER chaperone machinery and depends on disulfide bond formation as well as glycosylation. A model for E2 based on the structure of the envelope protein from tick-borne encephalitis virus (TBE; a member of the flavivirus genus)71 was proposed.72 According to this model, E2 forms an elongated and flat head-to-tail homodimer. The fact that the envelope protein of Semliki Forest virus,73 a more distantly related alphavirus, has a similar structure to the envelope proteins of TBE71 and dengue virus,74 suggests that HCV may have a similar surface architecture. However, virtually nothing is known about the actual structure of the HCV E1–E2 complex, and the processes that mediate viral attachment, entry, and fusion have only recently become amenable to systematic study (see below). As discussed above, the genes encoding the envelope glycoproteins E1 and E2 are particularly variable. A hypervariable region (HVR) of approximately 28 amino acids in the N-terminal domain of E2 has been termed HVR1.75,76 The HVR1 amino acid sequence differs by up to 80% among HCV isolates. Interestingly, despite high variability at the sequence level, the structure of this domain was found to be quite conserved.77 HVR1 appears to contain a neutralization epitope78 and variability, therefore, may be driven by antibody selection. Of note, the HVR1 could be deleted from infectious HCV cDNA clones without abrogating infectivity, although the mutant virus replicated poorly and compensating changes in E1 and E2 were selected upon passaging.79 These observations suggest a functional role of this domain, probably in virus entry into the host cell. A second hypervariable region, HVR2, has been described at amino acid positions 91–97 of genotype 1 E2 protein.
p7 p7 is a 63-amino-acid polypeptide that is often incompletely cleaved from E2. It has two transmembrane domains connected by a short hydrophilic segment which forms a cytoplasmic loop, and the N and C termini are oriented toward the ER lumen.80 Both transmembrane passages have been predicted to form a-helices, and the C-terminal transmembrane segment has been shown to function as an internal signal peptide. p7 of the related pestivirus BVDV is essential for the production of infectious progeny, but not for RNA replication.81 Similarly, HCV p7 is not required for HCV RNA replication because it is not present
in subgenomic replicons. However, it is essential for virus infectivity in vivo, as shown by genetic studies using infectious HCV cDNA clones.28 p7 has recently been reported to form hexamers and to possess ion channel activity.82,83 These properties suggest that p7 belongs to the viroporin family, could have an important role in viral particle maturation and release, and may represent an attractive target for antiviral intervention.
VIRION STRUCTURE Although exciting progress has recently been made with respect to related flavi-84–86 and alphaviral virion structures,73 HCV has not so far been conclusively visualized and its structure remains unknown. By analogy to these related viruses, it can be assumed that the core protein and the envelope glycoproteins E1 and E2 are the principal structural components of the virion. E1 and E2 are presumably anchored to a host cell-derived double-layer lipid envelope that surrounds a nucleocapsid composed of multiple copies of the core protein and encapsidating the genomic RNA. The basic biophysical properties of the HCV particle were revealed early on by experiments performed in chimpanzees. Infectivity was abolished by treatment with lipid solvents, indicating that the viral particle is enveloped.87 A rough estimate of virion size was obtained by filtration studies demonstrating that the particle is able to pass through 50 nm pore filters.88 Based on subsequent electron microscopy studies, HCV particles are believed to have a diameter of 40–70 nm.89,90 HCV circulates in various forms in the infected host, including virions bound to low-density (LDL) and very lowdensity lipoproteins (VLDL),91 which appear to represent the infectious fraction, virions bound to immunoglobulins, and free virions. In addition, non-enveloped nucleocapsids harboring HCV RNA have been described.92
NON-STRUCTURAL PROTEINS NS2-3 PROTEASE Cleavage of the polyprotein precursor at the NS2/NS3 junction is accomplished by a protease encoded by NS2 and the N-terminal one-third of NS3.93,94 NS2 is dispensable for replication of subgenomic replicons in vitro (see below) and is thus not essential for the formation of a functional replication complex. However, the NS2-3 protease activity is essential for the replication of full-length HCV genomes in vivo. Site-directed mutagenesis has shown that amino acids His 143 (i.e. His 952 of the HCV polyprotein), Glu 163 (i.e. Glu 972) and Cys 184 (i.e. Cys 993) are essential for catalytic activity.93,94 The importance of these amino acid residues is consistent with a thiol protease catalytic mechanism, but NS2-3 activity is stimulated by zinc or certain other divalent metal ions. Interestingly, the cellular chaperone Hsp90 was found to be essential for the activation of the NS2-3 protease.95 The membrane topology of NS2 has been controversial. It has been proposed that NS2 is a transmembrane protein with at least one and up to four transmembrane segments in the N-terminal domain.96,97 There are also data which suggest that the C terminus of NS2, after autocatalysis in the cytosol, may relocate to the ER lumen.97 Recombinant proteins lacking the N-terminal membrane domain of NS2 have been found to retain cleavage activity,
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allowing further characterization of this unique enzymatic activity.98,99 Indeed, recent efforts have led to the determination of a high-resolution structure for the NS2 protease domain, which reveals a dimeric structure with a thiol protease-like active site.100
NS3-4A COMPLEX A distinct serine protease located in the N-terminal one-third of NS3 is responsible for the downstream cleavage events in the nonstructural region9,101,102 (reviewed in 103). In addition, an RNA helicase/NTPase domain is found in the C-terminal two-thirds of NS3.104 The NS4A polypeptide functions as a cofactor for the NS3 serine protease and is incorporated as an integral component into the enzyme core. Complex formation occurs via a tight interaction of the 22 N-terminal residues of NS3 with 12 amino acid residues in the center of NS4A. NS3 by itself has no membrane anchor. The N-terminal domain of NS4A is strongly predicted to form a transmembrane a-helix responsible for membrane anchorage of the NS34A complex.105 The crystal structures of the serine protease106–108 and RNA helicase domains of NS3109,110 as well as the entire NS3 protein111 (Figure 8-4) have been elucidated. These enzymes are essential for viral replication and have emerged as prime targets for the design of specific inhibitors as antiviral agents112,113 (see below). The catalytic triad of the NS3 serine protease is formed by His 57 (i.e. His 1083 of the HCV polyprotein), Asp 81 (i.e. Asp 1107)
and Ser 139 (i.e. Ser 1165). Crucial determinants of substrate specificity include an acidic amino acid residue at the P6 position, a P1 cysteine (trans cleavage sites) or threonine (cis cleavage site between NS3 and NS4A), and an amino acid residue with a small side chain, i.e. alanine or serine, at the P1¢ position. A consensus cleavage sequence, therefore, would read D/E-X-X-X-X-C/T | S/AX-X-X. The three-dimensional structure of full-length NS3 revealed that a C-terminal b strand of the helicase domain lies within the active site of the serine protease domain, where it is expected to be located during the cis cleavage that separates NS3 from NS4A. This results in autoinhibition that is released upon trans substrate binding.111 Helicases catalyze the unwinding of doubled-stranded RNA or DNA into single-stranded nucleic acids. The energy required for this process is generated by hydrolysis of NTPs by an associated NTPase activity. Thus, the NS3 helicase couples unwinding of RNA regions with extensive secondary structures with NTP hydrolysis. The NS3 helicase is a member of the so-called helicase superfamily 2. These are also called DEXH/D helicases, according to a characteristic signature sequence in one of the essential enzyme motifs. It was recently shown that NS3 unwinds RNA through a highly coordinated cycle of fast ripping and local pausing that occurs with regular spacing along the duplex substrate, suggesting that nucleic acid motors can function in a manner analogous to cytoskeletal motor proteins.114
Figure 8-4. Three-dimensional structure of the HCV NS3-4A complex. The structure was determined using an engineered single-chain molecule consisting of NS3 and 14 NS4A residues known to activate the serine protease linked to the N terminus of NS3. The two bbarrels in the serine protease domain are shown in magenta and red, the helicase subdomains are shown in green, light blue and dark blue, and the central NS4A domain interacting with NS3 is shown in olive. Residues of the serine protease catalytic triad (His 57, Asp 81 and Ser 139) are shown in ball-and-stick representation, and the protease structural zinc ion is shown as a white sphere. The red spheres represent a phosphate molecule located at the NTP-binding site of the helicase. Note the interaction between the C terminus and the protease active site region. (Reproduced from111, with permission.)
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NS4B NS4B, a 27 kDa integral membrane protein, is the least characterized HCV protein.115 It is predicted to be a polytopic membrane protein with a cytoplasmic N-terminal region followed, depending on the prediction, by four or six transmembrane segments and a Cterminal region in the cytosol.115,116 It has been shown experimentally that the bulk of the protein is cytosolically oriented.115 Introduction of glycosylation acceptor sites at various positions of NS4B recently confirmed the presence of a predicted ER luminal loop around amino acid position 161.117 Surprisingly, the N terminus of NS4B was found to be translocated into the ER lumen at least partially, presumably by a post-translational mechanism.117 The NS4B proteins of HCV, pesti- and flaviviruses are similar in size, amino acid composition, and hydrophobic properties. No function, however, has yet been ascribed to NS4B in any of these related viruses. More recently, it was found by electron microscopy that expression of HCV NS4B induces the formation of a seemingly ERderived specific cellular membrane alteration, designated the membranous web, that harbors the viral replication complex53,118 (see below). Thus, a function of NS4B may be to induce the specific membrane alteration that serves as a scaffold for the HCV replication complex.
NS5A NS5A is a phosphoprotein of unknown structure and function. It is found in a basally phosphorylated form of 56 kDa and in a hyperphosphorylated form of 58 kDa. NS5A of HCV and BVDV, as well as NS5 of YFV, are phosphorylated by as yet unidentified serine/threonine kinases, suggesting that these proteins share a common function related to their phosphorylation state.119 Basal phosphorylation requires domains in the center and C terminus of
1
Domain I
C57 C59 C39 C80
Membrane anchor domain
NS5A. The centrally located serine residues 225, 229 and 232 (i.e. Ser 2197, Ser 2201 and Ser 2204 of the HCV polyprotein) are important for NS5A hyperphosphorylation (Figure 8-5). However, it is unknown whether these serine residues are actually phosphorylated or whether they affect phosphorylation indirectly. The only phosphoacceptor sites that have been mapped experimentally are serine residues 222 (i.e. Ser 2194 of the polyprotein)120 and 349 (i.e. Ser 2321) (genotype 1a HCV H strain).121 The cellular kinase(s) responsible for NS5A phosphorylation appear to belong to the so-called CMCG group of serine/threonine kinases to which casein kinase II, cyclin-dependent kinases and mitogen-activated protein kinases belong. However, the cellular kinase(s) responsible for NS5A phosphorylation remains elusive. Interestingly, adaptive mutations have been found to cluster in the central region of NS5A in the context of selectable subgenomic HCV replicons, suggesting that NS5A is involved – either directly or by interaction with cellular proteins and pathways – in the viral replication process. This observation, together with the modulation of NS5A hyperphosphorylation by nonstructural proteins 3, 4A and 4B,122,123 strongly supports the notion of NS5A being an essential component of the HCV replication complex. An N-terminal amphipathic a-helix mediates membrane association of NS5A.124–126 This helix exhibits a hydrophobic, tryptophanrich side embedded in the cytosolic leaflet of the membrane bilayer, while the polar, charged side is exposed to the cytosol. Thus, NS5A is a monotopic protein with an in-plane amphipathic a-helix as membrane anchor. Structure–function analyses demonstrated that this helix displays fully conserved polar residues at the membrane surface, which define a unique platform probably involved in specific protein–protein interactions essential for the formation of a functional HCV replication complex.126 Comparative sequence analyses and limited proteolysis of recombinant NS5A protein have
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Δ 235-282 237
276 ISDR
237
356
Putative NLS
Adaptive changes
222
342
302
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447
GFP insertions
354-362 384 418 349 Δ 399-441 (1a) 384-407
V3
PKR interaction domain Figure 8-5. Overview of the HCV NS5A protein. NS5A is drawn to scale as a box. Amino acid positions relate to the HCV Con1 sequence (genotype 1b; GenBank accession number AJ238799; add 1972 amino acids to obtain positions relative to the HCV polyprotein). The N-terminal amphipathic a-helix that mediates membrane association of NS5A, the region where cell culture-adaptive changes have been found to cluster in the replicon system, the so-called interferon sensitivity determining region (ISDR), the double-strand RNA-activated protein kinase (PKR) interaction domain, the putative nuclear localization signal (NLS), and variable region 3 (V3) are highlighted. The domain organization recently proposed by Tellinghuisen et al.127 is also shown. Cysteine residues 39, 57, 59 and 80, denoted by blue lines, coordinate one zinc atom per NS5A protein. Deletions identified in the replicon system are shown in light green.13,178,163 GFP insertion sites tolerated in the replicon system are highlighted by green lines.180 Mapped phosphoacceptor sites for genotype 1b (amino acid position 222)120 and the genotype 1a HCV H isolate (amino acid position 349)121 are highlighted in red. Dashed red lines denote serine residues that affect NS5A phosphorylation.
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recently led to a proposed domain organization of NS5A (Figure 85).127 The relatively highly conserved domain I immediately following the membrane-anchoring a-helix has been shown to contain four absolutely conserved cysteine residues that coordinate one zinc atom per NS5A protein.127 Mutation of these residues abolishes HCV RNA replication, indicating that the zinc is essential for NS5A structure and/or function. Thus, NS5A is a zinc metalloprotein. The structure of the NS5A domain I has recently been solved, revealing a completely novel protein fold, a new zinc coordination motif, and a rare cytoplasmic disulfide bond. The structure also defines surface properties that may be involved in NS5A dimer formation and NS5A interaction with viral and cellular proteins, membranes and RNA128 (Figure 8-6). HCV NS5A has attracted considerable interest because of its potential role in modulating the IFN response. Studies performed in Japan first described a correlation between mutations within a discrete region of NS5A, termed interferon sensitivity-determining region (ISDR) (Figure 8-5), and a favorable response to IFN-a therapy.129 These studies demonstrated that strains closely matching the prototype HCV genotype 1b (HCV-J) ISDR sequence correlated with IFN resistance. These findings were largely confirmed in Japan, but not in Europe and North America. The reasons for this discrepancy are not understood, but may involve differences in both doses and regimens of IFN treatment and the low prevalence of ‘mutant type’ HCV genotype 1b isolates in western countries. Even if a meta-analysis of published data seems to confirm an association of specific ISDR sequences with the IFN response,130 this remains a controversial issue that has thus far not translated into clinically
applicable predictors. The same is true for other regions of NS5A that have been associated with the response to IFN therapy, such as a variable region in the C-terminal domain of NS5A termed V3 (Figure 8-5). Interestingly, however, an interaction with and repression of the catalytic activity of PKR by NS5A has been found by biochemical, transfection, and yeast functional analyses.131 Mutations within the ISDR that were observed in clinically IFN-sensitive genotype 1b strains disrupted the ability of NS5A to interact with and repress PKR activity, supporting the notion that NS5A mediates HCV resistance to IFN through down-regulation of PKR.132 However, these findings are controversial, and numerous additional potential functions have recently been attributed to NS5A (reviewed in 60,133,134). However, similar to the core protein, only very few of these postulated interactions and functional properties have been validated in a meaningful context involving active HCV RNA replication or HCV infection in vivo.
NS5B HCV replication proceeds via synthesis of a complementary negative-strand RNA using the genome as a template and the subsequent synthesis of genomic positive-strand RNA from this template. The key enzyme responsible for both of these steps is the NS5B RdRp. This essential viral enzyme has been extensively characterized at the biochemical135–138 and the structural level139–142 and has emerged as a major target for antiviral intervention. The HCV NS5B protein contains motifs shared by all RdRps, including the hallmark GDD sequence within motif C, and, based on the similarity of the enzyme structure with the shape of a right hand, possesses the classic fingers,
ER lumen
Membrane anchor
Membrane anchor
Cytoplasm
II III
II III
Figure 8-6. Structure of HCV NS5A domain I. The dimeric form of NS5A domain I (amino acids 36–198) modeled in relation to the membrane at the site of RNA replication.128 The cytoplasmic and luminal leaflets of the membrane are indicated. The two NS5A domain I monomers are colored in blue and green. Also shown in blue and green are the N-terminal amphipathic membrane anchors of NS5A lying flat in the plane of the membrane.126 Zinc atoms coordinated by domain I are shown as red spheres. The hypothetical locations of domains II and III of NS5A are indicated by schematic spheres. This orientation of domain I places the largely basic surface towards the phospholipid head groups of the membrane and positions the large ‘claw’ or groove of the NS5A dimer away from the membrane, where it may interact with RNA. This figure was reproduced from an illustration kindly provided by Dr Timothy L. Tellinghuisen, Rockefeller University, New York, USA.
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UTP
C-ter tP
N-ter
palm and thumb subdomains. A special feature of the HCV RdRp is that extensive interactions between the fingers and thumb subdomains result in a completely encircled active site139–142 (Figure 87). This feature is shared by other RdRps, including those of the bacteriophage F6 and of BVDV.143,144 As with poliovirus RdRp,145–147 oligomerization of HCV NS5B has recently been reported to be important for cooperative RNA synthesis activity.148,149 Membrane association of the HCV RdRp is mediated by the Cterminal 21 aa residues, which are dispensable for polymerase activity in vitro. Membrane targeting occurs via a post-translational mechanism and results in integral membrane association of NS5B.150 These features, namely post-translational membrane targeting via a hydrophobic C-terminal insertion sequence; integral membrane association; and cytosolic orientation of the functional protein domain, define the HCV RdRp as a member of the so-called tailanchored proteins. The HCV RdRp insertion sequence crosses the membrane bilayer as a transmembrane segment,151 is essential for HCV RNA polymerase in cells, and is likely to possess additional functions apart from its membrane anchor function.152
Figure 8-7. Crystal structure of the catalytic domain of the HCV RNA-dependent RNA polymerase (140; PDB accession code 1GX6). Ribbon diagram of the NS5A-D55 protein complexed with UTP and Mn2+. a-Helices are colored blue, b-strands red, and connecting loops silver. The bound nucleotide and the side chains of the catalytic aspartic acids (Asp 220 and Asp 318) in the center of the structure are represented as ball-and-stick colored black and cyan, respectively. Mn2+ ions are shown as green spheres. Also labeled is the triphosphate (tP) moiety of a nucleotide bound to the ‘priming’ site. This figure was reproduced from an illustration kindly provided by Dr Felix A. Rey, Laboratoire de Virologie Moléculaire et Structurale, UMR 2472 CNRS/UMR 1157 INRA, Gif-sur-Yvette, France.
Table 8-2. In Vitro and in Vivo Models to Study HCV In vitro models In vitro transcription–translation Transient cellular expression systems Stably transfected cell lines (constitutive/inducible expression) Infection of primary hepatocytes and established cell lines Retroviral pseudoparticles displaying functional HCV glycoproteins Replicons (subgenomic/full-length; selectable/transient) Chimeric viruses (e.g. poliovirus—HCV) Related viruses (e.g. BVDV) In vivo models Transgenic mice Immunodeficient mice/hepatocellular reconstitution models Chimpanzee (Pan troglodytes) Tree shrew (Tupaia belangeri chinensis)? Related viruses (e.g. GBV-B in tamarins)
Given the lack of a robust cell culture system allowing natural infection, replication, and release of viral progeny, various in vitro and in vivo models have been used to study HCV (Table 8-2) (reviewed in 153).
results. In their present format some of these systems may be useful for neutralization assays, but not for a systematic investigation of the viral life cycle.154,155 An alternative approach involves the generation of cell lines constitutively or inducibly expressing viral sequences from chromosomally integrated cDNA.156 Moreover, viable chimeras of certain positive-strand RNA viruses, such as polio and Sindbis virus, with HCV genetic elements, such as the IRES157 or NS3,158 have been constructed and may facilitate the screening of selected antiviral compounds.
IN VITRO MODELS
The Replicon System
Infection of primary hepatocytes and established cell lines in vitro yielded only low-level replication and often poorly reproducible
The replicon system has revolutionized the investigation of HCV RNA replication.12 The prototype subgenomic replicon was a
MODEL SYSTEMS
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5¢ NCR
3¢ NCR
EMCV IRES
(U)n NS3
NeoR
A NS4 B
A
NS5
B
In vitro transcription
5¢ NCR
3¢ NCR
EMCV IRES NeoR
Figure 8-8. Prototype subgenomic HCV replicon.12 RNA is transcribed in vitro from a plasmid containing the HCV IRES followed by a neomycin resistance cassette, a second heterologous IRES from encephalomyocarditis virus (EMCV IRES), the HCV non-structural region (NS3 to NS5B), and the HCV 3’ NCR. RNA is subsequently transfected into HuH7 human hepatoma cells, followed by selection with G418 of clones harboring autonomously replicating subgenomic HCV RNA.
(U)n NS3
A NS4 B
A
NS5
B
Transfection
HuH-7 cells
Selection
Clones carrying HCV replicons
bicistronic RNA where the structural region of HCV was replaced by the neomycin phosphotransferase gene and translation of the non-structural proteins 3–5B was driven by a second, heterologous IRES from encephalomyocarditis virus (Figure 8-8). Using this approach it became possible, for the first time, to study efficient and genuine HCV RNA replication in HuH-7 human hepatoma cells in vitro. Interestingly, certain amino acid substitutions, i.e. cell cultureadaptive changes, can increase the efficiency of replication initiation by more than 105-fold.13,159 Adaptive changes cluster in certain regions, such as the center of NS5A (Figure 8-4), the C-terminal part of the NS3 serine protease and the N-terminal part of the NS3 RNA helicase domains, as well as two positions in NS4B.160 In NS5A these changes often affect serine residues required for hyperphosphorylation, suggesting that hyperphosphorylation of NS5A reduces HCV RNA replication.161–163 According to one model, hyperphosphorylation of NS5A reduces interaction with the human vesicleassociated membrane protein-associated protein A (hVAP-A).161 However, adaptive changes are likely to increase HCV RNA replication by additional, as yet unidentified mechanisms. In this context, it is interesting to note that there is an inverse correlation between mutations that permit efficient replication of HCV RNA in HuH-7 cells in vitro and productive replication in chimpanzees in vivo after intrahepatic inoculation.164 The replicon system has allowed genetic dissection of HCV RNA elements and proteins, provided material for biochemical and ultrastructural characterization of the viral replication complex, and facilitated drug discovery efforts (Table 8-3). Moreover, the replicon system has been exploited for analysis of the effect of cytokines on
134
Table 8-3. Lessons from the Replicon System Adaptive changes Cell-cycle dependence of HCV RNA replication Inverse correlation between replicon activity in vitro and HCV replication/particle formation in vivo Evaluation of new antiviral agents and antiviral resistance Effect of cytokines on HCV RNA replication Identification and characterization of the HCV replication complex Host factors Identification of essential RNA elements Requirements for infectious HCV particle formation
HCV RNA replication165,166 and the study of other aspects of the interaction between HCV and the host cell.167–169a Since the original reports of functional genotype 1b replicons, replicons for genotype 1a170 and 2a171, as well as derivatives expressing easily quantifiable marker enzymes in a separate cistron, have been made to facilitate genetic studies as well as drug screening and evaluation.172–174 In addition, full-length replicons and HCV genomes efficiently replicating in tissue culture have been developed,175–177 and the spectrum of permissive host cells has been expanded.178,179 Finally, replicons have been established that allow tracking of functional HCV replication complexes in living cells.180 One puzzling (and disappointing) observation was that full-length genome RNAs with adaptive mutations were incapable of producing infectious virus. This led investigators to favor the idea that
Chapter 8 REPLICATION AND PATHOGENESIS OF HEPATITIS C VIRUS
HuH-7 and other HCV permissive cell lines lacked some factor(s) necessary for particle formation and release. However, this does not appear to be the case, given recent results with a genotype 2a isolate from Japan, JFH-1. JFH-1 was isolated from a patient with acute fulminant hepatitis C, and JFH-1 subgenomic replicons can replicate efficiently in HuH-7 cells without adaptive mutations.171 Remarkably, full-length JFH-1 produces infectious virus.181,182 Chimeras consisting of the 5¢ and 3¢ NTRs and replicase region of JFH-1 (NS3-5B) and the C-NS2 region of other isolates will also sometimes produce cell culture infectious particles.183 One possibility, which seems to be growing in popularity, is that cell cultureadaptive mutations that promote efficient RNA replication may be deleterious in vivo because they compromise particle assembly and release. With these advances, the late (assembly and egress) and early (entry) events in HCV infection can now be studied. These steps can also be explored as possible new therapeutic targets.
IN VIVO MODELS The restricted host range of HCV has hampered the development of a suitable small animal model of viral replication and pathogenesis. Apart from a single report on the transmission of HCV to tree shrews (Tupaia belangeri chinensis),184 the chimpanzee (Pan troglodytes) is the only animal known to be susceptible to HCV infection.185 Indeed, the chimpanzee was essential in the early characterization of the agent of non-A, non-B hepatitis, and has allowed the determination of important aspects of HCV replication, pathogenesis and prevention. In this context, it would not have been possible to demonstrate the functionality of infectious clones of HCV without chimpanzees.11 In addition, recent studies in chimpanzees have provided new insight into the host immune response against hepatitis C,186–188 and the chimpanzee remains the only faithful model to test the immunogenicity and efficacy of vaccine candidates.189–191 However, ethical and financial restrictions limit the use of primates to highly selected experimental questions. Expression of HCV proteins in transgenic mice provided some insights into the pathogenesis of HCV-induced liver disease.55,56 However, expression of HCV proteins from chromosomally integrated cDNA does not appropriately reflect the viral life cycle, and studies on viral entry and replication are hardly conceivable in this system. GBV-B, the closest relative of HCV within the family Flaviviridae can be transmitted to tamarins (Saguinus sp.) and may represent a valuable surrogate model for HCV. Remarkably, GBV-B can be cultured in tamarin hepatocytes in vitro.192 In addition, infectious cDNA clones193 and replicons194 have been established for GBV-B. However, in tamarins GBV-B typically leads to self-limited infection without viral persistence unless animals are immunosuppressed or molecular clones are used.195 GBV-B can also be propagated in common marmosets (Callithrix jacchus), which are easier to breed in captivity, are smaller, and are already regularly used for drug metabolism, pharmacokinetic and toxicology studies.196 Progress in the development of a small animal model of HCV replication was achieved with the successful HCV infection of immunodeficient mice reconstituted with human hepatocytes.197 The properties of two different mouse strains, the Alb-uPA-transgenic and the immunodeficient SCID mouse, were combined to develop a model system that allows orthotopic engraftment of
human hepatocytes (Figure 8-9). Expression of the murine urokinase-type plasminogen activator under the transcriptional control of the albumin promoter (Alb-uPA) programs murine hepatocyte death, providing a suitable microenvironment for the engraftment and expansion of transplanted human hepatocytes. In homozygous animals, reconstitution with human hepatocytes was reported to reach >50% of the liver cell mass. In consequence, Alb-uPA homozygous animals were characterized by persistent high human albumin production. Inoculation with serum from patients with hepatitis C resulted in persistent HCV viremia in about 75% of mice with high-level human hepatocyte engraftment. HCV RNA could be detected by PCR for up to 35 weeks, with titers ranging from 3 ¥ 104 to 3 ¥ 106 copies/ml. These viral titers are similar to those found in infected humans. Moreover, an approximately 3-log rise in viral titers after inoculation, detection of viral negative-strand RNA in the liver, and the ability to serially passage the virus through several generations of animals provided convincing evidence for active replication and production of infectious viral progeny in this system. However, the handling of these fragile animals affected by major bleeding disorders and severe immunodeficiency (approximately 35% mortality in newborns) presents a non-trivial challenge and requires special expertise. Moreover, access to fresh human hepatocytes is limited for many investigators.
REPLICATION CYCLE The life cycle of HCV includes 1) binding to an as yet unidentified cell surface receptor and internalization into the host cell; 2) cytoplasmic release and uncoating of the viral RNA genome; 3) IRESmediated translation and polyprotein processing by cellular and viral proteases; 4) RNA replication; 5) packaging and assembly; and 6) virion maturation and release from the host cell (Figure 8-10).
RECEPTOR CANDIDATES CD81, a tetraspanin molecule found on the surface of many cell types, including hepatocytes,198 the low-density lipoprotein receptor (LDLR)199 and scavenger receptor class B type I (SR-BI)200 have, among others, been proposed as HCV receptors or components of a receptor complex. Both CD81 and SR-BI bind E2 and are currently viewed as necessary, but not sufficient, for HCV entry.14,15,201–203 Expression of CD81 in CD81-negative liver-derived cell lines confers susceptibility to HCV pseudoparticles (see below), and blocking antibodies against CD81 or SR-BI, recombinant CD81, or siRNA-mediated down-regulation of CD81 expression reduces infectivity. However, additional, as yet unidentified hepatocyte-specific factors are required for HCV entry. The LDLR has been attractive as a candidate receptor because infectious HCV has been reported to be associated with LDL or VLDL (see above). At present, however, it is unclear whether interaction of HCV with the LDLR can lead to productive infection. Cell lines without LDLR are still infectable with HCV pseudoparticles. HCV E2 also binds to DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) and L-SIGN (liver/lymph node-specific intercellular adhesion molecule-3grabbing integrin). The latter is a calcium-dependent lectin expressed
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Figure 8-9. A small animal model of HCV replication.197
SCID
Alb-uPA
Immunodeficient Alb-uPA transgenic mouse
Newborns with liver cell destruction
Human hepatocytes
Partial repopulation with human hepatocytes
Mouse with ‘chimeric’ liver
(+) RNA 1
3
5
3 3 5A
ER
2
C
p7
E1
2
5B
E2 4A 4B (-) RNA 4
3 5
6 (+) RNA
5 5
136
MW 3
Figure 8-10. Life cycle of HCV. 1, Virus binding and internalization; 2, cytoplasmic release and uncoating; 3, IRESmediated translation and polyprotein processing; 4, RNA replication; 5, packaging and assembly; 6, virion maturation and release. The topology of HCV structural and nonstructural proteins at the endoplasmic reticulum (ER) membrane is shown schematically. HCV RNA replication occurs in a specific membrane alteration, the membranous web (MW). Note that IRES-mediated translation and polyprotein processing, as well as membranous web formation and RNA replication, illustrated here as separate steps for simplicity, may occur in a tightly coupled fashion.
Chapter 8 REPLICATION AND PATHOGENESIS OF HEPATITIS C VIRUS
on liver sinusoidal endothelial cells that may facilitate the infection process by trapping the virus for subsequent interaction with the receptor.204–207 Identification and validation of HCV receptor candidates has been limited by the paucity of systems for analysis of the early steps of the viral life cycle. Given the lack of native HCV particles and efficient cell culture systems, various alternatives have been explored to study the early steps of HCV infection. Soluble C-terminal truncated versions of HCV envelope glycoprotein E2,198,200,205,208,209 liposomes reconstituted with HCV E1 and E2,210 and virus-like particles expressed in insect cells211,212 have been used to study HCV glycoprotein interactions with the cell surface. The production of viruslike particles has also been described in mammalian cells.213 However, it is unclear how virus-like particles produced in inset or mammalian cells will compare to authentic HCV virions. Pseudotyped vesicular stomatitis virus (VSV) or influenza virus particles have been reported incorporating chimeric E1 and/or E2 glycoproteins whose C-terminal transmembrane domains were modified to allow transport to the cell surface.208,214–216 However, such modifications may interfere with the multiple and complex roles of the E1 and E2 transmembrane domains69 and may perturb the conformation and functions of E1–E2 complexes. Therefore, the use of such pseudotypes as a tool to study HCV assembly and entry remains controversial.216 Against this background, the recent establishment of infectious retroviral pseudotypes displaying functional HCV glycoproteins as a robust model system for the study of viral entry represents a major breakthrough14,15 (Figure 8-11). HCV pseudoparticle infectivity is restricted primarily to human hepatocytes and hepatocyte-derived cell lines, and entry is pH dependent. Thus, HCV entry likely involves transit through an endosomal low-pH compartment and fusion with the endosomal membrane. The structural basis for low pH-induced membrane fusion has recently been elucidated for the dengue, TBE and Semliki Forest viruses.217–219 The envelope proteins of these related flavi- and alphaviruses possess an internal fusion peptide that is exposed during low pH-mediated domain rearrangement and trimerization of the protein. The scaffolds of these so-called class II fusion proteins are remarkably similar, suggesting that all members of the Flaviviridae, including HCV, could behave similarly.
CMV
MLV Gag-Pol Plasmid 2
CMV
E1
ψ
E2
Plasmid 1
CMV
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Plasmid 3
293T cells
ψ
CMV
E1
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HCV pseudoparticle Figure 8-11. Generation of infectious HCV pseudoparticles. Cotransfection of 293T human embryo kidney cells with plasmids allowing expression of (1) unmodified HCV E1-E2 glycoproteins, (2) retroviral core proteins, and (3) a packaging-competent green fluorescent protein (GFP) expression construct leads to secretion into the supernatant of pseudoparticles bearing HCV envelope glycoproteins instead of the retroviral envelope protein on their surface. CMV, cytomegalovirus promoter; y, retroviral packaging sequence.
REPLICATION COMPLEX The formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, and altered cellular membranes, is a hallmark of all positive-strand RNA viruses investigated thus far (see 220,221 for reviews). Depending on the virus, replication may occur on altered membranes derived from the ER,222–226 Golgi apparatus,227–229 mitochondria230 or even lysosomes.231 The role of membranes in viral RNA synthesis is not well understood. It may include (i) the physical support and organization of the RNA replication complex;147 (ii) the compartmentalization and local concentration of viral products;232 (iii) tethering of the viral RNA during unwinding;220 (iv) provision of lipid constituents important for replication;233,234 and (v) protection of the viral RNA from double-strand RNA-mediated host defenses or RNA interference. In the case of HCV, protein–protein interactions among HCV non-structural proteins have been described235,236 and determinants
for membrane association of the HCV proteins have been mapped. The membrane association of HCV proteins is schematically illustrated in Figure 8-12. For a more comprehensive review on the interactions of HCV proteins, including the structural proteins, with host cell membranes, see references.237,238 A specific membrane alteration, designated the membranous web, was recently identified as the site of RNA replication in HuH-7 cells harboring subgenomic HCV replicons118 (Figure 8-13). Formation of the membranous web could be induced by NS4B alone (see above), and it was very similar to the ‘sponge-like inclusions’ previously found by electron microscopy in the liver of HCV-infected chimpanzees. The membranous web was often found closely associated with the rough ER. Based on this observation, together with earlier studies demonstrating the co-localization of individually expressed HCV proteins with membranes of the ER,105,115,124,150 and data
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Figure 8-12. Membrane association of HCV proteins. Note that the topologies of NS2, NS4A and NS4B are currently under investigation and are illustrated only schematically. A recent study indicated that the C terminus of NS2 may be localized in the ER lumen, resulting in four transmembrane domains.97 Also, it was recently reported that the N terminus of NS4B can be at least partially translocated into the ER lumen.117
Cytosol NS5B NS3 p7
C
NS2
CC
NS5A C C
N
N
NS4B C
N N
N
C
NS4A
E1 E2
ER lumen
ER N
ER
N
M M A
B
Figure 8-13. HCV replication complex.118 (A) Low-power overview of a HuH-7 cell harboring a subgenomic HCV replicon. A distinct membrane alteration, named membranous web (arrows), is found in the juxtanuclear region. Note the circumscript nature of this specific membrane alteration and the otherwise unaltered cellular organelles. Bar, 1 mm. (B) Higher magnification of a membranous web (arrows) composed of small vesicles embedded in a membrane matrix. Note the close association of the membranous web with the rough endoplasmic reticulum. Bar, 500 nm. The membranous web harbors all HCV non-structural proteins and nascent viral RNA in HuH-7 cells harboring subgenomic replicons, and therefore represents the HCV RNA replication complex. N, nucleus; ER, endoplasmic reticulum; M, mitochondria.
indicating that HCV RNA replication takes place in a compartment that sustains endoglycosidase H-sensitive glycosylation,151 it is currently believed that the membranous web is derived from membranes of the ER. Ongoing studies are aimed at isolating and further characterizing this complex and at defining the viral and cellular processes involved in the formation of the membranous web. Recent studies demonstrate a complex interaction between HCV RNA replication and the cellular lipid metabolism, presumably via the trafficking and association of viral and host proteins with intracellular membranes. In this context, it was found, for example, that geranylgeranylation of one or more host proteins is required for HCV RNA replication.239,240 Such observations suggest that pharmacologic manipulation of lipid metabolism may have therapeutic potential in hepatitis C.
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EVOLVING THERAPEUTIC STRATEGIES In principle, each of the steps of the HCV life cycle illustrated in Figure 8-12 represents a target for antiviral intervention.241–243 Specific inhibitors of the biochemically and structurally wellcharacterized NS3 serine protease, as well as the RNA helicase/ NTPase and the NS5B RdRp, are currently being developed as antiviral agents, and the first candidates are already in early-phase clinical trials.112,113 Already at this early stage it becomes evident that the genetic variability of HCV represents a major challenge to the clinical development of specific enzyme inhibitors and that, similar
Chapter 8 REPLICATION AND PATHOGENESIS OF HEPATITIS C VIRUS
to HIV infection, combination therapy will be necessary for therapeutic success.244,245 In addition to these more classic pharmacological approaches, gene therapeutic strategies aimed at inhibiting HCV replication and gene expression are currently being explored in various experimental systems. These include, among others, antisense oligodeoxynucleotides, ribozymes, and small interfering RNAs. Moreover, based on the concept that a quantitatively and qualitatively insufficient CD4+ and CD8+ T-cell response may contribute to viral persistence, immunotherapeutic strategies aimed at enhancing the cellular immune response against HCV are currently being investigated. Apart from more efficient therapeutic strategies, the development and implementation of preventive measures is of paramount importance. The development of an effective recombinant vaccine has been hampered by the high genetic variability of HCV and the lack of a suitable cell culture infection system and small animal model.189–191 It has been shown, however, that vaccination with recombinant envelope proteins expressed in mammalian cells can protect chimpanzees from primary infection with a homologous virus isolate.246 The correlates of and requirements for a broader protection and a potentially neutralizing immune response still need to be defined, however. The potential of DNA vaccination to induce a humoral and cellular immune response is particularly interesting in this regard. Alternative currently pursued strategies include peptide and protein vaccines, dendritic cell-based vaccines, and virus-like particles. Although sterilizing immunity will probably be difficult to achieve, the aim of inducing a state of immunity that prevents the development of chronic infection appears more realistic. Along these lines, and in contrast to earlier more pessimistic views citing lack of protective immunity,247–249 more recent observations indicate that chimpanzees that clear infection do exhibit protective – albeit not sterilizing – immunity upon rechallenge,250,251 i.e. they show an attenuated course with rapid control of the rechallenge inoculum. In addition, studies in intravenous drug users have shown that there is some protective immunity in hepatitis C.252 This study showed that the risk of developing HCV viremia was lower for intravenous drug users who had successfully cleared a previous HCV infection than for those who had no evidence of previous HCV infection. In any case, it is likely that induction of both a humoral and a cellular immune response will be required for an effective HCV vaccine. Such a vaccine might also be useful therapeutically.253
PATHOGENESIS HCV infection is a highly dynamic process with a viral half-life of only a few hours and an average daily virion production and clearance of up to more than 1012.254 This high replicative activity, together with the lack of a proofreading function of the viral RdRp, provides the basis for the genetic variability of HCV. In addition, these findings are similar to the dynamics of HIV infection and provide, as discussed above, a rationale for the development and implementation of combination antiviral therapies. The mechanisms responsible for liver injury in acute and chronic hepatitis C are poorly understood.255–257 In acute HCV infection, liver cell damage coincides with the development of the host immune response and not with infection and viral replication. In
addition, persistent viral replication often occurs without evidence of liver cell damage, suggesting that HCV is not directly cytopathic. The immune response against HCV therefore plays a central role in the HCV pathogenesis of hepatitis C. HCV-specific major histocompatibility complex (MHC) class IIrestricted CD4+ helper T-cell258,259 and MHC class I-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses260,261 have been identified in patients with acute and chronic HCV infection. CTL-mediated lysis of virus-infected host cells may lead to clearance of the virus or, if incomplete, to viral persistence and eventually chronic hepatitis. Based on these observations and parallels in other viral diseases, viral persistence and immunologically mediated liver cell injury are important mechanisms leading to chronic hepatitis C.255 Patients who clear HCV infection have a more vigorous CD4+258,259 and CD8+ T-cell response early on.261 The role of specific CD4+ and CD8+ T-cell responses in control of HCV infection was elegantly illustrated by in vivo depletion studies in chimpanzees.187,188 Despite the presence of an immune response, however, HCV is rarely eliminated. Thus, HCV may overwhelm, not induce, or evade antiviral immune responses. Perhaps the simplest explanation is quantitative, based on the kinetics of infection relative to the induction of a CTL response during the early phase of infection. According to this model, viral persistence would be predicted if the replication rate of the virus exceeded the kinetics of the immune response. Indeed, HCV reaches high serum titers within 1 week of infection, whereas adaptive cellular immune responses are delayed by at least 1 and humoral immune responses by at least 2 months.262–264 The rate of increase of the viral titer slows only several weeks after infection, and HCV RNA titers decline after approximately 8–12 weeks, when the serum ALT levels peak.264 Studies performed in chimpanzees showed that the appearance of adaptive cellular immune responses and the induction of type II interferon, i.e. interferon-g, coincides with the decrease in HCV RNA titers.186,264–265 Interferon-g may have a direct antiviral effect, as it efficiently inhibits the replication of HCV replicons.166 However, the effector functions of HCV-specific T cells appear to be reduced.266 Most patients develop chronic infection with relatively stable viral titers, about 2–3 logs lower than in the acute phase. Only a small proportion of patients recover and test negative for HCV RNA using standard assays. Whether HCV is completely cleared after recovery, or whether trace amounts of virus persist, similar to hepatitis B virus, is debated.267 HCV-specific antibodies may disappear completely 10–20 years after recovery.268 Microarray analyses of serial liver biopsy samples in experimentally infected chimpanzees revealed that HCV induces the intrahepatic expression of many genes, including type I interferon, i.e. interferon-a and -b, responses.265 However, even if HCV RNA replication in vitro is efficiently inhibited by type I interferons,166 HCV seems to be resistant to these responses and frequently succeeds in establishing chronic hepatitis. As discussed above, HCV may have evolved numerous mechanisms to counteract the innate immune response, including interference with the interferon system at the induction,167–169a signaling269–271 and effector levels.131,132,272 In addition, HCV may interfere with natural killer (NK) cell functions.273,274 In this context, a recent large immunogenetic study revealed an association between a NK cell receptor (KIR2DL3
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allele)-HLA compound genotype and HCV clearance and clinical recovery, pointing toward a role of NK cells in early HCV infection.275 However, to be consistent with the repeated observation that the CTL response is less vigorous in chronically infected patients than it is during acute, self-limited infection, additional mechanisms must be involved. These may include the induction of peripheral tolerance or exhaustion of the T-cell response, infection of immunologically privileged sites, inhibition of antigen presentation, downregulation of viral gene expression, and viral mutations that abrogate, anergize or antagonize antigen recognition by virus-specific T cells.255 An impairment of dendritic cell function has been proposed,276 but this is controversial.277 There is some evidence that privileged sites may play a role, as HCV may infect extrahepatic cells and tissues. As mentioned above, the role of viral escape mutations and the quasispecies nature of HCV as a cause of viral persistence has attracted considerable interest. In this context, HCV escape to antibodies278,279 and T cells280–283 has been demonstrated both in humans and chimpanzees. The role of the humoral immune response in the natural course and pathogenesis of hepatitis C is not well understood. Recent studies using HCV pseudoparticles (see above) validated earlier studies demonstrating neutralizing of antibodies.284,285 However, the highest antibody titers are found in patients with chronic hepatitis C, and the role of antibodies capable of neutralizing a minor fraction of the HCV population is unknown.
5.
6.
7.
8.
9.
10.
11.
12.
13. 14.
15.
CONCLUSIONS AND PERSPECTIVES The development of powerful model systems has allowed us to systematically dissect important steps of the HCV life cycle. These efforts have translated into the identification of novel antiviral targets and the development of new therapeutic strategies, some of which are already in early-phase clinical evaluation. Much work remains to be done with respect to virion structure, the early and late steps of the HCV life cycle, the mechanism and regulation of RNA replication, and the pathogenesis of HCV-induced liver disease. Ultimately, a detailed understanding of the viral life cycle should result in innovative therapeutic and preventive strategies for one of the most common causes of chronic hepatitis, liver cirrhosis and HCC worldwide.
16.
17. 18.
19. 20.
21. 22.
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208. Flint M, Thomas JM, Maidens CM, et al. Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein. J Virol 1999;73:6782–6790. 209. Lozach PY, Lortat-Jacob H, De Lacroix De Lavalette A, et al. DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2. J Biol Chem 2003;278:20358–20366. 210. Lambot M, Fretier S, Op De Beeck A, et al. Reconstitution of hepatitis C virus envelope glycoproteins into liposomes as a surrogate model to study virus attachment. J Biol Chem 2002;277:20625–20630. 211. Triyatni M, Saunier B, Maruvada P, et al. Interaction of hepatitis C virus-like particles and cells: a model system for studying viral binding and entry. J Virol 2002;76:9335–9344. 212. Wellnitz S, Klumpp B, Barth H, et al. Binding of hepatitis C virus-like particles derived from infectious clone H77C to defined human cell lines. J Virol 2002;76:1181–1193. 213. Blanchard E, Brand D, Trassard S, et al. Hepatitis C virus-like particle morphogenesis. J Virol 2002;76:4073–4079. 214. Lagging LM, Meyer K, Owens RJ, Ray R. Functional role of hepatitis C virus chimeric glycoproteins in the infectivity of pseudotyped virus. J Virol 1998;72:3539–3546. 215. Matsuura Y, Tani H, Suzuki K, et al. Characterization of pseudotype VSV possessing HCV envelope proteins. Virology 2001;286:263–275. 216. Buonocore L, Blight KJ, Rice CM, Rose JK. Characterization of vesicular stomatitis virus recombinants that express and incorporate high levels of hepatitis C virus glycoproteins. J Virol 2002;76:6865–6872. 217. Bressanelli S, Stiasny K, Allison SL, et al. Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. EMBO J 2004;23:728–738. 218. Gibbons DL, Vaney MC, Roussel A, et al. Conformational change and protein–protein interactions of the fusion protein of Semliki Forest virus. Nature 2004;427:320–325. 219. Modis Y, Ogata S, Clements D, Harrison SC. Structure of the dengue virus envelope protein after membrane fusion. Nature 2004;427:313–319. 220. Egger D, Gosert R, Bienz K. Role of cellular structures in viral RNA replication. In: Semler B, Wimmer E, eds. Molecular biology of picornaviruses. Washington DC: ASM Press, 2002: 247–253. 221. Ahlquist P, Noueiry AO, Lee WM, et al. Host factors in positive-strand RNA virus genome replication. J Virol 2003;77:8181–8186. 222. Restrepo-Hartwig MA, Ahlquist P. Brome mosaic virus helicaseand polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral RNA synthesis. J Virol 1996;70:8908–8916. 223. Schaad MC, Jensen PE, Carrington JC. Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein. EMBO J 1997;16:4049–4059. 224. van der Meer Y, van Tol H, Locker JK, Snijder EJ. ORF1aencoded replicase subunits are involved in the membrane association of the arterivirus replication complex. J Virol 1998;72:6689–6698. 225. Rust RC, Landmann L, Gosert R, et al. Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex. J Virol 2001;75: 9808–9818. 226. Ritzenthaler C, Laporte C, Gaire F, et al. Grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes. J Virol 2002;76:8808–8819. 227. Mackenzie JM, Jones MK, Westaway EG. Markers for transGolgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells. J Virol 1999;73:9555–9567.
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228. Gazina EV, Mackenzie JM, Gorrell RJ, Anderson DA. Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae. J Virol 2002;76:11113–11122. 229. Krogerus C, Egger D, Samuilova O, et al. The replication complex of human parechovirus 1. J Virol 2003;77:8512–8523. 230. Miller DJ, Schwartz MD, Ahlquist P. Flock house virus RNA replicates on outer mitochondrial membranes in Drosophila cells. J Virol 2001;75:11664–11676. 231. Kujala P, Ikäheimonen A, Ehsani N, et al. Biogenesis of the Semliki Forest virus RNA replication complex. J Virol 2001;75:3873–3884. 232. Schwartz M, Chen J, Janda M, et al. A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids. Mol Cell 2002;9:505–514. 233. Wu S-X, Ahlquist P, Kaesberg P. Active complete in vitro replication of nodavirus RNA requires glycerophospholipid. Proc Natl Acad Sci USA 1992;89:11136–11140. 234. Ahola T, Lampio A, Auvinen P, Kääriäinen L. Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity. EMBO J 1999;18:3164–3172. 235. Shirota Y, Luo H, Qin W, et al. Hepatitis C virus NS5A binds RNA-dependent RNA polymerase NS5B and modulates RNAdependent RNA polymerase activity. J Biol Chem 2002;277:11149–11155. 236. Dimitrova M, Imbert I, Kieny MP, Schuster C. Protein–protein interactions between hepatitis C virus nonstructural proteins. J Virol 2003;77:5401–5414. 237. Dubuisson J, Penin F, Moradpour D. Interaction of hepatitis C virus proteins with host cell membranes and lipids. Trends Cell Biol 2002;12:517–523. 238. Moradpour D, Gosert R, Egger D, et al. Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex. Antiviral Res 2003;60:103–109. 239. Ye J, Wang C, Sumpter R Jr, et al. Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation. Proc Natl Acad Sci USA 2003;100:15865–15870. 240. Kapadia SB, Chisari FV. Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids. Proc Natl Acad Sci USA 2005;102:2561–2566. 241. Brass V, Blum HE, Moradpour D. Recent developments in target identification against hepatitis C virus. Expert Opin Ther Targets 2004;8:295–307. 242. Pawlotsky JM, McHutchison JG. Hepatitis C. Development of new drugs and clinical trials: promises and pitfalls. Hepatology 2004;39:554–567. 243. De Francesco R and Migliaccio G. Challenges and successes in developing new therapies for hepatitis C. Nature 2005;436: 953–960. 244. Trozzi C, Bartholomew L, Ceccacci A, et al. In vitro selection and characterization of hepatitis C virus serine protease variants resistant to an active-site peptide inhibitor. J Virol 2003;77:3669–3679. 245. Reiser M, Hinrichsen H, Benhamou Y, et al. Antiviral efficacy of NS3-serine protease inhibitor BILN-2061 in patients with chronic genotype 2 and 3 hepatitis C. Hepatology 2005;41:832–835. 246. Choo Q-L, Kuo G, Ralston R, et al. Vaccination of chimpanzees against infection by the hepatitis C virus. Proc Natl Acad Sci USA 1994;91:1294–1298. 247. Farci P, Alter HJ, Govindarajan S, et al. Lack of protective immunity against reinfection with hepatitis C virus. Science 1992;258:135–140. 248. Prince AM, Brotman B, Huima T, et al. Immunity in hepatitis C infection. J Infect Dis 1992;165:438–443.
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249. Lai ME, Mazzoleni AP, Argiolu F, et al. Hepatitis C virus in multiple episodes of acute hepatitis in polytransfused thalassaemic children. Lancet 1994;343:388–390. 250. Bassett SE, Guerra B, Brasky K, et al. Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection. Hepatology 2001;33:1479–1487. 251. Major ME, Mihalik K, Puig M, et al. Previously infected and recovered chimpanzees exhibit rapid responses that control hepatitis C virus replication upon rechallenge. J Virol 2002;76:6586–6595. 252. Mehta SH, Cox A, Hoover DR, et al. Protection against persistence of hepatitis C. Lancet 2002;359:1478–1483. 253. Nevens F, Roskams T, Van Vlierberghe H, et al. A pilot study of therapeutic vaccination with envelope protein E1 in 35 patients with chronic hepatitis C. Hepatology 2003;38:1290–1296. 254. Neumann AU, Lam NP, Dahari H, et al. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science 1998;282:103–107. 255. Cerny A, Chisari FV. Pathogenesis of chronic hepatitis C: immunological features of hepatic injury and viral persistence. Hepatology 1999;30:595–601. 256. Shoukry NH, Cawthon AG, Walker CM. Cell-mediated immunity and the outcome of hepatitis C virus infection. Annu Rev Microbiol 2004;58:391–424. 257. Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection. Nature Rev Immunol 2005;5:215–229. 258. Diepolder HM, Zachoval R, Hoffmann RM, et al. Possible mechanism involving T lymphocyte response to non structural protein 3 in viral clearance in acute hepatitis C virus infection. Lancet 1995;346:1006–1007. 259. Missale G, Bertoni R, Lamonaca V, et al. Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral cell-mediated immune response. J Clin Invest 1996;98:706–714. 260. Cerny A, McHutchinson JG, Pasquinelli C, et al. Cytotoxic T lymphocyte response to hepatitis C virus-derived peptides containing the HLA A2.1 binding motif. J Clin Invest 1995;95:521–530. 261. Lechner F, Wong DK, Dunbar PR, et al. Analysis of successful immune responses in persons infected with hepatitis C virus. J Exp Med 2000;191:1499–1512. 262. Thimme R, Oldach D, Chang KM, et al. Determinants of viral clearance and persistence during acute hepatitis C virus infection. J Exp Med 2001;194:1395–1406. 263. Thimme R, Bukh J, Spangenberg HC, et al. Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease. Proc Natl Acad Sci USA 2002;99:15661–15668. 264. Major ME, Dahari H, Mihalik K, et al. Hepatitis C virus kinetics and host responses associated with disease and outcome of infection in chimpanzees. Hepatology 2004;39:1709–1720. 265. Su AI, Pezacki JP, Wodicka L, et al. Genomic analysis of the host response to hepatitis C virus infection. Proc Natl Acad Sci USA 2002;99:15669–15674. 266. Appay V, Dunbar PR, Callan M, et al. Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nature Med 2002;8:379–385. 267. Pham TN, MacParland SA, Mulrooney PM, et al. Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C. J Virol 2004;78:5867–5874. 268. Takaki A, Wiese M, Maertens G, et al. Cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis C. Nature Med 2000;6:578–582.
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269. Heim MH, Moradpour D, Blum HE. Expression of hepatitis C virus proteins inhibits signal transduction through the Jak-STAT pathway. J Virol 1999;73:8469–8475. 270. Blindenbacher A, Duong FH, Hunziker L, et al. Expression of hepatitis c virus proteins inhibits interferon alpha signaling in the liver of transgenic mice. Gastroenterology 2003;124:1465–1475. 271. Duong FH, Filipowicz M, Tripodi M, et al. Hepatitis C virus inhibits interferon signaling through up-regulation of protein phosphatase 2A. Gastroenterology 2004;126:263–277. 272. Taylor DR, Shi ST, Romano PR, et al. Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science 1999;285:107–110. 273. Crotta S, Stilla A, Wack A, et al. Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein. J Exp Med 2002;195: 35–41. 274. Tseng CT, Klimpel GR. Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions. J Exp Med 2002;195:43–49. 275. Khakoo SI, Thio CL, Martin MP, et al. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science 2004;305:872–874. 276. Bain C, Fatmi A, Zoulim F, et al. Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection. Gastroenterology 2001;120:512–524. 277. Longman RS, Talal AH, Jacobson IM, et al. Presence of functional dendritic cells in patients chronically infected with hepatitis C virus. Blood 2004;103:1026–1029.
278. Shimizu YK, Hijikata M, Iwamoto A, et al. Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses. J Virol 1994;68:1494–1500. 279. Farci P, Shimoda A, Coiana A, et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science 2000;288:339–344. 280. Chang K-M, Rehermann B, McHutchison JG, et al. Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus. J Clin Invest 1997;100:2376–2385. 281. Tsai SL, Chen YM, Chen MH, et al. Hepatitis C virus variants circumventing cytotoxic T lymphocyte activity as a mechanism of chronicity. Gastroenterology 1998;115: 954–965. 282. Erickson AL, Kimura Y, Igarashi S, et al. The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes. Immunity 2001;15:883–895. 283. Seifert U, Liermann H, Racanelli V, et al. Hepatitis C virus mutation affects proteasomal epitope processing. J Clin Invest 2004;114:250–259. 284. Logvinoff C, Major ME, Oldach D, et al. Neutralizing antibody response during acute and chronic hepatitis C virus infection. Proc Natl Acad Sci USA 2004;101:10149–10154. 285. Yu MY, Bartosch B, Zhang P, et al. Neutralizing antibodies to hepatitis C virus (HCV) in immune globulins derived from anti-HCV-positive plasma. Proc Natl Acad Sci USA 2004;101:7705–7710.
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9
THE LIVER AND THE IMMUNE SYSTEM Percy A. Knolle Abbreviations APC Antigen-presenting cell IFN-g Interferon-g LSEC Liver sinusoidal endothelial cell MHC Major histocompatibility complex
NKT PGE2 TGF-b
Natural killer T lymphocytes Prostaglandin E2 Transforming growth factor-b
LIVER AND IMMUNE SYSTEM The connotation of the liver with the immune system usually comprises a number of observations, such as immune-mediated development of hepatitis during persistent viral infection, or autoimmunity on one side and the development of immune tolerance towards antigens delivered to the liver on the other. Whereas during immune-mediated damage the liver is believed to serve as a target for the immune response, the induction of immune tolerance in the liver is rather considered to be an active process attributing active immune-regulatory potential to the liver. This chapter will attempt to shed light on the molecular and cellular immune mechanisms active in the liver with respect to the induction of immunity as well as of immune tolerance.
FUNCTIONS OF THE IMMUNE SYSTEM The immune system is a remarkable defense mechanism to protect the organism against constant challenge by potentially pathogenic microorganisms. Genetically determined and acquired immunodeficiencies clearly illustrate the central role of the immune response in the survival of the organism. Thus, it is generally accepted that interaction with microorganisms has shaped the immune system in evolutionary terms. Although other roles for the immune system, such as the elimination of tumor cells, have been debated it has become clear that immune defense against pathogens must avoid attack against host antigens in order to prevent autoimmunity. However, immune tolerance towards autoantigens is an active process that requires constant ‘education’ of lymphocytes. To accomplish these complex features the immune system consists of highly specialized cell populations. In general, immune responses are shaped by the interaction between these cell populations in specific anatomic compartments. The immune system can generally be categorized into two parts: the innate immune response, which comprises cells such as NK cells, macrophages, granulocytes, dendritic cells, and soluble molecules such as complement, which display fast and antigen non-specific
TLR TNF VAP-1
Toll-like receptor Tumor necrosis factor Vascular adhesion protein 1
effector functions. The adaptive immune response is comprised of cells with a clonally restricted antigen-specific receptor, such as T and B cells. Activation of cells of the adaptive immune system requires a complex and cognate interaction with antigen-presenting cells. One of the most eminent features of the immune system is its dynamic. Being of bone marrow origin, immune cells continuously circulate through the body in order to retrieve information (antigen) and/or to exert effector function. Antigen-presenting cells, most importantly dendritic cells, patrol peripheral tissues and collect antigens. They migrate to secondary lymphoid tissue (lymph nodes) where, in a unique microenvironment, MHC-restricted presentation of antigen to naïve CD4 and CD8 T cells takes place. Depending on their activation status dendritic cells will then either induce tolerance in T cells or stimulate T-cell immunity. In recent years it has become increasingly clear that many different dendritic cell subpopulations exist.1 These subpopulations display different functional capacities, which further adds to the complex reaction pattern of the immune system. Dendritic cells have been found that continuously sample antigens from mucosal surfaces. Contrary to the belief that epithelial cells in the mucosa constitute a tight barrier that prevents (commensal) bacteria from entering the body, dendritic cells breach this barrier by forming transepithelial dendrites and continuously sample antigen and bacteria directly from the gut lumen or other mucosal surfaces.2,3 Moreover, the dendritic cell collecting antigen may not be identical to the dendritic cell that finally presents the antigen to T cells. It has become clear that there is a ‘division of labor’ among different dendritic cell subtypes. For instance, Langerhans’ cells reside in the skin, continuously collect antigen, and upon appropriate stimulation migrate into regional lymph nodes. However, it is not Langerhans’ cells that present skinderived antigen to T cells but a lymph node-resident CD8+ dendritic cell population, indicating that Langerhans’ cells transfer antigen to this apparently more specialized cell population.4 Finally, antigens may gain access directly to the organism. Following oral ingestion a rapid dissemination of antigen via the bloodstream is observed that leads to systemic activation of the immune system towards gut-derived antigens.5 Antigens may even gain access directly to lymph nodes using a conduit system that delivers them into certain anatomic compartments in the lymph node, where
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interaction with specialized dendritic cells occurs.6 All these results strongly support the notion that different dendritic cell subpopulations exist in different anatomic compartments and are shaped in their function by the unique microenvironment in order to achieve local and systemic immune surveillance. Immune tolerance in the organism is achieved via many different mechanisms. Clonal elimination of autoreactive T cells occurs early during T-cell ontogeny in the thymus. However, not all autoreactive T cells are eliminated here, and further control in the periphery is required. Many different mechanisms have been described as functioning in the maintenance of peripheral immune tolerance, such as clonal deletion, anergy and regulation/deviation. Central to all these mechanisms are antigen-presenting cells. Under immature steadystate conditions dendritic cells induce T-cell tolerance.7 It is difficult to study the mechanisms involved in tolerance induction by dendritic cells, because isolation and culture conditions modify the functional phenotype of the cells. Their capacity to induce T-cell tolerance in vivo under non-activating conditions has been clearly demonstrated.8 But what induces the switch from a tolerogenic to an immunogenic dendritic cell? Activation of the dendritic cell is the key to understanding their ability to trigger immunity. This activation can be achieved by a number of different molecular events, the most important probably being activation via CD40 ligation and via Toll-like receptors (TLR). Conserved microbial patterns are recognized by these pattern recognition receptors and initiate strong activation of the dendritic cell. This interaction appears to license the dendritic cells to educate T cells for subsequent immune effector function.9 Furthermore, CD40 ligation on dendritic cells or help from CD4 T cells seems to be critical for the induction of strong immunity by dendritic cells.10,11 Upon priming of naïve T cells in lymphatic tissue, activated antigen-specific T cells undergo clonal expansion and will eventually exit lymphoid tissue for recirculation via the bloodstream. T cells that have undergone appropriate activation display a migratory pattern different from that of naïve T cells, and will patrol peripheral tissues where they exert an effector function after antigenspecific stimulation. Given the ubiquitous distribution of immune cells, one would assume that immune reactions occur in a similar fashion regardless of anatomic localization. However, a wealth of experimental data rather supports the contrary: immune responses are strongly influenced by the local microenvironment.
FUNCTIONAL HEPATIC ANATOMY The liver is optimally structured to function as a metabolic organ, i.e. in the clearance of blood from macromolecules and the release of metabolic products from hepatocytes into the bloodstream. Blood from the gastrointestinal tract rich in nutrients and in microbial degradation products enters the liver via the portal vein, which drains after extensive ramifications into the so-called portal field, which is comprised of one portal venous vessel, one arterial vessel and a bile duct surrounded by connective tissue. Portovenous and arterial blood both drain into the hepatic sinusoids, which form a three-dimensional meshwork of vessels generating a mixed arteriovenous perfusion of the liver. This generates a microenvironment with a low oxygen pressure, and metabolically active hepatocytes
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have adapted to this unique situation. It is assumed that the hepatic microenvironment is further characterized by the presence of gutderived molecules, for example microbial degradation products such as endotoxin.12
HEPATIC CELL POPULATIONS: IMMUNE PHENOTYPE AND FUNCTION SINUSOIDAL CELL POPULATIONS Although hepatic sinusoidal cell populations (Kupffer cells, LSEC and stellate cells) contribute only to 6.3% of total liver volume they represent approximately 40% of the total number of hepatic cells, 26% of total membrane surface (mainly LSEC), 58% of total endocytotic vesicles (mainly LSEC) and 43% of total lysosomal volume (mainly Kupffer cells and LSEC) (Table 9-1).13
Kupffer Cells The hepatic macrophage population is named after the scientist von Kupffer. Kupffer cells are located predominantly in the periportal area.14 They are of bone marrow origin, as has been shown by the detection of recipient-derived macrophages in hepatic allografts.15 The lifespan of Kupffer cells appears to be more than 3 months.16 Under certain conditions proliferation of Kupffer cells is observed and can account for bone marrow-independent amplification of Kupffer cells.17 Depletion of Kupffer cells can be achieved through the application of gadolinium chloride or liposome-encapsulated chlodronate.18 Kupffer cell depletion does not by itself lead to liver damage, but affects certain hepatic immune functions (see below). Kupffer cells have three complex functions: (1) phagocytosis of particulate matter and uptake of macromolecules; (2) presentation of antigen; and (3) the release of soluble mediators. Together with the liver sinusoidal endothelial cells they form the reticuloendothelial cell system of the liver. They are efficient in phagocytosis of particulate matter and uptake of macromolecules via receptormediated endocytosis. Elimination of bacterial degradation products such as LPS is to a large extent achieved by Kupffer cells.19 Kupffer cells further contribute to the elimination of tumor cells,20 apoptotic cellular material21 and bacterial degradation products.22 As Kupffer cells are situated mainly in the periportal area, clearance of blood is achieved soon after entry into the hepatic microcirculation.23 The localization of Kupffer cells correlates with their function.24 Periportal Kupffer cells display high phagocytic capacity but
Table 9-1. Hepatic Cell Populations Hepatic cell population
Percent of liver volume*
Percent of liver cells
Kupffer cells LSEC Stellate cells Pit cells Hepatocytes
2.1 2.8 1.4 n.d. 78
15 19 5–8 n.d. 60
* Sinusoidal lumen 10.6%, space of Dissé 4.9%. (Adapted from 13)
Chapter 9 THE LIVER AND THE IMMUNE SYSTEM
Table 9-2. Soluble Mediators Released by Kupffer Cells Molecule
Function
Prostanoids IL-1 IL-1RA IL-6 IL-10 IL-12 IL-18 TNF-a TGF-b1 and TGF-b2 ROI NO
Modulation of immune function Inflammation Blockade of IL-1 activity Inflammation Anti-inflammatory activity Induction of IFN-g, immunity Induction of IFN-g, immunity Inflammation Fibrosis, anti-inflammatory activity Effector function, inflammation Effector function, vasorelaxation
Table 9-3. Phenotype of Kupffer Cells Reference 42 43 44 37, 45 36 46 47 48 49, 50
Molecule
Function
CD54 (ICAM) Fc-receptor
Cell adhesion Uptake of pathogens coated with antibodies Receptors for endotoxin Cell adhesion Co-stimulation Co-stimulation Co-stimulation Antigen presentation
CD14, TLR4, MD2 CD11a / CD11b CD40 CD80 CD86 MHC II
Reference 53 27 54 27, 55 56 56 57 56 57 27
51 52
low expression levels of MHC class II molecules and low release of mediators. In contrast, Kupffer cells located in the perivenous area show lower phagocytic capacity but express higher levels of MHC class II molecules.25 It has been demonstrated that Kupffer cells function as antigen-presenting cells for CD4 T cells by MHC class II restricted antigen presentation.26–29 Kupffer cells are endowed with a large number of receptors (see Table 9-3) that allow them to function as sentinel cells. In response to activation Kupffer cells release soluble mediators such as IL-1 and IL-6 that trigger hepatocellular expression of acute-phase proteins.30 Further mediators released by Kupffer cells include reactive oxygen species, eicosanoids, cytokines, chemokines, proteinases, nitric oxide (NO) and hemoxygenase (Table 9-2). However, the unique hepatic microenvironment, which is rich in bacterial degradation products and gut-derived antigens, appears to shape the immune function of Kupffer cells. Although Kupffer cells resemble macrophage populations in other anatomic sites there is a clear difference between them with respect to the release of soluble mediators after contact with pathogenic microorganisms.31 As already mentioned, Kupffer cells contribute to the clearance of bloodborne endotoxin. Interestingly, different sets of receptors are involved in the clearance and sensing of endotoxin by Kupffer cells. Scavenger receptors are functional in binding and endocytosis of endotoxin, whereas expression of the pattern recognition receptors CD14 and TLR4, in combination with the adaptor protein MD2, contributes to endotoxin-triggered Kupffer cell activation.22 In contrast to macrophages obtained from the peritoneal cavity, CD14 is not required for Kupffer cell activation by endotoxin.32 As portal venous blood contains bacterial degradation products,12 it seems plausible that protective mechanisms have evolved to prevent the inadvertent activation of immune reactions while conserving scavenger activity. In this respect, Kupffer cell activity towards endotoxin seems to be restricted. The high hepatic arginase activity that results in low arginin concentrations locally in the liver appears to limit Kupffer cell reactivity towards endotoxin.33 Furthermore, levels of reactive oxygen intermediates released by Kupffer cells, in contrast to macrophages derived from other locations, are rather low.34 In addition, Kupffer cells develop a refractory state after repetitive stimulation with endotoxin. After a first contact with endotoxin, the release of soluble mediators such as TNF-a by Kupffer cells is dramatically decreased.35 At the same time scavenger
activity, as determined by increased phagocytosis, is increased.35 These results demonstrate that local populations of innate immune cells have adapted their function to physiological needs. Further mechanisms may operate to restrict Kupffer cell reactivity to endotoxin. Kupffer cells release IL-10 after contact with endotoxin.36 IL10 is known to have anti-inflammatory effects, and the expression of IL-10 in Kupffer cells indeed controls reactivity to subsequent stimulations with endotoxin.37 The expression of IL-10 thus can be considered to function as a negative autoregulatory feedback loop. It is interesting to note that activation of Kupffer cells is not only achieved by direct contact with microbial products but may occur via the sympathetic nervous system, resulting in fast release of IL10.38 There is an increasing wealth of data suggesting a contribution of the autonomic nervous system to the control of immune responses in the liver. Adrenergic innervation appears to downregulate inflammatory immune responses in the liver, whereas peptidergic innervation aggravates immune-mediated liver injury.39 Whereas inadvertent reactivity of Kupffer cells to inflammatory stimuli appears to be controlled at multiple levels, their effector function is critically linked to activation via TLR, because their inability to react to TLR-4 ligands leads to failure to eliminate Gramnegative bacteria from the liver.40 However, Kupffer cells alone do not achieve elimination of bacteria, but require cooperation from neutrophils for efficient elimination of pathogens (see below). Kupffer cells may further engage in amplification of cell-mediated immune responses in the liver leading to organ pathology. Such a role has been described for ischemia–reperfusion injury, alcoholinduced liver injury and neutrophil-induced liver injury. Central to the deleterious function of Kupffer cells is the release of TNF-a, which exerts deleterious affects in the liver via TNF receptor 2 expressed on parenchymal cells.41 Clearly, the expression and release of proinflammatory mediators in the liver must be precisely controlled to avoid unnecessary damage secondary to immune activation.
NKT Cells The liver harbors a large number of lymphocytes that share the characteristics of T cells and NK cells. Characteristically, NKT cells express a T-cell receptor and a prototypical NK cell marker of the C type II lectin superfamily, i.e. NK1.1. Most NKT cells express an invariant T-cell receptor, Va14/Ja281 in the mouse and Va24/JaQ in humans, together with a skewed repertoire of TCRb chains, Vb8.2 in the mouse and Vb11 in humans.58 However, NK1.1+
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Table 9-4. Different Populations of NKT Cell
Repertoire Vb8.2 Vb diverse Vb diverse Phenotype NK receptor NK1.1 NK1.1+/Restriction And others MHC II Reactivity
I
II
Va14–Va18 Va3.2–Ja9/Va8Vb8 Va diverse
Va diverse
III
IV
CD4+ or CD8+
CD4+ or DN NK1.1 DX5
CD4+ or DN NK1.1+/-
CD8+, CD4+ or DN DX5+/-
CD1d MHC I
CD1d
MHC I
a-GalCer
Not determined
Self-agonist
Not determined
DN, double negative. (Adapted from58)
TCR+ cells are heterogeneous and can be classified according to their surface phenotype and their requirement for antigen-specific stimulation (Table 9-4). The classic NKT cells are CD1d-restricted but their natural ligands have not so far been identified. Most classic NKT cells respond to CD1d-restricted presentation of a-GalCer with fast release of soluble mediators. Most NKT cells develop in the thymus: only certain subpopulations appear to originate from other sites.59 Although the natural ligands for NKT cells are not known and their skewed expression of TCR genes imply a rather narrow ligand specificity, other ligands have been detected.60 The distinct properties of glycosphingolipid antigen recognition by classic NKT cells further suggests high ligand specificity.61 In contrast to conventional T cells, NKT cells are more numerous in the liver than in other organs. Expression of CD54 and CD11a is required for homing of NKT cells to the liver.62,63 For survival and expansion of NKT cells in the liver one single cytokine, IL-15, is most important. Interestingly, migration or retention of NKT cells to other organs is independent of CD11a.63 The hepatic cell population most important for recruitment of NKT cells is NK cells.64 In turn, Kupffer cells are operative in the recruitment of NK cells65 and dendritic cells66 to the liver. The molecular mechanisms underlying recruitment of monocytes and hepatic differentiation of monocytes into organ-resident Kupffer cells are still not entirely clear. After stimulation in vivo NKT cells undergo a wave of proliferation in the liver, spleen and bone marrow, accompanied by sustained and fast release of cytokines. Depending on the subtype, NKT cells may release IFN-g, TNF-a and/or IL-4. It has been assumed that release of these mediators shifts T helper immune responses into either the Th1 or the Th2 direction.58 Moreover, a contribution of NKT cells in many experimental disease models has been described, which suggests a role in the elimination of tumors, control of infection, mediation of autoimmunity, maintenance of tolerance and others.58 It is likely that the diverse subpopulations of NKT cells and the organ microenvironment influencing NKT cell function all contribute to modulation of local immune responses and are thus responsible for the observation of diverse functional phenotypes of NKT cell.
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Table 9-5. Expression of Receptors Involved in Receptor-Mediated Endocytosis Receptors expressed on LSEC
Ligands
Hyaluronan/ scavenger receptor Mannose receptor
Hyaluronan, oxidized LDL, advanced glycation end products, aminoterminal propeptide of type I and III collagen Lysosomal enzymes, tissue plasminogen acivator, carboxyterminal propeptides, mannose-containing structures Collagen a chain
76–79
Immunoglobulins
82, 83
Collagen a-chain receptor Fc-receptor (FcgR, FcRn)
Reference
80
81
Liver Sinusoidal Endothelial Cells (LSEC) LSEC form a thin but continuous layer between leukocytes passing the liver in the bloodstream from hepatocytes.67 In contrast to endothelial cells in other organs, they do not express tight junctions and are not separated from parenchymal tissue by a basement membrane. The space between hepatocytes and LSEC is called the space of Dissé, which contains abundant extracellular matrix produced by LSEC and is populated by the stellate cells that span the LSEC and control sinusoidal blood flow by contraction, leading to a reduction in sinusoidal diameter.68 LSEC possess enormous endocytotic capacity. Uptake of macromolecules from the blood is achieved mainly by receptor-mediated endocytosis and not pinocytosis or macropinocytosis in these cells. Via receptor-mediated binding LSEC immobilize particulate material but are unable to internalize molecules larger than 200 nm.69 The receptors active in endocytotic activity of LSEC and their main ligands are summarized in Table 95. The wide ligand range of these receptors ensures the effectiveness of LSEC scavenger function.70 This scavenger function is not observed in endothelial cells from other vascular beds and emphasizes the notion that unique local cell populations define liver
Chapter 9 THE LIVER AND THE IMMUNE SYSTEM
function.70 Scavenged molecules are quickly degraded by LSEC or are transported across the cell to neighboring hepatocytes in a transcytotic fashion.71,72 The molecular mechanisms determining lysosomal degradation or transcytosis in LSEC have not yet been identified. The scavenger function of LSEC may further be operative in the hepatotropism observed for certain viruses. Uptake of bloodborne virus by LSEC has been demonstrated for duck hepatitis B virus73 and for hepatitis C virus.74 LSEC express receptors for these viruses. In analogy to what has been observed for human immunodeficiency virus (HIV) binding to dendritic cells through DC-SIGN,75 binding of hepatotropic viruses to receptors on scavenger LSEC may target the virus to the liver and eventually lead to infection of hepatocytes in trans. LSEC have pores – so-called fenestrae – approximately 100– 150 nm in size67 which can be dynamically regulated by the actin cytoskeleton upon contact with substances such as alcohol or nicotine.84 Blood cells passing through the narrow hepatic sinusoids exert a ‘sinusoidal massage’, causing improved exchange of fluid between the sinusoidal lumen and the space of Dissé.67 Flexible macromolecules larger than 100 nm in diameter or rigid macromolecules larger than 12 nm are excluded from access to the space of Dissé via diffusion through the fenestrae, resulting in a ‘sieve’ function of LSEC.67 Larger molecules, such as chylomicrons exceeding 100 nm in size, must be metabolized by membrane-associated lipase85 before they can pass through fenestrae.86 Interestingly, the infection rate of hepatocytes by bloodborne adenovirus has been demonstrated to depend critically on the diameter of LSEC fenestrae.87 Alternatively, molecules may gain access to hepatocytes through receptormediated uptake by LSEC and subsequent transcytosis (see also above).88 LSEC constitutively express the molecules necessary to establish interaction with passenger leukocytes. Expression levels of CD54 and CD106 are linked to bacterial colonization of the gut, supporting the notion that bacterial degradation products derived from the gastrointestinal tract shape the hepatic microenvironment. LSEC further express two molecules that support the adhesion of leukocytes, i.e. VAP-1 and L-SIGN.89,90 These molecules are expressed in the liver on LSEC and in lymphatic tissue. MHC class I and low levels of MHC class II molecules are constitutively expressed, together with low levels of co-stimulatory molecules (CD80, CD86 and CD40).57
Dendritic Cells Dendritic cells are the prototypic antigen-presenting cells of the immune system. Depending on their maturation status (either immature, semi-mature or mature), they are considered to shape the immune response in the direction of either immune tolerance or immunity.1,91,92 Dendritic cells are found in the liver under normal steady-state conditions. They are found primarily in the portal tract, but are present in lower numbers in the periportal and perivenous areas.93 As the liver is a strongly vascularized organ, immune surveillance here by dendritic cells is different from immune surveillance in other sites such as the skin or the gut. Dendritic cells arrive in the liver via the bloodstream, and as a consequence of the slow blood flow within hepatic sinusoids have the opportunity to interact with sinusoidal lining cells. Within the liver dendritic cells translocate from hepatic sinusoids to the lymph, and
finally accumulate in draining celiac lymph nodes.94 The transition of dendritic cells from the bloodstream into adherent cells within the hepatic sinusoid appears to be accompanied by changes in their ultrastructural characteristics, which is suggestive of a maturation step.95 It is unclear whether changes in dendritic cell ultrastructure are paralleled by functional maturation. However, after transition from the hepatic sinusoid dendritic cells lose their phagocytic capacity. The preferential accumulation of circulating dendritic cells in the liver implies that these cells will engage in antigen collection within the liver. Indeed, after translocation from the hepatic sinusoid into the space of Dissé dendritic cells are observed to engage in close physical contact with hepatocytes and stellate cells.95 Although the total number of dendritic cells in liver is high compared to that of other parenchymal organs, their relative density in the liver is much lower.96 However, a fast turnover of dendritic cells may compensate for lower numbers. As already mentioned, constant recruitment of dendritic cells from the marginating blood pool may result in different migration kinetics of dendritic cells in the tissue. The composition of dendritic cell subtypes in the liver appears to differ from those in other organs such as the spleen.97 The liver bears more plasmacytoid dendritic cells, characterized by CD8a B220+ expression, than the spleen. However, it seems difficult to attribute functional capacity to dendritic cells purely on the basis of their phenotypic characteristics. It has become clear that subpopulations of dendritic cells are shaped by external factors, and changes in the pattern of surface molecule expression may in fact reflect local influences rather than the recruitment of lineage-dependent subpopulations of cells. A number of investigations have been performed that yielded extensive information on the phenotype of hepatic dendritic cells.97–99 A common result is that hepatic dendritic cells are rather immature compared to dendritic cells from other organs. In addition to the different distribution of dendritic cell subpopulations, hepatic dendritic cells show distinct immune-regulatory features.98 Dendritic cells isolated from liver show a reduced capacity to prime naïve allogeneic T cells compared to dendritic cells isolated from bone marrow. T cells primed by hepatic dendritic cells do not develop strong cytotoxic effector function.100 Furthermore, adoptive transfer of these in vitro-propagated hepatic dendritic cells leads to increased expression of IL-10 in lymphatic tissue.100 Similar results were obtained by a number of other investigators.97,99,101 Using a new technique to obtain dendritic cells from human liver, it was possible to investigate the immune function of human hepatic dendritic cells. These cells were less effective in T-cell stimulation than were dendritic cells isolated from the skin. Moreover, hepatic dendritic cells expressed significant amounts of IL-10, a cytokine known to mediate potent anti-inflammatory action.102 T cells primed by hepatic dendritic cells release IL-10 and IL-4 but no IFNg, which is suggestive of their ability to induce regulatory T cells. Because dendritic cells derived from other organs show different functional features it is most likely that dendritic cells entering the liver were modified by the microenvironment. Increasing the numbers of dendritic cells and NK cells by injection of flt3-ligand leads to modification of intrahepatic immune regulation. Liver transplants are typically well tolerated in rodent models of allotransplantation. However, if livers from flt3-ligand-treated animals were transplanted increased transplant rejection was observed, which
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suggests that flt3-ligand-induced changes in hepatic cell populations tip the balance from a tolerogenic to an immunogenic milieu.103 Another important feature of hepatic dendritic cells is their low expression of TLR4. Stimulation of hepatic dendritic cells with low concentrations of endotoxin does not lead to strong activation and induction of a mature phenotype, but rather results in a reduced capacity to induce T-cell priming and proliferation compared to dendritic cells isolated from spleen.104 Given the continuous presence of bacterial degradation products in portal venous blood, it seems important to prevent inadvertent immune activation locally in the liver. Dendritic cells in diseased liver are mainly found in the portal tract. Hepatic expression of the chemokine CCL21 stimulates increased recruitment of CCR7+ immune cells to the portal tract, which resembles tertiary lymphoid tissue.105 CCL21 recruits naïve T cells and dendritic cells, which both bear CCR7+, and thus promotes strong local interaction between these cell populations,106 similar to the role of CCR7 for recruitment of immune cells to lymphatic tissue.107 So far, little is known about the ability of migratory hepatic dendritic cells to stimulate T cells in the draining lymph node. It is interesting to note that the composition of subpopulations of dendritic cells in the hepatic draining lymph node does not reflect the subpopulation composition in the liver.108 In particular, dendritic cells in the draining hepatic lymph node had an activated mature phenotype, and only few cells had the surface phenotype characteristic of plasmacytoid dendritic cells.108
Stellate Cells Stellate cells are located in the space of Dissé between LSEC and hepatocytes. Stellate cells store 80% of retinoids in the body as retinyl palmitate within cytoplasmic vesicles. Following hepatic injury, stellate cells transdifferentiate into cells similar to myofibroblasts, with a profibrogenic phenotype depositing large amounts of extracellular matrix.109 They are further involved in regulating intrahepatic vascular resistance in response to a number of vasoactive substances.110 Although stellate cells may not have direct contact with passenger leukocytes in the sinusoid, they release a number of mediators that are important for local immune control. Several chemokines, such as MCP-1, MIP-2 and IL-8, are produced by stellate cells upon activation by proinflammatory cytokines.111 However, under resting conditions few if any chemokines are expressed by these cells, suggesting that they contribute only in inflammatory situations to the recruitment of leukocytes from the blood into the hepatic parenchyma. In addition to their important scaffolding function, stellate cells have the capacity to take up antigens by fluid-phase endocytosis, receptor-mediated endocytosis and phagocytosis.112 They further express low levels of MHC class I and II molecules and co-stimulatory molecules (CD80 and CD40), which are further increased following incubation with proinflammatory mediators (Table 9-6). Whereas resting stellate cells fail to engage in cognate interaction with naïve T cells, cytokine-stimulated stellate cells support the proliferation of CD4 T cells in a mixed lymphocyte reaction.112 In conclusion, stellate cells do not function as full antigen-presenting cells but can support ongoing inflammatory reactions by inducing T-cell stimulation and proliferation. Similar to Kupffer cells and LSEC, stellate cells express TLR4 and are thus responsive to stimulation by endotoxin, leading to
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Table 9-6. Immune Phenotype of Stellate Cells Surface molecule
Expressed in resting/activated stellate cells
Reference
CD54 (ICAM-1) CD106 (VCAM-1) CD40 CD80 MHC class I MHC class I NCAM
-/+ -/+ (+)/+ -/+ +/++ (+)/+ +/++
113, 116 113, 117 112 112 112 112 118
the release of chemokines.113 However, upon activation stellate cells also release potent anti-inflammatory mediators. Stellate cells treated in vitro with TNF-a or endotoxin or isolated from animals after bile duct ligation demonstrated a prominent increase in IL-10 release.114 Moreover, stellate cell activation results in the expression of transforming growth factor b (TGF-b), one of the most potent anti-inflammatory cytokines.49 Latent TGF-b-binding protein, expressed by trans-differentiating stellate cells, serves as a matrix to anchor latent TGF-b in hepatic tissue. The release of bioactive TGF-b then requires proteolytic cleavage of the binding protein.115
HEPATOCYTES Hepatocytes constitute the vast majority of hepatic cells. They are situated behind a physical barrier constituted by LSEC and stellate cells. However, a large body of evidence suggests that hepatocytes have direct access to molecules contained in portal venous blood as well as to passenger leukocytes in the hepatic sinusoid. In normal liver hepatocytes express few MHC I molecules and are negative for MHCI II. Other molecules relevant for interaction with T cells are either not expressed or expressed at very low levels, e.g. CD54, or co-stimulatory molecules such as CD80 and CD86. The main function of hepatocytes is metabolism. Consequently, enormous amounts of molecules are generated within hepatocytes and are subsequently released into serum or bile. Degradation of waste or toxic products also occurs in hepatocytes and gives rise to modifications in the structure of protein antigens released by hepatocytes. Furthermore, hepatocytes metabolize nutrients extracted from portal venous blood. Given the huge metabolic function of hepatocytes, it seems plausible that mechanisms have evolved to protect these cells from inadvertent immune attack.
Characteristics of Blood Flow and Leukocyte–Liver Cell Interaction in the Liver The liver holds a unique position with regard to the blood circulation. It receives venous blood draining from almost the entire gastrointestinal tract via the portal vein, and from the systemic circulation via the hepatic artery. More than 2000 l of blood stream daily through the human liver. Even with an average circulation time of 1 hour, peripheral blood leukocytes pass through the liver more than 12 times per day. These simple facts clearly demonstrate that the liver is a ‘meeting-point’ for antigens and leukocytes circulating in the blood. Hepatic sinusoids are narrow channels with an average diameter ranging from 7 to 12 mm. Leukocytes, having a mean
Chapter 9 THE LIVER AND THE IMMUNE SYSTEM
diameter of 10–12 mm, have to force their way through the sinusoidal meshwork in the liver.119 Moreover, blood flow in the liver is slow because of low-pressure perfusion. Interaction between passenger cells flowing through hepatic sinusoids and the sinusoidal cell populations is facilitated by these physical conditions.120 Expression of selectins by endothelial cells is typically required to engage in interaction with leukocytes in the bloodstream. Hepatic sinusoidal cell populations express little if any CD62E (E-selectin),121 which is instrumental in the recruitment of leukocytes to endothelial cells. Despite the absence of CD62E, recruitment of leukocytes to sinusoidal cells can be observed even under inflammatory conditions.122 LSEC rather express other molecules that mediate interaction with passenger leukocytes, notably L-SIGN90 and VAP-1.89 Blood flow in the liver is intermittent as a result of interaction of passenger leukocytes with sinusoidal cell populations.123 Kupffer cells have been reported to patrol the sinusoids at low speed and to temporarily block sinusoidal perfusion.124 This further facilitates the interaction between passenger leukocytes and sinusoidal cell populations. Given the large cumulative surface of LSEC and the high numbers of Kupffer cells, interaction of passenger leukocytes with these cell populations is most likely. Contact with other hepatic cell populations, such as stellate cells, hepatocytes or dendritic cells, is not excluded but is likely to occur at much lower frequencies.
IMMUNE FUNCTIONS OF THE LIVER Immune Tolerance in the Liver Among the many functions of the liver, clearance of the blood from macromolecules and its metabolization are important for the understanding of the liver as an immune-regulatory organ. Nutrients have to be extracted from portal venous blood and further used for hepatocellular metabolism, but at the same time the liver must eliminate toxic waste products and proinflammatory agents, such as endotoxin or other bacterial degradation products derived by translocation from the gut, from blood without eliciting an immune response to all these antigens. Induction of immune tolerance in the liver was reported in 1967 by Cantor et al. and in 1969 by Calne et al.,125,126 and since then by many other groups. Three main points demonstrate the ability of the liver to induce antigen-specific immune tolerance. 1. Liver transplants are accepted by the recipient’s immune system despite MHC discrepancy and even in the absence of immune suppression.125,126 2. Simultaneous transplantation of the liver and another organ from the same donor leads to increased graft acceptance of the cotransplanted organ. Further transplants from another donor led to graft rejection, demonstrating antigen-specific induction of immune tolerance by the transplanted liver.127 3. Drainage of a transplant directly into the portal vein, or the direct application of donor cells into the portal vein, led to increased acceptance of the graft.128–131 Although for a long time antigen-specific induction of immune tolerance in the liver was observed in the context of organ transplantation, it is clear from the physiological function of the liver that immune tolerance needs equally to be established for circulating antigens. Although the mechanisms involved in tolerance induction
do not necessarily need to be different for antigens produced or taken up by hepatic cell populations, it is evident that immune tolerance towards circulating antigens requires the participation of cells conveying this specific information, i.e. antigen-presenting cells.
The Hepatic Microenvironment Constitutive exposure to gut-derived bacterial degradation products in portal venous blood contributes to the unique hepatic microenvironment.12,132 Endotoxin not only induces the release of proinflammatory mediators from hepatic sinusoidal cell populations, but at the same time leads to the expression of a number of potent anti-inflammatory immunosuppressive mediators such as IL-10,36 TGF-b49 and certain prostanoids, such as PGE2.133,134 This creates a local environment that suppresses rather than stimulates immune responses in the liver. IL-10, TGF-b and PGE2 are known to ‘educate’ antigen-presenting cells such as dendritic cells and to induce a tolerogenic phenotype. Dendritic cells exposed to these mediators fail to induce immunity, but rather support the induction of tolerant T cells.135 It is difficult to separate the influence of specific hepatic antigen-presenting cell populations and the hepatic microenvironment on the induction of immune tolerance from each other, as the microenvironment certainly has a role in shaping the functional phenotype of hepatic antigen-presenting cell populations.
Induction of T-Cell Tolerance by AntigenPresenting Cells in the Liver Different hepatic antigen-presenting cell populations contribute to antigen-specific induction of tolerance in T cells.136 Although the relevance of each of the antigen-presenting cell populations has been described separately, more than one such population may be involved in the induction of tolerance in the liver. In fact, a number of determinants, such as the origin and quantity of antigen, may strengthen or diminish the contribution of individual antigenpresenting cell populations to tolerance induction (see below).
Hepatocytes Hepatocytes can serve as antigen-presenting cells and induce stimulation of naïve CD8 T cells in a transgenic mouse setting, where hepatocytes express a transgenic MHC I molecule that is recognized together with an endogenous peptide. Although antigen-specific stimulation of T cells by hepatocytes even in the absence of co-stimulatory molecules CD80/CD86 is rather effective during the first 3 days, T cells undergo apoptosis at later time points. Thus, clonal elimination of T cells is involved in the mediation of hepatic T-cell tolerance. As interaction with passenger T cells and antigen-presenting hepatocytes occurs even in the absence of local inflammation in the transgenic mouse model described above, hepatocytes may continuously contribute to shaping of the immune response.137 As hepatocytes do not have the capacity to cross-present exogenous antigens on MHC I to CD8 T cells, tolerance induction is limited to proteins expressed by hepatocytes but does not extend to antigens entering the body.
Hepatic Dendritic Cells As described above, immature hepatic dendritic cells contribute to the induction of T-cell tolerance. Hepatic dendritic cells may be
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Section I. Pathophysiology of the Liver
directly involved in the induction of T-cell tolerance by virtue of their tolerogenic phenotype, which may result from different subpopulations of dendritic cells being present in the liver or from the influence of the local microenvironment.97–99,101,102,104,138 On the other hand, dendritic cells may indirectly contribute to hepatic immune tolerance via the release of soluble mediators or the induction of regulatory T cells. Release of type I IFN from dendritic cells attenuates liver injury139 and at the same time promotes the induction of regulatory T cells.140 Activated CD8 T cells are found to undergo apoptosis in the liver.141 The trapping of CD8 T cells may occur antigen specifically or through CD54/CD106-dependent mechanisms.142–144 Using bone marrow chimeric animals it was possible to demonstrate that antigen-specific recruitment of CD8 T cells was achieved by nonmyeloid organ-resident cells, whereas elimination of CD8 T cells occurred through a bone marrow-dependent cell population.142
LSEC Similar to dendritic cells, LSEC have the capacity to prime CD4+ T cells, i.e. stimulation of cytokine release from naïve CD4+ T cells that have not previously encountered their specific antigen.145 Whereas dendritic cells require maturation and signals from the highly specialized lymphatic microenvironment in order to function as potent APC for naive CD4+ T cells,146 LSEC do not require maturation or migration into lymphatic tissue in order to gain APC function. This function of LSEC as sessile, organ-specific and constitutively active antigen-presenting cells is not shared by endothelial cells from other organs. Microvascular endothelial cells from the skin or the gut are unable to act as antigen-presenting cells for naive CD4+ T cells unless stimulated by proinflammatory cytokines such as IFN-g.147–149 In contrast to antigen presentation by dendritic cells, however, CD4+ T cells stimulated by antigenpresenting LSEC fail to differentiate into effector Th1 CD4+ T cells, but rather gain an immune-regulatory phenotype.145 CD4+ T cells primed by LSEC release large amounts of IL-4 and IL-10 following triggering by the T-cell receptor,145 which efficiently downregulates ongoing T cell-mediated immune responses (P. Knolle, unpublished results). Thus, antigen presentation by LSEC to naive CD4+ T cells may rather down-regulate Th1-type cell-mediated immune responses and at the same time stimulate Th2-type immune responses, leading to increased production of antibodies. Indeed, ineffective cell-mediated immune responses despite the presence of an efficient antibody response are observed during persistent infection of the liver with non-cytopathic viruses.150 Endothelial cells from other sites equally fail to lead to the development of fully differentiated effector Th1 CD4+ T cells.149,151 It is important to note that these cells lack the capacity to actively engage in immune modulation, as either endothelial cells or T cells have to be prestimulated in order to observe functional interaction, thus requiring other cell populations that drive the developing immune response. Together with the observation that intraportal injection of antigen leads to the development of T cells that release IL-4 and IL-10 upon restimulation,152 it can be assumed that LSEC rather than endothelial cells in other organs are involved in the induction of tolerance to intraportally applied antigens. Cytotoxic CD8+ T cells are of crucial importance for a successful immune response to infection with intracellular pathogens and
156
against the development of cancer cells. Presentation of antigen on MHC class I molecules to CD8+ T cells was believed to be restricted to those antigens synthesized de novo within the same cell. Although this allows for immune surveillance of parenchymal cells by CD8+ T cells, it is difficult to envisage how professional antigen-presenting cells, not infected by the pathogenic microorganism or not transformed into neoplastic cells, could in the first place induce a protective and efficient CD8+ T cell-mediated immune response. Thus, presentation of exogenous antigens on MHC class I molecules (termed cross-presentation) is obviously required. Initially identified by Bevan et al.,153 it was recently demonstrated that cross-presentation occurs in bone marrowderived antigen-presenting cells such as dendritic cells and macrophages, and in some instances in B cells.154,155 Cross-presentation by dendritic cells was shown to be necessary in order to mount an efficient CD8+ T cell-mediated immune response against virus infection, although not all virus infections appear to require cross-presentation by myeloid APC for the induction of immunity.156 It is therefore surprising to find that LSEC can efficiently crosspresent exogenous antigens on MHC class I molecules to CD8+ T cells.157 Cross-presentation by LSEC is characterized by a number of features: efficient uptake of antigen by receptor-mediated endocytosis; shuttling of antigen from endosome to cytosol for proteasomal degradation; TAP-dependent loading of processed peptides on de novo synthesized MHC class I molecules in the ER; and transport to the cell surface.157 LSEC require only 60–120 minutes to complete cross-presentation and to express peptide-loaded MHC class I molecules on the surface. Minute amounts of antigen, i.e. in the low nM range, are sufficient for cross-presentation by LSEC, suggesting an important role of cross-presenting LSEC in the hepatic immune response.157 LSEC not only cross-present antigen to armed effector CD8+ T cells, but have in fact the capacity to stimulate naïve CD8+ T cells.157 Following an encounter with cross-presenting LSEC in vitro, naïve CD8+ T cells release cytokines and begin proliferating. However, antigen-specific restimulation of these T cells revealed that they lost the ability to express effector cytokines such as IL-2 and IFN-g and that they lost their cytotoxic activity.157 In vivo it has been demonstrated that LSEC cross-present antigen to naive CD8+ T cells outside the lymphatic system. So far, stimulation of naïve T cells was believed to occur exclusively in the highly specialized lymphatic microenvironment . Following stimulation by cross-presenting LSEC, naïve CD8+ T cells start to proliferate locally in the liver. However, the outcome of cross-presentation by LSEC in vivo is the induction of systemic immune tolerance. Similar to CD8+ T cells stimulated by crosspresenting LSEC in vitro, CD8+ T cells in vivo lose the capacity to express effector cytokines and to exert cytotoxic activity against their specific target antigens once stimulated by cross-presenting LSEC.157 Deletion of antigen-specific CD8+ T cells occurs to some extent but is not the main mechanism of immune tolerance induced by LSEC. Mice rendered tolerant by LSEC cross-presenting a model antigen fail to develop an immune response against a tumor carrying this model antigen, which constitutes the prime target of the immune response in non-tolerant littermates, leading to immunity and tumor rejection in control animals.157
Chapter 9 THE LIVER AND THE IMMUNE SYSTEM
Concept of Organ-resident Antigen-Presenting Cells in the Liver LSEC represent a new type of organ-resident APC which is organ specific. In order to establish organ-specific control of immune responses, local presentation of antigen by resident APC has a number of advantages. 1. Dendritic cells take up antigen in the peripheral organs and, after appropriate stimuli, migrate to draining lymph nodes. During this journey they undergo functional maturation, which renders them potent APC once they arrived in the highly specialized and structured microenvironment of lymphatic organs. In contrast, LSEC simultaneously perform all the salient functions of an APC, i.e. uptake, processing and presentation of antigen, without the requirement for maturation. This ensures that antigen presentation of bloodborne antigens by LSEC occurs within a short time frame. 2. Although LSEC preclude access of bloodborne antigen-specific T cells to hepatocytes presenting the cognate antigen in the absence of local inflammation,158 it has been shown that armed effector cells can gain access to hepatocytes159 once LSEC can present the cognate antigen. Depending on the presence of sufficient numbers of armed effector T cells, antigen presentation by LSEC then apparently allows for immune surveillance of the liver. 3. Continuous culture of T cells or professional APC such as dendritic cells with immune suppressive mediators such as IL-10 or TGF-b in vitro gives rise to APC that induce T-cell tolerance rather than immunity.160,161 Situated in the hepatic sinusoid, sessile LSEC are continuously exposed to the unique hepatic microenvironment, which is especially rich in immune suppressive mediators. Incorporation of signals from an organ-specific microenvironment is clearly more prominent in sessile LSEC than in conventional APC that stay only for short periods in peripheral organs before migrating into lymphatic tissue. The unique hepatic microenvironment may thus have a considerable influence on the way immune responses are modulated by sessile LSEC. 4. Systemic distribution of antigen leads to the development of immune tolerance.162,163 Given the dual function of LSEC – fast and efficient presentation of bloodborne antigens and the induction of immune tolerance – the timing and distribution of an antigen appear to critically determine the outcome of the ensuing immune response. As dendritic cells require time for migration, maturation and induction of T-cell immunity in the lymphatic system,146 tolerance induction by LSEC can occur in a much shorter time frame. Immune tolerance ensues if antigen is first presented in the liver.157,164 Given the ever-changing nature of antigens released from metabolizing hepatocytes, tolerance induction by LSEC appears a useful mechanism to prevent immune attack against innocuous antigens released from hepatocytes. However, it is possible that LSEC contribute to the persistence of viral infection in hepatocytes, as abundant viral proteins are released from infected hepatocytes and can be taken up and presented by LSEC to T cells. Local presentation of antigen by LSEC may thus constitute a mechanism to balance the immune response in the liver and protect hepatocytes from immune-mediated damage.
LSEC are ideally positioned in the hepatic sinusoid to scavenge bloodborne antigens and to present these antigens to passenger T cells. Given the large volume of blood – containing both T cells and antigens – passing daily through the liver and the large cumulative surface of LSEC, the liver sinusoid appears to be a perfect ‘meeting point’ where immune responses towards bloodborne antigens can be shaped.
Implication of the Liver in Immune Surveillance The liver may not only serve as a target for immune responses generated in lymphatic tissue but may actively contribute to the modulation of immune responses. Such an active role in the shaping of local and systemic immune responses involves the induction of both immune tolerance137,157 and immunity.165 Antigens and pathogens are not contained entirely by local immune cell populations in the skin or the gut. Following oral ingestion of antigens, such antigens can be found in the systemic circulation within minutes, and within a few hours antigen-specific systemic activation of T cells is detected.5 Within minutes after application to the skin, antigens can be found in the liver, where they lead to the induction of cytokine release from local hepatic cell populations.165 The fact that antigens as well as pathogens overcome physical barriers and the hurdles of local immune surveillance operating at external body surfaces, together with their rapid systemic dissemination, necessitates a role for the liver in the containment of immune responses towards these antigens. The clearance function of the liver, which is mainly achieved by Kupffer cells and LSEC, is important to limit the systemic dissemination of pathogens and antigens. The largest source of environmental antigens and commensal microorganisms is the gut, and subsequently portal venous blood draining from the gastrointestinal tract into the liver. In the liver, the immune system has to discriminate between nutrient or innocuous antigens and (potentially) pathogenic microorganisms. As hepatic antigen-presenting cells, i.e. Kupffer cells, dendritic cells and LSEC, take up antigens and present them to the immune system, it is unlikely that maintenance of a tolerant state towards bloodborne antigens is simply achieved by ignorance of the immune system towards these antigens. It seems rather that T cells with specificity for antigens presented locally in the liver are actively tolerized by hepatic antigen-presenting cells. Hepatic dendritic cells show considerable plasticity, as they are activated upon encounter with microorganisms and subsequently induce pathogen-specific T-cell immunity.166 In contrast, LSEC do not undergo maturation upon stimulation with proinflammatory mediators,157 which suggests that antigen presentation by these cells always leads to the induction of T-cell tolerance. Hepatic dendritic cells and LSEC may thus constitute a functional framework of local antigen-presenting cells, where both cell populations induce tolerance towards soluble bloodborne antigens in the absence of inflammatory signals, but only hepatic dendritic cells upon sensing of ‘danger’ will contribute to the induction of specific T-cell immunity. The absence of functional plasticity in LSEC may operate to protect hepatocytes from inadvertent immune responses by constantly inducing T-cell tolerance at the sinusoidal level. Achieving immunity in the liver involves several cell populations. Bacteria are not eliminated simply by phagocytic uptake through Kupffer cells, but require the recruitment of neutrophils to Kupffer cells.167 Furthermore, hepatic dendritic cells interact with NKT cells
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in order to elicit sustained release of mediators promoting the development of specific T-cell immunity.168 Complex signaling involving chemokines and cytokines between sinusoidal cell populations and passenger leukocytes, leading to hepatic recruitment of immune cells, is operative in protective immune responses against viruses infecting the liver.169,170 Furthermore, CD1d-restricted NKT cells recognize bacterial antigens on antigen-presenting cells that have ingested infectious microorganisms and release mediators promoting pathogen-specific immune responses.171 But even IL-12 released from pathogen-activated dendritic cells is sufficient to activate NKT cells in the vicinity for the sustained release of soluble mediators.172 CD1d-restricted NKT cells recognize lipid autoantigens,173 and in a so-far uncharacterized fashion may contribute to local hepatic immune tolerance. Skewing of the microenvironment towards inflammation by local release of mediators such as IL-12 from dendritic cells or Kupffer cells may represent a pivotal point in deciding whether immune tolerance is locally maintained or whether immunity is induced.172 On the other hand, interaction of dendritic cells with distinct CD1 reactive T cells leads to maturation of dendritic cells, and may therefore contribute to early polarization of the immune response into immunity or immune tolerance.173 The continuous presence of a large population of lymphocytes in the liver may thus ensure efficient immune control, but at the same time lymphocytes must be actively kept in a tolerant state during physiological situations. In this respect it is worth mentioning that immune tolerance in T cells is induced through molecular mechanisms, which suggests a temporary nature to immune tolerance.174 Immune tolerance in the liver must then be regarded as a labile equilibrium requiring constant induction of T-cell tolerance through local antigen-presenting cell populations. Failure to deliver these tolerogenic signals is assumed to result in breakdown of hepatic tolerance and local induction of immunity. Antigen presentation by hepatic antigen-presenting cells not only has consequences for local control of immune responses in the liver, but even influences systemic immunity. Induction of antigen-specific T-cell tolerance by LSEC leads to outgrowth of tumor cells expressing the antigen initially presented by LSEC to T cells.157 Adoptive transfer of hepatic dendritic cells equally modulates systemic immune responses, rather favoring the Th2 type of response.100,104 However, it is not only the type and the functional status of the antigen-presenting cell itself that determines the outcome of immune responses. The decision whether to mount immunity or tolerance towards an antigen appears to depend on the anatomic location where the antigen is first encountered by the immune system. First encounter with the antigen in the liver leads to the induction of specific T-cell tolerance, whereas first encounter in lymphatic tissue gives rise to strong immunity.164 If antigen is sequentially encountered first in lymphatic tissue and then in the liver, T cells are not tolerized but mediate strong immunity, leading to hepatic tissue damage.164 It is intriguing to speculate that such mechanisms may be operative in the immunopathogenesis of persistent liver infection with viruses that show a strong hepatotropism and therefore fail to be presented to the immune system first in lymphatic tissue. However, so far there are no definitive experimental data supporting the notion that local hepatic immune control is responsible for deviating immune responses towards infectious microorganisms or hematogenously metastasizing tumor cells.
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The liver appears not only to promote tolerance towards soluble antigens, but under certain conditions even promotes local immunity at peripheral sites. Using a model of skin contact sensitivity it was demonstrated that the liver is involved in mediating the recruitment of antigen-specific T cells to the site of initial antigen exposure, i.e. the skin. Minutes after application to the skin, antigen is found to stimulate NKT cells in the liver to release IL-4, which in turn stimulates peritoneal B1 B cells to express IgM antibodies.165 Circulating IgM antibodies form complexes with the antigen at the site of initial antigen application, and trigger local complement activation and the release of vasoactive mediators that finally lead to Tcell recruitment.175 This example nicely illustrates the extraordinary position of the liver in scavenging antigens from the systemic circulation, and its function as a sentinel organ to detect the presence of foreign antigens. Central to this complex sentinel function is the concomitant presence of scavenger activity and sufficient numbers of lymphocytes that can engage in cognate interaction with local scavenger cells. In addition to the immune-regulatory function of the liver towards bloodborne soluble antigens, and thereby control of systemic immune responses, immune tolerance needs to be achieved towards antigens endogenously expressed by hepatic parenchymal cells. In contrast to the conventional immunologic view that initiation of immune responses is restricted to lymphatic tissue, a large body of experimental evidence suggests that hepatic cell populations induce immune tolerance towards endogenously produced antigens. Hepatocytes are sufficient in presenting antigen to T cells and eliciting T-cell tolerance through clonal elimination.137,164 Biliary epithelial cells function as antigen-presenting cells and modulate T-cell responses towards endogenous antigens.176 Such a regulatory function of biliary epithelial cells and hepatocytes suggests that immune responses towards tissue antigens are further modified once T cells have gained access to the tissue. Indeed, other studies have clearly demonstrated that parenchymal cells induce tolerance under steady-state circumstances that allow tissue access to lymphocytes.158,177–179 In conclusion, the liver not only serves as a target for the immune response during infection with microorganisms, but appears to be constantly involved in the modulation of local as well as systemic immune responses towards bloodborne antigens. Central to systemic modulation of immune responses is the scavenger activity of hepatic antigen-presenting cell populations and their unique functional immune phenotype: antigen-presenting organ-resident LSEC and hepatic dendritic cells mediate the induction of T-cell tolerance rather than immunity. Parenchymal liver cells are equally involved in attenuating immune responses towards tissue-specific antigens, thus preventing autoimmune reactivity, but these cells do not engage in the induction of tolerance towards soluble antigens as they lack the ability to present soluble exogenous antigens to T cells. The liver further contributes to the induction of immunity. Phagocytic hepatic cell populations eliminate pathogens in cooperation with neutrophils. Following activation by pathogenic microorganisms hepatic dendritic cells are activated, and in this activated functional state sensitively induce pathogen-specific immunity. Moreover, the large population of unconventional T cells in the liver contributes to early signaling and sustained release of mediators following specific recognition of antigens scavenged by the various antigen-presenting cell
Chapter 9 THE LIVER AND THE IMMUNE SYSTEM
populations of the liver, and thus contribute to the decision as to whether to mount immunity or immune tolerance towards bloodborne antigens.
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promotes the development of portal-associated lymphoid tissue in chronic inflammatory liver disease. Am J Pathol 2002;160:1445. Cyster JG. Chemokines and the homing of dendritic cells to the T cell areas of lymphoid organs. J Exp Med 1999;189:447. Forster R, Schubel A, Breitfeld D, et al. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 1999;99:23. Tanis W, Mancham S, Binda R, et al. Human hepatic lymph nodes contain normal numbers of mature myeloid dendritic cells but few plasmacytoid dendritic cells. Clin Immunol 2004;110:81. Friedman SL. Hepatic stellate cells. Prog Liver Dis 1996;14:101. Reynaert H, Thompson MG, Thomas T, et al. Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension. Gut 2002;50:571. Maher JJ. Interactions between hepatic stellate cells and the immune system. Semin Liver Dis 2001;21:417. Vinas O, Bataller R, Sancho-Bru P, et al. Human hepatic stellate cells show features of antigen-presenting cells and stimulate lymphocyte proliferation. Hepatology 2003;38:919. Paik YH, Schwabe RF, Bataller R, et al. Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells. Hepatology 2003;37:1043. Wang SC, Ohata M, Schrum L, et al. Expression of interleukin10 by in vitro and in vivo activated hepatic stellate cells. J Biol Chem 1998;273:302. Breitkopf K, Lahme B, Tag CG, et al. Expression and matrix deposition of latent transforming growth factor beta binding proteins in normal and fibrotic rat liver and transdifferentiating hepatic stellate cells in culture. Hepatology 2001;33:387. Hellerbrand S, Wang C, Tsukamoto H, et al. Expression of intracellular adhesion molecule 1 by activated hepatic stellate cells. Hepatology 1996;24:670. Knittel T, Dinter C, Kobold D, et al. Expression and regulation of cell adhesion molecules by hepatic stellate cells (HSC) of rat liver: involvement of HSC in recruitment of inflammatory cells during hepatic tissue repair. Am J Pathol 1999;154:153. Knittel T, Aurisch S, Neubauer K, et al. Cell-type-specific expression of neural cell adhesion molecule (N-CAM) in Ito cells of rat liver. Up-regulation during in vitro activation and in hepatic tissue repair. Am J Pathol 1996;149:449. Knolle PA, Gerken G. Local control of the immune response in the liver. Immunol Rev 2000;174:21. McCuskey RS, Reilly FD. Hepatic microvasculature: dynamic structure and its regulation. Semin Liver Dis 1993;13:1. Essani NA, McGuire GM, Manning AM, et al. Endotoxininduced activation of the nuclear transcription factor kappa B and expression of E-selectin messenger RNA in hepatocytes, Kupffer cells, and endothelial cells in vivo. J Immunol 1996;156:2956. Wong J, Johnston B, Lee SS, et al. A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature. J Clin Invest 1997;99:2782. MacPhee PJ, Schmidt EE, Groom AC. Intermittence of blood flow in liver sinusoids, studied by high-resolution in vivo microscopy. Am J Physiol 1995;269:G692. MacPhee PJ, Schmidt EE, Groom AC. Evidence for Kupffer cell migration along liver sinusoids, from high-resolution in vivo microscopy. Am J Physiol 1992;263:G17. Calne RY. Induction of immunological tolerance by porcine liver allografts. Nature 1969;223:472. Cantor H, Dumont A. Hepatic suppression of sensitization to antigen absorbed into the portal system. Nature 1967;215:744. Dahmen U, Qian S, Rao AS, et al. Split tolerance induced by orthotopic liver transplantation in mice. Transplantation 1994;58:1.
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128. Barker CF, Corriere JN Jr. Canine renal homotransplantation with venous drainage via the portal vein. Ann Surg 1967;165:279. 129. Boeckx W, Sobis H, Lacquet A, et al. Prolongation of allogeneic heart graft survival in the rat after implantation on portal vein. Transplantation 1975;19:145. 130. Gorczynski RM, Chan Z, Chung S, et al. Prolongation of rat small bowel or renal allograft survival by pretransplant transfusion and/or by varying the route of allograft venous drainage. Transplantation 1994;58:816. 131. May AG, Bauer S, Leddy JP, et al. Survival of allografts after hepatic portal venous administration of specific transplantation antigen. Ann Surg 1969;170:824. 132. Jacob AI, Goldberg PK, Bloom N, et al. Endotoxin and bacteria in portal blood. Gastroenterology 1977;72:1268. 133. Rieder H, Ramadori G, Allmann KH, et al. Prostanoid release of cultured liver sinusoidal endothelial cells in response to endotoxin and tumor necrosis factor. Comparison with umbilical vein endothelial cells. J Hepatol 1990;11:359. 134. Kuiper J, Zijlstra FJ, Kamps JA, et al. Identification of prostaglandin D2 as the major eicosanoid from liver endothelial and Kupffer cells. Biochim Biophys Acta 1988;959:143. 135. Steinbrink K, Wolfl M, Jonuleit H, et al. Induction of tolerance by IL-10-treated dendritic cells. J Immunol 1997;59:4772. 136. Crispe IN. Hepatic T cells and liver tolerance. Nature Rev Immunol 2003;3:51–62. 137. Bertolino P, Bowen DG, McCaughan GW, et al. Antigen-specific primary activation of CD8+ T cells within the liver. J Immunol 2001;166:5430. 138. Gorczynski L, Chen Z, Hu J, et al. Evidence that an OX-2positive cell can inhibit the stimulation of type 1 cytokine production by bone marrow-derived B7-1 (and B7-2)-positive dendritic cells. J Immunol 1999;162:774. 139. Trobonjaca Z, Kroger A, Stober D, et al. Activating immunity in the liver. II, IFN-beta attenuates NK cell-dependent liver injury triggered by liver NKT cell activation. J Immunol 2002;168:3763. 140. Dikopoulos N, Bertoletti A, Kroger A, et al. Type II,FN negatively regulates CD8+ T cell responses through IL-10producing CD4+ T regulatory 1 cells. J Immunol 2005; 174:99. 141. Huang L, Soldevila G, Leeker M, et al. The liver eliminates T cells undergoing antigen-triggered apoptosis in vivo. Immunity 1994;1:741. 142. Mehal WZ, Azzaroli F, Crispe IN. Antigen presentation by liver cells controls intrahepatic T cell trapping, whereas bone marrowderived cells preferentially promote intrahepatic T cell apoptosis. J Immunol 2001;167:667. 143. Mehal WZ, Juedes AE, Crispe IN. Selective retention of activated CD8+ T cells by the normal liver. J Immunol 1999;163:3202. 144. John B, Crispe IN. Passive and active mechanisms trap activated CD8+ T cells in the liver. J Immunol 2004;172:5222. 145. Knolle PA, Schmitt E, Jin S, et al. Induction of cytokine production in naïve CD4(+) T cells by antigen-presenting murine liver sinusoidal endothelial cells but failure to induce differentiation toward Th1 cells. Gastroenterology 1999;116:1428. 146. Cella M, Sallusto F, Lanzavecchia A. Origin, maturation and antigen presenting function of dendritic cells. Curr Opin Immunol 1997;9:10. 147. Cunningham AC, Zhang JG, Moy JV, et al. A comparison of the antigen-presenting capabilities of class IIM,HC-expressing human lung epithelial and endothelial cells. Immunology 1997;91:458. 148. Haraldsen G, Sollid LM, Bakke O, et al. Major histocompatibility complex class II-dependent antigen presentation by human intestinal endothelial cells. Gastroenterology 1998;114:649.
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149. Marelli-Berg FM, Hargreaves RE, Carmichael P, et al. Major histocompatibility complex class II-expressing endothelial cells induce allospecific nonresponsiveness in naïve T cells. J Exp Med 1996;183:1603. 150. Rehermann B. Immunopathogenesis of viral hepatitis. Baillières Clin Gastroenterol 1996;10:483. 151. Ma W, Pober JS. Human endothelial cells effectively costimulate cytokine production by, but not differentiation of, naïve CD4+ T cells. J Immunol 1998;161:2158. 152. Gorczynski RM. Adoptive transfer of unresponsiveness to allogeneic skin grafts with hepatic gamma delta + T cells. Immunology 1994;81:27. 153. Bevan MJ. Interaction antigens detected by cytotoxic T cells with the major histocompatibility complex as modifier. Nature 1975;256:419. 154. Heath WR, Kurts C, Miller JF, et al. Cross-tolerance: a pathway for inducing tolerance to peripheral tissue antigens. J Exp Med 1998;187:1549. 155. Kurts C. Cross-presentation: inducing CD8 T cell immunity and tolerance. J Mol Med 2000;78:326. 156. Sigal LJ, Crotty S, Andino R, et al. Cytotoxic T-cell immunity to virus-infected non-haematopoietic cells requires presentation of exogenous antigen. Nature 1999;398:77. 157. Limmer A, Ohl J, Kurts C, et al. Efficient presentation of exogenous antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T-cell tolerance. Nature Med 2000;6:1348. 158. Limmer A, Sacher T, Alferink J, et al. Failure to induce organspecific autoimmunity by breaking of tolerance: importance of the microenvironment. Eur J Immunol 1998;28:2395. 159. Ando K, Guidotti LG, Cerny A, et al. CTL access to tissue antigen is restricted in vivo. J Immunol 1994;153:482. 160. Groux H, O’Garra A, Bigler M, et al. AC,D4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 1997;389:737. 161. Jonuleit H, Schmitt E, Steinbrink K, et al. Dendritic cells as a tool to induce anergic and regulatory T cells. Trends Immunol 2001;22:394. 162. Jacobs MJ, van den Hoek AE, van de Putte LB, et al. Anergy of antigen-specific T lymphocytes is a potent mechanism of intravenously induced tolerance. Immunology 1994;82:294. 163. Liblau RS, Tisch R, Shokat K, et al. Intravenous injection of soluble antigen induces thymic and peripheral T-cell apoptosis. Proc Natl Acad Sci USA 1996;93:3031. 164. Bowen DG, Zen M, Holz L, et al. The site of primary T cell activation is a determinant of the balance between intrahepatic tolerance and immunity. J Clin Invest 2004;114:701. 165. Campos RA, Szczepanik M, Itakura A, et al. Cutaneous immunization rapidly activates liver invariant Valpha14 NKT cells stimulating B-1 B cells to initiate T cell recruitment for elicitation of contact sensitivity. J Exp Med 2003;198:1785. 166. Johansson C, Wick MJ. Liver dendritic cells present bacterial antigens and produce cytokines upon Salmonella encounter. J Immunol 2004;172:2496. 167. Gregory SH, Wing EJ. Neutrophil-Kupffer cell interaction: a critical component of host defenses to systemic bacterial infections. J Leukocyte Biol 2002;72:239. 168. Trobonjaca Z, Leithauser F, Moller P, et al. Activating immunity in the liver. I. Liver dendritic cells (but not hepatocytes) are potent activators of IFN-gamma release by liver NKT cells. J Immunol 2001;167:1413. 169. Salazar-Mather TP, Hamilton TA, Biron CA. A chemokine-tocytokine-to-chemokine cascade critical in antiviral defense. J Clin Invest 2000;105:985. 170. Salazar-Mather TP, Lewis CA, Biron CA. Type I interferons regulate inflammatory cell trafficking and macrophage inflammatory protein 1alpha delivery to the liver. J Clin Invest 2002;110:321.
Chapter 9 THE LIVER AND THE IMMUNE SYSTEM
171. Fischer K, Scotet E, Niemeyer M, et al. Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1drestricted T cells. Proc Natl Acad Sci USA 2004;101:10685. 172. Brigl M, Bry L, Kent SC, et al. Mechanism of CD1d-restricted natural killer T cell activation during microbial infection. Nature Immunol 2003;4:1230. 173. Vincent MS, Leslie DS, Gumperz JE, et al. CD1-dependent dendritic cell instruction. Nature Immunol 2002;3:1163. 174. Mueller DL. E3 ubiquitin ligases as T cell anergy factors. Nature Immunol 2004;5:883. 175. Askenase PW, Szczepanik M, Itakura A, et al. Extravascular Tcell recruitment requires initiation begun by Valpha14+ NKT cells and B-1 B cells. Trends Immunol 2004;25:441.
176. Kamihira T, Shimoda S, Nakamura M, et al. Biliary epithelial cells regulate autoreactive T cells: implications for biliary-specific diseases. Hepatology 2005;41:151. 177. Tafuri A, Alferink J, Moller P, et al. T cell awareness of paternal alloantigens during pregnancy. Science 1995;270:630. 178. Alferink J, Tafuri A, Vestweber D, et al. Control of neonatal tolerance to tissue antigens by peripheral T cell trafficking. Science 1998;282:1338. 179. Arnold B. Parenchymal cells in immune and tolerance induction. Immunol Lett 2003;89:225.
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10
MOLECULAR PATHOGENESIS OF HEPATOCELLULAR CARCINOMA Jack R. Wands and Darius Moradpour Abbreviations AFB1 aflatoxin B1 FZD7 Frizzled-7 GBV-A GB viruses A Grb2 growth factor receptor-bound protein 2 HBV hepatitis B virus HCC hepatocellular carcinoma HCV hepatitis C virus hTERT human telomerase reverse transcriptase iNOS inducible nitric oxide synthase
IP3R1/ inositol 1,4,5-triphosphate receptor types IP3R2 1 and 2 IRAK2 IL-1R-associated kinase 2 LOH loss of heterozygosity MHBst truncated middle hepatitis B surface antigen NTRK2 neurotropic tyrosine receptor kinase 2 p42MAPK1 p42 mitogen-activated protein kinase 1 PI3K phosphoinositide 3-kinase PKC protein kinase C
INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide.1,2 The incidence ranges from fewer than 10 cases per 100 000 population in North America and western Europe to 50–150 cases per 100 000 population in parts of Africa and Asia, where HCC is responsible for a large proportion of cancer deaths. A rise in the incidence of and mortality from HCC, most likely reflecting the increased prevalence of hepatitis C virus (HCV) infection, has recently been observed in most industrialized countries.3,4 The major etiologies of HCC are now well defined and include chronic viral hepatitis B, C and D, toxins and drugs (e.g. alcohol, aflatoxins, anabolic steroids), metabolic liver diseases (e.g. hereditary hemochromatosis, a1-antitrypsin deficiency) and, to an as yet less clearly defined extent, non-alcoholic fatty liver disease. On a global scale, chronic hepatitis B virus (HBV) and HCV infection account for well over 80% of HCCs. Hepatocarcinogenesis is a multistep process involving different genetic alterations that ultimately lead to malignant transformation of the hepatocyte.5–7 However, although significant progress has been made in recognizing the sequence of events involved in other forms of cancer, notably colorectal cancer and certain hematopoietic malignancies, the molecular contribution of the multiple factors and their interactions in hepatocarcinogenesis is still poorly understood. In fact, a picture of HCCs as genetically very heterogenous tumors is emerging. This is not unexpected, given the heterogeneity of etiologic factors implicated in HCC development, the complexity of hepatocyte functions, and the late stage at which HCCs are usually detected and analyzed. As shown in Figure 10-1, malignant transformation of hepatocytes may occur regardless of the
PKR RAR-b SERCA1 SH3 SITA TRUP VEGF WHV
RNA-activated protein kinase retinoic acid receptor-b sarco/endoplasmic reticulum calcium ATPase src homology 3 a-2,3-sialyltransferase thyroid hormone uncoupling protein vascular endothelial growth factor woodchuck hepatitis virus
etiologic agent through a pathway of increased liver cell turnover, induced by chronic liver injury and regeneration in a context of inflammation and oxidative DNA damage. This may result in genetic alterations, such as the activation of cellular oncogenes; the inactivation of tumor suppressor genes, possibly in cooperation with genomic instability, including DNA mismatch repair defects and impaired chromosomal segregation; overexpression of growth and angiogenic factors; and telomerase activation. Chronic viral hepatitis, alcohol, metabolic liver diseases such as hemochromatosis and a1-antitrypsin deficiency, as well as nonalcoholic fatty liver disease, may act predominantly through this pathway of chronic liver injury, regeneration, and cirrhosis. Accordingly, the major clinical risk factor for HCC development is liver cirrhosis, as 70–90% of HCCs develop in a cirrhotic liver. The HCC risk in patients with liver cirrhosis depends on the activity, duration and etiology of the underlying liver disease. It is particularly high in cirrhosis due to chronic viral hepatitis and hemochromatosis, followed, in descending order, by alcoholic cirrhosis, autoimmune hepatitis and primary biliary cirrhosis, and it is low in Wilson’s disease. Coexistence of etiologies, e.g. HBV and HCV co-infection, HBV infection and aflatoxin B1 (AFB1),8 or HCV infection and alcohol, increases the relative risk of HCC development.9 In general, HCCs are two to four times more frequent in males than in females, and the incidence increases with age. On the other hand, there is evidence that HBV – and possibly also HCV – may under certain circumstances play an additional direct role in the molecular pathogenesis of HCC. Moreover, well defined mutations in the p53 tumor suppressor gene induced by aflatoxin exposure are a prime example of how environmental factors contribute to tumor development. Finally, advances in our understanding of the molecular genetics of HCC has led to the identification
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of signal transduction pathways that are activated during hepatic transformation.
HEPATITIS B VIRUS An estimated 300–400 million individuals worldwide are chronically infected with HBV. Prevalence rates range from 0.1–1% of the general population in North America and western Europe to up to 20% in Southeast Asia and parts of Africa. Epidemiologic studies have convincingly shown that HCC is closely associated with chronic HBV infection.10 The incidence of HCC in chronically HBVinfected individuals is approximately 100 times higher than in the uninfected population, and the lifetime HCC risk of males infected at birth is estimated to approach 40%. Importantly, a study from Taiwan demonstrated a decline in the incidence of HCC in children
HCV HBV
Growth factor activation
Alcohol Chronic injury inflammation
Metabolic disorders
Regeneration
Environmental factors (AFB)
Genetic and/ or epigenetic alterations
Hepatocellular carcinoma Figure 10-1. Factors involved in the pathogenesis of hepatocellular carcinoma.
Primary 1 2 3 kb 23 9.8 6.6
166
1
Focus 2
3
1
Nude 2
3
after the implementation of a universal hepatitis B vaccination program.11 A common molecular mechanism for HBV-induced hepatocarcinogenesis has not yet been discovered.12 Most cases of HCC occur after many years of chronic hepatitis, which could provide the mitogenic and mutagenic environment to precipitate random genetic alterations and lead to the development of HCC. In this context, using an elegant transgenic mouse model, Nakamoto et al.13 showed that chronic immune-mediated liver cell injury is sufficient to cause HCC. Inhibition of cytotoxic T lymphocyte-induced apoptosis and chronic inflammation by neutralization of Fas ligand prevented HCC development in this model.14 On the other hand, several lines of evidence support a more direct oncogenic contribution of HBV to HCC development, as summarized below.
HBV DNA INTEGRATION HBV and retroviruses share a replication strategy that includes the reverse transcription of an RNA intermediate. However, in contrast to proviral integration into the host cell genome, HBV DNA integration is not part of the viral life cycle, but rather occurs as an epiphenomenon of HBV replication. Hepadnaviral DNA integration does not preserve the viral genome sequence, thereby rendering it impossible for the viral integrant to function as a template for virus replication. Integrated HBV sequences have been found in the majority of HCCs that develop in patients with chronic HBV infection (Figure 10-2). Expectations that there might be a common target sequence in the cellular DNA led investigators to examine the viral junction and flanking cellular DNA sequences of many different HBV integration sites. These studies have shown that integration was random within human chromosomes. HBV may therefore act as a non-selective insertional mutagenic agent. In addition, secondary chromosomal rearrangements associated with HBV DNA integration, such as duplications, translocations, and deletions, suggest that a major oncogenic effect of HBV DNA integration may be an increased genomic instability. Only a few examples of HBV DNA integration within or near known cellular genes have been documented. In this regard, characterization of a single HBV DNA integration in an early HCC revealed insertion into the retinoic acid receptor-b (RAR-b) gene,
Liver 1 2
3
Figure 10-2. Southern blot analysis of DNA isolated from a HCC (Primary), a cell line derived from the same tumor (Focus), and a Focus tumor grown in a nude mouse (Nude). DNA was digested with three different restriction enzymes, electrophoresed through an agarose gel, transferred to a nylon membrane, and probed with 32P-labeled HBV DNA. Note the stable integration pattern in the primary tumor, the derived cell line, and the nude mouse tumor. No HBV DNA integration was detectable in the non-tumorous liver (Liver).
Chapter 10 MOLECULAR PATHOGENESIS OF HEPATOCELLULAR CARCINOMA
resulting in overexpression of a truncated RAR-b with altered functions.15–17 Investigation of another HBV DNA integration site led to the identification of the human cyclin A gene.18 This integration resulted in an HBV PreS2/S-cyclin A fusion protein with increased stability. Constitutive expression of this stabilized cyclin A protein may have led to or contributed to increased cell proliferation. In another HCC, HBV DNA was found integrated into the gene encoding SERCA1 (sarco/endoplasmic reticulum calcium ATPase), which plays a pivotal role in regulating intracellular calcium levels as a second messenger for cell proliferation and death signals.19,20 Viral integration has also been associated with mutations in key regulatory genes, including neurotropic tyrosine receptor kinase 2 (NTRK2), IL-1R-associated kinase 2 (IRAK2), p42 mitogen-activated protein kinase 1 (p42MAPK1), inositol 1,4,5-triphosphate receptor types 1 and 2 (IP3R1/IP3R2), a-2,3-sialyltransferase (SITA), thyroid hormone uncoupling protein (TRUP), and human telomerase reverse transcriptase (hTERT).21 These genes are involved in cell signaling, proliferation, differentiation and survival. Based on sequence analyses of these and other viral cellular DNA integration sites, it appears that HBV insertional mutagenesis provides both specific and non-specific molecular mechanisms that contribute to hepatocarcinogenesis.
TRANS-ACTIVATION OF CELLULAR GENES BY HBV Mammalian hepadnaviruses contain a gene that can function as a transcriptional trans-activator. It is called the X gene, as its role in the viral life cycle is unknown. The X gene product, referred to as HBx, is a 154 amino acid protein that is essential for productive HBV infection in vivo. It can function as a trans-activator of various cellular genes associated with growth control. This observation has led to the hypothesis that HBx may be involved in the development of HBV-associated HCC.22 A plethora of additional functions, e.g. interference with cell cycle control, DNA mismatch repair and apoptosis, as well as numerous apparently unrelated cellular interaction partners, have been reported for HBx, primarily in heterologous overexpression systems.23 However, the function of HBx in the HBV life cycle and its role, in HCC development remains elusive. The failure of HBx to bind directly to any defined DNA sequences suggests that trans-activation does not involve a known direct DNA sequence-specific interaction. The biologic role of HBx may rather be mediated through an effect on cellular transcription factors. For example, it has been reported that HBx may trans-activate via a signal transduction pathway comprising 1,2-diacylglycerol, protein kinase C (PKC), and the transcription factors AP-1 (Jun-Fos), AP-2, and NF-kB.24 Alternatively, it was postulated that HBx may promote hepatocarcinogenesis through activation of the Ras-RafMAP kinase pathway. A role of HBx in HBV-associated hepatocarcinogenesis was further supported by the observation that certain X gene-transgenic mice develop HCC.25,26 However, other investigators have not found tumors in HBx-transgenic mice. This discrepancy may be explained by differences in the level and duration of X gene expression and the genetic background of the mouse strains used in these studies. It is of interest to note that the avian hepadnaviruses lack an X open reading frame. The fact that HCCs do not arise in the context
of chronic duck or heron hepatitis B virus infection supports an oncogenic role of HBx in tumor development. Studies suggested that HBx may, like gene products of other DNA tumor viruses, interact with p53 and thereby interfere with its functions.27 The p53 tumor suppressor gene modulates gene transcription and controls apoptosis, cell cycle progression, and DNA repair. HBx may block p53-mediated apoptosis and provide a clonal selective advantage to hepatocytes.28 However, it is currently unclear whether such an interaction occurs at HBx levels expressed in hepatocytes during natural HBV infection. Thus, further studies are needed to clarify this intriguing issue. Another HBV gene product that has been reported to possess trans-activational properties is a truncated form of the PreS2/S gene, referred to as MHBst (truncated middle hepatitis B surface antigen).22,29,30 The fact that a structural viral protein gains regulatory functions following truncation is unusual. However, truncated PreS2/S sequences have frequently been found in HBV DNA integration sites in HCC.31
CHRONIC HEPADNAVIRUS INFECTION AND HEPATOCELLULAR CARCINOMA HBV is a member of the Hepadnaviridae family. These hepatotropic DNA viruses share a similar virion structure, genetic organization and replication strategy. Although the evidence for a role of HBV in the pathogenesis of human HCC is convincing, the association of woodchuck hepatitis virus (WHV) with HCC development is even stronger.32 HCCs occur almost invariably in chronically WHVinfected woodchucks.33,34 In this context, it is of considerable interest that HCC develops more frequently also in woodchucks serologically recovered from WHV infection. WHV DNA was detected in a substantial number of such tumors by Southern blot analysis.35 This is reminiscent of the increased incidence of HCC found in HBsAg-negative individuals with serological evidence of past HBV infection.36 Investigation of hepadnaviral integration sites to identify cellular genes involved in HCC development was particularly rewarding in the case of HCCs associated with chronic WHV infection. Activation of myc family oncogenes, presumably resulting from cis- and trans-acting effects of integrated WHV regulatory elements, was found in the majority of these tumors.37,38 Analysis of WHV DNA integration sites in woodchuck HCCs has led to the identification of a second intron-less N-myc gene.39 WHV DNA integration, either upstream of N-myc2 or in the 3¢ non-coding region of N-myc2, was observed in a total of 27 of 66 (41%) woodchuck HCCs investigated in three studies.39–41 Interestingly, in a significant proportion of tumors where N-myc2 expression was up-regulated in the absence of a nearby viral integration, WHV DNA integration was found in a common chromosomal site located about 200 kb downstream of N-myc2, suggesting long-range protooncogene activation by the WHV enhancer.42 There is accumulating evidence that the level of replication induced by naturally occurring mutations in the precore and core promoter regions increases the risk of HCC.43–45 Thus, a high viral replication phenotype places the infected liver at greater risk for transformation. Finally, with the development of diagnostic techniques sensitive enough to detect very low levels (3.0 mg/dl (1 point), ALT >100 IU/l (1 point), pre-TIPS encephalopathy (1 point), and urgency of TIPS (2 points). Patients were divided into three groups (low risk – 0 points, medium risk – 1–3 points, high risk – 4–5 points) and survival was significantly worse in those who were considered high risk.25 These two models and the Child–Pugh score were used to predict survival following TIPS in a subsequent study.29 The concordance statistics for each of the models are shown in Table 17-2. All of the models are good at predicting 30-day mortality following a TIPS, whereas MELD is better at predicting long-term survival. Short-term survival has also been predicted using only bilirubin, the APACHE-II score, and the need for emergency TIPS.30,31 Irrespective of which tests are used to determine mortality, the information should be used to decide whether or not the risk of death is too great to perform the procedure in any environment other than a transplant center. Also, if the patient has a poor 1-year survival prognosis they should be referred to a transplant center following completion of the TIPS.
COMPLICATIONS (Table 17-3) Dysfunction is the most common complication of TIPS, and it is this that creates the need for frequent monitoring and reintervention to maintain shunt patency. TIPS dysfunction is said to occur when there is a loss of the portal system decompression that had been originally achieved by the shunt. Most physicians feel that when the HVPG rises to above 12 mmHg or the complication for which the TIPS was originally created recurs, TIPS dysfunction is present and intervention is required.32,33 TIPS dysfunction may be due to either thrombosis or endothelial hyperplasia. Thrombosis of the TIPS may occur within the first 24 hours after creation of the TIPS, and usually within the first few weeks. Thrombosis is observed in 10–15% of cases and is thought to be due to leakage of bile into the stent, hypercoagulable syndromes, or inadequate
Frequency (%) 10–15 18–78 33 1–2 ~1 Rare 150 mmol/L), which is associated with a high mortality rate, and thus lactulose, like all drugs, has side effects that may be associated with morbidity and mortality.146
TREATMENTS THAT REDUCE AMMONIA PRODUCTION AND ABSORPTION IN THE GUT Non-absorbed disaccharides, such as lactulose or lactitol, have been tested as treatments for episodes of HE.70,72,126–128,136,137,147–156 Their mechanism of action is complex. First, they are quite effective osmotic laxatives, but the production of an acidic environment after metabolism in the colon is felt to be a crucial aspect of their efficacy in HE.157–163 This luminal acidification has been shown to trap ammonia in the colon and promote its incorporation into bacterial proteins.163 However, the acidification theory has been questioned in one study.159 The possibility that lactulose may inhibit intestinal glutaminase activity has been raised as another possible mechanism of action.164 Increased glutaminase activity in the small intestine has been noted in patients with advanced cirrhosis, possibly providing another explanation for the blood ammonia-lowering action of lactulose.165 Although these possible mechanisms of action of lactulose are interesting, the fact remains that its credentials as the standard of care in the treatment of HE are very weak (Tables 18-13 and 1814): so weak, in fact, that in this age of evidence-based medicine lactulose would be considered an unproven therapy.137 The recent systematic review of all poorly absorbed disaccharides seriously questions the overall validity of lactulose as a standard of care for HE. In the only placebo-controlled trials of lactulose in the treatment of HE there was no statistical superiority of lactulose over placebo. Somewhat larger RCTs comparing lactulose to neomycin Table 18-13. RCTs of Lactulose versus Placebo for Overt HE Study
n
Comparator
Number improved/total lactulose vs placebo Crossover no difference in efficacy 10/14 vs 7/12 Crossover no difference in efficacy 5/9 vs 6/9
Elkington147
7
Sorbitol
Simmons132 Rodgers148
26 6
Glucose Sorbitol
Germain133
18
Saccharose
Table 18-14. RCTs of Lactulose versus Treatment for Minimal HE Study
n
Comparator
Number improved/total lactulose vs no tx
Watanabe150 Li132 Phiman153
36 86 26
No Tx No Tx No Tx
10/22 vs 3/14* 26/48 vs 11/38* 8/14 vs 0/12*
*Lactulose significently better than no treatment
320
(usually with added sorbitol)126–128 have been stated to show an equivalence in efficacy of these two agents. However, virtually none of these studies had sufficient patients to demonstrate equivalence by today’s standards, as equivalence studies require far more patients than studies where one therapy is superior to another. Moreover, poorly absorbed antibiotics in some of the RCTs were more effective than lactulose.126 Exactly how lactulose came to be the standard of care is difficult to comprehend, but would appear to have been more because of its lower toxicity relative to neomycin rather than any demonstrated superior efficacy. The call for placebo-controlled trials to clarify the efficacy of lactulose (or lactitol) in the treatment of HE137 awaits the resolution of the ethical constraints in performing such trials. The solution may be to study patients with stage II HE, as has been done by a number of investigators already. This alleviates the major problems encountered with more severe HE in patients with advanced cirrhosis, where there is concern that failure to treat the HE may be associated with unacceptable risks to the patient.166
Poorly Absorbed Antibiotics Despite the lack of definite proof that neomycin was more effective than placebo, it is still used for the treatment of HE and needs to be discussed.124,125 The mechanism of action of neomycin is at least twofold. First, the antibiotic suppresses the growth of aerobic intestinal flora, which in turn reduces ammonia production in the gut.167,168 More interestingly, neomycin was noted in a number of reports to induce malabsorption and villous atrophy in the small intestine.169,170 If one proposes that neomycin actively helps HE, then a reduction of intestinal glutaminase activity may be an important mode of action of the drug. Weber et al.171 demonstrated in dogs that the small bowel contributes quantitatively more ammonia to the portal vein than does the colon. This ammonia arises primarily from mucosal glutaminase activity, and if neomycin reduces glutaminase activity this would reduce ammonia production. Also, the possibility that small bowel bacterial overgrowth in cirrhotic patients was treated by oral neomycin therapy has also been considered.172 A number of studies have been performed comparing rifaximin to either other antibiotics or lactulose/lactitol (Table 18-15). Like neomycin, rifaximin is considered non-absorbable from the intestine. However, only a tiny amount is absorbed, unlike the 3% or so of neomycin.173,174 Rifaximin has a much broader range of antibacterial action, yet has not been reported to cause fungal overgrowth.173,174 Enteric flora develop a resistance to this antibiotic by a non-plasmid mediated mechanism, but the resistance is insufficient to prevent antibacterial action by the high levels of rifaximin in the gut. A cycling system of 2 weeks on and 2 weeks off rifaximin for 6 months has been tested for long-term control of HE.175 Apparently this works well, which may be useful for long-term treatment. Most studies, however, have been shorter, and all the data have found equivalence between rifaximin and neomycin or lactulose or lactitol.129–131,176–183 One study compared rifaximin to lactitol in over 102 patients and confirmed the efficacy of rifaximin reported in earlier studies.184 Some of the design issues mentioned with other treatments of HE also apply to rifaximin. Most comparison trials have had insufficient numbers to compare equivalency with other treatments. This is a moot point, as most other therapies are of
Chapter 18 HEPATIC ENCEPHALOPATHY
Table 18-15. RCTs of Non-absorbable Disaccharide vs Antibiotics for Overt HE Study
n
Disaccharide/Other
Antibiotic/Other
Number improved/total
Conn128 Atterbury127 Orlandi126 Russo154 Blanc125 Bucci176 Fera177 Festi131 Massa178 Song179 Longuerico180 MAS184
33 47 190 15 60 58 40 21 40 64 27 103
Lact/placebo Lact/placebo Lactulose Lactulose Lactitol Lact + placebo Lact + placebo Lactulose Lact + placebo Lactitol Lact + placebo Lact + placebo
Neomycin/sorbitol Neomycin/sorbitol Neomycin/MgSo4 Ribostamycin Vancomycin Rifaximin/sorbitol Rifaximin/placebo Rifaximin Rifaximin/sorbitol Rifaximin Rifaximin/placebo Rifaximin/placebo
15/18 vs 13/15 19/23 vs 20/24 28/91 vs 34/82 7/8 vs 5/7 20/29 vs 21/31 Equivalent results 16/20 vs 20/20 Equivalent results 18/20 vs 20/20 18/25 vs 31/39 2/13 vs 8/14 41/53 vs 40/50
unproven value. At this point it is important to state that although most treatments of HE have not been proved effective in placebocontrolled RCTs, this is not to say that they are without effect. The probability is that most have some efficacy, but deficiencies in study design have led to equivocal results in many trials. A well designed placebo-controlled trial is still awaited. Other antibiotics have been used to treat HE. Metronidazole became very popular in the early 1980s as a result of a study published by Morgan et al.185 They reported a similar efficacy for metronidazole compared to neomycin despite 90% absorption of the drug in the upper intestine. In many centers metronidazole is the preferred second-line drug if lactulose in ineffective or poorly tolerated. However, like most treatments for HE it has not been compared in efficacy to placebo. Also, the number of patients in Morgan et al.’s trial was insufficient to declare equivalency of treatment effect. Nonetheless, as with lactulose, many physicians are passionate believers in metronidazole as an effective (if unproven) therapy for HE. Accumulation of metronidazole in advanced liver disease is a significant risk. CNS toxicity and peripheral neuropathy are well recognized side effects, especially if long-term (>2 weeks) therapy is used. Vancomycin was used extensively after a report by Tarao et al.186 that it had efficacy in lactulose-resistant HE. Its popularity as perhaps the third-line drug after lactulose and metronidazole faded with the advent of vancomycin-resistant enterococcus. Most physicians voluntarily ceased its use for this indication.
DIETARY PROTEIN RESTRICTION Returning to the theme of lessons learned from earlier treatment trials in HE, dietary protein restriction has an interesting history. The precipitation of overt episodes of HE by dietary protein loading was first reported in the 1950s.17,18 Many patients in subsequent RCTs had prior portosystemic shunts and were felt to be exquisitely sensitive to even normal dietary protein intake.75 Accordingly, in many trials a standard 40 g/day protein intake was employed,155,158 in addition to whatever therapies were being investigated for efficiency in the treatment of HE. Although it was not the aim of these studies, the basic concept was to establish whether agents such as neomycin or lactulose could improve oral protein tolerance in the long term. What was actually tested was the ability of these treatments to improve an episode of HE. As few placebo-controlled trials
were performed, it is difficult to gauge whether protein restriction or the active agent under test was responsible for any improvement in HE. In comparison studies, for instance, the efficacy of neomycin versus lactulose was not actually investigated.155,158 Instead, the addition of these agents to dietary protein restriction was tested in patients experiencing a bout of overt HE. Because dietary protein overload was frequently the dominant precipitating factor in these episodes,75 one can see the problems created by the use of standardized oral protein restriction for all patients. Indeed, one wonders whether the failure of agents such as neomycin or lactulose to be proved superior to placebo124,125,132,133 in RCTs was in part due to an improvement in HE in the placebo arm using oral protein restriction alone. Dietary protein restriction still is used in patients with recurrent bouts of overt HE,187 despite strong statements from experts that this mode of therapy should not be employed.188 Dietary protein restriction below the maintenance level (e.g. 0.8 g/kg/day) may in fact lead to lean body mass catabolism and may ultimately increase the nitrogen load to the systemic circulation. Patients truly found to have thresholds for the induction of HE below maintenance daily protein requirements should be treated with alternative protein sources, such as amino acid formulations.189 Vegetable protein85 or enteral branched-chain amino acid-supplemented regimens190 may permit an adequate daily intake of protein without aggravating HE. Recently a study was published showing that emergence from a bout of overt HE was not delayed if a normal protein diet was delivered in addition to standard HE therapy, and so the efficacy of such therapy in patients receiving a standard protein diet should be examined.191 Before summarizing the role of protein restriction in the treatment of HE it is worth remembering that we frequently provide minimal nutritional support to patients in the first few days of a bout of severe HE. One of the potential virtues of the branched-chain amino acid-enriched formulation was the perceived need to rapidly initiate and then maintain adequate nutritional support in such patients. The data on the efficacy of these formulations are controversial.139,192–197 Suffice it to say at this point that the data in support of these special parenteral formulations as a specific treatment for HE are not compelling. Currently, oral protein restriction is not advocated as a treatment for HE. Of necessity it occurs in the first few days of treatment of severe disease, but should virtually be
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never used in long-term care. If oral dietary protein intolerance is documented then a vegetable protein diet is recommended,198,199,200 with additional pectin or fiber.201–203 Branched-chain amino acidenriched supplements47,110,204 or formulations are still useful in highly selected protein-intolerant patients, but again at a high financial cost.205,206
DISACCHARIDASE INHIBITORS Inhibitions of disaccharidase enzyme activity in the small intestine have been reported in at least two trials to be efficacious in the treatment of HE.78,207 This approach essentially causes carbohydrate malabsorption, which in many ways works in an equivalent fashion to non-absorbable disaccharides such as lactulose. Both trials featured patients with relatively mild HE, but improvements were fairly well documented. The most recent study exclusively enrolled diabetic patients with HE and demonstrated an improvement in glycemic control as well.78 As both trials were double-blind RCTs they should be given attention, especially as they both had a placebo-controlled arm. However, as is often the case in HE trials, the characterization of the patients enrolled in the study is rather sparse (e.g., how many on therapy before enrollment, etc.). In addition, as in all studies with treatment agents that cause diarrhea, one has concerns about the blinding process. Nonetheless, we may see more advances in this form of treatment in the years to come.
PROBIOTICS As in many disciplines in medicine, there has been renewed interest in modifying the gut flora to treat HE.208–212 Early studies with Lactobacillus acidophilus appear to have been discouraging, but may have been confounded by the difficulty of maintaining this bacterium in the gut flora.213 Another study included neomycin, which might have contributed to the reported modest efficacy.214 Repeated oral administration of encoated Enterococcus SF was deemed as effective as lactulose in both short-term215 and one long-term controlled trial.216 This type of bacterium is fermentative, lactic-acid producing, urease negative, and resistant to certain antibiotics. More studies are beginning to appear in this interesting approach to HE therapy.
PROMOTION OF WASTE NITROGEN EXCRETION Hyperammonemia in patients with advanced liver disease arises by multiple mechanisms. Prevention of the generation and absorption of ammonia in the gut has been the primary focus of the commonly used treatments for HE (e.g. lactulose, neomycin etc.); however, another approach to this problem is to promote the excretion of waste nitrogen. This can be approached by enhancing what remains of the liver’s capacity for ureagenic and glutamine synthesis, or by providing substances that fix ammonia and are excreted in the urine. More drugs working along these lines are being developed. Some of the existing data will be reviewed here. L-Ornithine–L-Aspartate (LOLA) Apart from its catchy name, there are many virtues to this therapy for HE. First, its potential mechanism of action is quite well understood and it promotes ureagenesis and glutamine synthetase activity in the liver.217 There is also evidence that it promotes glutamine
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synthesis and possibly protein anabolism in skeletal muscle.218 Most importantly from the clinical perspective, this drug has been tested in both a parenteral and oral form for efficacy in HE in placebocontrolled randomized trials.142,219 The study of infused LOLA is perhaps one of the better studies to be published for decades.142 It clearly demonstrated significant improvement in stage II HE using multiple measures. Interestingly, it also demonstrated a 40% improvement rate in the placebo arm of the study. Several other trials have been performed with this agent versus placebo with, by now, a total enrollment of 370 or more patients.220 The drug has not officially been released for treatment of HE in any country, which is unfortunate given the findings in the above studies.
Sodium Benzoate Metabolic fixation of ammonia using sodium benzoate to form hippurate, originally used to promote alternative paths of waste nitrogen excretion in patients with inborn errors of urea synthesis, has been explored as a treatment for HE.221 The only published RCT compared sodium benzoate to lactulose in the treatment of overt episodes of HE.222 The two agents were judged to be equivalent in efficacy, but the study may have lacked the numbers to prove equivalency by today’s standards. Its low cost and availability in oral and parenteral form suggests that sodium benzoate should be studied further as a treatment for HE. Unfortunately, it has an unpleasant taste and its sodium content is a concern in patients with advanced liver disease. A number of physicians use this agent at a dose of 5 g orally twice daily as a second-line drug to treat HE. They obtain it from a commercial pharmacy.
CORRECTION OF NEUROTRANSMITTER ABNORMALITIES IN THE BRAIN As numerous abnormalities in brain chemistry were identified in HE primarily in animal models, therapy was designed to try and connect these derangements. Some agents showed promise, but currently none is used frequently. Consequently, even though there is a large body of information on these potential approaches to the treatment of HE only selective comments will be made.
Flumazenil The reversal of HE in an animal model without access to prescription benzodiazepine drugs was interpreted to indicate that the accumulation of endogenous benzodiazepines might be a factor in HE.50 This possibility was later examined in a series of clinical studies.223–225 The subsequent discovery of benzodiazepines detected by assays in cirrhotic patients verified to be drug free supported this hypothesis.60,73,226 Because flumazenil is a very selective benzodiazepine antagonist it was logical to perform studies to evaluate its efficacy as a treatment for HE. Table 18-16 briefly outlines the reported efficacy in patients with overt and minimal HE, respectively.52–59 Unlike non-absorbable disaccharides, the efficacy of flumazenil appeared to be far more demonstrable in overt versus minimal HE. This tends to suggest that minimal HE is not mediated to any significant degree by endogenous benzodiazepines. Although the consistent reporting of improvements in overt HE with flumazenil is encouraging, it has not translated into widespread use of this agent. There are valid reasons for this, not the least of
Chapter 18 HEPATIC ENCEPHALOPATHY
Table 18-16. RCTs of Flumazenil versus Placebo for Overt and Minimal HE
Study
n
Pomier-Layrargues53 Cadranel55 Gyr56 Barbaro52 Zhu57 Lacetti58
21 18 49 527 25 21
(A) Overt Study design Crossover Crossover Parallel Crossover Parallel Parallel
Improvement in HE Drug vs placebo 5/11 vs 0/11* 6/10 vs 0/8* 7/28 vs 0/21* 66/265 vs 9/262* 3/13 vs 0/12* 5/11 vs 0/10
*Signifies superior to placebo.
Table 18-17. Current Status of BCAA Supplementation in HE Parenteral use of branched-chain amino acid-enriched formulation is rare Enteral supplements of BCAAs in a standard diet are still used selectively In general enteral studies show benefit in terms of preventing or ameliorating HE while improving total protein intake The cost and skepticism regarding efficacy has limited use of this therapy in the USA
Table 18-18. More Common Reasons for HE Resistant to Treatment (B) Minimal
Kapczinski54 Gooday138 Amodio138 Dursun59
20 10 13 40
Crossover Crossover Crossover Parallel
No significant effect Flumazenil superior No effect 8/20 vs 0/20* Flumazenil superior ? All minimal HE
which is a lack of a long-acting oral preparation to treat patients who respond to an initial dose.8 Also, many physicians believe that the flumazenil response rate is not only modest (~30%), but may be due to occult ingestion of prescription benzodiazepines. There is good evidence against the latter, but the problem with detection assays for endogenous and pharmaceutical benzodiazepines is that both are variably detected and indeed may be identical.60 One assumes that this form of treatment will be used in selected cases in the ICU, but will not enter the regular HE treatment arena until the identity and source of endogenous benzodiazepines are clarified and a long-acting antagonist becomes available. One interesting aspect to flumazenil treatment trials in HE is that it illustrates that placebo-controlled trials can be performed in very severe HE if an ultrafast therapy (working in minutes/hours) is being tested.
Branched-Chain Amino Acid (BCAA)-Enriched Formulations This form of therapy has been subjected to detailed analysis.139,192–197 The results of these trials can be summarized by stating that the parenteral form of therapy has never been validated as a treatment for HE. In contrast, the enteral form of BCAA supplements has some reasonable evidence in its favor.205,206 Whether the gains in preventing HE occurrences are worth the cost is debatable. As mentioned earlier, vegetable protein-based diets are now employed for patients with dietary intolerance to a typical American diet. The evidence in favor of this approach is not strong either. The extensive systematic review by Ahls-Nielsen et al.139 is excellent reading for those interested in this field. Rather than trying to repeat their excellent review, the salient points are shown in Table 18-17.
Dopaminergic Agents Also the subject of a recent systematic review,140 dopaminergic agents have a very limited role to play in the management of HE. However, the data available do not preclude some benefit from this
End-stage liver disease only – still should be able to rouse Excessive purgation leading to dehydration/free water loss Failure to identify and treat sepsis Ileus, especially in association with azotemia (may need dialysis) Long-acting sedative drug intake Undiagnosed concomitant CNS problem, e.g. hypothyroidism Too effective portosystemic shunt procedure Profound zinc deficiency
form of treatment. L-Dopa and bromocriptine, the dopaminergic agents used in these trials, may have a role to play in the management of the apparently newly discovered cirrhotic patient with extrapyramidal symptoms and no overt signs of HE.79,80 It is distinctly possible that more rigorously designed studies might in future change the view that these therapies are ineffective.
Opiate Antagonists No RCTs of narcotic antagonist treatment of HE have been published. Data from animal models of HE show a reversal of disease with naloxone.66,227 Moreover, there is considerable evidence of accumulation of endogenous opiates, particularly enkephalins, in patients with advanced liver disease.228 Opiate antagonists may play a significant role in pruritus in patients with cholestasis,68,69 but the jury is still out with regard to HE. The rapidity of action of narcotic antagonists such as naloxone would make it appear to be relatively easy to perform an RCT of this type of therapy.
Zinc Repletion Two major studies have been published regarding the effect of zinc supplementation on HE,229–231 but after one encouraging report229 enthusiasm for the treatment has waned.230 In resistant cases zinc has merit as a treatment, especially if leukocyte zinc levels are low.232 A report appeared of HE that was resistant to all therapy until zinc repletion was tried.233
HE RESISTANT TO THERAPY Having discussed most of the agents used over the years to treat HE, a few comments about resistant HE are in order (Table 18-18). It is actually very rare to be unable to rouse a patient from severe HE, no matter how bad their liver function has become. It may be difficult to keep patients out of overt bouts of HE, but at least temporary arousal should be achievable with one or more of the
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treatments outlined in the previous sections. If no arousal is seen a number of explanations should be considered. The first and perhaps most common contributory factors to resistant HE are inadvertent dehydration and untreated sepsis. The former has already been mentioned and is a particular problem in patients with advanced liver disease. If serum sodium levels are in the high normal to above normal range the patient may be dehydrated, leading to resistant HE. Intravenous D5W will generally rouse these patients. Untreated sepsis prevents emergence from severe HE and should be vigorously sought in all patients with severe HE who do not arouse after 3–4 days of treatment. The combination of renal failure and ileus, together with severe spontaneous bacterial peritonitis, often results in HE resistant to therapy. Indeed, little other than enemas can be given in this situation. Hemofiltration may be needed to control the situation until bowel function returns. Occasionally long-lasting metabolites of sedative drugs can lead to protracted HE. Drug screens can help in identifying this population, even though we still cannot distinguish between endogenous and pharmaceutical benzodiazepines. Coincidental hypoadrenalism and hypothyroidism can give the appearance of resistant HE and need to be uncovered before liver transplantation is attempted. Sustained HE after a portosystemic shunt can be improved by measures that reduce the shunt size or flow. Finally, severe zinc deficiency has been reported to cause resistant HE.233 Small bowel loss of zinc can occur with excessive purgation.
LIVER SUPPORT SYSTEMS The quest for liver support systems that either stabilize or correct some of the manifestations of liver failure continues.234 Most of the literature on this topic features uncontrolled trials, which initially show promise. Subsequently, the support systems often disappear from view or fail to perform satisfactorily in an RCT.235–242 The molecular absorbent recirculating system (MARS) seems to have broken this trend, with the report of Heeman et al.,235 who reported apparent benefit using this device in patients with acute or chronic liver failure. The study was terminated prematurely because the MARS-treated group were doing ‘too well’ and thus the study was underpowered. Nonetheless, it represents a positive outcome in an area where few studies show clear benefit in favor of liver support devices. One other device, the so-called Biologic DT system, also seemed to be effective in one study.243 The ‘mistake’ with testing of these systems was their original use in end-stage liver disease patients in either acute or chronic liver failure.234 As mentioned earlier, it is difficult to perform controlled trials in this population. The acute-on-chronic liver failure syndrome, especially if the liver support system is applied earlier in the course of liver failure, may represent good testing situations with liver support systems. Interestingly, in virtually all reports the improvement in HE with these systems is striking.244,245 Survival, however, is the usual endpoint in such studies, and a survival benefit has been difficult to demonstrate. Systematic reviews of the work to date have appeared in the literature,246,247 and the current status of these systems is briefly described in Table 18-19.
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Table 18-19. Current Status of Liver Support Systems in HE Reasonably good evidence exists that these devices improve HE Most studies have been in very advanced liver failure. This results in multiple uncontrollable events affecting outcome The molecular albumin recirculating system (MARS) has been the most tested system to date. It shows promise as a safe and fairly effective liver support system Early use in acute on chronic liver failure may be associated with a better outcome The precise mechanism whereby these systems improve HE is not known
Table 18-20. Issues of Liver Transplantation and HE HE is not included in the MELD score No priority is given to patients with severe recurrent or resistant HE Failure to show HE independently predicts survival may be related to its close association with liver function status Older data on the failure of HE to predict survival in advanced liver disease patients may have occurred because of overstaging of HE to improve priority listing New association of the independent predictive power of HE on survival may be more valid in the MELD era. However, HE status needs to be reported more exactly
CLOSURE OF PORTOSYSTEMIC SHUNTS Despite the paucity of signs of HE in most patients with portosystemic shunts who do not have liver disease, there are those with liver disease who experience severe and recurrent bouts of HE attributable to portosystemic shunting.85 The classic patient has excellent liver synthetic function and yet has bouts of severe HE. When this type of patient is encountered, imaging studies of the abdomen should be performed to identify large collateral vessels. Closure of these vessels by either radiological or surgical interventions is sometimes feasible.248–255 However, before proceeding to attempt these maneuvers it is best to consult a transplant surgeon to ensure that this will not preclude or interfere with later liver transplantation. Reduction of pre-existing transhepatic stent diameters, or closure of large collateral vessels, can have a dramatic effect on the course of HE.249
LIVER TRANSPLANTATION Although it is not commonly the primary indication for liver transplantation, HE is generally improved by a successful graft. Table 1820 lists some of the current issues regarding HE and liver transplantation. Rather than reiterate these points in detail, the issue that will be discussed is the timing of referral for transplantation assessment in patients with drug and alcohol abuse problems. Successful completion of a rehabilitation program to qualify for liver transplantation requires well preserved cognitive skills, which may be limited because of HE. Tragically, we see too many patients with drug problems referred when they already have difficult-to-control HE. At present, recurrent or persistent HE does not give patients
Chapter 18 HEPATIC ENCEPHALOPATHY
priority for liver transplantation. Moreover, the impact of HE episodes can be such that patients often fail to complete drug rehabilitation programs satisfactorily.
SUMMARY The terminology of HE has officially been changed, and this system should be used in future publications in this field. After years of discord there is general agreement that cerebral edema is present to some degree in all types of HE. Ammonia, either via its own action or via astrocyte function, is the major theory under review in studies on the pathogenesis of HE. Other theories thought to be distinct are now being found to fit into this unifying hypothesis of disturbances in cellular function centering on the astrocyte. The state of affairs as regards evidence in support of most of the therapies for HE is poor. Newer perspectives are being brought to bear on improving study design for future trials. New treatments are being developed that will benefit from better designed RCTs in the future.
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181. Pedretti G, Calzetti C, Missale C, Fiackadon F. Rifaximin versus neomycin on hyperammonemia in chronic portal systemic encephalopathy of cirrhosis: a double-blind randomized trial. Ital J Gastroenterol 1991;23:175–178. 182. Williams R, James OE, Warners TW, Morgan MY. Evaluation of the efficacy and safety of rifaximin in the treatment of hepatic encephalopathy: a double-blind, randomized, dose finding multicenter study. Eur J Gastroenterol Hepatol 2000;12:203–208. 183. Puxeddu A, Quartini M, Massimetti A, Ferrieri A. Rifaximin in the treatment of chronic hepatic encephalopathy. Curr Med Res Opin 1995;13:274–281. 184. Mas A, Rodes J, Sunyer L, et al. Comparison of rifaximin and lactitol in the treatment of acute hepatic encephalopathy: results of a randomized, double-blind, double-dummy, controlled clinical trial. J Hepatol 2003;38:51–58. 185. Morgan M, Read AE, Speller PCE. Treatment of hepatic encephalopathy with metronidazole. Gut 1982;23:1–7. 186. Tarao K, Ikeda T, Hayashi K, et al. Successful use of vancomycin hydrochloride in the treatment of lactulose resistant chronic hepatic encephalopathy. Gut 1990;31:702–706. 187. Soulsby CT, Morgan MY. Dietary management of hepatic encephalopathy in cirrhotic patients: survey of current practice in United Kingdom. Br Med J 1999;318:1391. 188. Plauth M, Merli M, Kondrup J, et al. ESPEN guidelines for nutrition in liver disease and transplantation (Consensus statement). Clin Nutr 1997;16:43–55. 189. Plauth M, Merli M, Kondrup J. Management of hepatic encephalopathy. N Engl J Med 1997;337:1921–1922. 190. Seymour CA, Whelan K, Dietary management of hepatic encephalopathy. Br Med J 1999;318:1364–1365. 191. Cordoba J, Lopez-Hellin J, Planas M, et al. Normal protein diet for episodic hepatic encephalopathy: results of a randomized study. Hepatology 2004;41:38–43. 192. Erikkson LS, Conn HO. Branched-chain amino acids in hepatic encephalopathy. Gastroenterology 1990;99:604–607. 193. Gluud C. Branched-chain amino acids for hepatic encephalopathy? Hepatology 1991;13:812–813. 194. Naylor CD. Branched-chain amino acids in hepatic encephalopathy. Continuing controversy (letter; comment). Int J Technol Assess Health Care 1991;7:648–650. 195. Ferenci P. Branched-chain amino acids in hepatic encephalopathy. Gastroenterology 1990;98:1395–1396. 196. Erikkson LS, Conn HO. Branched-chain amino acids in the management of hepatic encephalopathy an analysis of variants. Hepatology 1989;10:228–246. 197. Naylor CD, O’Rourke K, Detsky AS, Baker JP. Parenteral nutrition with branched-chain amino acids in hepatic encephalopathy. A meta-analysis. Gastroenterology 1989;97:1033–1042. 198. Shaw S, Worner TM, Lieber CS. Comparison of animal and vegetable protein sources in the dietary management of hepatic encephalopathy. Am J Clin Nutr 1983;38:59–63. 199. Keshavarzian A, Meek J, Sutton C, et al. Dietary protein supplementation from vegetable sources in the management of chronic portal systemic encephalopathy. Am J Gastroenterol 1984;79:945–949. 200. Uribe M, Dibildox M, Malpica S, et al. Beneficial effect of vegetable protein diet supplemented with psyllium plantago in patients with hepatic encephalopathy and diabetes mellitus. Gastroenterology 1985;88:901–907. 201. Herman R, Shakoor T, Weber FL. Beneficial effect of pectin in chronic hepatic encephalopathy. Gastroenterology 1987;92:1795. 202. Garcia-Compean D, Uribe M. Fiber rather than protein determines tolerance to nitrogen load in chronic portal systemic encephalopathy: a randomized trial. Hepatology 1987;7:1034. [abstract]. 203. Uribe M, Conn HO. Dietary management of portal–systemic encephalopathy. In: Conn HO, Bircher J, eds. Hepatic
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encephalopathy: Syndromes and therapy. Bloomington, IL: Medi-Ed Press, 1994: 331–349. Marchesini G, Dioguardi FS, Bianchi GP, et al. Long-term oral branched-chain amino acid treatment in chronic hepatic encephalopathy. A randomized double-blind casein-controlled trial. The Italian Multicenter Study Group. J Hepatol 1990;11:92–1001. Egberts EH, Schomerus H, Hamster W, Jurgens P. Branchedchain amino acids in the treatment of latent portosystemic encephalopathy. A double blind placebo controlled crossover study. Gastroenterology 1985;88:887–895. Horst D, Grace ND, Conn HO, et al. Comparison of dietary protein with an oral, branched chain enriched amino acid supplement in chronic portal–systemic encephalopathy. Hepatology 1984;4:279–287. Uribe M, Moran S, Poo JL, et al. Beneficial effect of carbohydrate maldigestion induced by a disaccharidase in inhibitor (AO-128) in the treatment of chronic portal–systemic encephalopathy. A double-blind randomized controlled trial. Scand J Gastroenterol 1998;33:1099–1106. Solga SF. Probiotics can treat hepatic encephalopathy. Med Hypoth 2003;61:307–313. Bongaerts G, Severijinam R, Timmerman H. Effect of antibiotics, prebiotics and probiotics in treatment for hepatic encephalopathy. Med Hypoth 2005;64:64–68. Jic L, Zhang MH. Comparison of probiotics and lactulose in the treatment of hepatic encephalopathy in rats. World J Gastroenterol 2005;14:908–914. Liu Q, Duan ZP, Ha da K, et al. Symbiotic modulation of gut flora on minimal hepatic encephalopathy in patients with cirrhosis. Hepatology 2004;39:1441–1449. Solga SF, Diehl AM. Gut flora based therapy in liver disease? The liver cares about the gut. Hepatology 2004;39:199–200. Macbeth WA, Kass EH, McDermott WV Jr. Treatment of hepatic encephalopathy by alteration of intestinal flora with lactobacillus acidophilus. Lancet 1965;Vol 1, 399–403. Reed AE, McCarthy CF, Heaton KW, Laidlaw J. Lactobacillus acidophilus in treatment of hepatic encephalopathy. Br Med J 1966;1:1267–1269. Loguerico C, Del Vecchio Blanco C, Coltarti M. Enterococcus lactic acid bacterial strain SF 68 and lactulose in hepatic encephalopathy: A controlled trial. J Int Med Res 1987;15:335–343. Loguerico C, Abbiati R, Ranaldi M, et al. Long-term efficacy of enterococcus fecium SF 68 versus lactulose in the treatment of patients with cirrhosis. J Hepatol 1995;23:39–46. Kircheis G, Quack G, Erbler H. L-ornithine-L-aspartate in the treatment of hyperammonemia and hepatic encephalopathy. In: Conn HO, Bircher J, eds. Hepatic encephalopathy: Syndromes and therapies. Bloomington, IL: Medi-Ed Press, 1994: 373–383. Kircheis G, Wettstein M, Dahl S, Haussinger D. Clinical efficacy of L-ornithine-L-aspartate in the management of hepatic encephalopathy. Metab Brain Dis 2002;17:453–462. Stauch S, Kircheis G, Adler F, et al. Oral L–ornithine-L-aspartate therapy of chronic hepatic encephalopathy: results of a placebocontrolled double-blind study. J Hepatol 1998;28:856–864. Delcker AM, Jalan R, Schumacher M, Comes G. L-Ornithine-Laspartate vs placebo in the treatment of hepatic encephalopathy: a meta-analysis of randomized placebo-controlled trials using individual data. Hepatology 2000;32:310A [abstract]. Mendenhall CL, Rouster S, Marshall L, Weisner R. A new therapy for portal systemic encephalopathy. Am J Gastroenterol 1986;82:540–543. Sushma S, Dasarathy S, Tandon RK, et al. Sodium benzoate in the treatment of acute encephalopathy: a double blind randomized trial. Hepatology 1982;16:138–144. Mullen KD, Martin JV, Mendelson WB, et al. Evidence for the presence of benzodiazepines receptor binding substances in
224.
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239. 240.
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cerebrospinal fluid of a rabbit model of hepatic encephalopathy. Metab Brain Dis 1989;4:253–260. Basile AS, Parnell L, Jasuri T, et al. Brain concentrations of benzodiazepines are elevated in an animal model of hepatic encephalopathy. Proc Natl Acad Sci USA 1990;87:5263–5267. Mullen KD, Szauter KM, Kaminsky Russ K. Endogenous benzodiazepine activity in physiological fluids of patients with hepatic encephalopathy. Lancet 1990;336:81–83. Basile AS, Hughes RD, Harrison PM, et al. Elevated brain concentrations of 1:4-benzodiazepines in fulminant hepatic failure. N Engl J Med 1991;325;473–478. Yurdaydin C, Li Y, Ha JH, et al. Brain and plasma levels of opioid peptides are altered in rats with thioacetamide-induced liver failure: implications for the treatment of hepatic encephalopathy. J Pharmacol Exp Ther 1995;273:185–192. Yurdaydin C, Karavelioglu D, Onaran O, et al. Opioid receptor ligands in human hepatic encephalopathy. J Hepatol 199;29:796–801. Reding P, Duchateau J, Bataile C. Oral zinc supplements improve hepatic encephalopathy. Results of a randomized controlled trial. Lancet 1984;2:493–495. Riggio O, Oriosto F, Merli M, et al. Short-term oral zinc supplementation does not improve chronic hepatic encephalopathy: results of a double-blind crossover trial. Dig Dis Sci 1991;36:1204–1208. Riggio O, Merli M, Capocaccia L, et al. Zinc supplementation reduces blood ammonia and increased liver transcarbamylase activity in experimental cirrhosis. Hepatology 1992;16:785–788. Keeling PW, Jones RB, Hilton PJ, Thompson RH. Reduced leucocyte zinc in liver disease. Gut 1980;21:561–564. Van der Rijt CCD, Scholm SW, Wchat H, et al. Overt hepatic encephalopathy precipitated by zinc deficiency. Gastroenterology 1991;100:1114–1118. Mullen KD, Dasarathy S. MARS – Does it stand the test of time? Metab Brain Dis 2004;19:223–228. Mitzner SR, Klammt S, Peszynski P, et al. Improvement of multiple organ functions in hepatorenal syndrome during albumin dialysis with the molecular adsorbent recirculating system. Ther Apher 2001;5:417–422. O’Grady JG, Gimson AE, O’Brien CJ, et al. Controlled trials of charcoal hemoperfusion and prognostic factors in fulminant hepatic failure. Gastroenterology 1988;94:1186–1192. Hughes RD, Pucknell A, Routley D, et al. Evaluation of the Biologic-DT sorbent–suspension dialyser in patients with fulminant hepatic failure. Int J Artif Org 2001;24:434–442. Ellis AJ, Hughes RD, Nicholl D, et al. Temporary extracorporeal liver support for severe acute alcoholic hepatitis using the BioLogic-DT. Int J Artif Org 1999;22:27–34. Wilkinson AH, Ash SR, Nissenson AR. Hemodiabsorption in treatment of hepatic failure. J Transpl Coord 1998;8:43–50. He JQ, Chen CY Deng JTT, et al. Clinical study on the treatment of fatal hepatitis with artificial liver support system. Chin Crit Care Med 2000;12:105–108. Ellis AJ, Hughes RD, Wendon JA, et al. Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure. Hepatology 1996;24:1446–1451. Stevens C, Busuttil R, Han S, et al. An interim analysis of a phase II/III prospective randomized multicenter controlled trial of the Hepat Assist Bioartificial Liver Support System for the treatment of fulminant hepatic failure. Hepatology 2001;4 [abstract]. Mitzner SR, Stange J, Klammt S, et al. Improvement of hepatorenal syndrome with extracorporeal albumin dialysis MARS: results of a prospective, randomized, controlled clinical trial. Liver Transpl 2000;6:287–289. Kramer L, Gendo A, Madl C, et al. A controlled study of sorbent suspension dialysis in chronic liver disease and hepatic encephalopathy. Int J Artif Org 2001;24:434–442.
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245. Sen S, Jalan R, Williams R. Extracorporeal albumin dialysis in acute-on-chronic liver failure: will it stand the test of time? Hepatology 2002;36:1014–1016. 246. Liu JP, Gluud LL, Als-Nielsen B, Gluud C. Artificial and bioartificial support systems for liver failure. In: The Cochrane Library, Issue 1, 2004. Chichester: John Wiley & Sons. 247. Kjaergard LL, Liu J, Als-Nielsen B, Gluud C. Artificial and bioartificial support systems for acute and acute-on-chronic liver failure: a systematic review. JAMA 2003;289:217– 222. 248. Clarke B, Ellis MJ, Leung V, et al. Reversal of hepatic encephalopathy and alteration in amino acid profiles after blocking a surgical splenorenal shunt by interventional radiological techniques. J Hepatol 1989;8:325–329. 249. Hiroka A, Kubose K, Hamada M. Hepatic encephalopathy due to intrahepatic portosystemic venous shunt successfully treated by interventional radiology. Intern Med 2004;44:212–216.
250. Sachdev A, Duseja A. Decompressive shunts and hepatic encephalopathy. Indian J Gastroenterol 2003;22(Suppl 2): S21–S24. 251. Ong JP, Mullen KD. Hepatic encephalopathy. Eur J Gastroenterol Hepatol 2001;13:325–334. 252. Gerbes AL, Waggershauser T, Holl J, et al. Experiences with novel techniques for reduction of start flow in transjugular intrahepatic portosystemic shunts. Z Gastroenterol 1998;36:373–377. 253. Rossle M, Siegerstetter V, Huber M, Ochs A. The first decade of the transjugular intrahepatic portosystemic shunt (TIPS): State of the art. Liver 1998;1:73–89. 254. Nishie A, Yoshimitsu K, Honda H. Treatment of hepatic encephalopathy by retrograde and transcaval coil embolization of an ilea vein-to-right gonadal vein portosystemic shunt Cardiovasc Intervent Radiol 1997;20:222–224. 255. Mullen KD. Hepatic encephalopathy after portosystemic shunts: Any clues from TIPS. Am J Gasterol 1995;70:531–533.
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19
ASCITES Guadalupe Garcia-Tsao Abbreviations AFB acid fast bacilli Catn catheterization COX-2 cyclooxygenase-2 CT computed tomography FHVP free hepatic vein pressure HVPG hepatic venons pressure gradient IV intravenous
LVP NO PCD PRA PTFE PVS
large-volume paracentesis nitric oxide post-paracentesis circulatory dysfunction plasma renin activity polytetrafluoroethylene peritoneovenous shunt
INTRODUCTION Ascites is the accumulation of fluid in the peritoneal cavity and its most common cause is cirrhosis. That fluid accumulates in the abdominal cavity has been known since ancient times, and it was Hippocrates who recognized that ascites (from the Greek askos, meaning a leather bag used to carry wine, water or oil) derived from a diseased liver and that it carried a grim prognosis.1 The development of ascites is one of the complications that marks the transition from compensated to decompensated cirrhosis. Other complications that mark this transition are variceal hemorrhage, hepatic encephalopathy or jaundice; however, ascites is the most common.2,3
EPIDEMIOLOGY At the time of diagnosis, ascites is present in 20–60% of patients with cirrhosis, depending on the referral pattern.2,4 In prospective studies of patients with compensated cirrhosis of all etiologies, the cumulative probability of developing ascites ranges from 35 to 50% in 5 years (Figure 19-1),2,5 not unlike the annual incidence rate of around 6% in patients with viral cirrhosis.6 In a large cohort of patients with decompensated HCV-related cirrhosis, ascites was the most frequent first decompensating event, occurring in 48% of cases.3
PATHOGENESIS In cirrhosis, leakage of ascites into the peritoneal space occurs as a result of sinusoidal hypertension, which in turn results from hepatic venous outflow block secondary to regenerative nodules and fibrosis. The other essential factor in the pathogenesis of cirrhotic ascites is plasma volume expansion, through sodium and water retention, which allows for the replenishment of the intravascular volume, thereby maintaining the formation of ascites (Figure 19-2).
RAAS SAAG SBP TIPS WHVP
renin-angiotensin-aldosterone system serum ascites albumin gradient spontaneous bacterial peritonitis transjugular intrahepatic portosystemic shunt wedged hepatic vein pressure
SINUSOIDAL HYPERTENSION Similar to gastroesophageal varices, in which a minimal portal pressure gradient of 10–12 mmHg is needed for their development, the development of ascites also seems to require a minimal portal pressure gradient of 12 mmHg.7,8 In a recent consensus conference on portal hypertension, a portal pressure gradient of 10 mmHg or more was defined as ‘clinically significant portal hypertension’ because complications of cirrhosis do not occur below this threshold pressure.9
SODIUM RETENTION Sinusoidal hypertension alone is not sufficient to maintain ascites formation. Without replenishment of the intravascular space, leakage of fluid into the peritoneal cavity would be a self-limited process. Replenishment of the intravascular space, that is, plasma volume expansion, is accomplished through sodium retention, which has been shown to precede the development of ascites.10 Splanchnic and systemic arteriolar vasodilatation is the most likely mechanism that leads to sodium retention.11 This vasodilatation results in a reduction of the ‘effective’ arterial blood volume, which in turn leads to stimulation of neurohumoral systems, specifically the renin–angiotensin–aldosterone system (RAAS), the sympathetic nervous system, and the non-osmotic release of antidiuretic hormone. Activation of RAAS and the sympathetic nervous system results in sodium retention and, in extreme cases, renal vasoconstriction. Activation of antidiuretic hormone leads to free water retention and hyponatremia.12 An increased production of the vasodilator nitric oxide (NO) is currently considered the main cause of splanchnic and systemic vasodilatation in cirrhosis. In fact, NO blockade has been shown to raise systemic blood pressure, increase sodium excretion and decrease the volume of ascites in experimental cirrhosis and portal hypertension while down-regulating RAAS activation.13,14 The administration of vasodilators such as prazosin, angiotensin receptor blockers and phosphodiesterase inhibitors to cirrhotic patients leads to further RAAS activation,15–17 with associated sodium retention,15,17 ascites,15 and decreases in creatinine clearance.16
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The presence of normal or low levels of plasma renin activity in some patients with cirrhosis and ascites suggests that in some cases sodium retention occurs unrelated to vasodilatation.
CLINICAL FEATURES The most frequent symptoms associated with the presence of ascites are increased abdominal girth (described as tightness of the belt or garments around the waist) and recent weight gain.18 Ascites induces abdominal distention, but this sign in itself has a poor specificity19 as other conditions, including obesity, gas, tumors, and pregnancy, will also induce abdominal distention. When present in small to moderate amounts, ascites can be identified by bulging flanks, flank dullness and shifting dullness. The last two are the most sensitive
100 0 Ascites
90
Jaundice
80
Encephalopathy GI hemorrhage
xxxxxx
70
50 40 30 20 10 0 20
40
60
DIAGNOSIS Abdominal ultrasonography is the most cost-effective and least invasive method to confirm the presence of ascites and is therefore considered the gold standard in its diagnosis. It can detect amounts as small as 100 ml,22 and even as small as 1–2 ml when Morison’s pouch and the pelvic cul-de-sac are scanned.23 Abdominal ultrasound is also useful in determining the best site to perform a diagnostic or therapeutic paracentesis, particularly in patients with a small amount of ascites or in those with loculated ascites. Additionally, although not very sensitive in the diagnosis of cirrhosis, ultrasound is the most useful initial test to investigate the presence of hepatic vein obstruction, an important and frequently overlooked cause of ascites.23 Therefore, in patients with new onset of ascites abdominal ultrasound should always include Doppler examination of the hepatic veins.
DIFFERENTIAL DIAGNOSIS
60
0
physical maneuvers in the diagnosis of ascites.19,20 Ascites can be classified into three grades:21 1, mild ascites only detectable by ultrasound examination; 2, moderate ascites manifest by moderate symmetrical distention of the abdomen; and 3, large or gross ascites with marked abdominal distention.
80
100
120
140
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xxxxxx Figure 19-1. Probability of developing decompensated cirrhosis in 257 patients with compensated cirrhosis of different etiologies. Of the complications of portal hypertension, ascites is the most frequent decompensating event.5
Although cirrhosis is the cause of ascites in more than 75% of patients, other causes, such as peritoneal malignancy (12% of cases), cardiac failure (5%) and peritoneal tuberculosis (2%)24 should be considered in the differential diagnosis of ascites. A diagnostic paracentesis should be the first test performed in the diagnostic workup of a patient with ascites. It is a safe procedure with a very low incidence of serious complications, mostly transfusion-requiring hematomas that occur at a rate of 0.2–0.9%.25,26 Renal dysfunction appears to be more predictive of bleeding complications than clotting abnormalities, and therefore coagulopathy is not a contraindication to perform a diagnostic paracentesis.25,26 Care should always be taken to avoid abdominal wall collaterals and to avoid the area of the inferior hypogastric artery, which lies midway between the anterior superior iliac spine and the
Figure 19-2. Pathophysiology of cirrhotic ascites. Two main pathogenic mechanisms in the formation of ascites are sinusoidal hypertension and vasodilatation leading to activation of neurohumoral systems and sodium and water retention.
Cirrhosis
Intrahepatic resistance
Sinusoidal pressure
Ascites
334
Arteriolar resistance (vasodilation)
(HVPG ≥ 10 mmHg)
Sodium and water retention
Effective arterial blood volume
Activation of neurohumoral systems (aldosterone, renin angiotensin, epinephrine)
Chapter 19 ASCITES
three main causes of ascites – cirrhosis, peritoneal pathology (malignancy or tuberculosis) and heart failure – can be distinguished by combining the results of both the SAAG and the ascites total protein content, and the workup of the patient with ascites can thus be further refined (Figure 19-4). In cirrhosis, the SAAG is high and
30
HVPG (mmHg)
pubic tubercle. Technically, the midline below the umbilicus is often recommended as a site for paracentesis because of its presumed avascularity. However, in patients with portal hypertension this area is commonly vascular,27 and therefore the flanks are preferable. Uncomplicated ascitic fluid is transparent, straw colored to slightly yellow. The presence of blood in a non-traumatic tap (in which blood does not clot) may indicate the presence of malignant ascites. Milky fluid is indicative of chylous ascites, and although cirrhosis is the most common cause of non-surgical chylous ascites it represents only 0.5–1% of cases of cirrhotic ascites.28 Ascites total protein and serum ascites albumin gradient (SAAG) are two inexpensive tests that, taken together, are most useful in determining the etiology of ascites and hence in guiding the workup of patients with ascites. A high (>2.5 g/dl) ascites total protein occurs in peritoneal processes (malignancy, tuberculosis) owing to leakage of high-protein mesenteric lymph from obliterated lymphatics and/or from an inflamed peritoneal surface. A high ascites total protein also occurs in cases of postsinusoidal or posthepatic sinusoidal hypertension when sinusoids are normal and protein-rich lymph ‘leaks’ into the peritoneal cavity.29,30 In hepatic cirrhosis an abnormally low protein content of liver lymph has been demonstrated as a result of deposition of fibrous tissue in the sinusoids (‘capillarization of the sinusoid’) that renders the sinusoid less leaky to macromolecules.31,32 On the other hand, the SAAG, which involves measuring the albumin concentration of serum and ascitic fluid specimens and subtracting the ascitic fluid value from the serum value, has been shown to correlate with hepatic sinusoidal pressure (Figure 19-3).33,34 A SAAG cut-off value of 1.1 g/dl has been shown to be the best to distinguish patients in whom ascites is secondary to liver disease and those with malignant ascites.35 Interestingly, this cut-off corresponds to a portal pressure gradient of 11–12 mmHg,33 the threshold pressure necessary for the development of ascites in cirrhotic patients (Figure 19-3). Therefore, the
11
20
10
y = 7.08X + 3.62 0 0.0
Figure 19-3. A direct, very significant correlation between the serum–ascites albumin gradient (SAAG) and the hepatic venous pressure gradient (HVPG), a measure of sinusoidal pressure, is present.33 A cut-off SAAG of >1.1 g/dl has been identified as one that distinguishes ascites secondary to sinusoidal hypertension from that secondary to peritoneal causes. This cut-off corresponds to an HVPG of 11 mmHg, identified by other studies as the threshold pressure for the formation of ascites.
Peritoneum SAAG < 1.1
Normal ‘leaky’ sinusoid Ascites protein > 2.5
Peritoneal lymph Ascites protein > 2.5
Sinusoidal hypertension –Cirrhosis –Late Budd Chiari
Post-sinusoidal hypertension –Congestive heart failure –Constrictive pericarditis –Early Budd Chiari syndrome –Veno-occlusive disease
Peritoneal pathology –Malignancy –Tuberculosis
–Echocardiogram –Right heart catheter and HVPG
3.0
SAAG (g/dl)
‘Capillarized’ sinusoid Ascites protein < 2.5
–Ultrasound or CT scan –Endoscopy
2.0
1.1
Source of ascites
Hepatic sinusoids SAAG > 1.1
1.0
Figure 19-4. Differential diagnosis of ascites depending on the source of ascites. The serum– ascites albumin gradient (SAAG) is high (>1.1 g/dl) when the source is hepatic sinusoids and low when the source is other than the sinusoids. Ascites total protein is high (>2.5 g/dl) when ascites is coming either from normal ‘leaky’ sinusoids or from the peritoneum. Workup is directed accordingly.
–Cytology/AFB –Laparoscopy with peritoneal biopsy
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ascites total protein is low; in posthepatic or postsinusoidal causes of portal hypertension (e.g. heart failure, constrictive pericarditis), SAAG is high and ascites total protein is high; and in ascites secondary to peritoneal causes, SAAG is low and ascites total protein is high (Figure 19-5).36–38 In patients with mixed ascites (e.g. cirrhosis with superimposed peritoneal malignancy) the SAAG is high and the ascites protein is low, that is, the findings of ascites due to cirrhosis predominate.38 Of note, massive hepatic metastasis can lead to the development of ascites, but as the mechanism of ascites formation is sinusoidal hypertension, these cases of ‘malignant ascites’ will have a high SAAG.37 The definitive test to determine whether ascites is the result of sinusoidal hypertension is to actually measure hepatic sinusoidal pressure. The hepatic venous pressure gradient (HVPG), obtained by subtracting the free hepatic vein pressure (FHVP) from the wedged hepatic vein pressure (WHVP), is a measure of sinusoidal pressure. In cases of cirrhotic ascites the HVPG will be ≥10–12 mmHg. In cases of cardiac ascites, both the WHVP and the FHVP will be elevated (reflecting elevated systemic pressures) and hence the HVPG will be normal. In cases of peritoneal ascites (i.e. malignancy or tuberculosis), all hepatic venous pressure measurements (WHVP, FHVP and HVPG) will be normal, unless the patient has coexisting cirrhosis or heart failure. When performed properly, HVPG measurements are reproducible and safe.39 Additionally, hepatic vein catheterization for measurement of hepatic vein pressures allows for the performance, in the same procedure, of a transjugular liver biopsy that will further define the etiology of ascites.
ASSOCIATED CONDITIONS Hyponatremia develops in approximately 30% of cirrhotic patients with ascites and is defined as a serum sodium concentration 80% in initial series to a current rate of 10–20%.51
EPIDEMIOLOGY SBP is the most common type of infection in hospitalized cirrhotic patients, occurring in about 9% of cases and accounting for about 25% of all infections.52 The prevalence of SBP appears to be lower in the outpatient setting, where a 3.5% rate has been reported in patients undergoing serial therapeutic paracenteses.53 In prospective studies, the 12-month probability of developing a first episode of SBP in cirrhotic patients with ascites has ranged between 11%54 and 29%,55 incidence that is highly dependent on ascites total protein content (0% in patients with an ascites protein >1 g/dl vs 20% in patients with an ascites protein 1.1 g/dl) SAAG and a low ascites protein (250/mm3 has been identified as having the greatest diagnostic accuracy.58 In hemorrhagic ascites (i.e. ascites red blood cell count >10 000/mm3), subtracting one PMN for every 250 red blood cells will correct for the excess blood in ascites.
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Section III. Clinical Consequences of Liver Disease
To maximize the possibilities of isolating an infecting organism, both ascites and blood bacteriological cultures should be performed whenever SBP is suspected.57
TREATMENT Once an ascites PMN count >250/mm3 is detected, antibiotic therapy needs to be started before obtaining the results of ascites or blood cultures. Antibiotic therapy would also be justified in patients in whom ascites cultures are twice positive despite a PMN count 4 mg/dl and evidence of renal impairment at baseline (BUN >30 mg/dl and/or creatinine >1.0 mg/dl).71 It is therefore this subgroup of patients in whom albumin can be recommended.72 On the other hand, there is a subgroup of patients with SBP – those with community-acquired infection, no encephalopathy and normal renal function – that have a 100% cure
Figure 19-6. Algorithm in a patient with suspected spontaneous bacterial peritonitis (SBP). Once a polymorphonuclear cell count (PMN) >250/mm3 is detected, antibiotic treatment should be initiated. If ascites culture is positive (in a patient with an initial PMN count 250/mm3. If the count is still 250
No action
Treat
Culture negative
Culture positive
No action
Repeat tap
Culture negative Continue therapy
PMN < 250
PMN > 250
No action
Treat
Culture negative
Culture positive
No action
Treat
Culture positive
Chapter 19 ASCITES
rate and 100% survival with antibiotic therapy alone.69 Albumin would not be indicated in these patients.72 A control paracentesis performed 48 hours after starting therapy is recommended to assess the response to therapy and the need to modify antibiotic therapy (depending on the isolation of a causative organism) and/or to initiate investigations to rule out secondary peritonitis.57 This failure of initial therapy occurs in up to 23% of cases. In the presence of an obvious clinical improvement, control paracentesis may not be necessary. Intravenous antibiotics can be safely switched to oral after 2 days of therapy and once a response to therapy is demonstrated by a decrease in ascites PMN.73
bleeding and/or who have not had a previous episode of SBP, particularly as the rate of infection in this patient population is low.53 Limiting the use of prophylactic antibiotics to those at the highest risk of developing SBP is important, given the increased rate of infections with quinolone-resistant and trimethoprim–sulfamethoxazole-resistant organisms observed in patients on long-term norfloxacin prophylaxis.52,79 The results of placebo-controlled trials of antibiotic prophylaxis performed in populations at a higher risk of developing SBP, identified by low ascites protein, high serum bilirubin and a low platelet count,80 are awaited. Investigations of nonantibiotic measures to prevent SBP and other bacterial infections in cirrhosis are ongoing.72
PROPHYLAXIS In patients who survive an episode of SBP the 1-year cumulative recurrence rate is high, at about 70%. Recurrence, particularly from Gram-negative organisms, is significantly and markedly lower with the use of oral norfloxacin at a dose of 400 mg/day.74,75 It is therefore essential that patients surviving an episode of SBP be started on antibiotic prophylaxis to prevent recurrence. The use of weekly quinolones is not recommended, as they have been shown to be less effective in preventing SBP recurrence and are associated with a higher rate of development of quinolone-resistant organisms.75 Prophylaxis should be continuous until the disappearance of ascites (i.e. patients with alcoholic hepatitis), death or transplant. Another group of cirrhotic patients in whom antibiotic prophylaxis should be used routinely is those admitted with gastrointestinal (GI) hemorrhage, in whom the rate of bacterial infection is as high as 45%. In these patients, short-term antibiotic prophylaxis has been shown to be effective not only in reducing the rate of bacterial infections, but also in reducing in-hospital mortality76 and variceal rebleeding.77 The preferred antibiotic is norfloxacin at a dose of 400 mg orally twice a day for 7 days (or less if the patient is to be discharged from the hospital). Data from the only placebo-controlled trial of primary prophylaxis of SBP78 are inconclusive, and therefore there is insufficient information to support the use of long-term antibiotic prophylaxis in outpatient cirrhotic patients with ascites who do not have GI
TREATMENT OF ASCITES The treatment of cirrhotic ascites is important, not only because it improves the patient’s quality of life, but because SBP, one of the most lethal complications of cirrhosis, does not occur in the absence of ascites. Therapies for ascites include sodium restriction, diuretics, large-volume paracentesis (LVP), the transjugular intrahepatic portosystemic shunt (TIPS) and the peritoneovenous shunt (PVS) (Figure 19-7). The development of ascites in a cirrhotic patient denotes a poor prognosis and is an indication to initiate liver transplant evaluation. Therefore, transplantation constitutes the ultimate treatment for ascites and its complications. Ascites responds appropriately in 80–90% of patients upon the attainment of a negative sodium balance through the use of sodium restriction and/or diuretics. Even though this treatment takes longer and may have a higher complication rate than LVP, it still constitutes the mainstay of therapy, given its general applicability, low cost and ease of administration. In fact, the categorization of cirrhotic patients with ascites is based on their response to diuretic therapy. In this scheme, uncomplicated ascites assumes an uninfected ascites with a good response to diuretics, and refractory ascites assumes either diuretic-resistant ascites (ascites that is not eliminated even with maximal diuretic therapy) or diuretic-intractable ascites
Transplant
Figure 19-7. Different treatments of ascites placed in the context of its pathophysiology.
Cirrhosis
Intrahepatic resistance
Sinusoidal pressure
Arteriolar resistance (vasodilation)
Albumin TIPS PVS
TIPS
Diuretics Ascites PVS
LVP
Sodium and water retention Sodium restriction
Effective arterial blood volume
Activation of neurohumoral systems (aldosterone, renin, angiotensin, epinephrine)
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(ascites that is not eliminated because maximal doses of diuretics cannot be reached given the development of renal and/or electrolyte abnormalities).81
SODIUM RESTRICTION Sodium restriction is recommended in all cirrhotic patients with ascites. Although dietary sodium should be restricted to levels lower than urinary sodium excretion, sodium restriction to ~90 mEq/day (i.e. 2 g sodium/day or 5.2 g dietary salt/day, considering that 1 mEq sodium = 23 mg sodium = 58.5 mg dietary salt) is a realistic goal, particularly in the outpatient setting.21 Further restriction of sodium is unrealistic and difficult to achieve. Patients with a baseline urinary sodium excretion >50 mEq/24 hours, an infrequent setting, may respond to salt restriction alone. There are virtually no complications associated with sodium restriction. However, clinicians should be cautious about the nutritional status of patients on sodium restriction, as the non-palatability of a salt-restricted diet may lead to an inadequate food intake. In these cases, liberalizing sodium restriction and adding diuretics is preferable to compromising the already compromised nutrition of the cirrhotic patient with ascites.
DIURETICS Spironolactone is the diuretic of choice. Even though loop diuretics such as furosemide are more potent natriuretics, randomized trials have shown a significantly lower efficacy of furosemide used alone than of spironolactone alone82,83 or the combination spironolactone/furosemide.82 When furosemide is used alone, sodium that is not reabsorbed in the loop of Henle is taken up at the distal and collecting tubules as a result of the hyperaldosteronism present in most cirrhotic patients with ascites. Therefore, furosemide should not be used as the sole agent in the treatment of cirrhotic ascites. Diuretic therapy can be initiated with spironolactone alone or with spironolactone plus furosemide. Both schemes are equally effective; however, dose adjustments are needed more frequently in patients in whom treatment is initiated with combination therapy because of more rapid increases in blood urea nitrogen and/or decreases in serum sodium.82,84 Therefore, and particularly in the outpatient setting, it is preferable to initiate therapy with spironolactone alone at a daily dose of 100 mg orally, and to increase it in a stepwise fashion to a maximum of 400 mg/day. Because spironolactone takes several days to take effect, it can be administered in a single daily dose and adjustments should only be made after the patient has been ≥4 days on a stable dose. If weight loss is not optimal or if hyperkalemia develops, furosemide should be added at an initial single daily dose of 40 mg, increased in a stepwise fashion to a maximum of 160 mg/day. An insufficient diuretic response necessitating increases in the dose of diuretics is defined as a weight loss 50% to >1.5 g/dl) diuretics should be temporarily discontinued and restarted at a lower dose once creatinine returns to baseline. Patients who develop hyponatremia (serum sodium 40 mmHg or a decrease in CPP 30 is over 60%.395,396 A transplant psychologist and an ethicist should assist in decision making when a patient presents with an intentional acetaminophen overdose or history of drug or alcohol abuse. Many international liver transplant centers have reported average survival after transplantation for ALF of about 65%, which compares favorably with medical management.348,392,394,397–403 In one of the largest studies404 a number of static and dynamic variables were
Table 21-19. Effects of Prostaglandin E on Survival in ALF Author
n
Drug and route of administration
Sinclair390 Bernau432
17 22 (HBV) 22 (drug/ indet.) 22 20 41 13
PGE1 IV, followed by PGE2 p.o. PGE IV PGE IV
O’Brien433,434 Sheener435 Sterling391
PGE1 IV PGE1 IV PGE2 IV vs placebo PGE1 IV vs placebo
Survival (%) 71 13 13 72 100 40 vs. 38 60 vs. 50
HBV, ALF due to hepatitis B virus; drug/indet., ALF due to idiosyncratic drug reaction or indeterminate etiologies.
evaluated as predictors of outcome after transplantation in 100 patients with ALF. In patients with ALF unrelated to acetaminophen (n = 79) the etiology was an important predictor of survival, with 100% of those with Wilson’s disease surviving 2 months after transplant compared to only 12.5% of those with drug-induced ALF. Of the dynamic variables, an elevated serum creatinine predicted poor outcome, as did grade III/IV encephalopathy (80% survival for those with grade I/II vs 56% for grade III/IV).405–407 Survival after liver transplantation is also adversely influenced by the utilization of suboptimal organs (fatty liver, ABO-incompatible liver).394,405,408 The outcome of liver transplantation in children is similar to that in adults.348,409–411 Although experience worldwide with adult-to-adult living donor liver transplantation is limited, the procedure has been used in occasional patients with ALF.412,413 The major hindrance to its widespread use in ALF, however, will probably remain the amount of time required to evaluate a potential donor, which often requires several days. Auxiliary liver transplantation, in which a donor liver (whole or partial) is heterotopically implanted below the native liver to provide support while regeneration of the native liver occurs, has also been explored in patients with ALF.414–416 Withdrawal of immunosuppression after regeneration of the native liver causes rejection and atrophy of the donated liver, and obviates the need for long-term immunosuppression. In one recent series, complete regeneration of the native liver was noted in 68% of 22 patients with ALF who received an auxiliary transplant.83
PROGNOSIS AND NATURAL HISTORY Patients with ALF have one of three outcomes: spontaneous recovery, liver transplantation, or death. As of June 2004, the US Acute Liver Failure Study Group Registry of nearly 700 patients recorded that roughly 45% recovered spontaneously, 25% underwent liver transplantation (of whom 13% died), and 30% died without transplantation (WM Lee, personal communication). The overall survival, with or without liver transplantation and for all major etiologies, has steadily improved with time (Figure 21-16) and is currently 66% in the US Registry. The ability to predict which patient with ALF will recover spontaneously with medical management, and who will die without transplantation, remains of paramount importance. Although liver transplantation offers hope of survival from ALF, the decision to transplant introduces the need for lifelong immunosuppression, an operative mortality of up to 30%, and the use of a scarce resource.417 Thus, universal liver transplantation for ALF cannot be endorsed. Although mortality from all causes of ALF parallels the depth of hepatic encephalopathy (>80% mortality rates for grade III/IV encephalopathy),6 spontaneous recovery occasionally follows even deepest hepatic coma;418 thus, more accurate predictors of outcome are needed. Several groups have proposed guidelines with which to select a patient for liver transplantation (Table 21-20). The most widely accepted were proposed by O’Grady et al. in 1989,3 and have become known as the King’s College criteria. Based on a retrospective review of 588 patients with ALF who were managed medically between 1973 and 1985, these authors identified poor prognostic variables in patients with ALF due to acetaminophen overdose and
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Section III. Clinical Consequences of Liver Disease
other etiologies by multivariate analysis, and then applied the variables to a test group of 175 patients who were seen between 1986 and 1987. For patients with acetaminophen overdose, acidosis (arterial pH 100 seconds, serum creatinine >3.4 mg/dl, and grade III/IV hepatic encephalopathy was highly associated with mortality without liver transplantation. Fulfillment of one of these criteria predicted 77% of the total deaths in the test group. In the
Hepatitis A
Hepatitis B
Acetaminophen
Non A non B, indeterminate
Halothane
80 Transplantation
Survival (%)
60
40
20
0 ’73-’76 ’77-’79 ’80-’82 ’83-’85 ’86-’88 ’89-’91 ’91-’93 Year Figure 21-16. Improving survival of patients with ALF at the King’s College Hospital Liver Failure Unit according to etiology. Survival represents patients with grade III/IV hepatic encephalopathy who received liver transplants as well as those who spontaneously recovered. (From Williams R, Wendon, J. Indications for orthotopic liver transplantation in fulminant liver failure. Hepatology 1994; 20:S5, with permission.)
group with ALF from non-acetaminophen etiologies, three static variables obtained on admission (etiology, age, and duration of jaundice to onset of encephalopathy >7 days), and two dynamic variables obtained during the evolution of liver failure (peak bilirubin and prothrombin time), predicted poor prognosis. The presence of a prothrombin time of >100 seconds or, in patients with a prothrombin time 7 days, prothrombin time >50 seconds, bilirubin >17.4 g/dl, predicted over 96% of the fatalities in the test group. The predictive accuracy of the King’s College criteria has been substantiated by other groups.86,419–421 In a recent study from Pittsburgh, however, failure to fulfill the King’s College criteria did not predict survival,422 and in the ALF Study Group Registry, the King’s College criteria predicted patient mortality with a sensitivity of only 12%.8 Other indices have been developed to improve on the King’s College criteria. One scheme was developed from a retrospective analysis of survival from hepatitis B and non-A, non-B viral ALF, with subsequent prospective validation.423 The risk score was derived from bilirubin, white blood cell count, prothrombin time, ALT, and duration of illness before encephalopathy, and demonstrated a predictive accuracy of nearly 90%. More recently, the APACHE (Acute Physiology and Chronic Health Evaluation) II score was prospectively evaluated in patients with ALF due to acetaminophen overdose.424 Calculated after the first 24 hours following admission, this scoring system is widely used to estimate risk of hospital death, and is based upon easily obtained parameters.395 An APACHE II score of >15 was highly predictive of death or the need for liver transplantation, even though prothrombin time and bilirubin, two most important indicators of hepatic necrosis, are not part of the score. Coagulation parameters also have been recommended as the best prognostic indicators for mortality in patients with ALF (Table 2120). The prothrombin time is probably the most reliable, widely
Table 21-20. Schemes for Predicting Mortality and Need for Liver Transplantation in ALF Test
Etiology of ALF
Criteria for liver transplantation
Reference
King’s College Criteria
APAP
Arterial pH < 7.30 or all of the following: PT > 100 s, and Creatinine >3.4 mg/dl, and Grade 3/4 encephalopathy PT >100 s (INR > 6.5) or any three of the following: NANB/drug/halothane etiology, Jaundice to encephalopathy > 7 d, Age < 10 or > 40 yrs PT > 50 s Bilirubin > 17.4 mg/dl Age < 30 yrs: factor V < 20% or Any age: factor V < 30% and grade 3/4 encephalopathy Gc level < 34 g/ml (normal 280–560 g/ml) Hepatocyte necrosis > 70% See reference > 1.2 mmol/l > 3.5 mmol/l Score > 15
O’Grady, 19893
Non-APAP
Factor V (Clichy Criteria)
Viral
Unbound serum Gc protein Liver biopsy Severity index Arterial phosphate Serum lactate APACHE II score
Mixed Mixed HBV, NANB APAP APAP APAP
APAP, acetaminophen; HBV, hepatitis B virus; NANB, non-A, non-B viral hepatitis; mixed, mixed etiologies.
404
Bernuau, 1986, 199129,400 Lee, 1995421 Donaldson, 1993419 Takahashi, 1994423 Schmidt, 2002196 Bernal, 2002427 Mitchell, 1998424
Chapter 21 ACUTE LIVER FAILURE
80 Survived
Died
Factor V (%)
60
40
20
0 0
1
2
3
4
Days after admission Figure 21-17. Sequential factor V levels in 22 patients with acetaminopheninduced ALF according to prognosis. Factor V levels recovered to within a normal range (60–150%) within 4 days of admission in survivors, whereas patients who died had no significant recovery of factor V levels. (From Pereira L, Langley P, Hayllar K, et al. Coagulation factor V and VIII/V ratio as predictors of outcome in paracetamol induced fulminant hepatic failure: relation to other prognostic indicators. Gut 1992;33:98, with permission.)
available parameter.3,425 Bernuau et al.29 first proposed that factor V, a liver-derived coagulation factor with a short half-life (12–24 hours), may be a more accurate indicator of the need for liver transplantation than the prothrombin time, which tends to become markedly elevated relatively late in ALF, and is disproportionately elevated in certain etiologies of ALF.417 Based on experience with HBV-induced ALF, these ‘Clichy criteria’ were later refined in a prospective study of patients with viral ALF. In patients with stage III/IV hepatic encephalopathy a factor V level of =10 mEq/day
0.2
=3 ml/min 0.2 1.5 mg/dl) that does not meet the criteria for type 1
Major criteria Low glomerular filtration rate, as indicated by serum creatinine > 1.5 mg/dl or 24-h creatinine clearance < 40 ml/min Absence of shock, ongoing bacterial infection, fluid losses and current treatment with nephrotoxic drugs No sustained improvement in renal function (decrease in serum creatinine to 1.5 mg/dl or less or increase in creatinine clearance to 40 ml/min or more) following diuretic withdrawal and expansion of plasma volume with 1.5 l of a plasma expander Proteinuria < 500 mg/day and no ultrasonographic evidence of obstructive uropathy or parenchymal renal disease Additional criteria Urine volume < 500 ml/day Urine sodium < 10 mEq/l Urine osmolality greater than plasma osmolality Urine red blood cells less than 50 per high-power field Serum sodium concentration < 130 mEq/l *All major criteria must be present for the diagnosis of hepatorenal syndrome. Additional criteria are not necessary for the diagnosis, but provide supportive evidence.
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Section III. Clinical Consequences of Liver Disease
tional status and encephalopathy, although some patients with HRS may show only moderate liver failure. In general, patients with type 1 HRS have more severe liver failure than those with type 2 HRS.
In some patients HRS develops without any identifiable precipitating factor, whereas in others it occurs in close chronologic relationship with bacterial infections, particularly spontaneous bacterial peritonitis.12,62,76,77 Approximately one-third of patients with spontaneous bacterial peritonitis develop an impairment of renal function during treatment with non-nephrotoxic antibiotics and in the absence of shock.76 This impairment in renal function is of functional origin and occurs in the setting of a further decrease in effective arterial blood volume of patients with ascites, as indicated by a marked activation of vasoconstrictor systems, and increased serum and ascitic fluid levels of cytokines.76,77 In approximately one-third of patients developing renal failure after spontaneous bacterial peritonitis, the impairment in renal function is reversible after resolution of infection. However, in the remaining patients it is not, and meets the criteria for HRS (type 1 in most cases). Patients who develop type 1 HRS after spontaneous bacterial peritonitis have a dismal outcome, with an in-hospital mortality close to 100%.76,77 The administration of albumin at the time of diagnosis of the infection and 2 days later (1.5 g/kg and 1 g/kg body weight, respectively) prevents the development of HRS and improves survival in these patients.78 Spontaneous bacteremia also is associated with an increase in the prevalence of HRS comparable to that of spontaneous bacterial peritonitis. The prevalence of HRS is lower during other infections, such as pneumonia, urinary tract infections, and lymphangitis. Although uncommon, HRS has been reported after therapeutic paracentesis without plasma expansion.79 This is one of the reasons that the administration of IV albumin is recommended when largevolume paracentesis is performed.80 Gastrointestinal bleeding has been classically considered a precipitating factor of HRS.2 However, the development of renal failure after this complication is not very common in patients with cirrhosis (approximately 10%) and occurs mainly after hypovolemic shock, in most cases associated with ischemic hepatitis, which suggests that renal failure in this setting is frequently related to the development of acute tubular necrosis (ATN) and is not functional in origin.81 Diuretic treatment has also been classically described as a precipitating factor of HRS, but there is no clear evidence to support such a pathogenic relationship.
DIAGNOSIS The diagnosis of HRS is currently based on several diagnostic criteria (Table 22-4).62 The minimum level of serum creatinine required for the diagnosis is 1.5 mg/dl, because most patients with cirrhosis with a serum creatinine above 1.5 mg/dl have a GFR below 30 ml/min (Figure 22-5).65 In patients receiving diuretics, serum creatinine measurement should be repeated after diuretic withdrawal because in some patients serum creatinine may increase slightly during diuretic therapy due to volume depletion. Because no specific laboratory tests are available for the diagnosis of HRS and patients with advanced cirrhosis may develop renal failure of other etiologies (prerenal failure due to volume depletion, acute tubular necrosis (ATN), drug-induced nephrotoxicity, and glomerulonephritis), the most important step in the diagnosis of
424
Glomerular filtration rate (ml/min)
PRECIPITATING FACTORS
200
150
100
50
0 0
2
4
6
8
Serum creatinine (mg/dl) Figure 22-5. Correlation between glomerular filtration rate measured by inulin clearance and serum creatinine concentration in patients with cirrhosis and ascites.
HRS is to rule out renal failure secondary to volume depletion or intrinsic renal disease.62 Gastrointestinal fluid losses, due to vomiting or diarrhea, or renal fluid losses, due to excessive diuresis, should be sought in all patients with cirrhosis presenting with renal failure. If renal failure is secondary to volume depletion, renal function improves rapidly after volume repletion and treatment of the precipitating factor. Shock is another common condition in patients with cirrhosis that may lead to renal failure due to ATN. Whereas hypovolemic shock due to gastrointestinal bleeding is easily recognized, the presence of septic shock may be more difficult to diagnose because of the paucity of symptoms of bacterial infection in some patients with cirrhosis. Moreover, arterial hypotension due to the infection may be erroneously attributed to the underlying liver disease. In some patients with septic shock oliguria is the first sign of infection. These patients may be misdiagnosed as having HRS if signs of infection (cell blood count, examination of ascitic fluid) are not sought. On the other hand, as discussed earlier, patients with cirrhosis and spontaneous bacterial peritonitis may develop renal failure during the course of the infection, in the absence of septic shock.76–78 Renal failure in these patients may either improve with the antibiotic therapy or evolve into a true HRS, even after the infection has been resolved. The administration of NSAIDs is another common cause of acute renal failure in patients with cirrhosis and ascites, which is clinically indistinguishable from a true HRS.81–83 Therefore, use of these drugs should always be ruled out before the diagnosis of HRS is made. Likewise, patients with cirrhosis are at high risk of developing renal failure due to ATN when treated with aminoglycosides.84,85 Because of this high risk of nephrotoxicity and the existence of other effective antibiotics (i.e. third-generation cephalosporins) treatment with aminoglycosides should be avoided in patients with chronic liver disease. Finally, patients with cirrhosis may also develop renal failure due to glomerulonephritis.5 In these cases, proteinuria and/or hematuria are almost constant and provide a clue for the diagnosis, which may be confirmed by renal biopsy in selected cases.
Chapter 22 RENAL DYSFUNCTION IN CIRRHOSIS: PATHOPHYSIOLOGY, CLINICAL FEATURES AND THERAPY
FACTORS INVOLVED IN FUNCTIONAL RENAL ABNORMALITIES IN CIRRHOSIS CIRCULATORY ABNORMALITIES
in arterial blood flow. This phenomenon is thought to be a homeostatic mechanism to protect the intestine against edema formation. This protective mechanism is not operative in chronic portal hypertension, and arteriolar resistance is reduced and not increased. The resultant increases in capillary pressure and filtration may be important factors in the formation of ascites in cirrhosis. The mechanism(s) by which portal hypertension induces splanchnic arteriolar vasodilatation is not completely understood, although a number of vasoactive mediators have been proposed (see below).
HEPATIC AND SPLANCHNIC CIRCULATION The existence of cirrhosis causes marked structural abnormalities in the liver that result in severe disturbance of intrahepatic circulation, causing increased resistance to portal flow and subsequent hypertension in the portal venous system. Progressive collagen deposition and the formation of nodules alter the normal vascular architecture of the liver. Moreover, selective deposition of collagen in the space of Dissé may constrict the sinusoids, resulting in further mechanical obstruction to flow. In addition to this passive resistance to portal flow there is an active component of intrahepatic resistance, which is due to the contraction of hepatic stellate cells (myofibroblast-like cells) present in sinusoids and terminal hepatic venules.5,86,87 The contraction of these cells is affected by endogenous vasoconstrictors and can be modulated by vasodilators and drugs that antagonize the vasoconstrictor factors.5,88–90 Moreover, there is a strong body of evidence indicating that despite the overproduction of the vasodilator nitric oxide (NO) in the systemic circulation in cirrhosis, the production of NO in the intrahepatic circulation is reduced and contributes further to the increased intrahepatic resistance characteristic of cirrhotic livers. Interestingly, in vivo gene transfer of neuronal nitric oxide synthase (NOS) isoform to cirrhotic rat livers increases NO production from endothelial cells and hepatic stellate cells, and significantly decreases portal pressure. Portal hypertension induces profound changes in the splanchnic circulation. Classically, portal hypertension was believed to cause changes only in the venous side of the splanchnic circulation. However, studies in experimental animals indicate that portal hypertension also causes marked changes in the arterial side of the splanchnic vascular bed. In the venous side, the main changes consist of increased pressure and the formation/opening of a portocollateral circulation, which causes the appearance of gastroesophageal varices and shunting of blood from the portal venous system to the systemic circulation, which in turn are responsible for gastrointestinal bleeding and hepatic encephalopathy, respectively. On the arterial side there is marked arterial vasodilatation, which increases portal venous inflow. This high portal venous inflow plays an important role in the increased pressure in the portal circulation and may explain, at least in part, why portal pressure remains increased despite the development of portocollateral circulation. This arteriolar vasodilatation is also responsible for marked changes in splanchnic microcirculation that may predispose to increased filtration of fluid. It has been shown that the increases in intestinal capillary pressure and lymph flow that occur in chronic portal hypertension (i.e. cirrhosis) are much greater than those caused by an acute increase in portal pressure of the same magnitude (i.e. acute portal vein obstruction). This is probably due to a loss of the normal autoregulatory mechanism of the splanchnic microcirculation. The acute elevation of venous pressure in the intestine elicits a strong myogenic mechanism, which leads to a reduction
Several lines of evidence indicate that portal hypertension is a major factor in the pathogenesis of ascites. First, patients with early cirrhosis without portal hypertension do not develop ascites or edema. Moreover, a certain level of portal hypertension is required for ascites formation. Ascites rarely develops in patients with portal pressure below 12 mmHg, as assessed by the difference between wedged and free hepatic venous pressure (normal portal pressure: 5 mmHg).5,91,92 Second, cirrhotic patients treated with surgical portosystemic shunts for the management of bleeding gastroesophageal varices have a much lower risk of developing ascites than do patients treated with procedures that obliterate gastroesophageal varices but do not affect portal pressure (i.e. sclerotherapy, esophageal transection). Finally, reduction of portal pressure with side-to-side or end-to-side portocaval anastomosis or TIPS is associated with an improvement of renal function and suppression of antinatriuretic systems.93 The mechanism(s) by which portal hypertension contributes to renal functional abnormalities and ascites and edema formation is not completely understood, yet several pathogenic mechanisms have been proposed: 1. Alterations in the splanchnic and systemic arterial circulation that would result in activation of vasoconstrictor and antinatriuretic systems and subsequent renal sodium and water retention; 2. Hepatorenal reflex due to increased hepatic pressure which would cause renal sodium and water retention; 3. Putative antinatriuretic substances escaping from the splanchnic area through portosystemic collaterals that would have a sodium-retaining effect in the kidney. Most data from experimental and human cirrhosis supports the first of these three potential mechanisms (see below for further discussion).
SYSTEMIC CIRCULATION The development of portal hypertension is associated with marked hemodynamic changes not only in the hepatic and splanchnic circulation, as discussed in the previous section, but also in the systemic arterial circulation. These changes, which have been well characterized in human and experimental cirrhosis, consist of reduced systemic vascular resistance and arterial pressure, increased cardiac index, increased plasma volume, and activation of systemic vasoconstrictor and antinatriuretic factors. The decrease in arterial pressure and the stimulation of these vasoactive systems are more marked as the disease progresses.94 Numerous studies have presented evidences that all these hemodynamic changes of cirrhosis are related to an arterial vasodilatation located mainly in the splanchnic circulation.94,95 Whether or not arterial vasodilatation occurs also in non-splanchnic territories is still controversial. Some studies using
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Section III. Clinical Consequences of Liver Disease
duplex Doppler have found arterial vasodilatation and reduced vascular resistance in the upper and lower limbs, whereas others have reported that blood flow in these arterial beds is normal or even reduced relative to the increased cardiac output.69,73 Whether located exclusively or predominantly in the splanchnic circulation, this arterial vasodilatation causes an abnormal distribution of blood volume, which results in a reduction of the blood volume in the central arterial tree that is sensed by baroreceptors (known as effective arterial blood volume).95,96 This may explain why in most patients with cirrhosis and ascites systemic vasoconstrictor factors remain activated despite an increased plasma volume that in normal conditions would suppress the activation of these systems. The reduction in central blood volume correlates directly with systemic vascular resistance and inversely with portal pressure, indicating that the greater the vasodilatation and the pressure in the portal system, the lower the central blood volume.95 The crucial role played by the reduced central blood volume in the activation of vasoconstrictor systems has been further corroborated by studies showing that improvement of central blood volume by the combination of expansion of plasma volume or head-out water immersion and administration of vasoconstrictor agents, suppresses the activation of vasoconstrictor systems.97–99 Despite extensive investigation, the mechanism(s) responsible for arterial vasodilatation in cirrhosis is not completely understood. Several explanations have been proposed, including opening of arteriovenous fistulas, reduced sensitivity to vasoconstrictors, dysfunction of the autonomic nervous system, and increased circulating levels of vasodilator substances.94 This latter mechanism seems to be the most important and has been extensively investigated. Proposed mediators of this vasodilatation include glucagon, vasoactive intestinal peptide, prostaglandins, natriuretic peptides, platelet-activating-factor, substance P, calcitonin gene-related peptide (CGRP), adrenomedullin, endocannabinoids, and carbon monoxide, but their role in the pathogenesis of vasodilatation is either minor or still unclear. At present, most available data, obtained mainly from experimental cirrhosis, indicate that nitric oxide (NO) is the main mediator of arterial vasodilatation in cirrhosis (Table 22-5)
Table 22-5. Evidence for a Role of an Increased Vascular Production of Nitric Oxide (NO) in the Pathogenesis of Arterial Vasodilatation and Subsequent Sodium and Water Retention in Cirrhosis Experimental cirrhosis Reversal of the impaired pressor response to vasoconstrictors of isolated aortic rings or splanchnic vascular preparations by NOS inhibition Enhanced vasodilator response to NO-dependent vasodilators Increased pressor effect of systemic NOS inhibition Increased NO synthesis in vascular tissue Normalization of the hyperdynamic circulation, activity of antinatriuretic systems and sodium and water retention by chronic NOS inhibition Increased expression of NOS isoenzymes in vascular tissue Human cirrhosis Correction of the arterial hyporesponsiveness to vasoconstrictors by NOS inhibition Enhanced vasodilatory response to NO-dependent vasodilators Increased plasma levels of NO and NO metabolites Increased NO in the exhaled air Increased NOS activity in polymorphonuclear cells and monocytes
426
(reviewed in 100). NO synthesis from arterial vessels of cirrhotic animals is markedly increased compared to that from control animals. This increased NO synthesis appears to occur in the whole vascular bed, except for the intrahepatic circulation, where NO synthesis is reduced (as discussed previously), but predominates in the splanchnic territory.101 Among the different isoforms of NO synthase, the constitutive form appears to be the one responsible for the increased NO synthesis, although a role for the inducible and neuronal isoforms has also been proposed.102 The observation that the normalization of NO synthesis in experimental cirrhosis by the administration of inhibitors of NO synthesis is associated with a marked improvement of splanchnic and systemic hemodynamics, suppression of the increased activity of the renin–angiotensin–aldosterone system and AVP concentration, increased sodium and water excretion, and a reduction in or the disappearance of ascites provides a strong argument in favor of the important role of NO overproduction in the pathogenesis of circulatory dysfunction and subsequent functional renal abnormalities in cirrhosis (Figure 22-6).103 The mechanism of the increased NO synthesis in cirrhosis is not well established. Because arterial vasodilatation occurs predominantly in the splanchnic circulation, a local factor acting in the mesenteric arterial vascular compartment is a very likely mechanism. Endotoxin or other products derived from the activity of the intestinal flora has been proposed as a possible mechanism, as intestinal bacterial orvergrowth, translocation of bacteria from the intestinal lumen into mesenteric lymphatics, or the absorption of bacterial products such as endotoxin, that stimulate the vascular synthesis of NO, are common in cirrhosis with portal hypertension.104 An alternative hypothesis is an activation of the nonadrenergic non-cholinergic nervous system. This is a sensitive system, present in the intestine and other organs and tissues, which responds to mechanical and chemical stimuli and releases neurotransmitters leading to vasodilatation by calcitonin gene-related peptide, substance P, and nitric oxide. During the last few years evidence has been presented indicating that the pathogenesis of the circulatory dysfunction in cirrhosis, particularly in the later phases of disease, is more complex than previously thought. The traditional concept is that the deterioration of systemic hemodynamics in cirrhosis is the consequence of a progression of the splanchnic arterial vasodilatation during the course of the disease as a result of the progression of portal hypertension and liver failure. However, this has not been confirmed in patients with HRS, the extreme expression of circulatory dysfunction in cirrhosis. Studies in these patients suggest that impairment in circulatory function in advanced cirrhosis is due to both a progression of arterial vasodilatation and an impairment in cardiac function.75
NEUROHUMORAL SYSTEMS The functional renal abnormalities that occur in cirrhosis are the result of a complex interplay between different systemic and local (intrarenal) neurohumoral systems. Some of these systems play a pathogenic role in the functional renal abnormalities seen in cirrhosis, whereas others represent defensive mechanisms designed to maintain renal function within normal levels. In this section, the former are referred to as effector mechanisms and the latter as
Chapter 22
3.0 p 2 ¥ ULN Features of both hepatocellular and cholestatic patterns
Jaundice; itching; nausea, anorexia, when very severe
Mixed
Patterns of injury
Hepatocytic “swelling” with foamy appearance of cytoplasm and centrally located nuclei Apoptotic bodies— hepatocyte dropout with minimal inflammation No or minimal fibrosis
Reye’ s syndrome Acute fatty liver of pregnancy Inborn or other acquired defects in mitochondrial function—fatty acid oxidation and/or ATP production
Normal liver No biliary dilatation Normal pancreas Normal spleen No PHT No changes to suggest chronic liver disease or cholecystitis
Nausea, anorexia Vomiting Confusion Somnolence (hepatic encephalopathy) Serum ALT, AST 5–25 ¥ ULN Serum AP 1–3 ¥ ULN Serum TBR, DBR variable, often normal Resembles acute viral hepatitis
Microvesicular
Variable amounts of neutral fat accumulation in hepatocytes, usually mainly in zones 3 and 2 Hepatocyte nuclei pushed to periphery of cells by macrovesicular steatosis Apoptotic bodies, hepatocyte dropout Variable inflammation Variable fibrosis, usually pericellular Lipogranulomas common in zone 3
Alcoholic liver disease Liver disease associated with metabolic syndrome: NAFL, NASH Inborn or other acquired defects in normal hepatic lipid metabolism
Diffuse, generalized hepatomegaly Increased echogenicity (US) Decreased attenuation (CT) No biliary dilatation Normal or “fatty” pancreas No changes to suggest chronic liver disease or cholecystitis
Serum ALT, AST 1–5 ¥ ULN Serum AP 1–3 ¥ ULN Serum TBR, DBR variable, usually normal Resembles alcoholic hepatitis
Asymptomatic Upper abdominal discomfort, heaviness Nausea, anorexia
Mixed micro/macrovesicular
Steatosis
Section IV. Toxin Mediated Liver Injury
Stop offending drug N-acetylcysteine for acetaminophen, Prednisolone, 20–30 mg/d, azathioprine, 1–2 mg/kg/d, for severe immunoallergic disease
Follow “Hy’ s rule”: ~10% develop jaundice ~10% of those which develop jaundice die If FHF develops, case-fatality rate for non-acetaminophen cases is ~75%; For acetaminophen cases is ~25% A minority (perhaps ~15–30%) with smoldering presentations may develop bridging fibrosis or cirrhosis Triggering of ongoing AI hepatitis by drugs is very rare ( 180 days to resolve Stop offending drug Ursodeoxycholic acid, 20– 30 mg/kg/d Cholestyramine, phenobarbital, (rifampicin) for severe itching Stop offending drug Prednisolone, 20–30 mg/d, Azathioprine, 1–2 mg/kg/d, for severe immunoallergic disease Ursodeoxycholic acid, 20– 30 mg/kg/d Cholestyramine, phenobarbital (rifampicin) for severe itching A mixture of the prognoses listed to the left.
Variable course Usually more protracted than hepatocellular, but less than cholestatic.
Full recovery No progression to chronic liver disease
Stop offending drug Supportive care, nutrition Urgent liver transplant for severe disease with grade 3–4 encephalopathy
Rapid improvement in symptoms, signs, and labs with >50% decreases within 8–30 days
Variable, depending upon underlying conditions and duration and nature of prior injury
Stop offending drug Supportive care, nutrition Consider prednisone (20–40 mg/d), pentoxfylline (400 mg tid) for severe disease (DF > 32 or renal insufficiency)
Variable, depending upon drug accumulation, half life Often, underlying alcohol or metabolic syndrome effects persist
AI, autoimmune; ALT, alanine aminotransferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; DBR, direct-reacting bilirubin; FHF, fulminant hepatic failure; TBR, total bilirubin; ULN< upper limit of normal.
Rapid improvement in symptoms, signs, and lab tests, with >50% decreases within 8–30 days
Typical course after inciting agent stopped
Chapter 26
DRUG-INDUCED LIVER INJURY
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underlying pathogenesis involves primarily apoptosis and/or necrosis of hepatocytes, serum ALT and AST levels are markedly elevated. In the case of acute poisoning of intrinsic hepatotoxins, such as acetaminophen, carbon tetrachloride, or other halogenated hydrocarbons, the elevations of serum aminotransferases may be extreme (more than 100 times the upper limit of normal (ULN). For the larger number of drugs that produce idiosyncratic, unpredictable, non-dose dependent DILI, the degree of elevation of serum ALT and AST generally is less marked (10–25 times the ULN). The serum alkaline phosphatase (AP) is generally normal or mildly elevated (less than twice the ULN). Serum total and direct bilirubin are variable. They may remain normal, although with the more severe forms of injury they are invariably increased. The degree of increase in serum bilirubin may be extreme, and it is one of the negative prognostic factors for hepatocellular-type injury. Hepatobiliary–pancreatic imaging in such injury shows a normal liver or diffuse homogeneous hepatomegaly. For some drugs, changes compatible with diffuse fatty change (Table 26-3) or phospholipidosis (Table 26-4) may be present. Of particular importance, especially when patients are jaundiced, is the lack of evidence of dilatation of the biliary tree or cholecystitis. Of course, pre-existing gallstones may be present, making it somewhat more difficult to arrive at a correct diagnosis. Some drugs, such as acetaminophen, can also cause acute pancreatic, myocardial, or renal injury. If pancreatitis occurs, the pancreas on imaging studies generally shows diffuse enlargement or edema. Typically, changes suggestive of chronic underlying liver disease are absent, although there is nothing about pre-existing liver disease that prevents patients from developing DILI. Therefore, such changes may be present. The major considerations for the differential diagnosis of acute hepatocellular injury due to drugs include acute ischemic liver injury, acute viral hepatitis, which may be due to any of the agents that are capable of causing this syndrome (see Chapters 30–35), acute congestive hepatitis, including Budd–Chiari syndrome, autoimmune hepatitis, or hepatic decompensation due to Wilson’s disease.
Table 26-3. Some Drugs and Chemicals that Produce Hepatic Steatosis Microvesicular
Macrovesicular or mixed micro-/macrovesicular
Aflatoxin b1 Amiodarone L-Asparaginase Aspirin Chloroform Cocaine Coumadin Deferoxamine Didanosine Ethanol
FIAU Halothane Methotrexate Minocycline Mitomycin Tamoxifen Tetra-, trichloroethylene Tetracyclines Valproic acid
CHOLESTATIC PATTERN OF INJURY
Table 26-4. Some Drugs and Chemicals that may Produce Mallory Bodies Amiodarone Diethylstilbestrol 4,4¢-Diethylaminoethoxyhexestrol Ethanol
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The typical findings on liver biopsy, when performed during acute hepatocellular injury due to drugs, are variable and highly dependent upon the offending agent. The most common hepatotoxic drug, namely acetaminophen, causes acute necrosis first and foremost in zone 3 of the hepatic acinus. When very severe, necrosis extends into and through zone 2 as well (see Figure 26-5). Other common findings include variable inflammation of the portal tracts, often with a considerable number of polymorphonuclear or eosinophilic forms. Acute granulomas may also occur. Indeed, drug-induced liver injury is one of the common causes of granulomas in the liver.97–99 Some drugs and chemicals are well known to produce fatty change in the liver. Usually this is primarily in zone 3, although it is certainly not restricted to this zone. All of the features of steatohepatitis may sometimes be present. In most instances of hepatocellular injury, particularly when it has been sudden and acute in onset, there is a rapid improvement in symptoms, signs, and laboratory features when the offending agent is discontinued. This does not always occur, however, and in rare individuals drugs appear to be capable of triggering the development of self-perpetuating autoimmune hepatitis.96,100 The short- and long-term prognosis of hepatocellular type injury follows ‘Hy’s rule’. This was popularized by Hyman Zimmerman, a clinical hepatologist with special interest in drug-induced liver injury.95 Hy’s rule states that about 10% of patients with druginduced liver injury of the hepatocellular type develop jaundice, and that, among those who do, about 10% will die of drug-induced liver injury. The case fatality rates for persons who develop fulminant hepatic failure due to drugs is very high (around 75%) for drugs other than acetaminophen. In contrast, the case fatality rate for acetaminophen-induced fulminant hepatic failure is much lower, with only about 25% of patients dying and/or requiring liver transplant. For the most part there is no specific therapy for drug-induced liver injury, beyond identifying the offending agent and stopping its use. It is clear that acute acetaminophen overdose should be treated immediately with N-acetylcysteine. For adults with acetaminophen ingestion less than 24 hours before presentation, a loading dose of 140 mg/kg/body weight should be given, followed by 70 mg/kg every 4 hours for 17 doses, starting 4 hours after the loading dose. It has been suggested that N-acetylcysteine may be of benefit in other forms of fulminant hepatic failure, and indeed there seems little to be lost by administering it in other forms. Particularly when hepatocellular type injury is severe, and/or when it is accompanied by evidence of immunoallergic features, a corticosteroid, such as prednisolone (20–30 mg/day), and azathioprine (1–2 mg/kg body weight per day) often are given as well.
Glucocorticoids Griseofulvin Nifedipine Tamoxifen
The typical presentation of cholestatic hepatitis due to drugs is jaundice and itching. Nausea, anorexia, or vomiting typically occur only when the reaction is very severe. The typical laboratory features are those of any cholestatic syndrome, with elevations primarily in serum AP, which is more than twice the ULN, and serum total and direct bilirubin, which also are at least twice the ULN. In the pure cholestatic case, serum aminotransferases are normal or only mildly elevated, and certainly less than three times the ULN.
Chapter 26 DRUG-INDUCED LIVER INJURY
The typical hepatobiliary–pancreatic imaging findings in cholestatic DILI are chiefly important by showing no evidence of biliary dilatation and no pancreatic abnormalities. The liver is usually normal or nearly normal, and there is nothing to suggest chronic liver disease or cholecystitis (see Table 26-2). The major differential diagnosis for cholestatic DILI includes biliary obstruction due to gallstones, tumors, strictures, or pancreatic diseases, and autoimmune disorders that affect chiefly the bile ducts, such as primary biliary cirrhosis or primary sclerosing cholangitis. There are also ‘overlap’ syndromes of autoimmune cholangitis and autoimmune hepatitis. These are considered in greater detail in Section VI (Immune diseases). Typical findings on liver biopsy in cholestatic DILI are the presence of bile in hepatocytes, bile plugs in canaliculi, and hepatocyte swelling in zone 3 (Figure 26-8). Bile lakes or other features of extrahepatic obstruction are absent, and as a rule there are no findings of acute cholangitis or pericholangitis, such as one would expect to see in bacterial ascending cholangitis. The typical course of cholestatic hepatitis is quite different from that of hepatocellular DILI in being more protracted. In fact, it is not uncommon for signs and laboratory worsening to continue after the drug has been stopped, sometimes for as long as 30–60 days. There is gradual improvement thereafter, unless the offending agent or another like it is readministered. However, this can require as long as 180 days. There are rare instances in which the disease does not resolve but rather goes on to produce the adult vanishing bile duct syndrome, sometimes with progression to secondary biliary cirrhosis.99 The usual therapy of cholestatic DILI is to stop the offending drug and administer ursodeoxycholic acid. In light of growing evidence that higher doses of this agent are more effective in chronic cholestatic disorders such as primarily biliary cirrhosis or primary sclerosing cholangitis, it is our recommendation that the drug be given at a dose of 20–30 mg/kg/day in two divided doses. If the itching is severe the usual treatment is cholestyramine, but this must be given at times other than when ursodeoxycholic acid or other drugs are administered, because it will bind the drugs and prevent their absorption. We generally recommend that the cholestyramine be administered in the morning, when there is maximal turnover of the biliary pool. Phenobarbital and/or rifampicin can be helpful for severe itching, although both of these drugs, especially rifampicin may to cause hepatotoxicity on their own.
‘MIXED’ PATTERN OF INJURY This pattern, as the name implies, involves features both of hepatocellular and cholestatic injury (see Table 26-2). The typical clinical presentation is nausea, anorexia, and vomiting when severe. There is also jaundice and itching. The typical laboratory findings are for serum aminotransferase levels to be greater than three times the ULN and for serum AP and total and direct bilirubin to be more than twice the ULN. The biopsy features are also a mixture of features described above for the two types of injury. The considerations for differential diagnosis must include ischemic hepatitis, acute congestive hepatitis, acute viral hepatitis, autoimmune hepatitis, or overlap syndromes of autoimmune cholangitis and hepatitis, hepatic decompensation due to Wilson’s
A
B
Figure 26-8. (A) Cholestatic injury. This biopsy from a patient who became jaundiced while taking the NSAID nabumetone has relatively ‘bland’ cholestasis with numerous canalicular bile plugs (arrows), but relatively little hepatocellular injury. The peak serum bilirubin in the patient was 110 mg/dL. (B) Cholestatic injury. This biopsy from a patient who became jaundiced after a course of amoxicillin shows a combined hepatocellular and cholestatic injury with canalicular bile plugs (arrows) as well as hepatocyte injury, apoptosis and dropout with Kupffer cell hypertrophy and lymphocytic inflammation, producing disarray of the liver cell plates.
disease, primary biliary cirrhosis, primarily sclerosing cholangitis, and biliary obstruction due to gallstones, tumors, strictures, or primary pancreatic diseases. The typical treatments are the same as already described for hepatocellular and cholestatic injuries. The typical course is somewhat longer than for hepatocellular injury, but somewhat shorter than for typical cases of pure cholestatic DILI.
STEATOSIS (FATTY LIVER) As shown in Table 26-2, there are two major types of disease that produce primarily fatty change in the liver, namely, pure small droplet fat (microvesicular) and fewer large droplet fat (macrovesicular), although the latter is usually associated with at least a mild degree of microvesicular steatosis as well.
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Section IV. Toxin Mediated Liver Injury
Microvesicular steatosis is due principally to mitochondrial toxicity, leading both to a deficiency in mitochondrial b-oxidation of free fatty acids and to critical compromise of mitochondrial ATP production. Patients with these defects commonly present with nausea, anorexia, vomiting, confusion, or coma, the latter due to hepatic encephalopathy with prominent and severe hyperammonemia. They often have significant lactic acidosis, owing to the critical defect in mitochondrial respiration and oxidative phosphorylation. The typical laboratory features are moderate to marked increases in serum aminotransferases; serum AP is normal or only slightly increased, and serum bilirubin levels are variable, depending on the severity of the injury. Typical hepatobiliary pancreatic imaging studies in patients with microvesicular steatosis show a normal liver, no biliary dilatation, normal pancreas and spleen, and nothing to suggest portal hypertension or chronic liver disease. The major differential diagnosis is Reye’s syndrome or inborn or other acquired defects in mitochondrial function, particularly fatty acid metabolism or ATP production. The findings on liver biopsy are remarkably mild (Figure 26-9). In order to see the lipid clearly, it may be necessary to perform oil red O staining on frozen sections. The reason is that there is diffuse lipid accumulation in very small droplets, often smaller than the limit of resolution by light microscopy. There is no displacement of hepatocytic nuclei, such that the lipid may not be apparent in formalin-fixed tissue stained in the routine way. There is minimal inflammation, although apoptotic bodies and evidence of hepatocytic dropout may be present, and there is usually no fibrosis. The usual course is one of rapid improvement if the inciting agent is stopped. However, some patients have such severe defects that they may be unable to recover unless they receive urgent liver transplantation. Certainly, all such patients who might be transplant candidates and who develop higher grades of hepatic encephalopathy should rapidly be transferred to a transplant center. If patients can be nursed successfully through the acute phase of disease, complete
recovery with no progression to chronic liver disease will ensue. Examples of drugs that produce microvesicular steatosis are summarized in Table 26-3.
MACROVESICULAR OR MIXED MICRO- AND MACROVESICULAR STEATOSIS The accumulation of fat is probably the most common liver abnormality. Potential causes of fatty liver are manifold and discussed in greater detail in Chapter 55. Drugs and chemicals are among the important causes of fatty liver. Indeed, if one considers ethanol as a drug, they are probably the most common causes. Most people with fatty liver due to alcohol or other conditions that produce macrovesicular steatosis are asymptomatic. When the fatty deposition is severe hepatomegaly ensues, and patients may have upper abdominal discomfort and a sense of heaviness. It is rare for more severe symptoms, such as nausea, anorexia, vomiting, jaundice, etc., to occur. Laboratory studies may be entirely normal or may show mild increases in serum aminotransferases. Serum AP may be slightly increased; g-glutamyl transpeptidase is usually elevated more. Typical findings on hepatobiliary–pancreatic imaging are diffuse, generalized hepatomegaly. Ultrasound shows evidence of increased echogenicity, whereas CT scanning shows a decrease in hepatic attenuation. There is generally no biliary dilation and the pancreas is normal, or may show increased echogenicity indicative of a fatty deposition in the pancreas. In addition to heavy alcohol use, macrovesicular steatosis is commonly caused by liver disease associated with the metabolic syndrome (non-alcoholic fatty liver and non-alcoholic steatohepatitis, see Chapter 55). The typical findings on liver biopsy in drug-induced macrovesicular steatosis are indistinguishable from those caused by alcohol or by non-alcoholic fatty liver. It is common for patients to have these changes owing to an element of alcohol and nonalcoholic fatty liver plus one or more drugs. Mallory bodies may develop as a result of alcoholic or non-alcoholic steatohepatitis, and have been associated with several drugs (see Table 26-4). The usual therapy is to stop the offending drug. However, if the fatty change is mild and asymptomatic, and if the drug is essential for other reasons, such as methotrexate for the management of rheumatoid arthritis or psoriasis, the decision may be made to continue the drug with careful monitoring. In addition to the histopathological features already described, drugs can cause the accumulation of phospholipids in hepatocytes and other cells (Table 26-5), vascular lesions in the liver, including peliosis hepatis (Table 26-6), sinusoidal obstruction or veno-occlusive disease, and arterial vascular compromise, which is manifest as a syndrome that resembles sclerosing cholangitis.
PREDICTABLE VS UNPREDICTABLE DILI
Figure 26-9. Microvesicular steatosis in a child taking valproic acid. Most of the hepatocytes have small vacuoles of fat, and there has been liver cell dropout with Kupffer cell hypertrophy and a mild lymphocytic infiltrate.
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Another useful way to categorize DILI is as predictable or ‘intrinsic’ injury versus unpredictable or ‘idiosyncratic’. By far the most important example of the former is acetaminophen, which, by mechanisms already described, will produce liver injury in virtually everyone who takes a sufficient dose. Examples of other drugs or toxins that act similarly are listed in Table 26-7. Most drugs, however, cause DILI unpredictably and in only a small percentage of subjects. Such reactions are called idiosyncratic
Chapter 26 DRUG-INDUCED LIVER INJURY
reactions. These are further subdivided according to whether they are accompanied by immunoallergic manifestations or not. Such manifestations include fever, peripheral eosinophilia, skin rash, arthralgia, arthritis etc. As shown in Table 26-7, many drugs are recognized to be capable of causing idiosyncratic DILI either with or without an immunoallergic phenotype, stressing the importance of genetic host factors in modulating the response to injury (see Figure 26-2). The mechanisms that probably give rise to immunoallergic injury are summarized in Figure 26-10. According to this view, drugs may give rise to antigens, by binding to host proteins (perhaps altering them), which are recognized as foreign and against which the host’s immune system mounts a T- or B-lymphocyte response. Because such neoantigens are displayed on hepatocytes, where most drug metabolism occurs, the net effect may resemble autoimmune hepatitis. Indeed, ingestion of drugs appears to trigger autoimmune hepatitis in rare individuals.96,100
Table 26-5. Some Drugs that Produce Phospholipidosis [All amphiphilic drugs] Amantadine Amikacin Amiodarone Amitryptiline Chloramphenicol Chlorcyclizine Chloripramine Chloroquine Chlorpheniramine Chlorpromazine Desipramine
Gentamicin Imipramine Iprindole Ketoconazole Mepacrine Promethazine Propranolol Sulfamethoxazole-trimethoprin Thioridazine Trimipramine Trippelennamine
Table 26-6. Some Drugs and Chemicals that may Produce Peliosis Hepatis Anabolic steroids Arsenic Azathioprine Contraceptive steroids Danazol Diethylstilbestrol Estrone
Glucocorticoids Medroxyprogesterone Tamoxifen Thioguanine Thorotrast Vinyl chloride Vitamin A excess
Parent drug Bioactivation
DILI DUE TO SPECIFIC AGENTS ANESTHETICS Of the agents currently in use to induce and maintain anesthesia, it is only the halogenated volatile agents that have clinically significant
Modified protein Kupffer cell
Native protein
Ag-processing APC
Modified protein
Reactive metabolite
Dendritic cell
Helper T cell MHC class 2
APC Processed Ag T cell activation + proliferation
Cytotoxic T cells
B cells producing antibodies
T cell-mediated killing of hepatocytes
Figure 26-10. Likely mechanisms for pathogenesis of drug-induced immunoallergic hepatitis.
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Section IV. Toxin Mediated Liver Injury
Table 26-7. Classification of DILI: Comparison of Intrinsic (Predictable) vs Idiosyncratic (Unpredictable) DILI Variable
Type of drug-induced liver injury Intrinsic
Idiosyncratic With immunoallergic features
Without immunoallergic features
Predictability/dose dependence
High/yes All subjects given high doses will develop hepatotoxicity Regularly produced in experimental animals
Low/slight or nil Most subjects will not develop hepatotoxicity, regardless of dose Not reproducible in experimental animals
Low/slight or nil Most subjects will not develop hepatoxicity, regardless of dose Not reproducible in experimental animals
Associated features
Toxic damage to other tissues also occurs Drug-induced renal, pancreatic injury common
Fever, skin rash, peripheral adenopathy, eosinophilia Development of autoantibodies (ANA, ASMA), hyperglobulinemia
No extrahepatic manifestations of immunoallergic responses
Underlying risk factors
Induction (without inhibition) of enzymes that increase formation of toxic intermediates Conditions that decrease metabolism, detoxification, and removal of toxic intermediates
Allergic diathesis Other host genetic factors presumed to play a role such as presence of certain HLA types, factors that influence Th1 vs Th2 phenotypes, etc. Women more susceptible than men (as for most AI diseases)
Other host genetic factors presumed to play a role, such as genetic variations that influence expression of phase 1–3 enzymes of drug metabolism Presence of underlying liver disease, especially chronic viral hepatitis in subjects receiving HAART and fatty liver in subjects receiving methotrexate or dugs implicated in producing steatohepatitis.
Typical pattern of injury
Hepatocellular, acute
Hepatocellular, acute Less often, cholestatic or mixed
Hepatocellular, acute Less often, cholestatic or mixed
Response to rechallenge
Reproduced promptly and dependably
Very rapid recurrence (1–3 doses)
Variable—may be delayed for several weeks, usually more rapid than the initial episode
Examples of inciting agents
Acetaminophen Amanita toxins Bromobenzene Carbon tetrachloride Chloroform Halothane White phosphorus
Amoxicillin/clavulanic acid Alphamethyldopa Diclofenac Doxycycline Fenofibrate Halothane Hydralazines Minocycline Nitrofurantoin Penicillins Phenlbutazone Phenytoin Quinidine Statins (very rarely)
Amoxicillin/clavulanic acid Chlorpromazine (other phenothiazines) Enflurane Fluroxene Glitazones (rosi-, pio, troglitazone) Isoniazid Nifedipine Penicillins Phenelzine Phenylbutazone Propylthiouracil Statins (rarely) Sulfonylureas Quinidine
Typical duration of exposure, prior to onset
Very brief ( cholestatic Cholestatic > hepatitic
Not reported to cause hepatotoxicity Ethosuximide (Zarontin), gabapentin (Neurontin), levetriacetam (Keppra), phenobarbital* primidone (Mysoline), tiagabine (Gabitril), zonisamide (Zonegram) *Phenobarbital activates orphan nuclear receptor CAR and exerts well known proliferative effects on hepatocytes.
The clinical symptoms usually manifest within 1–8 weeks of drug exposure and include fever, malaise, lymphadenopathy, splenomegaly, and rash. Serum aminotransferases are elevated 2–100-fold (ALT>AST) and AP two- to eightfold.125,127,128 Leukocytosis and atypical lymphocytes suggesting mononucleosis and eosinophilia are common, with a lupus-like syndrome and pseudolymphoma reported occasionally. Other organ system toxicities can include interstitial nephritis, myositis and rhabdomyolysis, pneumonitis, and marrow suppression. The clinical presentation can also simulate viral hepatitis. When liver biopsies are performed, the histology shows a panlobular mixed mononuclear and polymorphonuclear infiltrate with prominent eosinophilia. In 10% of cases cholestasis is the predominant finding. The findings are not specific, however. Therapy is discontinuation of the drug, which in most cases leads to resolution of toxicity. However, once liver failure develops, the case:fatality ratio can be as high as 40%. Because of cross-reactivity with carbamazepine and oxcarbamazepine,126,129 these latter agents should not be used to replace phenytoin for seizure control in subjects who have experienced symptomatic phenytoin toxicity. A phosphate ester prodrug of phenytoin, fosphenytoin, developed for parenteral administration,130 should also be avoided.
Carbamazepine Like phenytoin, carbamazepine can also cause asymptomatic mild elevations in serum GGTP (64%) and AP (14%) that do not require discontinuation of therapy.127 However, aminotransferase elevations, seen in 22% of patients, may indicate susceptibility to develop the more serious idiosyncratic hypersensitivity reaction. A Swedish analysis131 estimated the risk to be about 1 in 6000, which is more common than with phenytoin. The hypersensitivity reaction is also due to formation of a reactive metabolite, probably an unstable
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Section IV. Toxin Mediated Liver Injury
epoxide formed by CYP3A4.132 Drug toxicity usually occurs within 8–16 weeks of therapy and presents with fever, rash, and peripheral eosinophilia. Marrow suppression, nephritis, and pneumonitis can also occur. Carbamazepine is more likely than phenytoin to cause a pure cholestatic pattern of hepatotoxicity, which occurs in 30% of reactions. A mixed pattern of liver injury with AP, bilirubin, and aminotransferase elevations occurs in 50% of cases. A predominantly hepatocellular injury may have a worse prognosis.127 Consistent with the cholestatic clinical picture, histopathology commonly demonstrates a granulomatous reaction with eosinophilia.133 Resolution of injury takes several weeks after drug withdrawal. Because the injury is immune mediated rechallenge is not recommended, and both phenytoin and oxcarbamazepine should also be avoided.129,134
serum aminotransferases. Valproate therapy has also been reported to decrease serum albumin concentrations by up to 30% without apparent toxicity in a small study of children with severe neurologic disabilities.143 Because of the potential of valproate to damage mitochondrial function, L-carnitine was suggested as a protective agent144 and may improve survival with severe valproate hepatotoxicity,145 especially if given intravenously. Oral supplementation of 100 mg/kg/day is recommended for infants and young children taking valproate, and for patients with symptomatic hyperammonemia or multiple risk factors for hepatotoxicity.146 Prophylactic use of L-carnitine decreases the risk of valproate hepatotoxicity and is recommended.
Clonazepam Oxcarbazepine This keto analog of carbamazepine, first introduced in 1990 in Denmark, has recently become available in most countries, including the USA. It is considered to be a safe and useful anticonvulsant135 with fewer P450-related drug interactions than carbamazepine.122 However, it has also been reported to cause acute liver failure due to hypersensitivity,136 with a similar clinical presentation to that of carbamazepine and phenytoin.134
Valproic Acid This may be the most widely prescribed anticonvulsant worldwide,137 and in general is considered very safe, with the incidence of hepatotoxicity in adults and children older than 2 years being approximately 1 in 35 000.138 However, in children under 2, especially if taking other anticonvulsants, the incidence may be as frequent as 1 in 600. It is also clear that patients with genetic mitochondrial enzyme defects are at greater risk,139 most likely because of its depletion of coenzyme A levels and its metabolism via mitochondrial oxidation. Hepatotoxicity is the most common serious toxicity of the drug, and usually occurs within the first 3–6 months of therapy.127 Valproate’s hepatotoxicity is most likely dose related,139 although epidemiologic studies have suggested that other host factors and polypharmacy may be more important.138 Up to 40% of patients have transient asymptomatic ALT elevations that improve with dose reduction.127 High doses of drug, in addition to young age and polypharmacy, are significantly associated with higher excretion of thiol conjugates of the toxic valproate metabolite (E)-2,4-diene VPA.140 Therefore, ALT monitoring is recommended for the first 6 months of therapy and after dose increases. Patients taking olanzepine in addition to valproate had higher ALT elevations than with either drug alone.141 Although no specific degree of ALT elevation has been identified as an indicator of impending hepatic failure, a greater than threefold elevation should prompt drug cessation. If fever, nausea, vomiting, and abdominal pain accompany laboratory evidence of developing hepatic failure and poor seizure control, then liver failure will probably become irreversible. The characteristic histopathology of valproate hepatoxicity is that of microvesicular steatosis, similar to Reye’s syndrome, seen mainly in zones 2 and 3.127 These changes may occur without toxicity. In one recent report 61% of patients on long-term valproate had sonographic evidence of fatty liver,142 with the majority having normal
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Although the medication database www.epocrates.com lists hepatotoxicity as a serious adverse effect of clonazepam, no hepatotoxicity is mentioned as an adverse event in the manufacturer’s drug insert (www.pdr.net), and only one case report could be found in the literature.147 Therefore, it is unlikely that clonazepam has any significant hepatotoxicity.
Felbamate When this aromatic compound was approved for use in 1993, it was the first new anticonvulsant approved in the US since the introduction of valproate in 1978. However, during its first year of use an incidence of hepatic failure of 1 in 6000 (and an aplastic anemia incidence of 1 in 3000) prompted restriction of its use to severe epilepsy not responding to other agents.122 However, felbamate is still considered an important drug, with over 8000 patients treated annually in the USA. By 1996, 36 cases of hepatotoxicity had been collected by the FDA, with five deaths.148 However, since then no further cases of hepatotoxicity have been reported. The mechanism of toxicity appears to involve the formation of an aldehyde monocarbamate149 that is activated to atropaldehyde.127,150,151 Because felbamate is usually given with other anticonvulsants and is a CYP3A4 substrate, studies of drug interactions have been carried out. A recent in vivo study152 suggests that felbamate may heteroactivate CYP3A4 to promote the formation of carbamazepine-10,11epoxide when these agents are used together. The paucity of reported cases makes it difficult to describe the clinical characteristics and histopathology of felbamate hepatotoxicity. However, presentation occurs between 3 weeks and 6 months, with a possible female preponderance.127
Lamotrigine This chlorinated phenyltriazine anticonvulsant has been in use for over a decade, and the first case of fulminant hepatic failure due to it was reported in 1995.153 Another severe case of an 8-year-old boy who recovered was reported in 1998.154 Despite over 2 million prescriptions written,122 only nine cases of hepatotoxicity have been reported so far in the literature155 and most were on polytherapy. The most common adverse event of lamotrigine is skin rash,156,157 which occurs in 3–10% of patients127 and can be a severe Stevens–Johnson syndrome. Whether the metabolism of lamotrigine to a reactive arene oxide158 is responsible for both the cutaneous toxicity and the rare hepatotoxicity is not yet clear.
Chapter 26 DRUG-INDUCED LIVER INJURY
Topirimate
Pergolide
This anticonvulsant, also marketed to prevent migraines, is related to carbonic anhydrase inhibitors and can cause a metabolic acidosis. It has been considered very safe with few side effects (the majority being CNS), especially if started at low doses and increased slowly ( cholestatic hepatitic, ?Cholestatic cholestatic, hepatitic too new, incidence unknown cholestatic > hepatitic very low incidence too new, incidence unknown hepatitic
Antiparisitics Thiabendazole (Mintezol) Mebendazole (Vermox) Albendazole (Albenza)
cholestatic>hepatitic ≠alts ≠alts
Antimalarials Pyrimethamine/sulfadoxine (Fansidar) Amodiaquine (not available in U.S.)
very rare very rare
Antitubercular Isoniazid Rifampicin Pyrazinamide Ethambutol (Myambutol) Dapsone Rifapentine (Priftin) Ethionamide (Trecator-SC)
Idiosyncratic hepatitic Idiosyncratic hepatitic Idiosyncratic hepatitic doubtful ≠alts Idiosyncratic hepatitic Idiosyncratic hepatitic
Not reported to cause hepatotoxicity Antifungals: amphotericin (only mild ≠ALTs), clotrimazole (Mycelex), miconzaole (Monistat), nystatin (Mycostatin) Antimalarials: chloroquine (Aralen), hydroxychloroquine (Plaquenil), primaquine, mefloquine (Lariam +/-≠LFTs), atovaquone/proguanil (Malarone +/-≠LFTs), pyrimethamine (Daraprin) Antiparasitics: pentamidine (Pentam), atovaquone (Mepron), praziquantel (Biltricide), pyrantel (Antiminth), ivermectin (Stromectol +/-≠LFTs), nitzoxanide (Alina) Antitubercular: streptomycin, rifabutin (Mycobutin), cycloserine (Seromycin +/-≠LFTs)
The mechanism of ketoconazole liver injury appears to involve formation of an N-deacetyl metabolite that is converted to a toxic dialdehyde by the flavin-containing mono-oxygenases.214,215 Treatment is drug cessation, and ursodeoxycholate may help prevent progressive cholestatic injury.216 Itraconazole, a less potent CYP3A inhibitor than ketoconazole,206 also causes less hepatotoxicity.211 In a pharmaceutical database study of over 54 000 itraconazole and fluconazole users,217 ‘serious adverse liver events’ were reported in only 1 in 30 000 prescriptions for either drug. If itraconazole was given as ‘pulse’ therapy (1 week/month ¥ 3), then no serious hepatotoxicity was found.218 However, three cases of cholestatic liver injury were reported in patients taking itraconazole long term. These patients presented with jaundice, and ductopenia was noted in two of the three biopsies.219 Focal nodular hyperplasia has been linked to itraconazole in one patient who had been taking the drug for 4 months.220 Fluconazole is considered very safe, with only 5% of 562 children developing transient elevations of serum ALT.221 In some reports of DILI, fluconazole was found to have been administered with nitrofurantoin222 or amphotericin B.223 Voriconazole is probably still too
new for its incidence of hepatotoxicity to be known, and no literature citations have been found. However, recent safety reviews suggest monitoring liver tests as well as visual change when using voriconazole.224,225
Terbinafine This allylamine antifungal has replaced pulse intraconazole for the treatment of onychomycosis, and is widely advertised. The incidence of hepatobiliary dysfunction in postmarketing surveillance has been reported to be as low as 1 in 40 000.211,226 However, recent case reports,227–229 and many others too numerous to cite, suggest a higher incidence with a predominance of cholestatic reactions, including one liver transplant patient who was initially thought to have acute rejection 5 years after his transplant.230 Toxicity can be seen as early as 1 week after starting the drug.229 The mechanism of injury might be the formation of an N-dealkylated allylic aldehyde that is conjugated with GSH and transported across the canalicular membrane.228,231
Griseofulvin This older antifungal agent has been the mainstay of therapy for tinea capitis, but may now be supplanted by the newer antifungals that require only 2–3 weeks of therapy instead of 6 weeks.232 Although GI side effects of the drug are common, only one case report from 1976 describes griseofulvin hepatotoxicity.233 A more recent prospective study210 showed no liver test abnormalities in 74 patients treated for 3 months.
Caspofungin This new echinocandin antifungal is the first of its kind to be approved and is only available for intravenous use. It can be used by itself or with liposomal amphotericin B, or with voriconazole for refractory invasive aspergillosis and candidiasis in immunosuppressed patients,224,234–236 but is not effective for cryptococcus. The drug inhibits fungal cell wall b-(1,3)-glucan synthesis. It appears to be metabolized by hepatic P450s and may inhibit CYP3A4.237 Caution is therefore required when it is used with ciclosporin A238 and other calcineurin inhibitors. However, nelfinavir did not alter its pharmacokinetics.239 Because of a paradoxical loss of efficacy against Candida spp. at high concentrations,237 it may best be used for complicated infections in combination. Because phase 1 and 2 trials commonly reported elevated liver enzymes234, its long-term safety and incidence of hepatotoxicity are still to be determined.
Flucytosine This oral antifungal, available since the 1970s, is known to cause elevations of serum ALT in 5–15% of patients.204 Its mechanism of hepatic injury is unknown, but appears to be dose-related.240 Its main use currently is for the treatment of severe fungal infections. It has been used successfully in combination with fluconazole241 for cryptococcosis in a liver transplant patient. Because its use is so limited, it is doubtful that its mechanism of hepatotoxicity will ever be known.
Antimalarials The toxicities of most antimalarials, such as chloroquine and hydroxychloroquine, are chiefly neurologic and hematologic.
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However, pyrimethamine/sulfadoxine242,243 and amodiaquine242,244 have been associated with DILI and ALF when the drugs have been continued after the onset of jaundice. The incidence of serious hepatotoxicity is estimated to be 1 in 11 000–15 000.242 Amodiaquine is not available in the USA, but is widely used in countries with endemic malaria where drug-resistant strains are a problem.245,246
Benzimidazole Antiparasitics Thiabendazole, mebendazole, and albendazole all seem to cause elevations of serum ALT at times,247 but only thiabendazole, available since 1964, has been reported to cause a cholestatic hepatitis that has led to ductopenia and cirrhosis.248–251 The incidence appears to be low, but has not been determined, and no recent case reports have appeared.
Isoniazid, Rifampicin, and Pyrazinamide Isoniazid (INH), available since the 1960s, is well known to be a hepatotoxic drug that causes an idiosyncratic hepatitic reaction leading to overt clinical hepatitis in 0.3–1.0% of patients when used as monotherapy.252,253 It is the second most common drug responsible for ALF requiring liver transplantation in the US.1 Hepatotoxicity increases when used in combination with other agents, such as rifampicin253 and pyrazinamide.254 Despite the risks, INH continues to be used because it is still the most effective agent for Mycobacterium tuberculosis therapy.252 For patients with latent TB infection the use of INH monotherapy for 6 or 9 months is considered the therapy of choice,255 but compliance is often low256 (8%), to which the use of pyrazinamide may have contributed. A study from India also implicated the use of pyrazinamide.254 Other variables, including female gender, underlying liver disease related to alcohol, hepatitis B and C, and malnutrition have all been variably implicated in increasing the incidence of severe hepatotoxicity.252,254 A case of hyperacute liver failure in a young patient also receiving carbamazepine was recently reported.271 In an effort to avoid overt hepatitis, complex algorithms have been developed255 that include baseline laboratory tests for all but the most healthy young (10 times ULN, especially with lactic acidosis, HAART and other potentially hepatotoxic drugs must be stopped.
Nucleoside Analogue Reverse Transcriptase Inhibitors (NRTI) The most ominous toxic effect of NRTIs is the development of mitochondrial toxicity that leads to lactic acidosis and liver failure, similar to Reye’s syndrome.294 It is thought that depletion of mitochondrial DNA by NRTIs causes the mitochondrial dysfunction. This supposition was strengthened by findings that stavudine, didanosine, or zalcitabidine, which are known to deplete mtDNA in cultured hepatocytes,296 are associated with lower mtDNA in liver biopsy samples and higher serum lactate levels in HIV-HCV coinfected patients, compared with patients on zidovudine, lamivudine, and abacavir, which do not deplete mtDNA.297 Hepatitis C infection by itself might cause mitochondrial dysfunction,298 but it
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Section IV. Toxin Mediated Liver Injury
is not yet clear whether co-infection increases the risk of mitochondrial toxicity. Stavudine is associated with more cases of lactic acidosis than other NRTIs,299 but all have been associated with cases of microvesicular steatosis and liver failure.292 Some experts281,294,299,300 advise the use of coenzyme-Q (30–60 mg tid), carnitine (1–3 g/day), riboflavin (50 mg/day), and/or thiamine (100 mg/day) in the event of lipoatrophy or severe lactic acidosis, but most of these interventions have not been studied in the setting of liver failure.
Protease Inhibitors (PI) The introduction of PIs in the 1990s marked the beginning of highly active HAART and control of HIV. All of the PIs (see Table 26-11) are used in combination with other antivirals, and increased serum ALT or AST more than five times ULN occurred in 1–9.5% of patients during the original registration trials.293 Ritonavir used in higher doses caused liver injury more frequently than the other agents, and is now given in lower doses in combination with other PIs. A characteristic of all PIs is that they are metabolized by and are inhibitors of hepatic CYPs, primarily 3A4, and ritonavir is the most potent.301 Furthermore, all except possibly indinavir, appear to be mechanism-based inhibitors of the P450, which means they irreversibly inactivate the enzyme. Nelfinavir may also affect CYP2C19.301a Indinavir also causes a reversible inhibition of UDPGT that leads to benign unconjugated hyperbilirubinemia in 12% of patients. Because ritonavir inhibits CYP3A so well, it is given with lopinavir in one of the more commonly used PI combinations, to prevent lopinavir’s metabolism. Liver toxicity with this combination is not higher than with nelfinavir-based HAART.302 The mechanism whereby PIs cause liver injury is not yet known,293 nor whether asymptomatic ALT elevations promote more rapid progression of fibrosis in patients co-infected with hepatitis B or C. Because PIs are always given in combination with other agents to patients with other causes of liver injury, sorting this out will be complex. However, inhibition of CYPs by PIs causes the most important clinically relevant ADRs. One example is the effect of lopinavir/ritonavir therapy for HIV in liver transplant patients who are receiving tacrolimus and must have profound dose reductions in that immunosuppressive agent.303
Non-nucleoside Reverse Transcriptase Inhibitors (NNRTI) Of the three available NNRTIs, nevirapine and efavirenz have been utilized to a much greater extent than delavirdine, possibly because of the latter’s being an inhibitor of several CYPs.304 A number of large cohort studies have shown that, when given with NRTIs and/or PIs, nevirapine is two to three times as likely as efavirenz to cause increases in serum ALT more than five times ULN.291,305–310 The incidence across multiple studies with nevirapine was 10%,307 with clinical symptoms in 4.9%. The hepatotoxicity with both nevirapine and efavirenz was often delayed, being recognized 3–9 months (median 5.5) after therapy began,310 and was more common in patients coinfected with hepatitis B and C. Another study has suggested that HAART regimens that contain nevirapine are associated with more rapid progression of hepatic fibrosis in hepatitis C patients,311 and that co-infected patients on PIs do better than those on NNRTIs.
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Nevirapine has also been reported to cause severe hepatotoxicity and cutaneous reactions, both when used alone and when used with other antivirals for postexposure prophylaxis in non-HIV patients.312 These authors recommended avoiding nevirapine in prophylaxis regimens. The mechanism of hepatotoxicity for the NNRTIs may involve an idiosyncratic response with immune features, and this may be why the hepatotoxicity is worse in non-HIV subjects and is delayed in HIV patients.
Nucleotide Reverse Transcriptase Inhibitors Tenofovir is effective against HIV and does not appear to deplete mitochondrial DNA.313 Both tenofovir and adefovir are effective against hepatitis B.314,315 Although the Epocrates database lists hepatotoxicity as a serious reaction for both drugs, no citations could be found and adverse liver effects are not mentioned in the package inserts for either drug.
Cidofovir This anti-CMV agent, approved to treat CMV retinitis in HIV patients, causes nephrotoxicity and neutropenia as its major side effects (www.pdr.net). Hepatotoxicity is mentioned in the Epocrates database, but no citations could be found. Almost all of the anti-CMV agents appear to be devoid of hepatotoxicity.
ANTIMICROBIALS – ANTIBACTERIALS (Table 26-12)
Penicillins and Cephalosporins In general, the b-lactam antibiotics and structurally related cephalosporins have a very low incidence of hepatotoxicity, most of which is idiosyncratic with immune features that can manifest as either primarily hepatitic, primarily cholestatic, mixed, and/or granulomatous.204,247 That the same agent can present in several ways suggests that host factors must dictate the type of allergic injury. b-Lactamase-resistant agents, and penicillins with b-lactamase inhibitors, such as sulbactam and clavulanate, more often cause a cholestatic picture,316a–d occasionally with a prolonged clinical course.316e The incidence of DILI with amoxicillin alone is 1 in 30 000, but with clavulanate is 1 in 6000.316d If older patients are given repeated courses, the incidence may be as high as 1 in 1000. Piperacillin with another b-lactamase inhibitor, tazobactam, was noted to cause only mild increases in serum ALT in clinical trials,317 and no reports of cholestatic injury have yet been reported. Flucloxacillin, one of the earlier b-lactamase-resistant agents, had a very high incidence of chronic cholestasis318 and is no longer used in the USA. Only oxacillin and dicloxacillin are available in the US, with no serious hepatotoxicity listed for them in the Epocrates database. Yet, recent reports318,319 oxacillin of used intravenously in children suggest that hepatotoxicity cannot be discounted. For the cephalosporins, most of the reports of hepatotoxicity are from the 1980s.204 Ceftriaxone, which is excreted into bile, has been associated with the formation of biliary sludge and stones.320 In general, the cephalosporins have only a minor risk of hepatotoxicity. Although cefaclor and cefdinir are listed in the Epocrates database as causing cholestatic jaundice and hepatitis, no literature citations were found.
Chapter 26 DRUG-INDUCED LIVER INJURY
Table 26-12. Antibacterial Drugs and DILI Reported to cause hepatotoxicity Penicillins Ampicillin + sulbactam (Unasyn) Amoxicillin +clavulanic acid (Augmentin) Oxacillin Ticarcillin (Ticar) +clavulanic acid (Timentin) Piperacillin +tazobactam (Zosyn)
hepatitic > cholestatic cholestatic > hepatitic hepatitic > cholestatic cholestatic, granulomatous cholestatic, granulomatous unknown cholestatic ≠hepatitic or mixed
Cephalosporins Cefaclor (Ceclor) Cefdinir (Omnicef ) Ceftriaxone
possibly cholestatic possibly hepatitic biliary sludge
Macrolides Erythromycin Clarithromycin (Biaxin) Azithromycin (Zithromax) Telithromycin (Ketek)
cholestatic cholestatic cholestatic cholestatic
Quinolones Trovafloxacin (trovan) Probably all others
immune mediated FHF hepatitic or mixed
Sulfonamides Trimethoprim/sulfamethoxazole Sulfisoxazole (Gantrisin) Sulfadiazine
Immune hepatitic (esp HIV) cholestatic > hepatitic cholestatic > hepatitic
Tetracyclines Tetracycline Doxycycline Demeclocycline (Declomycin) Minocycline (Minocin, Vectrin)
mitochondrial in high doses hepatitic none reported autoimmune hepatitis, hepatitic
Other antimicrobials Nitrofurantoin Nalidixic acid Quinupristin/dalfopristin (Synercid) Fosfomycin (Monurol)
Acute hepatitic & chronic fibrosis possible cholestasis (one case) possible cholestasis ≠ALT
Not reported to cause significant hepatotoxicity Penicillins Pen V-K, ampicillin, nafcillin, mezlocillin (Mezlin) Cephalosporins Almost all first, second, and third-generation agents Other antimicrobials Chloramphenicol, aztreonam (Azactam, ±≠ALT), ertapenem (Invanz, ±≠ALT), meropenem (Merrem), clindamycin, metronidazole, tinidazole (Tindamax), furazolidine (Furoxone-not avail in US), vancomycin, daptomycin (Cubicin, ±≠ALT), imipenem/cilastin (Primaxin, ±≠ALT), linezolid (Zyvox), iodoquinol (Yodoxin), rifaximin (Xifaxan)
Macrolides Hepatotoxicity from erythromycin has been known for decades and can occur with either erythromycin base or any of the salts.204 Erythromycin toxicity is predominately cholestatic, owing to an idiosyncratic immunoallergic reaction. Often the clinical presentation will occur well after treatment has ended. Fever, jaundice, right upper quadrant pain, and nausea can present like acute cholecysti-
tis. Eosinophilia is often present. Fortunately the incidence is low (1 in 30 000).321a Although recovery can take many weeks, rarely is it fatal. Similar cholestatic presentations, including occasional fatalities, have been reported recently for clarithromycin,321b,322,323 azithromycin,324a–b and roxithromycin.324c However, the incidence of hepatotoxicity appears to be lower with the newer macrolides.325 Telithromycin, a new ketolide antibiotic that is a structural analog of erythromycin, was FDA approved in April 2004 to treat resistant Streptococcus pneumoniae respiratory infections and sinusitis.326,327 No reports of hepatotoxicity have appeared, although increased serum ALTs and hepatic dysfunction are listed as adverse reactions in the Epocrates database, and two instances have been entered into the US drug-induced liver injury network registry. Erythromycin and troleandomycin, a related macrolide no longer available, are well known to be potent inhibitors of CYP3A species and the p-glycoprotein transporter. Clarithromycin is also such an inhibitor.328,329 In fact, erythromycin and clarithromycin cause adverse drug interactions much more frequently than they cause cholestatic liver injury, especially with immunosuppressive agents such as ciclosporin A330,331 and tacrolimus,332 which require CYP3A metabolism. Azithromycin, roxithromycin, and dirithromycin are much weaker inhibitors of CYP3A.333 A potential link between CYP3A inhibition and cholestatic liver injury was identified when erythromycin and troleandomycin were found to block canalicular bile acid efflux in human hepatocytes much more effectively than the newer macrolides.334
Quinolones The fluoroquinolones are considered to be relatively safe antibiotics,335 with only trovafloxacin identified as having an incidence of hepatotoxicity (1 in 7000)204 appreciable enough to limit its use to serious infections in hospitalized patients. With trovafloxacin, the patients who developed ALF appeared to have a hypersensitivity reaction and were on medication for more than 14 days. However, case reports of hepatic failure have been published for most of the fluoroquinolones,336–339 and the Epocrates database lists increased serum ALTs as occurring with all of them.
Sulfonamides All the sulfonamides have been associated with reports of hepatotoxicity, usually thought to be idiosyncratic with immunoallergic features.204 A cholestatic clinical presentation is most common, usually with rash, fever, and eosinophilia. One case of intrahepatic cholestasis with phospholipidosis has been reported.340 Trimethoprim/sulfamethoxazole (TMP/SMX) is one of the oldest and most widely prescribed antibiotic combinations. The incidence of hepatotoxicity in the general population must be very low, because only occasional reports of hepatitis, ALF, and cholestatic disease appeared in the literature in the 1970s and 1980s.204 However, several recent case reports of liver failure341–343 serve to remind us of this potential. In HIV patients, the use of TMP/SMX has been noted to lead to a much higher incidence of allergic reactions (~20%) than in nonHIV patients.204,344–346 Because TMP/SMX is considered the best therapy for the treatment and prevention of Pneumocystis jiroveci pneumonia, desensitization protocols were developed347 and efforts
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Section IV. Toxin Mediated Liver Injury
made to determine the cause of hypersensitivity. A slow acetylator status may contribute348 by allowing more drug to be activated by CYPs to sulfamethoxazole hydroxylamine.204 Co-treatment with CYP3A inducers leads to more measurable hydroxylamine, and inhibitors decrease this metabolite.349 No consensus has yet been reached to explain HIV-induced sensitivity. Interestingly, TMP/ SMX is widely used in developing countries as chronic prophylaxis against opportunistic infections, with some success,350–352 and no mention is made of severe allergic or hepatotoxic reactions with the use of the drug in these populations.
Tetracyclines The original descriptions of hepatotoxicity due to tetracyclines were in patients receiving high doses intravenously.204,247 The clinical presentation was similar to Reye’s syndrome, with ALF, renal failure, and acidosis. Serum ALTs were generally not very high (crystalline)
≠ALT, rare hepatitic, cholestatic rare autoimmune picture rare acute hepatitic/cholestatic
Bosentan (Tracleer)
Although these drugs are relatively new, there have already been a number of reports suggesting that losartan can cause a mainly idiosyncratic hepatitic reaction,400,401 and candesartan,402 irbesartan,403 and valsartan404 have been reported to cause cholestatic hepatitis. Onset of clinical illness was always just a few weeks after starting therapy and resolution was relatively rapid after discontinuing the drug. The incidence of this reaction appears to be low.
≠ALT, hepatitic, non-immune
Ca2+ channel blockers Nifedipine Diltiazem (Cardizem) Verapamil
Other antihypertensive Hydralazine
inhibitors have been shown to have a low incidence of causing cholestatic hepatitis,128,187 including captopril,395,396 lisinopril,397 fosinopril,398 and ramipril.399 Whether the other congeners can cause this picture is not known. Patients who developed cholestasis were generally middle-aged and had been taking the drug for between 4 and 8 weeks. Most cases displayed a long recovery time. The sole fatality was a patient who had been continued on lisinopril for 3 weeks after developing jaundice, whose death was attributed to a perforated ulcer while his cholestasis was improving.397 Currently there are no known risk factors for developing cholestasis.
very rare immune hepatitic, granulomas ≠ALT, too new to characterize
Not reported to cause significant liver injury Antiplatelet/Anticoagulant/Thrombolytics Atteplase (Activase), clopidogrel (Plavix), anagrelide (Agrylin), dypridamole (Persantine), eptifibatide (Integrilin), cilostazol (Pletal), tirofiban (Aggrastat), abciximab (Reopro), fondaparinux (Arixta), bivalirudin (Angiomax), argatroban, antithrombin III (Atnativ), anistreplase (Eminase) streptokinase (Kabikinase), urokinase (Abbokinase), reteplase (Retevase), tenecteplase (TNKase) Antiarrhythmics Adenosine, bretylium, sotalol (Betapace), ibutilide (Corvert), moricizine (Ethmozine), mexiletine (Mexitil), disopyramide (Norpace), flecanide (Tambocor), dofetilide (Tikosyn) b-Blockers Almost all except labetolol appear without significant toxicity Ca2+ channel blockers All but verapamil and diltiazem appear safe Cholesterol lowering Gemfibrazole (Lopid), ezetimibe (Zetia) Diuretics All loop and thiazide appear without significant hepatotoxicity Other antihypertensive Minoxidil (Loniten), eplerenone (Inspra), treprostinil (Remodulin), epoprostenol (Flolan), fenoldapam (Corlopam), doxazosin (Cardura) clonidine (Catapres), terazosin (Hytrin), guanabenz (Wytensin), prazosin (Minipress), nesirtide (Natrecor)
The highly effective and widely used iodinated benzofuran antiarrhythmic amiodarone has long been known to cause liver injury.187 Its pulmonary toxicity is more serious, but elevations in serum aminotransferases or AP are common.405,406 Amiodarone was the drug most commonly associated with liver injury in one tertiary hepatology referral center.364 The spectrum of amiodarone liver injury is broad. An acute hepatitis can occur within 24 hours of starting parenteral therapy,407 but the incidence of this is difficult to assess because the drug is usually used during cardiac arrests and the majority of patients do not survive. Asymptomatic liver enzyme elevations occur in about 25% of patients on oral therapy, usually detected 10 months after exposure, with mean ALT (104 IU/mL) greater than mean AST (aspartate aminotransferase) (89 IU/ml), and generally normal AP and bilirubins. Although adaptation may occur, with normalization of values on continued use, the drug is often stopped because of toxicities to other organs and death from heart disease.405 Between 1 and 3% of patients develop symptomatic hepatitis, with hepatomegaly that resolves relatively quickly on drug withdrawal. The drug and its metabolite remain in liver and plasma for long periods and can cause persistent abnormalities for many months after cessation of therapy.405 The most ominous form of liver injury with amiodarone is the development of cirrhosis that has been termed pseudo-alcoholic based on the findings of Mallory’s hyaline, PMNs, and steatosis,408 and this can occur even with low doses of the drug.409 Regular monitoring of serum ALT is recommended, especially if doses greater than 400 mg/day are used. Decreasing the dose or stopping if ALTs are more than three times the ULN, and performing a liver biopsy if elevations persist, are also recommended. A recent prospective study of serum amiodarone levels in 125 patients suggested that only 6% will have serum ALTs more than three times the ULN if amiodarone levels are 60% is reported within 24 hours, and 91% by 5 days.40 Interestingly, the toxic components of impila have been identified as ATR and carboxyatractyloside, which decomposes to ATR. Antioxidant therapies, including S-adenosyl-L-methionine or betaine have been suggested for the management of toxicity caused by either herb, but are of unproven benefit at present.
BLACK COHOSH Black cohosh, Cimicifuga racemosa, is a leafy, cylindrical black rhizome with white flowers native to Canada and the US, and cultivated in Europe. The medicinal portions are in the fresh and dried roots. Black cohosh has many common names, including black snake root, rattleroot, rattleweed, squaw root, bugbane, bugwort, cimicifuga and richweed. It is used primarily for the relief of menopausal symptoms, but is also used to treat rheumatism, bronchitis, and as
a weight loss aid. Two cases of hepatotoxicity associated with black cohosh have recently been reported. In the first, a 47-year-old woman presented with jaundice, pruritus, elevated serum levels of alanine aminotransferase (ALT), serum bilirubin, and INR 1 week after taking black cohosh for relief of menopausal symptoms.45 Other potential causes of acute liver disease were excluded by serologic, biochemical and radiologic examination. The patient’s condition rapidly deteriorated and an orthotopic liver transplantation was performed. Histologic evaluation of the explanted liver revealed severe hepatitis and multiacinar dropout.45 The patient recovered post transplant without event. In the second reported case, a 52year-old woman presented with deep jaundice, elevated levels of ALT, bilirubin, INR, and alkaline phosphatase 4 weeks after discontinuing use of a herbal preparation she had used for 3 months to treat severe tinnitus.46 The preparation contained ground ivy, golden seal, ginkgo, oat seed and black cohosh. The patient’s condition deteriorated and she developed encephalopathy and hepatorenal failure. She underwent liver transplantation with an uneventful postoperative course. Evaluation of the explant revealed massive hepatic necrosis. It is not certain that black cohosh was the hepatotoxic agent in the above-mentioned cases, as neither preparation was evaluated for the presence of contaminants, and one contained several different herbal agents.47,48 The active ingredients in black cohosh are triterpenes, quinolizidine alkaloids, and phenylpropane derivatives. To date, no studies have implicated these agents in hepatic injury.49 However, the severity of the hepatic injury in these two cases cannot be ignored. Further study of black cohosh is needed to determine what, if any, role it plays in the development of hepatotoxicity.
CAMPHOR OIL Camphor oil is extracted from the camphor tree, Cinnamomum camphora, indigenous to Vietnam and an area extending from southern China to southern Japan. It is used externally as a bronchial secretolytic and hyperemic for cough and bronchitis, rheumatism and arrhythmia. One case of hepatotoxicity has been reported involving a 2-month old girl treated with a camphor-containing cold remedy applied to the skin.50 In this case, the infant, with recent swelling in the right inguinal area, was taken to her local hospital. She was noted to be malnourished due to use of an improperly diluted infant formula and was admitted for nutritional support. During routine laboratory evaluation to monitor for refeeding syndrome, abnormal aminotransferase values were noted. On physical examination, a soft liver edge was palpated 1.5 cm below the costal margin. Viral hepatitis as a cause was ruled out. On questioning, the mother admitted to applying generous amounts of Vicks VapoRub to the baby’s chest and neck three times a day for 5 days. Liver tests returned to normal after application of the rub was discontinued. A second report by Jimenez et al. involved a case of oral ingestion of camphor resulting in toxicity resembling Reye’s syndrome.51 In this report, a 6-month-old child was evaluated for a 2-day history of cough and fever, diagnosed with pneumonia, and treated with ampicillin by his private physician. The following day the infant was lethargic, with worsening pulmonary manifestations and radiographic evidence of bilateral diffuse interstitial infiltrates. Six hours later the infant was unrousable, the liver was palpated 3 cm below the costal margin, and a diagnosis of Reye’s syndrome was suspected.
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On transfer to a tertiary care center the infant was comatose, had an elevated white count with bandemia, aminotransferase levels of 750–1000 U/l, an elevated serum bilirubin level and a prolonged prothrombin time. Liver biopsy results did not show the characteristic pleomorphic, greatly swollen mitochondria with absent dense bodies and stranding of mitochondrial matrix typically seen in fatal Reye’s syndrome; however, given the clinical and neurologic syndrome, a presumptive diagnosis of Reye’s syndrome was made. On questioning the family, it was learned that the infant was regularly treated with a home remedy containing camphor and alcohol. Although the infant’s liver function improved over the next few days, his neurologic status did not and EEG studies revealed an absence of electrical activity. The infant died on the fifth hospital day as a result of cardiac arrest.51 Camphor is a cyclic terpene compound that is a constituent of several medications, including salves, ointments and oral cold remedies. When rubbed on the skin, camphor is a rubefacient which causes local irritation to the skin, thereby blocking pain by ‘counterirration’ (affects the same segmental central nervous system level as that inducing the original pain).52,53 Camphor can be absorbed through the skin, mucous membranes and placenta, leading to significant hepatoneurotoxicity, occasionally culminating in hepatic encephalopathy.54,55 Ingestion of small doses of camphor, characterized by abrupt onset of nausea and vomiting followed by agitation and seizures, can be fatal in young children. The mechanism by which camphor leads to hepatotoxicity is unclear. Ordinarily, camphor is metabolized in the liver and excreted in the urine as an inactive glucuronide compound. Although the exact hepatotoxic metabolite is unknown, it is felt that infants are particularly susceptible to camphor hepatotoxicity because of their immature hepatic detoxification mechanisms.56,57 As a result, it has been recommended that camphor-containing cold remedies (Vicks VapoRub, BenGay, Afrin saline mist) should not be used in children under the age of 2 years.
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the second a senna alkaloid, have also been associated with the development of chronic hepatitis.59,60 Interestingly, in the first report the authors attributed the hepatotoxicity to the dioctyl sulfosuccinate component rather than the anthraquinone. Cascara is approved by the FDA for use as a laxative and is widely used in the US without hepatic sequelae. Nevertheless, it appears that in extremely rare instances cholestatic hepatitis may occur.
CHAPARRAL Chaparral leaf, Larrea tridentate, is commonly known as creosote bush or greasewood. It is a desert plant indigenous to the southwestern US and Mexico. Native Americans grind the leaf and use it as a tea for a variety of ailments, including the common cold, bone and muscle pain, chicken pox, cancer, tuberculosis, venereal diseases and snake bites.2 Currently, chaparral is packaged as capsules, tablets or balms and used as a ‘liver tonic’ and as treatment for skin lesions. Sheikh et al. reviewed all 18 cases of chaparral-induced injury reported to the FDA since 1990.61 Thirteen of these displayed hepatic injury, ranging from mild hepatitis to cirrhosis and even fulminant hepatic failure. Although the hepatic manifestations were heterogeneous, the predominant pattern of liver injury was cholestatic, with elevations in serum aminotransferases, bilirubin and alkaline phosphatase levels. Only a few patients progressed to cirrhosis, and two required orthotopic liver transplantation (OLT) for hepatic failure. The latter two patients had used chaparral for more than a year, whereas in the remainder it had been used for 1–6 months. The pathophysiology of chapparal toxicity is unknown. It contains a mixture of flavonoids, amino acids, ligans and volatile oils.62 The active ingredient is nordihydroguaiaretic acid (NDGA), which may inhibit cyclooxygenase or cytochrome P450 activity, or act via an immune-mediated mechanism.63,64 In addition, chaparral metabolites exhibit estrogen activity which may contribute to hepatotoxicity.65,66
CASCARA
CHASO AND ONSHIDO
Cascara sagrada is derived from the dried bark of the bush or tree Rhamnus purshiana. The plant is indigenous to the western part of North America and is also cultivated in Canada and eastern Africa. Cascara is used for relief of constipation, hemorrhoids, and as a rectoanal postoperative treatment. The active constituent is an anthracene derivative (O- and C-glycosides). Right upper quadrant pain, jaundice, ascites and portal hypertension were reported in a 48-year-old man 3 days after ingesting cascara sagrada for laxative purposes.58 Liver biopsy revealed moderate portal inflammation with eosinophils and plasma cells, and mild portal–portal bridging fibrosis without cirrhosis. Bile duct proliferation and bile stasis were also noted. The patient recovered fully 3 months after discontinuation of cascara. No other cause for hepatotoxicity was identified and a presumed diagnosis of cascara hepatotoxicity was made. The pathogenesis of cascara hepatotoxicity is unknown, but it is assumed that anthracene glycosides are involved. The temporal association of the ingestion of cascara with the symptoms and liver biopsy evidence of moderate inflammation with lymphocytes, plasma cells and eosinophils suggests an immune-mediated process. Two anthraquinones, the first, Doxidan (a combination of danthron 1,8-hydroxy anthraquinone and dioctyl calcium sulfosuccinate), and
Chaso and Onshido are two widely available Chinese herbal weight loss aids. The manufacturers report that Chaso contains green tea, cassia torae semen, leaves of lotus, Fructus lycii, Fructus crataegi and chrysanthemum flowers. Onshido contains extract of Gynostemma pentaphyllum makino, green tea, aloe, Fructus crataegi and raphani semen. In 2002, 12 Japanese patients (six using Chaso and six using Onshido) presented with symptoms of severe fatigue and anorexia 5–40 days after ingesting the herbs.67 Most mistook their symptoms for those associated with weight loss and did not seek immediate medical attention. On presentation, the levels of the aminotransferases and bilirubin were increased, and the INR was significantly elevated. Two patients developed hepatic encephalopathy and one received a liver transplant 8 days after admission. Another patient died 45 days after admission secondary to intestinal bleeding and infection; the remaining 10 patients improved after discontinuation of the herbals, with the liver biochemical tests all returning to normal.67 On analysis of the ingredients contained in the preparations, N-nitroso-fenfluramine was detected. Fenfluramine was once prescribed for weight loss but was withdrawn from clinical use because of severe cardiac complications. Although fenfluramine was not identified in either Chaso or Onshido, data suggest that N-
Chapter 27 HEPATOTOXICITY OF HERBAL PREPARATIONS
nitroso compounds have been linked with hepatic carcinogenesis.68 The mechanism of injury is felt to be direct hepatotoxicity. Since the publication of this report, 21 cases of Chaso-induced hepatitis and 135 cases of Onshido-induced hepatitis were reported to the Ministry of Health, Labor and Welfare in Japan, most occurring between April and August 2002.69 The herbals have subsequently been removed from the market in the US.
COMFREY AND PYRROLIZIDINE ALKALOIDS (PA) Comfrey, Symphytum officinale, is a plant indigenous to Europe and temperate Asia. The medicinal portions are the leaves and fresh roots, which are used externally for bruises, sprains, rheumatism and pleuritis. The active ingredients, pyrrolizidine alkaloids, are the most important plant toxins associated with liver disease. Heliotroprium, Senecio, Crotalaria and t’u-san-chi’ (Compositae) species are most often responsible for liver injury. Hepatotoxicity related to PAs was first described 70 years ago as Senecio (mate tea) disease in South Africa.70–73 Reports of Jamaican children developing hepatomegaly and evidence of decompensated cirrhosis with ascites after the ingestion of ‘bush tea’ soon followed.74,75 Later, epidemics were reported from India and Afghanistan.76,77 PAs are dose-dependent hepatotoxins that typically cause veno-occlusive disease (VOD). VOD may present as acute, subacute or chronic liver injury, characterized by abdominal distention, hepatomegaly, ascites and edema. Pathophysiologically, non-thrombotic obliteration of terminal centrilobular veins develops, reminiscent of the Budd–Chiari syndrome and leading to disruption of hepatic blood flow.75 High doses of PAs are often responsible for acute liver injury, and the long-term use of relatively low-dose PA has been associated with insidious hepatotoxicity. Recovery from acute disease is the rule, but liver failure with resultant 20–40% mortality has been reported.78 The mechanism of PA-induced hepatotoxicity is unclear. Numerous articles suggest a toxic mechanism which is reproducible in animals and related to biotransformation of alkaloids by cytochrome P450, forming pyrrole derivatives which serve as alkylating agents.79,80 Toxicity can be augmented by the concomitant use of phenobarbital via the induction of cytochrome P450. Discontinuing the herbal can result in resolution of symptoms in some patients, but those with acute or chronic liver failure may require liver transplantation.
Dai-Saiko-to (Sho-Saiko-to, TJ-9, Xiao-ChaiHu-Tang) Dai-Saiko-to is a Japanese kampo formula consisting of seven herbs that has been used since AD100 for the treatment of fatigue, fever, dyspepsia, gallstones, and recently, chronic liver disease.81 DaiSaiko-to differs from Sho-Saiko-to only in the proportion of the herbal constituents, which include bupleurum, pinellia, jujube, ginseng, ginger rhizome, glycyrrhiza and scutellaria.81 Itoh et al. reported four cases of worsening of the aminotransferase levels, which improved with cessation of the drug and recurred with rechallenge.82 In 1997 a case of autoimmune hepatitis was reported that was possibly induced by Dai-Saiko-to.33 The patient was a 55-yearold woman who initially presented with fatigue, fever and abnormal liver tests (ALT 866 IU/l, total bilirubin 13 mg/dl, alkaline phosphatase 317 IU/l). Autoimmune markers and hepatitis serologic
tests were negative; liver histology revealed chronic hepatitis. Within 4 weeks the symptoms and biochemical abnormalities had resolved spontaneously and the ALT values remained normal for 5 years. Subsequently, an increase in the ALT level to 300 IU/l was noted and liver histology revealed chronic hepatitis with severe steatosis and a lipogranuloma. The patient was treated with Dai-Saiko-to and 2 weeks later developed fatigue, fever, an elevated ALT level of 390 IU/l and an antinuclear antibody titer of 1:2560. An International Autoimmune Hepatitis (AIH) diagnostic score of 18 supported the diagnosis of autoimmune disease, and a strongly positive lymphocyte stimulation test for Dai-Saiko-to (340%) suggested this herb was responsible.83 Abnormalities in the ALT values returned to normal after treatment with prednisolone. The mechanism by which the hepatic abnormalities occur is unclear. It has been postulated that scutellaria, which has been implicated in four other cases of hepatotoxicity, might be the culprit.84 Likewise, the saponin in bupleurum, suggested to have toxic effects on cell membranes, may also be involved.2
GERMANDER Germander, Teucrium chamaedrys, is a sub-shrub with a short-lived, main root from which long-reaching thin branched roots grow. It is indigenous to the Mediterranean region as far as the Urals. The active ingredients are diterpenes, iridoide monoterpenes, caffeic acid derivatives and flavonoids. Germander is felt to have cholagogic and antiseptic properties, and is ingested as a capsule or tea to treat dyspepsia, fever, gout and obesity. Following large-scale marketing as a weight loss aid in France in 1992, 30 cases of acute, chronic and fulminant hepatitis were reported.35,85 Most affected patients were women attempting to lose weight, most of whom ingested 600–1600 mg daily. The clinical syndrome, characterized by markedly elevated levels of aminotransferases and bilirubin, and impaired hepatic synthetic function, began approximately 2 months after ingestion. The range of histologic findings included mild chronic hepatitis, fibrosis, cirrhosis, and in some cases acute midzonal hepatocellular necrosis. Those patients without cirrhosis completely recovered after discontinuation of the herb. Analysis of Teucrium chamaedrys revealed the presence of a number of furan-containing neoclerodane diterpenoids which are well-known powerful carcinogens.86–88 In rat hepatocytes, these constituents are oxidized by cytochrome P450 3A4 to reactive metabolites which bind to proteins, deplete cellular glutathione and protein thiols, and cause plasma membrane blebbing and cell disruption.87 Two cell culture studies suggest that germander induces apoptosis after the formation of reactive metabolites.89,90 These reports suggest a reactive metabolite as the mechanism of injury for germander; however, an autoimmune mechanism was proposed after antimicrosomal epoxide hydrolase autoantibodies were found in the sera of some patients.91
GREATER CELANDINE Greater Celandine, Chelidonium majus, is a plant found throughout Europe and the temperate and subarctic regions of Asia. The root is harvested between August and October. Isoquinolone alkaloids and caffeic acid derivatives are felt to be the active ingredients. Celandine is believed to have mild analgesic, central sedative, chol-
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agogic and antimicrobial effects, and is used to treat biliary colic, cholelithiasis, jaundice, gastroenteritis, and diffuse liver and gallbladder complaints. Ten cases of acute hepatitis induced by celandine have been reported.34 All of the patients were women, with the onset of symptoms and moderate elevations of liver chemistries noted approximately 3 months after ingestion of the herb. A cholestatic pattern was noted in five patients; low titer antinuclear antibodies were noted in eight, and portal inflammation with bridging fibrosis and eosinophilic infiltrates was seen on liver biopsy in most of them. Liver chemistries returned to normal 2–6 months after cessation of the herb. The mechanism of action of celandine hepatotoxicity is unknown, but is considered to be idiosyncratic in nature owing to the variable latency period and the lack of dose dependence.31,37 Some have suggested an immune response owing to the presence of serum autoantibodies and eosinophilic infiltrates on liver biopsy, but these findings can be non-specific. More than 20 alkaloids with biologic activity have been identified in celandine; however, the toxic component has yet to be identified.
Ju Bu Huan Ju Bu Huan, Lycopodium serratum, is a plant found worldwide. It is believed to have sedative, analgesic and antispasmodic effects and has been used for more than a millennium as a sleeping aid. Several cases of acute and chronic hepatitis associated with ju bu huan have been published in the literature.92,93 The largest report describes seven patients who developed hepatitis a mean of 20 weeks after ingestion.92 Abdominal pain, constitutional symptoms, jaundice, hepatomegaly and pruritus were characteristic, as were elevations in the aminotransferase and alkaline phosphatase levels. Liver histology generally revealed periportal necrosis and cholestasis. In one patient eosinophilic inflammation was noted, and in another moderate periportal fibrosis, lymphocytic inflammation, focal hepatocellular necrosis, and microvesicular steatosis were present. Symptoms and abnormal laboratory tests resolved within 8 weeks after discontinuation of the herb. Horowitz et al. suggest that the clinical spectrum of ju bu huan toxicity may vary with the mode of ingestion: specifically, the usual dose versus an acute overdose.93 Acute overdose appears to be associated with flaccid weakness, lethargy and respiratory depression (noted in three children), whereas long-term use presents as acute hepatitis without neurologic findings. The mechanism of ju bu huan hepatotoxicity is not fully understood. An L-alkaloid, L-tetrahydropalmatine, which is structurally similar to pyrrolizidine and berberine alkaloids, may be the toxic agent. Long-term berberine use is associated with hyperbilirubinemia in animals, which may be caused by displacement of bilirubin from albumin or disruption of bilirubin conjugation.37,93 Although the exact mechanism of action of ju bu huan is unknown, the presence of fever, skin rash and eosinophilia in some patients suggests an immune-mediated mechanism.
Kava Kava is a rhizome of the pepper plant Piper methysticum. The plant is a 2–3-m tall bush indigenous to the South Sea Islands. The extract of the dried rhizome contains kava lactones, which have central muscle-relaxant, anticonvulsive and antispasmodic effects. The herb
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also has hypnotic/sedative, analgesic and psychotropic effects, which explain its worldwide use as a therapy for anxiety and tension. Four cases of acute hepatitis and 18 cases of acute liver failure have been reported in connection with its use.94–96 In one report, a 33-year-old woman took Laitan (a sleeping aid containing 210 mg of kava lactones) daily for 3 weeks.94 Two months later she restarted the kava preparation. Three weeks later, 1 day after ingesting 60 g of alcohol, she developed malaise and jaundice. Serum levels of the aminotransferases, bilirubin and alkaline phosphatase were elevated 60-, 15- and 3-fold, respectively. Other forms of acute hepatitis were excluded. Liver biopsy revealed inflammation of the portal tracts, bridging necrosis and canalicular cholestasis. The patient recovered completely within 8 weeks of cessation of kava. A lymphocyte transformation test indicated strong and concentration-dependent T-cell reactivity to Laitan (but not the herb Exsepta, which she was also using over the previous 2 months). Phenotyping of cytochrome P4502D6 activity with debrisoquine showed that the patient was a poor metabolizer. The authors interpreted these data as suggesting that the patient had an immune-mediated reaction to a metabolite of kava. In a second report, a 50-year-old man presented to his physician complaining of jaundice, fatigue, and dark urine of 1 month’s duration.96 He reported taking four capsules of kava extract daily (210–280 mg of kava lactones) for 2 months to treat anxiety. He was on no other medications and did not consume alcohol. Serum aminotransferase levels were elevated >50-fold; alkaline phosphatase, bilirubin and prothrombin time were also elevated. Ultrasound of the liver revealed mild hepatomegaly but no evidence of ascites or portal vein thrombosis. Viral hepatitis was excluded with blood tests. Within 48 hours the patient’s clinical condition deteriorated, he developed stage IV encephalopathy, and received an orthotopic liver transplant 2 days later. Histology of the explant revealed extensive hepatocellular necrosis and extensive lobular and portal infiltration with lymphocytes and eosinophils. The exact mechanism of kava hepatotoxicity is unknown. Some have suggested that a relatively common genetic polymorphism of drug metabolism, CYP2D6 deficiency, may predispose to kava hepatotoxicity.94,97 However, further study is required before this mechanism can be considered definitive.
Ma Huang Ma-huang, Ephedra sinica, is a 30-cm tall, lightly branched shrub which is found mainly in Mongolia and the bordering area of China. The dried young branchlets, which are harvested in the autumn, are considered the medicinal parts. Ma-huang is generally used as a tea for cough and bronchitis, as a weight loss aid, and as an energy enhancer. Instances of ma-huang hepatotoxicity have been reported. In the first case, a 33-year-old woman presented with nausea, vomiting, jaundice and abdominal pain 3 weeks after taking a herbal preparation containing ma-huang.98 She reportedly continued using the preparation despite symptoms, until jaundice developed. Serum aminotransferase and bilirubin levels were elevated without signs of chronic liver disease, and the presumptive diagnosis was acute viral hepatitis. She was admitted to hospital 1 week later with increasing jaundice, worsening aminotransferase levels, and evidence of autoimmune disease with an ANA titer of 1:160 and an antismooth muscle antibody titer of 1:80. Viral hepatitis serologies were nega-
Chapter 27 HEPATOTOXICITY OF HERBAL PREPARATIONS
tive. Liver biopsy demonstrated diffuse hepatic necrosis with occasional eosinophils and plasma cells in the portal tracts. On further questioning, the patient admitted taking another dose of the herbal preparation after her first hospital visit. Symptoms resolved and the liver panel returned to normal 4 months after discontinuation of ma-huang. In a second case, a 58-year-old woman with obesity presented with a history of 4 months of jaundice, fatigue, nausea and abdominal pain of unclear etiology.99 Initial evaluation revealed elevated levels of the aminotransferases, elevated bilirubin, negative viral hepatitis serologies, a negative ANA, an antismooth muscle antibody titer of 1:320, and normal abdominal CT imaging studies. Medication history revealed that she had used ma-huang as a weight loss aid for 4.5 months. Liver biopsy revealed severe infiltration with polymorphonuclear leukocytes, moderate fibrosis and lobular necrosis. She was treated with steroids for presumed autoimmune hepatitis but subsequently developed encephalopathy and was referred for liver transplant evaluation. While awaiting liver transplant, her status improved and she was discharged in a stable condition. The active ingredient in ma-huang is ephedrine, a sympathomimetic used in western medicine to treat asthma and also used as a central nervous system stimulant. Well-known side effects include nervousness, palpitations, headache and insomnia. The exact mechanism by which ephedrine induces hepatotoxicity is unknown, but the presence of autoimmune markers in both cases suggests an immune-mediated process, either as a primary effect or through unmasking of an underlying autoimmune diathesis. In the first case presented above, the concomitant use of other plant extracts in the preparation raises the possibility of herb–herb interaction or other contamination. In both cases the women were obese, and it is possible that non-alcoholic fatty liver disease may have contributed to the liver injury.
MARGOSA OIL (NEEM) Antelaea azadirachta (neem) and Azadirachta indica (margosa oil) are indigenous to the woods of India and Sri Lanka. The bark, leaves and seeds of the deciduous tree are the medicinal portions. Azadirachta indica and Antelaea azadirachta are commonly used in India, Sri Lanka, Burma, Indonesia, Thailand and Malaysia for inflammatory and febrile illnesses, as well as dyspeptic symptoms and worm infestations. There have been several case reports of toxic encephalopathy among infants and young children given small amounts of oral margosa oil.100 Although the oil is generally used externally, some traditional practices include giving small oral amounts to infants and children. Several children presented to physicians with vomiting, drowsiness, tachypnea and recurrent seizures. Laboratory studies revealed a leukocytosis, abnormal liver tests, severe metabolic acidosis, and hepatic lesions consistent with Reye’s syndrome. Supportive care and control of seizure activity led to resolution of symptoms in most children, but a few developed neurologic deficits and some died as a result of hepatic failure. Animal studies of margosa oil ingestion indicate that the injury sequence begins with the rapid development of mitoses and binucleated cells, followed by mitochondrial injury, swelling, and pleomorphism within the nuclei of hepatocytes.101,102 Proliferation
and hypertrophy of the endoplasmic reticulum and subsequent microvesicular steatosis have also been noted. It has been suggested that margosa oil is a mitochondrial uncoupler, increasing mitochondrial respiration and decreasing intramitochondrial ATP.101 These effects may be due to changes in fatty acid metabolism that result in a change in the proportion of acid-soluble and acid-insoluble coenzyme A esters. Even though some suggest that supplementary therapy with L-carnitine and coenzyme A may be useful in the management of margosa oil-induced Reye’s syndrome, avoidance of oral use of this herbal product is clearly prudent.100
MISTLETOE, SKULLCAP, VALERIAN Mistletoe, Viscum album, is a semiparasitic round bush that grows on deciduous trees found primarily in Europe. The medicinal portions include the leaves, stem, and pea-sized berries. Mistletoe has been widely used to treat many illnesses, including degenerative inflammation of the joints, hypertension, asthma, vertigo, diarrhea, epilepsy and nervousness. One case of presumed mistletoe hepatitis was reported in a 49-year-old woman who presented with nausea, general malaise and right upper quadrant pain.103 Aminotransferase levels were elevated, hepatitis B surface antigen was not detected, the cholecystogram was normal, and liver biopsy revealed mild inflammation. Two years later she presented with a similar illness, and questioning revealed that both episodes were preceded by the ingestion of a herbal remedy containing kelp, motherwort, skullcap and mistletoe.103 A challenge test established that the herb was responsible for the symptoms. At the time, mistletoe was the only herb known to contain a potential toxin, lectin, and it was therefore singled out as the causative agent. Later evaluation of the herbal compound suggested that mistletoe was probably not an ingredient, casting doubt on the association between mistletoe and hepatitis. Skullcap, Scutellaria, is a perennial herb 60 cm in height and thickly covered with simple and glandular hairs, which is indigenous to North America and cultivated in Europe. The herb is pulverized and used as a sedative, an antispasmodic and an anti-inflammatory agent, and it is thought to inhibit lipid peroxidation. Valerian, Valeriana officinalis, is a short, cylindrical rhizome with bushy round roots indigenous to Europe and the temperate regions of Asia. It is widely cultivated in England, France, Japan and the US, and is used to treat conditions such as nervousness, insomnia, lack of concentration, headache, and nervous stomach cramps. Often both valerian and skullcap are contained in the same preparation used to relieve stress. Several cases of acute hepatitis have been reported with the use of herbal preparations containing skullcap and valerian. Four cases of jaundice, abdominal pain and dark urine have been reported in women taking two different herbal preparations, Kalms and Neurelax, for relief of stress.84 One woman developed ascites and encephalopathy necessitating intensive medical support. Available liver biopsy tissue revealed a range of abnormalities, from moderate acute hepatitis to bridging fibrosis, to advanced fibrosis and cirrhosis. In all four women the aminotransferase levels returned to normal and symptoms resolved with discontinuation of the herbal compound. Several other cases of jaundice have been reported to the Welsh Drug Information Centre of ingestion of preparations containing skullcap, valerian or both.84 As with mistletoe, the association with hepatitis is presumed; direct experimental evidence of toxicities due to these herbals is currently lacking.
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PENNYROYAL Pennyroyal, Mentha pulegium, is a downy perennial which grows in western, southern and central Europe, Asia, Iran and Ethiopia. It is naturalized in North America. The medicinal portion, the essential oil, is extracted from the fresh plant or dried aerial parts. For centuries it was used as an abortifacient and as a pesticide against fleas, and because it is a source of intoxication it continues to be widely used.104 Current medical uses include digestive disorders, liver and gallbladder disease, amenorrhea, gout, colds, and skin disease. Most cases of hepatotoxicity have been reported in women who use pennyroyal to induce menstruation or abortion.104 In one instance, a 24-year-old woman ingested pennyroyal extract and black cohosh root for 2 weeks in an attempt to induce abortion. When this failed, she ingested additional, unknown amounts of these herbals over a short period. Soon after ingestion she developed abdominal cramping, chills, vomiting and syncope, with difficulty in being roused. Paramedics discovered the patient to be in cardiopulmonary arrest an estimated 7.5 hours after acute ingestion. She was intubated, successfully resuscitated and admitted to the intensive care unit. CT of the abdomen revealed a possible ruptured ectopic pregnancy. Aminotransferase levels, bilirubin and prothrombin time were elevated, and over the next 36 hours the patient developed signs of multiorgan failure and disseminated intravascular coagulation. The patient was comatose and unresponsive to all stimuli, anoxic encephalopathy was confirmed by CT, life support was discontinued and she died within 48 hours of acute ingestion. Two infants developed multiorgan failure after ingestion of mint tea containing pennyroyal, one with confluent hepatic necrosis noted at autopsy.105 Unlike most herbal preparations, the mechanism of pennyroyal hepatotoxicity is well known. The main constituent is R(+)-pulegone, which is oxidized by cytochrome P450 to menthofuran.106 Pulegone depletes hepatic glutathione by the formation of electrophilic metabolites, whereas menthofuran is directly toxic to hepatocytes. As a result of the glutathione loss, replacement of sulfhydryl groups by administering N-acetylcysteine has been advocated as a therapy. As menthofuran toxicity is not greatly affected by glutathione loss, the benefits of this therapy may only be evident in the early phases of pennyroyal poisoning. Despite this, N-acetylcysteine is recommended in cases where more than 10 ml of pennyroyal are ingested.
CONCLUSION The use of herbal preparations to treat various medical conditions is increasing. Among most consumers of herbals there is an implicit assumption of safety, and therefore physician consultation is not sought. Over the past decade, as the popularity of herbal remedies has increased, so have reports of toxicity. The scope of hepatotoxicity ranges from asymptomatic elevations in liver biochemistries to chronic hepatitis, cirrhosis and fulminant hepatic failure. Unfortunately, herbal preparations are neither regulated nor standardized, thereby making precise identification and quantification of ingredients or possible contaminants extremely challenging. This significantly hampers the ability to definitively assign causality to a particular herb when evidence of hepatic injury is observed. In most instances of reported toxicity no attempt at phytochemical analysis is made, but rather, a presumptive association is based on temporal
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relationships and occasionally, unintentional rechallenge. Despite these drawbacks, the volume of consistent reporting of herb-related hepatotoxicity mandates serious consideration. It is essential that increased public awareness regarding the potential toxicity of herbals be maintained, as well as improved agricultural monitoring, appropriate regulatory systems, and improved scientific evaluation of the potential benefits and hazards of herbal preparations.
REFERENCES 1. Eisenberg DM, Davis RD, Ettner SL, et al. Trends in alternative medicine use in the United States, 1990–1997: results of a follow-up national survey. JAMA 1998;280:1569–1575. 2. Stedman C. Herbal hepatotoxicity. Semin Liver Dis 2002;22:195–206. 3. Strader DB. Understudied populations with hepatitis C. Hepatology, 2002;36(Suppl 1): S226–236. 4. Seeff LB, Lindsay KL, Bacon BR, et al. Complementary and alternative medicine in chronic liver disease. Hepatology 2001;34:595–603. 5. Winslow LC, Kroll DJ. Herbs as medicines. Arch Intern Med 1998;158:2192–2199. 6. Brown JS, Marcy SA. The use of botanicals for health purposes by members of a prepaid health plan. Res Nurs Health 1991;14:339–350. 7. Anand KK, Singh B, Saxena AK, et al. 3,4,5-Trihydroxy benzoic acid (gallic acid), the hepatoprotective principle in the fruits of Terminalia belerica bioassay-guided activity. Pharmacol Res 1997;36:315–321. 8. Basaga H, Poli G, Tekkaya C, et al. Free radical scavenging and antioxidative properties of ‘silibin’ complexes on microsomal lipid peroxidation. Cell Biochem Funct 1997;15:27–33. 9. Halim AB, el-Ahmady O, Hassab-Allah S, et al. Biochemical effect of antioxidants on lipids and liver function in experimentally induced liver damage. Ann Clin Biochem 1997;34:656–663. 10. Kayano K, Sakaida I, Uchida K, et al. Inhibitory effects of the herbal medicine Sho-saiko-to (TJ-9) on cell proliferation and procollagen gene expressions in cultured rat hepatic stellate cells. J Hepatol 1998;29:642–649. 11. Lang I, NeKam K, Gonzalez-Cabello R, et al. Hepatoprotective and immunological effects of antioxidant drugs. Tokai J Exp Clin Med 1990;15:123–127. 12. Lin CC, Lin WC, Yang SR, et al. Anti-inflammatory and hepatoprotective effects of Solanum alatum. Am J Chin Med 1995;23:65–69. 13. Miguez MP, Anundi I, Sainz-Pardo LA, et al. Hepatoprotective mechanism of silymarin: no evidence for involvement of cytochrome P450 2E1. Chem Biol Interact 1994;91:51–63. 14. Mira L, Silva M, Manso CF. Scavenging of reactive oxygen species by silibinin dihemisuccinate. Biochem Pharmacol 1994;48:753–759. 15. Pietrangelo A, Borella F, Casalgrandi G, et al. Antioxidant activity of silybin in vivo during long-term iron overload in rats. Gastroenterology 1995;109:1941–1949. 16. Pines M, Knopov V, Genina O, et al. Halofuginone, a specific inhibitor of collagen type I synthesis, prevents dimethylnitrosamine-induced liver cirrhosis. J Hepatol 1997;27:391–398. 17. Bateman J, Chapman RD, Simpson D. Possible toxicity of herbal remedies. Scot Med J 1998;43:7–15. 18. Harper J. Traditional Chinese medicine for eczema. Br Med J 1994;308:489–490. 19. De Smet P. Adverse effects of herbal remedies. Adv Drug React Bull 1997;183:695–698.
Chapter 27 HEPATOTOXICITY OF HERBAL PREPARATIONS
20. Kew J, Leigh PN, Playford ED, et al. Arsenic and mercury intoxication due to Indian ethnic remedies. Br Med J 1993;306:506–507. 21. Keen RW, Deacon AC, Delves HT, et al. Indian herbal remedies for diabetes as a cause of lead poisoning. Postgrad Med J 1994;70:113–114. 22. Karliova M, Treichel U, Malago M, et al. Interaction of Hypericum perforatum (St John’s wort) with cyclosporin A metabolism in a patient after liver transplantation. J Hepatol 2000;33:853–855. 23. Cheng TO. Warfarin–danshen interaction. Ann Thorac Surg 1999;67:894. 24. Fugh-Berman A. Herb–drug interactions. Lancet 2000;355:134–138. 25. Shaw D, Leon C, Kolev S, et al. Traditional remedies and food supplements. A 5-year toxicological study (1991–1995). Drug Safety 1997;17:342–356. 26. Miller LG. Herbal medicinals: selected clinical considerations focusing on known or potential drug–herb interactions. Arch Intern Med 1998;158:2200–2211. 27. Rose KD, Croissant PD, Parliament CF, et al. Spontaneous spinal epidural hematoma with associated platelet dysfunction from excessive garlic ingestion: a case report. Neurosurgery 1990;26:880–882. 28. Janetzky K, Morreale AP. Probable interaction between warfarin and ginseng. Am J Health Syst Pharm 1997;54:692–693. 29. Homma M, Oka K, Ikeshima K, et al. Different effects of traditional Chinese medicines containing similar herbal constituents on prednisolone pharmacokinetics. J Pharm Pharmacol 1995;47:687–692. 30. Chen MF, Shimada F, Kato H, et al. Effect of glycyrrhizin on the pharmacokinetics of prednisolone following low dosage of prednisolone hemisuccinate. Endocrinol Jpn 1990;37:331–341. 31. Farrell GC. Drug-induced liver disease. Edinburgh: Churchill Livingstone, 1994: 513–518. 32. Kao WF, Hung DZ, Tsai WJ, et al. Podophyllotoxin intoxication: toxic effect of Bajiaolian in herbal therapeutics. Hum Exp Toxicol 1992;11:480–487. 33. Kamiyama T, Nouchi T, Kojima S, et al. Autoimmune hepatitis triggered by administration of an herbal medicine. Am J Gastroenterol 1997;92:703–704. 34. Benninger J, Schneider HT, Schuppan D, et al. Acute hepatitis induced by greater celandine (Chelidonium majus). Gastroenterology 1999;117:1234–1237. 35. Larrey D, Vial T, Pauwels A, et al. Hepatitis after germander (Teucrium chamaedrys) administration: another instance of herbal medicine hepatotoxicity. Ann Intern Med 1992;117:129–132. 36. Mattei A, Rucay P, Samuel D, et al. Liver transplantation for severe acute liver failure after herbal medicine (Teucrium polium) administration. J Hepatol 1995;22:597. 37. Chitturi S, Farrell GC. Herbal hepatotoxicity: an expanding but poorly defined problem. J Gastroenterol Hepatol 2000;15:1093–1099. 38. Larrey D, Pageaux GP. Hepatotoxicity of herbal remedies and mushrooms. Semin Liver Dis 1995;15:183–188. 39. Stickel F, Egerer G, Seitz HK. Hepatotoxicity of botanicals. Public Health Nutr 2000;3:113–124. 40. Popat A, Shear NH, Malkiewicz I, et al. The toxicity of Callilepis laureola, a South African traditional herbal medicine. Clin Biochem 2001;34:229–236. 41. Popat A, Shear NH, Malkiewicz I, et al. Mechanism of impila (Callilepis laureola)-induced cytotoxicity in Hep G2 cells. Clin Biochem 2002;35:57–64. 42. Mokhobo KP. Herb use and necrodegenerative hepatitis. S Afr Med J 1976;50:1096–1099. 43. Stewart MJ, Steenkamp V. The biochemistry and toxicity of atractyloside: a review. Ther Drug Monit 2000;22:641–649.
44. Obatomi DK, Brant S, Anthonypillai V, et al. Toxicity of atractyloside in precision-cut rat and porcine renal and hepatic tissue slices. Toxicol Appl Pharmacol 1998;148:35–45. 45. Whiting PW, Clouston A, Kerlin P. Black cohosh and other herbal remedies associated with acute hepatitis. Med J Aust 2002;177:440–443. 46. Lontos S, Jones RM, Angus PW, et al. Acute liver failure associated with the use of herbal preparations containing black cohosh. Med J Aust 2003;179:390–391. 47. Thomsen M, Vitetta L, Sali A, et al. Acute liver failure associated with the use of herbal preparations containing black cohosh. Med J Aust 2004;180:598–9; author reply 599–600. 48. Vitetta L, Thomsen M, Sali A. Black cohosh and other herbal remedies associated with acute hepatitis. Med J Aust 2003;178:411–412. 49. Huntley A, Ernst E. A systematic review of the safety of black cohosh. Menopause 2003;10:58–64. 50. Uc A, Bishop WP, Sanders KD. Camphor hepatotoxicity. South Med J 2000;93:596–598. 51. Jimenez JF, Brown AL, Arnold WC, et al. Chronic camphor ingestion mimicking Reye’s syndrome. Gastroenterology 1983;84:394–398. 52. Antman E, Jacob G, Volpe B, et al. Camphor overdosage. Therapeutic considerations. NY State J Med 1978;78:896–897. 53. Skoglund RR, Ware LL Jr, Schanberger JE. Prolonged seizures due to contact and inhalation exposure to camphor. A case report. Clin Pediatr (Phila) 1977;16:901–902. 54. Weiss J, Catalano P. Camphorated oil intoxication during pregnancy. Pediatrics 1973;52:713–714. 55. Lahoud CA, March JA, Proctor DD. Campho-Phenique ingestion: an intentional overdose. South Med J 1997;90:647–648. 56. Riggs J, Hamilton R, Homel S, et al. Camphorated oil intoxication in pregnancy; report of a case. Obstet Gynecol 1965;25:255–258. 57. Robertson JS, Hussain M. Metabolism of camphors and related compounds. Biochem J 1969;113:57–65. 58. Nadir A, Reddy D, Van Thiel DH. Cascara sagrada-induced intrahepatic cholestasis causing portal hypertension: case report and review of herbal hepatotoxicity. Am J Gastroenterol 2000;95:3634–3637. 59. Beuers U, Spengler U, Pape GR. Hepatitis after chronic abuse of senna. Lancet 1991;337:372–373. 60. Tolman KG, Hammar S, Sannella JJ. Possible hepatotoxicity of Doxidan. Ann Intern Med 1976;84:290–292. 61. Sheikh NM, Philen RM, Love LA. Chaparral-associated hepatotoxicity. Arch Intern Med 1997;157:913–919. 62. Gordon DW, et al. Chaparral ingestion. The broadening spectrum of liver injury caused by herbal medications. JAMA 1995;273:489–490. 63. Katz M, Saibil F. Herbal hepatitis: subacute hepatic necrosis secondary to chaparral leaf. J Clin Gastroenterol 1990;12:203–206. 64. Capdevila J, Gil L, Orellana M, et al. Inhibitors of cytochrome P-450-dependent arachidonic acid metabolism. Arch Biochem Biophys 1988;261:257–263. 65. Smith BC, Desmond PV. Acute hepatitis induced by ingestion of the herbal medication chaparral. Aust NZ J Med 1993;23:526. 66. Obermeyer WR, Musser SM, Betz JM, et al. Chemical studies of phytoestrogens and related compounds in dietary supplements: flax and chaparral. Proc Soc Exp Biol Med 1995;208:6–12. 67. Adachi M, Saito H, Kobayashi H, et al. Hepatic injury in 12 patients taking the herbal weight loss AIDS Chaso or Onshido. Ann Intern Med 2003;139:488–492. 68. Hasegawa R, Futakuchi M, Mizoguchi Y, et al. Studies of initiation and promotion of carcinogenesis by N-nitroso compounds. Cancer Lett 1998;123:185–191.
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69. Ministry of Health, L.a.W., Hepatic injury in cases taking selfimported healthfoods or non-approved drugs. L.a.W. Ministry of Health, Editor. 2002. 70. Ridker PM, McDermott WV. Comfrey herb tea and hepatic veno-occlusive disease. Lancet 1989;1:657–658. 71. Bach N, Thung SN, Schaffner F. Comfrey herb tea-induced hepatic veno-occlusive disease. Am J Med 1989;87:97–99. 72. Weston CF, Cooper BT, Davies JD, et al. Veno-occlusive disease of the liver secondary to ingestion of comfrey. Br Med J (Clin Res Ed) 1987;295:183. 73. Smith LW, Culvenor CC. Plant sources of hepatotoxic pyrrolizidine alkaloids. J Nat Prod 1981;44:129–152. 74. Tyler VE. Herbal medicine in America. Planta Med 1987;53:1–4. 75. Valla D, Benhamou JP. Drug-induced vascular and sinusoidal lesions of the liver. Baillières Clin Gastroenterol 1988;2:481–500. 76. Datta DV, Khuroo MS, Mattocks AR, et al. Herbal medicines and veno-occlusive disease in India. Postgrad Med J 1978;54:511–515. 77. Tandon BN, Tandon HD, Tandon RK, et al. An epidemic of veno-occlusive disease of liver in central India. Lancet 1976;2:271–272. 78. Steenkamp V, Stewart MJ, Zuckerman M. Clinical and analytical aspects of pyrrolizidine poisoning caused by South African traditional medicines. Ther Drug Monit 2000;22:302–306. 79. DeLeve LD, McCuskey RS, Wang X, et al. Characterization of a reproducible rat model of hepatic veno-occlusive disease. Hepatology 1999;29:1779–1791. 80. Wang X, Kanel GC, DeLeve LD. Support of sinusoidal endothelial cell glutathione prevents hepatic veno-occlusive disease in the rat. Hepatology 2000;31:428–434. 81. Shimizu I. Sho-saiko-to: Japanese herbal medicine for protection against hepatic fibrosis and carcinoma. J Gastroenterol Hepatol 2000;15(Suppl):D84–90. 82. Itoh S, Marutani K, Nishijima T, et al. Liver injuries induced by herbal medicine, syo-saiko-to (xiao-chai-hu-tang). Dig Dis Sci 1995;40:1845–1848. 83. Johnson PJ, McFarlane IG. Meeting report: International Autoimmune Hepatitis Group. Hepatology 1993;18:998–1005. 84. MacGregor FB, Abernethy VE, Dahabra S, et al. Hepatotoxicity of herbal remedies. Br Med J 1989;299:1156–1157. 85. Castot A, Larrey D. Hepatitis observed during a treatment with a drug or tea containing wild germander. Evaluation of 26 cases reported to the Regional Centers of Pharmacovigilance. Gastroenterol Clin Biol 1992;16:916–922. 86. Kouzi SA, McMurtry RJ, Nelson SD. Hepatotoxicity of germander (Teucrium chamaedrys L.) and one of its constituent neoclerodane diterpenes teucrin A in the mouse. Chem Res Toxicol 1994;7:850–856. 87. Lekehal M, Pessayre D, Lereau JM, et al. Hepatotoxicity of the herbal medicine germander: metabolic activation of its furano diterpenoids by cytochrome P450 3A depletes cytoskeleton-
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associated protein thiols and forms plasma membrane blebs in rat hepatocytes. Hepatology 1996;24:212–218. Loeper J, Descatoire V, Letteron P, et al. Hepatotoxicity of germander in mice. Gastroenterology, 1994. 106(2): p. 464–472. Feldmann G. Liver apoptosis. J Hepatol 1997;26(Suppl 2):1–11. Fau D, Lekehal M, Farrell G, et al. Diterpenoids from germander, an herbal medicine, induce apoptosis in isolated rat hepatocytes. Gastroenterology 1997;113:1334–1346. De Berardinis V, Moulis C, Maurice M, et al. Human microsomal epoxide hydrolase is the target of germanderinduced autoantibodies on the surface of human hepatocytes. Mol Pharmacol 2000;58:542–551. Woolf GM, Petrovic LM, Rojter SE, et al. Acute hepatitis associated with the Chinese herbal product jin bu huan. Ann Intern Med 1994;121:729–735. Horowitz RS, Feldhaus K, Dart RC, et al. The clinical spectrum of Jin Bu Huan toxicity. Arch Intern Med 1996;156:899–903. Russmann S, Lauterburg BH, Helbling A. Kava hepatotoxicity. Ann Intern Med 2001;135:68–69. Kraft M, Spahn TW, Menzel J, et al. Fulminant liver failure after administration of the herbal antidepressant Kava-Kava. Dtsch Med Wochenschr 2001;126:970–972. Escher M, Desmeules J, Giostra E, et al. Hepatitis associated with Kava, a herbal remedy for anxiety. Br Med J 2001;322:139. Teschke R, Gaus W, Loew D. Kava extracts: safety and risks including rare hepatotoxicity. Phytomedicine 2003;10:440–446. Nadir A, Agrawal S, King PD, et al. Acute hepatitis associated with the use of a Chinese herbal product, ma-huang. Am J Gastroenterol 1996;91:1436–1438. Borum ML. Fulminant exacerbation of autoimmune hepatitis after the use of ma huang. Am J Gastroenterol 2001;96:1654–1655. Lai SM, Lim KW, Cheng HK. Margosa oil poisoning as a cause of toxic encephalopathy. Singapore Med J 1990;31:463–465. Koga Y, Yoshida I, Kimura A, et al. Inhibition of mitochondrial functions by margosa oil: possible implications in the pathogenesis of Reye’s syndrome. Pediatr Res 1987;22:184–187. Sinniah R, Sinniah D, Chia LS, et al. Animal model of margosa oil ingestion with Reye-like syndrome. Pathogenesis of microvesicular fatty liver. J Pathol 1989;159:255–264. Harvey J, Colin-Jones DG. Mistletoe hepatitis. Br Med J (Clin Res Ed) 1981;282:186–187. Anderson IB, Mullen WH, Meeker JE, et al. Pennyroyal toxicity: measurement of toxic metabolite levels in two cases and review of the literature. Ann Intern Med 1996;124:726–734. Bakerink JA, Gospe SM Jr, Dimand RJ, et al. Multiple organ failure after ingestion of pennyroyal oil from herbal tea in two infants. Pediatrics 1996;98:944–947. Khojasteh-Bakht SC, Chen W, Koenigs LL, et al. Metabolism of (R)-(+)-pulegone and (R)-(+)-menthofuran by human liver cytochrome P-450s: evidence for formation of a furan epoxide. Drug Metab Dispos 1999;27:574–580.
Section IV. Toxin Mediated Liver Injury
28
OCCUPATIONAL AND ENVIRONMENTAL HEPATOTOXICITY Thomas D. Schiano and Kristel Hunt Abbreviations ALAD d-aminolevulinic acid dehydratase BEC biliary epithelial cells CAA chloroacetaldehyde CCA chromate copper arsenate CCl4 carbon tetrachloride CEO chloroethylene oxide CSI chemical substance inventory CYP2E1 cytochrome P450 DAPM 4,4¢-diaminodiphenylmethane DDT dichlorodiphenyltrichloroethane DMAC dimethylacetamide
DMF EPA GGTP IARC ICC MDA MDI NICC NIOSH NTP
dimethylformamide environmental protection agency g-glutamyltransferase international agency for research on cancer Indian childhood cirrhosis methylenedianiline 4,4¢-methylenediphenyl di-isocyanate idiopathic copper toxicosis national institute for occupational safety and hazard national toxicology program
INTRODUCTION The pattern of injury of various hepatotoxins and the actual hepatotoxins encountered, both at home and in the workplace, have changed considerably over time. Traditionally recognized occupational hepatotoxins, such as carbon tetrachloride, are now only rarely encountered, largely because of increased awareness among physicians and those workers potentially exposed. It remains difficult to definitively prove the hepatotoxicity of a specific chemical or environmental agent, as the effects are extremely diverse and range from mildly abnormal liver chemistry tests to the development of fulminant liver failure, cirrhosis, and liver cancer. As of December 2004 there were over 25 million organic and inorganic substances registered with the Chemical Abstracts Service, with over 8 million commercially available. Of those, just over 235 000 are regulated through various national and international registries, including the US Toxic Substances Control Act (TSCA) Chemical Substance Inventory (CSI), the Occupational Safety and Hazard Administration (OSHA), the Environmental Protection Agency (EPA), the National Toxicology Program (NTP), and the National Institute for Occupational Safety and Hazard (NIOSH), among others.1 The latest update of the Pocket Guide to Chemical Hazards from February 2004 lists 677 industrial chemicals, with 228 of them listed as capable of causing liver injury.2 Although many of these industrial and occupational toxins are capable of damaging the liver, they rarely do so in the course of typical exposures: the lungs, skin, kidneys, or bone marrow are the more important targets of industrial toxins.1 A list of websites to
OSHA PAs PCBs PVC TCDD TNT TSCA VC VOD
occupational safety and hazard administration pyrrolizidine alkaloids polychlorinated biphenyls polyvinyl chloride tetrachlorodibenzodioxins trinitrotoluene toxic substances control act vinyl chloride veno-occlusive disease
access information on potential occupational and industrial toxins is given in Table 28-1. The first part of this chapter will review known human occupational, chemical, and heavy metal hepatotoxins and their patterns of injury; the remainder will review the pathophysiology and clinical manifestations of specific environmental hepatotoxins: some well known chemical hepatotoxins and their occupational uses are shown in Table 28-2.
TYPES OF EXPOSURE Inhalation Most industrial exposures occur via inhalation, and most of the chemicals are lipid soluble and able to passively cross membrane barriers. Some volatile hepatotoxins may produce severe ocular, mucous membrane and skin irritation that precludes prolonged inhalational hepatotoxicity. Inhalational exposures are fairly well regulated, with well established maximum concentrations that can be tolerated for 40 hours per week without toxic effects.1
Ingestion Uncommon in the workplace, ingestion typically occurs in the domestic setting. Although most ingestions occur accidentally, there are now increasing concerns about intentional contamination of food items by toxic agents that occur in the industrial setting through bioterrorism. At home, toxic ingestion occurs either by accident or intentionally.1 Some household products that contain hepatotoxic chemicals are listed in Table 28-3.
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Table 28-1. Websites to Obtain Information on Potential Occupational and Industrial Toxins American Chemical Society http://www.chemistry.org/portal/a/c/s/1/home.html American College of Occupational and Environmental Medicine Physicians http://www.acoem.org Association for Occupational and Environmental Clinics http://www.aoec.org/ EPA http://www.epa.gov/fedrgstr/EPA-PEST/2005/March/Day11/p4466.htm Haz-Map. Occupational Exposure to Hazardous Agents (National Library of Medicine) http://hazmap.nlm.nih.gov International Agency for Research on Cancer http://www.1arc.fr/ National Center for Toxicological Research of the Food and Drug Administration http://www.fda.gov/nctr National Institute for Occupational Safety and Health of the Centers for Disease Control and Prevention http://www.cdc.gov/niosh/homepage.html National Institute of Environmental Health Sciences of the National Institutes of Health http://www.niehs.nih.gov National Toxicology Program http://ntp-server.niehs.nih.gov University of Kansas Resource and Learning Center. Environmental, Industrial and Occupational Toxicology Information http://library.kumc.edu/omrs/subjects/toxic.html
Table 28-3. Household Products that may be Hepatotoxic Household use
Chemical nature
Carburetor cleaner Christmas tree lights Dry cleaning fluids Furniture polishes and waxes
Chlorobenzene Methylene chloride Chlorinated aliphatic compounds Antimony Nitrobenzene Chlorobenzenes
Moth balls Paint products: Brush cleaners Paints Plasticizers, lacquers, resins Removers, paint, wax Plastic menders, greasers Plasticizers Shoe cleaners Spray repellant Stamping inks Toilet bowl blocks
Cresols Arsenic Varied Chlorinated aliphatic compounds, dimethylformamide Ethylenedichloride Phthalates Aniline Nitrobenzene Vinyl chloride Phenol Paradichlorobenzene
From Zimmerman HJ. Occupational hepatotoxicity. In: Zimmerman HJ. Hepatotoxicity: the adverse effects of drugs and other chemicals on the liver. Philadelphia: Lippincott Williams & Wilkins, 1999
Table 28-2. Some Important Chemicals, Their Uses and Associated Hepatotoxicity Chemical
Uses
Hepatic responses
Arsenic and inorganic salts Beryllium
As pesticides and alloys; in production of dyes, ceramics, drugs, fireworks, paint, petroleum, ink, and semiconductors In alloys, cathode ray tubes, ceramics, electrical equipment, gas mantles, missiles, nuclear reactors, and refractory materials As degreasers, fat processors, fire extinguisher, fumigant, production of solvents: in fluorocarbons, ink, insecticides, lacquer, propellants, refrigerants, rubber and wax As solvent, degreaser, cement component; in production of adhesives, deodorants, detergents, emulsions, fats, glue, lacquer, oil, paint, polish, shoe cream, varnish remover, waxes; in histology laboratories In munitions, pyrotechnics, explosives, smoke bombs, fertilizers, rodenticides, bronze alloys, semiconductors, and luminescent coatings As copper etcher, forensic and biology laboratory chemical; in batteries, colored glass, disinfectants, drugs, dyes, explosives, matches, photography chemicals, and tanneries In cable insulation, dyes, electric equipment, herbicides, lacquers, paper treatment, plasticizers, resins, rubber textiles, flameproofer, transformers, and wood preservation Contaminant of commercial preparations of 2,4,5-trichlorophenoxyacetic acid, polychlorinated biphenyls, and other chlorinated compounds As dry-cleaning agent, fumigant, solvent, degreaser; in production of gaskets, lacquers, paints, phosphorus, resins, varnish, wax As solvent, degreaser, chemical intermediate, fumigant; in production of cellulose esters, gums, rubber, soap, vacuum tubes, wax, wool As explosive As chemical intermediate and solvent; in production of polyvinyl chloride and resins
Acute hepatocellular injury cirrhosis; angiosarcoma Granulomata
Carbon tetrachloride
Dioxane
Phosphorus (yellow)
Picric acid (2,4,6trinitrophenol) Polychlorinated biphenyls 2,3,7,8-Tetrachlorodibenzo-p-dioxin Tetrachloroethane Tetrachloroethylene 2,4,5-Trinitrotoluene Vinyl chloride
Acute hepatocellular injury; cirrhosis
Subacute hepatocellular injury
Acute hepatocellular injury
Acute hepatocellular injury
Subacute hepatocellular injury; cirrhosis
Porphyria cutanea tarda Acute hepatocellular injury Acute hepatocellular injury Acute and subacute hepatocellular injury Fibrosis, non-cirrhotic portal hypertension, cirrhosis, angiosarcoma, carcinoma
Adapted from Gitlin N. Clinical aspects of liver diseases caused by industrial and environmental toxins. In: Zakim D, Boyer TD. Hepatology: A Textbook of Liver Disease, 2nd edn. Philadelphia: WB Saunders Company; 1990.
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Mucosal Absorption The chemical structure and lipid or water solubility of the toxin are the major determinants of absorption.1 Hepatotoxic agents are infrequently absorbed in this manner.
picion needs to be maintained, especially if the temporal relationship is appropriate.
HEPATOTOXIC CHEMICALS
DIAGNOSIS OF CHEMICAL-INDUCED LIVER INJURY
HALOGENATED AROMATIC HYDROCARBONS Polychlorinated Biphenyls
When patients present with liver dysfunction or abnormal liver chemistry tests, it often requires a high index of suspicion to recognize that this may be the result of exposure to an occupational or environmental toxin. Anecdotal reports tend to concentrate on unique, severe, or fatal outcomes, so the overall incidence of environmental and occupational hepatotoxic injury is probably underestimated. Many cases probably go unrecognized because they are never suspected, not properly investigated, or simply not reported.3 A simple severe exposure often leads to an acute clinical presentation, whereas prolonged exposure of a lesser degree may lead to subacute or chronic liver disease.3 The first clinical manifestations of hepatotoxicity can often be non-specific, with constitutional symptoms occurring much earlier than jaundice. The patient may otherwise be healthy but exposed to a known environmental hepatotoxin. When there are no other risk factors for chronic liver disease the diagnosis is usually easy to make in this setting, but unfortunately this scenario is very uncommon. A comprehensive history should be taken in any patient presenting with an acute or chronic cryptogenic liver disease to exclude an occupational or industrial exposure. Occupations that may entail exposure to potentially hepatotoxic chemicals are listed in Table 28-4. Other causes of liver disease must be excluded by careful assessment of clinical, radiologic, biochemical, and serologic testing. Patients with any form of chronic liver disease may develop decompensated liver disease in the presence of a toxic exposure. Some uncommon histological patterns of liver injury, such as angiosarcoma, hepatoportal sclerosis, zonal necrosis, and vascular injury, should raise the suspicion of occupational or environmental hepatotoxicity. The major goals of the clinical evaluation of patients with suspected chemical-induced liver dysfunction are to recognize that this is indeed what is occurring by establishing that there are sufficient criteria to assign a causal relationship to a particular agent. At the same time, other causes of acute and chronic liver diseases must be excluded, as occupationally and environmentally induced liver disease can clinically and histologically mimic almost any known liver disease (Table 28-5). Some drugs and chemicals may themselves be deposited in the liver. Usually this is an incidental finding and has little clinical significance apart from indicating prior exposure. Examples include gold compounds, thorium dioxide, and titanium.
Polychlorinated biphenyls (PCBs), with their significant flame-retardant and insulating properties, have been used since the 1930s in a
Management Early recognition is of utmost importance in the management of chemical-induced hepatotoxicity, as it is in drug hepatotoxicity. As with drug hepatotoxicity, continued exposure to the toxic agent once jaundice appears may lead to acute liver failure and hepatic decompensation. In this regard, anticipation and proactively seeking evidence for hepatotoxicity with the appropriate agencies, such as the Poison Control Center or OSHA, is imperative. Even when a substance is not known to cause hepatotoxicity a high index of sus-
Table 28-4. Occupations that Entail Exposure to Hepatotoxic Chemicals Artificial pearl makers Airplane hanger employees Burnishers Cement (rubber, plastic) makers Chemists Chemical industry workers Chlorinated rubber makers Cobblers Color makers Core makers Degreasers Dry cleaners Dye makers Dyers Electrical transformer and condenser makers Electroplaters Enamel makers Extractors, oil and fats Fire extinguisher makers Galvanizers Garage workers Gardeners (insecticides) Gas (illuminating) workers Glass makers Glue workers Ink makers Insecticide makers Insulators (wire) Lacquer makers and lacquerers Leather workers Linoleum makers Paint removers makers and users Paraffin workers Perfume makers Petroleum refiners Pharmaceutical workers Photographic material workers Polish (metal) makers and users Printers Refrigerator workers Resins (synthetic makers) Rubber workers Shoe factory workers Soap makers Spreaders (rubber works) Straw hat makers Thermometer makers Tobacco dinicotizers Varnish workers Waterproofers Wax makers From: Zimmerman HJ. Occupational hepatotoxicity. In: Zimmerman HJ. Hepatotoxicity: the adverse effects of drugs and other chemicals on the liver. Philadelphia: Lippincott Williams and Wilkins, 1999
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Table 28-5. Liver Injury Patterns for Occupational and Environmental Toxins Category
Examples
Acute hepatocellular injury:
Arsenic, cocaine, copper, halogenated aliphatic hydrocarbons, nitrobenzene, pyrrolizidine alkaloids, tetrachlorethane, TNT Ferrous sulfate, phosphorus Beryllium Arsenic, carbon tetrachloride, mushroom poisoning Arsenic, thorotrast, vinyl chloride Thorotrast Berxllium, copper, cyanobacterial toxins, methylene dianiline, paraquat, toxic rapeseed oil 1,1,2,2-tetrachloroethane, arsenic, cadmium, carbon tetrachloride, pyrrolizidine alkaloids, TNT Vinyl chloride Beryllium, copper Aflatoxin, thorotrast Arsenic, copper, vinyl chloride Copper 1,1,1-trichloroethane, 1,1,2,2tetrachloroethane, alfatoxins, carbon tetrachloride, cocaine, dimethylacetamide, dimethylformamide, margosa oil toxicity, phosphorus Thorotrast, vinyl chloride
Zone 1 Zone 2 Zone 3 Angiosarcoma Cholangiocarcinoma Cholestasis
Cirrhosis
Fibrosis, septal and subcapsular Granulomas Hepatocellular carcinoma Hepatoportal sclerosis Mallory’s hyaline Microvesicular steatosis
Peliosis hepatis Pigment deposition: Anthracite Gold Thorotrast Titanium Veno-occlusive disease
Noted in coal miners Gold compounds used in the treatment of arthritis Occupational exposure Pyrrolizidine alkaloids
variety of industrial applications. Concern about their presence in the environment began in the 1960s, when significant concentrations of PCBs were found in wildlife in Sweden. Subsequent research has shown their long-term persistence and bioaccumulation in the food chain, especially in the fatty tissues of fish and sea mammals. Since the use of these compounds was banned by the US EPA in 1977, the concentrations of PCBs have drastically declined in all environmental reservoirs.3,4
Acute Effects In the 1940s a number of cases of acute PCB toxicity were reported, with symptoms of anorexia, fatigue, nausea, peripheral edema, and rarely jaundice; over 50% of individuals, however, died of acute or subacute hepatic necrosis or of subsequent cirrhosis. Histologically this was described as severe zone 3 necrosis. Since then, there have been two epidemics of acute PCB toxicity, both as a result of ingestion of contaminated rice oil: in 1968 in Japan (Yusho), and in 1979 in Taiwan.5 Both epidemics were associated with liver dysfunction and chloracne, as well as low birthweights as a result of fetal exposure. Although elevated mortality from liver diseases was noted within 3 years of exposure, more recent studies have attributed the hepatotoxicity to contaminating dibenzofurans (PCDFs) instead.4,5
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Chronic Effects Given the significant carcinogenicity of PCBs in laboratory animals and the initial reports of increased rates of liver cancer in the setting of acute toxicity, in the 1980s PCBs were classified as probable human carcinogens by all major registries. A wealth of literature on occupational exposure, primarily from workers employed in electrical equipment manufacturing with years of PCB exposure and more than 30-year follow-up, however, has not shown any increase in acute or chronic health effects beyond skin and eye irritation and perhaps transient liver enzyme elevation.6 In particular, no increased risks of liver cancer – or any other tumors, for that matter – have been observed.6,7 Based on these data, it is unlikely that PCB exposure from environmental sources can pose a significant risk to humans, and indeed, in 2003, the EPA officially downgraded the perceived cancer risk of PCBs.8 As potent inducers of the enzyme P450 system, however, PCBs can significantly enhance the hepatotoxic effects of other chemical agents or drugs.
Tetrachlorodibenzodioxins (TCDD) Dioxins are a family of chlorinated aromatic compounds formed primarily during combustion of chlorine-containing materials such as PCBs, chlorophenols, and phenoxy herbicides. Like PCBs, they can persist in the environment for years. In experimental animals, dioxins are powerful hepatotoxins with potent P450-inducing abilities and strong carcinogenic potential. Humans, however, are more protected from the hepatotoxic effects than animals because of a lower affinity with dioxin in human cytoplasmic receptors.9 The major identified and chronicled dioxin exposures resulted from the explosion of a herbicide factory in Seveso, Italy, in 1976, and through dioxin-contaminated herbicide use during the Vietnam war (‘Agent Orange’). In the former, progression to porphyria cutanea tarda occurred in some individuals. Despite no early evidence for liver injury, there has been an increased incidence of cholangiocarcinoma and hepatocellular carcinoma, especially in women, in the Seveso cohort after 20 years of follow-up; no direct causal effect of dioxins has been formally established, however.9 No demonstrable hepatic injury or predisposition to liver cancer has been found among the military personnel exposed to Agent Orange, despite high TCDD levels found in blood.10
NITROAROMATIC COMPOUNDS A variety of nitroaromatic compounds have been shown to be hepatotoxic in humans, including nitrobenzene and dinitrobenzene, dinitrophenol, and perhaps one of the best-known hepatotoxins, trinitrotoluene (TNT). Tight regulation of their use in the workplace has dramatically limited the number of any recent occurrences. Nitrobenzene leads to zonal necrosis and is often used as a hepatotoxin in experimental animals. It is primarily used in the production of aniline, and rare instances of human hepatotoxicity have occurred in the industry as a result of inhalation of toxic fumes. A potent hepatic carcinogen in laboratory animals, nitrobenzene is deemed to be a potential human carcinogen as well.2 Dinitrophenol is used in the dye industry and in the manufacturing of photograph developers, as well as a fungicide. The most common human exposure is in the occupational setting by inhalation or dermal contact of workers involved in manufacturing of the
Chapter 28 OCCUPATIONAL AND ENVIRONMENTAL HEPATOTOXICITY
compound; there were reports of dinitrophenol toxicity via ingestion in the 1930s, when it was used in the treatment of obesity. Despite its potential for accumulation in the environment, there have been no reports of environmental toxicity in humans. Its mechanism of injury is primarily through its ability to interfere with oxidative phosphorylation, resulting in cholestasis and, rarely, hepatic necrosis.3 Trinitrotoluene (TNT) has been used primarily in the military setting as an explosive in bombs and grenades. The first reports of TNT hepatotoxicity date back to World War I, when severe liver disease with more than a 25% fatality rate was reported among munitions workers in England; in the US, over 400 people were purported to have died as a result of TNT-related liver injuries and aplastic anemia. Another peak of hepatotoxicity occurred during World War II, again through exposure to TNT-containing shells.3,11 Since then, the incidence of toxic effects associated with handling of TNT has decreased sharply, thanks to enhanced protective measures and better ventilation. The manifested hepatic toxicity is typically subacute, with symptoms presenting months after exposure. Initial symptoms of anorexia, nausea, and fatigue that may progress to jaundice are associated with minimal liver enzyme abnormalities. Thereafter, symptoms tend to evolve into one of two classic patterns: some patients develop massive hepatic necrosis within days of the initial symptoms and succumb to liver failure; others develop portal hypertension and ascites, with liver injury ranging from acute or subacute hepatic necrosis to macronodular cirrhosis.3,11 The proposed mechanism of injury is primarily through an increase in free radical levels (superoxide and hydrogen peroxide). The large variability in apparent susceptibility also suggests an idiosyncratic preferential conversion of TNT to toxic metabolites in certain predisposed individuals.
NITROALIPHATIC COMPOUNDS (NITROPARAFFINS) The nitroparaffins (nitromethane and nitroethane, 1-nitropropane and 2-nitropropane) are used primarily as industrial solvents in inks, paints, adhesives, varnishes, polymers, and synthetic materials; 2nitropropane is also used as a component in explosives and propellants, as well as in fuels for internal combustion engines. The primary routes of exposure are inhalation, ingestion, and dermal contact. In the 1970s and 1980s several cases of fulminant liver failure were attributed to 2-nitropropane; since its classification in 1999 by the International Agency for Research on Cancer (IARC) as a possible human carcinogen, no further cases have been reported. In general, nitroparaffins appear less toxic than nitroaromatic compounds.3
CHLORINATED ETHYLENES Vinyl Chloride (VC) Vinyl chloride (VC), historically used as a solvent, propellant, and refrigerant, is now primarily used for the production of polyvinyl chloride (PVC), a material used to manufacture automotive parts and accessories, furniture, packaging materials, pipes, wall coverings, and wire coatings. During the first five decades of its use, no clinically significant toxicity was noted. However, in 1974 a series of cases of angiosarcoma occurring in workers in rubber plants exposed
to VC was reported – a landmark discovery in occupational medicine. Despite its known toxicity, vinyl chloride remains a crucial component of the plastics industry: in 1998, 27 million tons of PVC was produced, accounting for 20% of all plastics production.12 The main route of occupational exposure to VC is via inhalation, and occurs primarily in VC manufacturing plants. Since its recognition in the 1970s as a significant hepatocarcinogen, workplace exposure standards have been tightly regulated, and indeed there have been only rare recent reports of VC-related occupational hepatotoxicity since that time. The exposure of the general population is primarily through accidental spills into the environment, or by chronic exposure to the fumes in the vicinity of VC industry or waste disposal sites. Small amounts of vinyl chloride can also be found in foodstuffs, cosmetic or pharmaceutical products in certain types of PVC packaging, although typically not in sufficient concentrations to lead to significant toxicity.12
Mechanism of Injury After inhalation, VC undergoes oxidation by the cytochrome P450 (CYP2E1) system to form chloroethylene oxide (CEO), which then rapidly forms chloroacetaldehyde (CAA). Both CEO and CAA can alkylate nucleic acid bases and form DNA adducts with strong mutagenic and promutagenic properties. In highly exposed workers with angiosarcoma, point mutations (primarily base-pair substitutions) have been detected both in p53 and K-ras oncogenes.13
Differences in Susceptibility There are significant interspecies and interindividual variations in both CYP2E1 and the glutathione S-transferase isoenzymes (involved in the detoxification process of CEO and CAA), leading to large differences in the dose required to produce VC-related hepatotoxicity. Younger age and female gender, as well as certain HLA alleles, may be associated with higher levels of toxicity. Recent studies have demonstrated clear synergistic effects of both alcohol and viral hepatitis in the setting of VC exposure, leading to a substantially higher risk of hepatocellular carcinoma and cirrhosis in exposed patients who are infected with either hepatitis B or C virus and who consume excessive amounts of alcohol.14 Several other environmental toxins have also been shown to work synergistically with VC: concurrent aflatoxin and PCB exposures have both led to increased rates of liver tumor formation.15
Acute and Chronic Effects The acute effects of vinyl chloride are primarily central nervous system related. With massive exposure, VC leads to cardiac arrhythmias and cardiovascular collapse. More chronic exposure, of the order of months to years, can lead to so-called ‘vinyl chloride disease’, including disorders of skin and connective tissue such as Raynaud’s phenomenon, neurologic symptoms such as headaches, dizziness and blurry vision, as well as hepatosplenomegaly associated with various types of liver pathology.16 The earliest findings of VC exposure are focal hepatocellular hyperplasia and focal mixed hyperplasia. Subcapsular fibrosis, progressive portal fibrosis, and an increase in intralobular connective tissue are precursors to the development of angiosarcoma, which is frequently multicentric.17 Indeed, the association between vinyl
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chloride and angiosarcoma is well described, with over 200 documented cases via either environmental or occupational exposure (Figure 28-1). Other substances associated with angiosarcoma formation include thorium dioxide, long-term exposure to inorganic arsenic, Fowler’s solution in the treatment of psoriasis, and androgens. Angiosarcoma is often peripherally located in the liver and may be associated with a hepatic bruit. Liver fibrosis is over five times more common in individuals exposed to vinyl chloride, and is often associated with significant portal hypertension in the absence of cirrhosis (hepatoportal sclerosis) (Figure 28-2).18,19 Substantially higher risks of hepatocellular carcinoma have also been shown in all the
major cohorts, with a standardized mortality ratio for HCC-related deaths of 1.35 in a pooled analysis.20–24
AROMATIC HYDROCARBONS Aromatic hydrocarbons such as benzene, toluene, xylene, and naphthalene are typically used as solvents and glues and in the manufacture of plastics. In general they are not associated with significant hepatotoxicity, although minor liver enzyme elevations and hepatic steatosis have been described. A recent report has described selflimited hepatitis in association with toluene handling.25
HALOGENATED ALIPHATIC HYDROCARBONS Carbon Tetrachloride (CCl4)
Figure 28-1. Angiosarcoma. Liver needle biopsy showing anastomosing vascular channels that are lined by atypical endothelial cells (arrow). Hematoxylin and eosin, original magnification 20¥. (Courtesy of Dr M.I. Fiel.)
Figure 28-2. Hepatoportal sclerosis. Photomicrograph of wedge biopsy of liver showing a portal tract (arrow) with pronounced herniation of portal veins (asterisks) into adjacent parenchyma. Marked centrilobular sinusoidal dilation is also present (arrowheads). In other areas, portal tracts are severely fibrotic and the portal veins are sclerotic. Hematoxylin and eosin, original magnification 10¥. (Courtesy of Dr M.I. Fiel.)
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CCl4 is the best-studied occupational hepatotoxin. It provides the prototype for several toxic phenomena: severe liver injury with zone 3 necrosis and steatosis, injury greatly enhanced by alcohol, and injury caused primarily by free radical metabolites causing direct damage to hepatocyte membranes. Until the 1920s, when its potent hepatotoxicity was first noted, it was widely used as a cleaning agent, solvent, fire extinguisher, and grain fumigant, as well as an intermediate for the synthesis of chlorofluorocarbons. Carbon tetrachloride poisoning typically follows inhalation of the vapor in poorly ventilated spaces, but can also occur after ingestion of contaminated foodstuffs or ground water, or via skin absorption.1,3 It is no longer produced on a large scale in the US, and thus occupational and household exposures typically only occur in countries that still allow its use.26–28 Industrial exposure usually occurs by inhalation of the fumes in a poorly ventilated environment. Alcohol ingestion and the use of barbiturates appear to enhance hepatic susceptibility to CC14, possibly via their effects on the P450 system.1,3
Acute Effects Acute carbon tetrachloride poisoning is manifested as a multisystem disorder, with early CNS, renal, and gastrointestinal toxicity. The majority of the deaths attributable to acute CCl4 toxicity are due to acute tubular necrosis and renal failure that frequently lead to pulmonary edema and congestive heart failure. Gastrointestinal symptoms, including nausea, vomiting, diarrhea, and abdominal pain, sometimes also associated with hemorrhagic gastritis, typically occur within the first 24 hours. Features of liver disease manifest within 24–48 hours, with massive hepatomegaly, jaundice, and a bleeding diathesis with spontaneous hemorrhage; ascites and hepatic encephalopathy occur in severe cases.3 The aminotransferases rise to extremely high levels, with AST higher than ALT. The bilirubin levels also soar, reflecting hepatocyte necrosis, hemolysis, disruption of the cytochrome P450 system, and an impaired clearance of bilirubin by the kidneys. In survivors recovery is rapid, with normalization of liver enzymes within 2 weeks. If the hepatic injury is so severe as to result in death, it will typically occur within the first 10 days. The histologic findings in acute toxicity are primarily those of zone 3 necrosis and steatosis, preceded by prominent ballooning and swollen granular cytoplasm with pale nuclei.3 Treatment is mainly supportive.
Mechanism of Injury Great intra- and interspecies variability in hepatotoxic potential has been noted, owing primarily to variability in the mixed-function
Chapter 28 OCCUPATIONAL AND ENVIRONMENTAL HEPATOTOXICITY
oxidase system (P450 cytochrome system). In general, males as well as older individuals have been found to be more susceptible to injury. CC14 appears to damage the hepatocellular membrane, with a resultant loss of enzymes and a flux of ions out of the hepatocyte. The CC14 is then metabolized to a toxic free radical by cytochrome P450 in the endoplasmic reticulum. The toxic free radical, via perioxidation of lipids, disrupts membranes further and interferes with the synthesis and transport of lipoproteins, causing fat to accumulate within the hepatocyte.29
1,1,1-Trichloroethane This was widely recognized as a hepatotoxin during the 1940s, when it was used as a solvent for varnish in airplane covers. It is still used as a solvent, and there have been rare reports of its association with fatty liver and cirrhosis.30,31
1,1,2,2-Tetrachloroethane 1,1,2,2-Tetrachloroethane is used as a chemical intermediate, primarily in the manufacture of trichloroethylene and tetrachloroethylene. There have been many reports of human hepatotoxicity causing abnormal liver chemistry tests and acute hepatitis with chronic exposure.1,3
Tetrachloroethylene (Perchloroethylene) This compound is used primarily in dry cleaning and textile processing, as a chemical intermediate in the production of fluorocarbons, and as a metal degreasing agent. It is also used as a solvent, as a pesticide intermediate, and as an antihelminthic.1,3 Transient hepatotoxicity in humans has occurred with single exposures, and abnormal liver enzymes and cirrhosis have been observed with chronic long-standing exposure.
N-SUBSTITUTED AMIDES Dimethyl Acetamide Dimethylacetamide (DMAC) is used as a solvent in the manufacturing of synthetic fibers, some resins and plastics, as well as in film and coating formulations. It is readily absorbed by inhalation as well as dermally. Acute toxicity leads to elevated transaminase levels and hepatomegaly, with focal necrosis seen on liver biopsy.32 Chronic exposures have resulted in microvascular steatosis as well as lipofuscin/hemosiderin accumulation in Kupffer cells, with minimal recovery after withdrawal of the toxin. Biological monitoring of DMAC in exposed workers by measurement of its urinary metabolite (monomethyl acetamide) is recommended.33
Dimethylformamide Dimethylformamide (DMF) has excellent solvent properties and has been used extensively in the production of synthetic leather and resin. Hepatotoxicity occurs in a dose–response fashion after exposure through inhalation, ingestion, and/or skin exposure. Acute exposure typically leads to modest elevations in aminotransferase levels which resolve after withdrawal of the exposure. Chronic exposure leads to microvesicular steatosis, and in severe cases hepatic necrosis can also occur. Both hepatitis B virus and alcohol use have been shown to have a synergistic effect in causing hepatotoxicity with DMF exposure.34,35
METHYLENEDIANILINE Methylenedianiline (MDA) was the agent responsible for a wellknown epidemic of hepatic injury in Epping, UK, in 1965, known as Epping jaundice.36 Bread baked from flour contaminated with MDA caused cholestatic hepatitis in 84 people. Within hours of consuming the bread, two-thirds of the affected individuals developed abdominal pain, fever and rash, and ultimately jaundice. The remaining one-third had minimal or no clinical symptoms. Elevations in serum bilirubin and alkaline phosphatase levels were pathognomonic, but marked transaminase elevations were seen in some cases as well. A similar mixed cholestatic–hepatocellular injury pattern was seen on liver biopsy, with predominant bile stasis, portal inflammatory infiltrates, and varying degrees of hepatocyte necrosis. The majority of patients recovered within 4–6 weeks, but some had prolonged jaundice lasting a few months; several patients had slightly elevated aminotransferase levels persisting for up to 2 years. A follow-up study nearly 40 years later found no increase in mortality rates among the people exposed, and no cases of death specifically from chronic liver disease or from liver cancer.37 Additional reported exposures to MDA have occurred in the occupational setting, with reports of toxicity following ingestion, inhalation, and dermal contact. In each case the hepatitis was self-limited. In rats, glutathione depletion has been shown to drastically enhance and accelerate MDA hepatotoxicity.38 4,4¢-Methylenedianiline (4,4¢-diaminodiphenylmethane; DAPM) is an aromatic diamine used in the production of polyamides, epoxy resins, and 4,4¢-methylenediphenyl di-isocyanate (MDI), the most widely used isocyanate in the production of polyurethanes. These polymers are made into insulation materials, seat cushions, wire coatings, aircraft parts, and medical devices, such as dialysis tubing, orthopedic and odontologic implants, intra-aortic balloons, and vascular grafts.39 In humans, accidental or acute occupational exposure causes jaundice, cholangitis, cholestasis, toxic hepatitis, and skin rash. Studies have demonstrated that acute administration of DAPM to rats produced striking changes in several biliary constituents, followed by necrosis and sloughing of biliary epithelial cells (BEC), cholestasis, and cholangitis. Effects on the biliary system occurred prior to hepatocellular injury, as evidenced by minimal increases in serum indicators of hepatic injury. Furthermore, alterations in biliary constituents were preceded by ultrastructural changes in BEC mitochondria and loss of luminal microvilli, whereas morphologic effects on tight junctions between BEC or between hepatocytes were absent.40 The identity of the DAPM metabolite(s) responsible for injury to BEC remains unknown.
PESTICIDES Dichlorodiphenyltrichloroethane (DDT) DDT, first introduced during World War II, was the first insecticide to be used on a mass scale. As it was highly beneficial for food production, billions of pounds were released into the environment over the next 30 years. Its favorable properties of extreme stability and slow biodegradability also led to its persistence in the environment; indeed, studies have shown that residual compound can be found as long as 10 years after a single application. It is found in the fat stores of animals and is processed up the food chain, leading to significant concentrations of DDT in humans.3
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Acute hepatotoxicity has occurred in rare accidental ingestions of large amounts of DDT, leading to massive hepatic necrosis and hepatic failure. No clear hepatic injury patterns have been described after decades of occupational exposure, and despite its perceived carcinogenicity (it was banned in the US in 1972 for that reason), no clear relationship between tissue DDT levels and cancer risk has been found.41 Similarly to PCBs, DDT is a powerful cytochrome P450 inducer and can enhance the hepatotoxicity or carcinogenicity of other chemicals or drugs.
Paraquat (Dichlorodiphenyltrichloroethane) Paraquat, a bipyridyl, is a widely used herbicide. There are no known hepatic sequelae of chronic paraquat exposure: all known cases of hepatotoxicity have occurred in the setting of substantial paraquat ingestion, usually in a suicide attempt, or rarely through massive skin exposure. The hepatic manifestations of paraquat poisoning usually present within 24–48 hours, and manifest initially as hepatocellular injury leading to zone 3 necrosis. If the individual survives the initial attack, the lesion becomes primarily cholestatic, with destruction of bile ducts. Paraquat poisoning has a high case fatality rate – 50–70% – usually due to initial overwhelming systemic toxicity or to the rapid development of pulmonary fibrosis over the ensuing weeks.3,42
Chlordecone (Kepone) Despite significant hepatic injury and carcinogenicity in laboratory animals exposed to chlordecone, substantial occupational exposures have not resulted in irreversible liver damage. Massive exposure in a manufacturing plant in Virginia in 1970 led to substantial neurologic and testicular damage as well as hepatosplenomegaly – only mild liver dysfunction and minimal steatosis were found on liver biopsy. With cessation of exposure, the liver dysfunction normalized.3,43
METALS Arsenic Arsenic was the first chemical agent with ascribed hepatotoxicity to be identified and was an important early model of experimental liver disease in animals.3,44 Naturally occurring arsenic is ubiquitous in soils, water, and even foods. In industry, arsenic is an essential ingredient in insecticides, herbicides and fungicides, wood preservatives, and dyestuffs, and also is a staple in veterinary medicine for the treatment of parasitic diseases. Chronic arsenic exposure in vineyard workers, miners, and farmers was a frequent cause of chronic liver disease in the early 20th century, but since its identification as a hepatotoxin occupational toxicity has significantly decreased.44
Mechanism of Injury Most of the toxicity of arsenic stems from its ability to inactivate thiol-containing enzymes and substitute arsenate for phosphate groups in a number of biochemical reactions, thus interfering with several key processes such as cellular respiration, gluconeogenesis, glucose uptake, and glutathione metabolism.44 The mechanism for the carcinogenic effects of arsenic is not completely understood, but is probably a combination of induced chromosomal aberrations, oxidative stress, and altered growth factors. Additional mechanisms,
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including gene amplification, altered DNA repair, enhanced cell proliferation, and suppression of the p53 oncogene, are currently under investigation.45 It is noteworthy that the carcinogenicity of arsenic compounds has not been reproducible in animal models, lending support to individual variations in susceptibility to arsenic toxicity. In additional, other host factors, such as age, sex, and nutritional status (in particular selenium deficiency), appear to be important.46
Acute Effects Acute arsenic poisoning is generally due to accidental or planned ingestion. Although larger doses (1–3 g) are common, severe toxicity can occur with as little as 1 mg. Ingestion of toxic quantities leads to symptoms within 30–60 minutes, with prominent gastrointestinal, neurological, and vascular effects and concomitant hepatic injury; death can ensue in 1–3 days. Histologically there is severe steatosis and varying degrees of necrosis, with preferential distribution in either zones 1 or 3.44
Chronic Effects Historically, occupational arsenic exposure occurred mainly through the widespread use of arsenical pesticides in agriculture and vineyards. Chromate copper arsenate (CCA) – in treated wood in homes and as playground equipment – has recently come under intense media scrutiny, leading to the involvement of the US EPA. Arsenic fumes are encountered in mining, smelting, and metallurgy, as well as in decorative glass-making. More recently, arsenic has become an essential component of semiconductor chips. In industries with known arsenic exposure workers are usually closely monitored to prevent clinical toxicity. The majority of human arsenic exposure is through the use of contaminated water. In certain areas of the world, such as West Bengal, India, and Bangladesh, it is an enormous health hazard, with millions of people exposed to unacceptably high levels of arsenic. Epidemiologic studies from these regions have documented the dose-related effects of arsenic on the endocrine, cardiovascular, and central nervous systems, and especially its tumorigenic effects on skin, bladder, lung, and liver.47–49 Hepatic lesions attributable to chronic arsenic ingestion include hepatoportal sclerosis,50 cirrhosis, and angiosarcoma,51 as well as a possible association with hepatocellular carcinoma.52
Phosphorus Yellow phosphorus has long been known to be a hepatotoxin and was used experimentally over a century ago. Acute poisoning with phosphorus, a primary constituent of matches and firecrackers, was not infrequent in the occupational or household setting until its ban in 1942. Since then, the toxicity has occurred primarily by ingestion of rat poison or firecracker contents, either accidentally or with suicidal intent. Acute phosphorus poisoning is a severely toxic syndrome, with initial severe gastrointestinal manifestations such as nausea, vomiting, gastroduodenal ulceration, and neurologic involvement, followed by a latent relatively symptomatic stage and then by hepatic and renal failure. Diagnosis is suggested by a garlic odor on the breath. Histological findings include a characteristic periportal fatty infiltration and hepatic necrosis.3 Aminotransferases are elevated two- to sixfold. Timely gastric lavage is essential.
Chapter 28 OCCUPATIONAL AND ENVIRONMENTAL HEPATOTOXICITY
Beryllium Beryllium is used predominantly in the manufacture of electronics and electrical equipment, and in defense and nuclear weapons technology. Historically, exposures have been by inhalation, with only rare case reports now that industrial hygiene has significantly improved. The two primary lesions are zone 2 necrosis and granulomatous lesions. Beryllium compounds have led to granulomatous lesions in both the liver and the lung.3
Iron Though not directly toxic as an organic molecule, iron in excess concentrations exhibits its hepatotoxicity by the formation of free radicals and lipid peroxidation, resulting in hepatocyte membrane destruction and necrosis. Acute toxicity is seen primarily in the home, occurring either via accidental ingestion (especially among children) or as a suicidal gesture. Increased tissue loads over a prolonged period can lead to the most important chronic toxicity of iron, hemosiderosis. It also contributes to the hepatic injury in alcoholic liver disease, non-alcoholic steatohepatitis, as well as in viral hepatitis.3
Mechanisms of Injury In acute overdose ferrous and ferric ions react with lipid peroxidases, producing free radicals. In contrast to most other toxic agents, the hepatic necrosis in iron-induced hepatotoxicity occurs mostly in zone 1. The periportal region may be primarily affected owing to its high concentrations of both iron and oxygen, substrates for free radical generation. Similarly, in chronic injury iron accumulates primarily in the hepatocytes of zone 1. Initially, cytosolic storage of ferritin and hemosiderin is maximized, but once the tissue stores reach their capacity, free iron is released to produce injury both via the formation of free radicals as well as via catalysis of hydroxyl radical formation.53
Clinical Manifestations Hepatic injury typically develops within 1–3 days after overdose. It is usually characterized by jaundice, elevated aminotransferase levels, and hypoprothrombinemia; in patients with hepatic failure the mortality rate is approximately 50%. However, the majority of patients presenting with iron poisoning will have minimal liver damage. It appears that iron exhibits dose-related toxicity: doses less than 20 mg/kg have been shown to produce no significant injury, whereas doses between 20 and 60 mg/kg can result in mild to moderate injury and doses between 60 and 200 mg/kg in serious injury; doses above 200 mg/kg are likely to lead to death.54 Orthotopic liver transplantation has been performed in the setting of acute iron intoxication.55
Copper Despite its widespread use in electrical wiring and electronics, and in transportation and industrial machinery, household and environmental exposures lead to more episodes of copper toxicity than occupational exposures. Indeed, the only documented occupational hepatotoxicity has been among vineyard workers using a copper solution (Bordeaux mixture) as a fungicide. In the home, exposure can occur from copper-containing supplements and medications, intrauterine conceptive devices, or from drinking water contami-
nated by copper pipes or plumbing components. Copper tubing has led to poisoning in hemodialysis patients. The majority of acute toxicity has occurred with either accidental or intentional (suicidal) ingestion of large amounts of copper.56
Mechanism of Injury The mechanism of copper hepatotoxicity is similar to that of iron toxicity and is caused primarily by free radical production leading to peroxidative injury of hepatocellular membrane lipids. Exposure of DNA to hydrogen peroxide in the presence of a copper– metallothionein complex results in the induction of a variety of types of oxidative damage, including DNA strand breaks and base modifications.
Acute Effects The early clinical manifestations of acute copper toxicity include dysgeusia, nausea, vomiting, and burning abdominal pain; jaundice accompanied by elevated aminotransferase levels, and hepatomegaly occur in approximately a quarter of patients. In severe cases, renal and hepatic failure and shock are the primary causes of death. Histological findings include centrilobular zonal necrosis, cholestasis, and dilated central veins with bile thrombi.57
Chronic Effects Several disease entities are attributable to chronic copper exposure, including Indian childhood cirrhosis (ICC), and idiopathic copper toxicosis (NICC). The role of copper exposure in both ICC and NICC, early childhood diseases that lead to cirrhosis, has been thoroughly investigated. The first reports of ICC were published in 1960, but the disease was not clearly defined until the 1980s, with histologic criteria that included necrosis of hepatocytes with ballooning, Mallory’s hyaline, pericellular intralobular fibrosis, and inflammatory inflammation in the context of a massive copper overload. Minimal fatty changes were observed, and cholestasis was noted only at a very advanced stage. Epidemiologic studies linked the disease to non-breastfed infants in a number of rural Indian villages and related the increase in copper intake to copper utensils used in milk preparation, as well as contamination of drinking of water by copper.58 Since then, similar cases of childhood cirrhosis related to copper exposure have been reported throughout the world, including an epidemiological study of 138 deaths due to infantile cirrhosis in rural Tyrol, Austria, between 1900 and 1974.59 Again, the copper intake was linked both to brass utensils and to high copper levels in the ground water. Current thinking on the pathogenesis of this disease involves a ‘’two-hit’ hypothesis: either excess copper ingestion in the setting of genetic predisposition (no specific mutations have so far been identified, but there is clear familial clustering beyond common copper exposure), or the synergistic effects of environmental toxins. Remarkably, with the copper content of drinking water now tightly regulated in Europe, no further cases of NICC have occurred since the 1970s.60 In vineyard workers exposed to the aerosolized Bordeaux mixture, the primary liver injury occurred in the form of hepatoportal sclerosis and non-caseating granulomas. Despite its carcinogenicity in laboratory animals, copper toxicity has not been linked to liver tumors.
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Lead
Mechanism of Injury
The majority of adult lead exposure is occupational, occurring in workers involved in lead smelting and refinement, and in the manufacture of batteries, pigments, solder, car radiators, and ceramic ware with lead glaze. The lead content of paint was unregulated until 1977, leading to significant exposures both in construction workers and in the home, especially among children. In addition, illegally distilled alcohol (‘moonshine’) is an important source of lead intoxication: data show that habitual users of moonshine are significantly more sensitive to lead exposure, which may lead to considerable blood lead levels and resultant hepatotoxicity.61
Cadmium is a well established hepatotoxin in laboratory animals. Cadmium binds to sulfhydryl groups on critical molecules in mitochondria, leading to oxidative stress, changes in mitochondrial permeability, and mitochondrial dysfunction. Cadmium-dependent activation of Kupffer cells and free radical generation, resulting in the release of a large number of proinflammatory and cytotoxic mediators, leads to further hepatic injury, with subsequent hepatic degeneration, necrosis, and cirrhosis.65 Cadmium’s ability to oxidate nucleic acids and alter DNA repair mechanisms has rendered it a carcinogen, and it is associated with increased rates of lung and renal cell carcinoma in humans.
Mechanism of Injury The mechanism of injury in lead toxicity is complex, but is based primarily on its action as an electropositive metal. As such, it has a high affinity for negatively charged sulfhydryl groups, leading to the inhibition of sulfhydryl-dependent enzymes such as d-aminolevulinic acid dehydratase (ALAD) and ferrochelatase in heme synthesis. The divalent lead acts competitively with calcium in mitochondrial respiration, and interferes with various energy and transport systems. It can also affect nucleic acids by mechanisms that are not yet fully understood. Recently, much research has been concentrated on ALAD polymorphisms and the way they modify lead toxicokinetics, ultimately influencing individual susceptibility to lead poisoning.62
Clinical Manifestations Classic acute cadmium poisoning, known in Japan as itai-itai disease, is characterized by multiple fractures, osteomalacia, osteoporosis, and renal disease. No acute cadmium-related hepatotoxicity has been described. Chronic high-level cadmium exposure has been associated with aminotransferase elevations but non-specific findings on liver histology; its importance lies mainly in its ubiquitous presence in the environment, given its strong synergistic effects with other hepatotoxins and viral hepatitis. Environmental exposure in the absence of underlying liver disease has led to no demonstrable hepatotoxicity so far.65
Toxic Rapeseed Oil Clinical Manifestations Lead poisoning is manifested primarily by damage to the central and peripheral nervous systems, renal toxicity, and anemia; it is also a frequent cause of colicky abdominal pain in children. If diagnosed early, most of the effects are reversible, but chronic high-level exposure can lead to irreversible CNS and kidney damage. No acute lead hepatotoxicity has been reported in the literature, but high blood levels (usually above 75–80 mg/dl) were associated with liver injury in a series of patients from Spain.63 Liver biopsies showed mild centrilobular hepatitis with fatty infiltration and hemosiderin deposition. In that series, markers for lead (blood and urinary lead levels, ALAD inhibition) were more correlated with the degree of liver injury than were liver chemistry tests. Withdrawal of lead exposure or, in severe cases, chelation therapy, led to rapid resolution of the liver dysfunction.
Cadmium The major source of cadmium toxicity is through occupational exposure among individuals engaged in cadmium production and in the industrial use of cadmium in the production of alloys, pigments, and batteries. Over the last century there has been a dramatic increase in environmental contamination with cadmium owing to its presence in household waste and industrial emissions. Soil is commonly contaminated via atmospheric emission and sewage, as well as through the use of cadmium-containing phosphate fertilizers. Cadmium is readily absorbed by plants grown in the contaminated soil, particularly tobacco, grain, rice, and vegetables. Over 50% of the total body cadmium is stored in the liver and kidneys, tightly bound to metallothionein in an intracellular complex.64
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In 1981 a massive intoxication of tens of thousands of people occurred in Spain following the consumption of adulterated rapeseed oil.66 The syndrome was referred to as the toxic-epidemic syndrome, and more than 350 people died. Initially there was acute multisystem involvement, with fever, arthraglia, rash, myalgia, conjunctivitis, digestive symptoms, and abdominal or chest pain, or both. Eosinophilia, raised levels of transaminases, alkaline phosphatase, g-glutamyl transpepidase, and IgE were commonly found. Three to 18 months after the attack episode a third of the patients who survived the initial toxicity developed scleroderma, sicca syndrome, alopecia, pulmonary hypertension, and respiratory failure. Hepatic involvement occurred in 25% of the victims, especially women in the fourth decade of life. Most of the patients with hepatic injury were asymptomatic.66 Elevation of g-glutamyl transpepidase was universal, and elevated alkaline phosphatase and transaminase levels were common. Histologic features included hepatocyte degeneration, cholestasis, a mixed cellular infiltration with a high cosmophil component, and acidophilic bodies – all similar to a drug-induced cholestatic hepatitis. Long-term histologic reassessment 30 months after the initial poisoning showed that the eventual prognosis could be forecast according to the initial biochemical abnormalities. Those patients with an initial transient elevation of transaminase levels recovered within 2 months. Those with initially raised transaminase and alkaline phosphatase levels mostly recovered or had evidence of a mild ongoing hepatitis. Of the patients who initially had jaundice, half continued to have an abnormal liver biochemistry profile at 30 months, and their histologic picture was suggestive of chronic cholestasis. Many of these patients also had evidence of scleroderma, and there was an increased incidence of HLA-DR3 and HLA-DR4 among them.66
Chapter 28 OCCUPATIONAL AND ENVIRONMENTAL HEPATOTOXICITY
ENVIRONMENTAL HEPATOTOXINS MUSHROOM POISONING Of the 5000 known species of mushroom, fewer than 100 are poisonous to humans and fewer than 10 of these are deadly. The gathering and consumption of wild mushrooms, a traditional social practice in western Europe, has become increasingly popular in the United States. Although the number of fatal cases per year reported in this country does not approach the 50–100 deaths occurring annually in western Europe, fatal mushroom poisoning remains a serious public health concern.67 The incidence of mushroom poisoning has been increasing in the United States since the 1970s because of the increasing popularity of natural foods and the popularization of the gourmet qualities of wild mushrooms. Poisonings often occur in amateur mushroom hunters who fail to distinguish between edible and non-edible varieties. In addition, immigrants may come from areas where only edible lookalikes exist. Unsupervised children and persons looking for hallucinogenic substances may be poisoned as well. Many mushroom poisonings involve young children left unattended outdoors who are later found with mushrooms in their mouth. These exposures are rarely serious, because most lawn mushrooms are innocuous, medical evaluation is quickly sought, and fresh specimens of the mushroom are usually available for analysis. Adult exposures tend to be more serious because the mushrooms usually have been eaten in large quantities and were collected in the forest, where poisonous species are more likely to exist. Adults often present much later after ingestion and may eat more than one species of mushroom, which may lead to a confusing clinical presentation.67
Amanita Poisoning Most severe mushroom poisonings are caused by the Amanita species, which contain amatoxin, one of the most potent toxins known. In the United States, most poisonings occur along the cool coastal woodlands of the Pacific Northwest, where Amanita species are most common. Hospitalizations for mushroom poisoning in California have occurred more frequently in the warmer months and reach their peak in August. In general, however, the peak season for wild mushrooms typically coincides with the rainy season, which in California usually is from late October through March. This discordance may in large part be caused by the mistaken ingestion of poisonous Agaricus campestris species, which results in GI symptoms and dehydration. Amanita species also have been identified in the Blue Ridge Mountains of the northeast, as well as in Pennsylvania, New Jersey, suburban New York State, and the Gulf Coast.67 Of the three common Amanita species, A. phalloides, A. verna, and A. virosa, A. phalloides (death cap) has been responsible for more than 90% of fatalities. The mature A. phalloides mushroom can be easily mistaken for similar-appearing non-toxic mushrooms, even by experts. Amanita species tend to grow in oak woodlands or in close proximity to oak trees, and are commonly mistaken for edible species; they have no characteristic smell or taste, and cooking does not destroy their toxin. Color varies with weather, soil, and the age of the mushroom. Amanita can be identified by the presence of white gills (Figure 28-3) underneath the cap, an annulus at
Cap
Annulus Stem
Volva
A
1 cm
B
Figure 28-3. (A) Amanita phalloides. (B) Lepiota helveola. (Adapted from reference 67.)
the top of the stalk, and a pouch at the base of the mushroom. Mushrooms that are cut off at ground level are often misidentified, as this characteristic pouch goes undetected. As Amanita is a mycorrhizal fungus living symbiotically with the roots of the host tree, it cannot be destroyed unless the tree itself is killed.67 A. phalloides exerts its hepatotoxicity through two distinct toxins, phalloidin and amatoxin. Phalloidin is a cyclic heptapeptide that causes irreversible polymerization of G-actin to F-actin, resulting in the ultimate disruption of hepatocyte cell membranes and cell death. Amatoxins are thermostable octapeptides that bind with a subunit of RNA polymerase II and interfere with mRNA synthesis. They are not destroyed by cooking or gastric acidity. The inability to produce vital structural proteins results in cell necrosis, with tissues having high rates of protein synthesis being primary targets for the toxin. The liver and kidney are most commonly involved, although the brain and pancreas also are affected. Amatoxin concentration in mushrooms varies by species, season, region, and local conditions. Although the amount of amatoxin per mushroom is difficult to quantify, a bite-sized piece of one mushroom can contain a lethal dose of toxin. Varying amounts of amatoxin are present in other potentially lethal mushrooms, such as some Galerina and Lepiota species. Fifteen to 20 of these mushrooms can constitute a potentially fatal dose.67 Amatoxins are readily absorbed from the intestinal epithelium and weakly bound by plasma proteins. They penetrate cells rapidly and, in the case of hepatocytes, are transported across the cell membrane via bile transport carriers. In the liver, massive hepatocellular damage results in centrilobular necrosis (Figure 28-4). Sixty per cent of absorbed amatoxin is excreted via the bile and returns to the liver via the enterohepatic circulation, resulting in continued toxin exposure. GI epithelium and the proximal and distal convoluted tubules of the kidney also are severely affected.67
Clinical Presentation Poisoning with non-lethal poisonous mushrooms produces crampy abdominal pain, nausea, emesis, and watery diarrhea soon after ingestion. With Amanita poisoning patients exhibit signs and symptoms that typically occur in stages. The lethal dose is about 50 g,
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tocyte membranes, or via interruption of the enterohepatic recirculation of amatoxin.68 Mortality rates have decreased with improved supportive care, but death still occurs in 20–30% of cases. Once stage 3 hepatic encephalopathy is reached, the patient is unlikely to survive without liver transplantation.
Lepiota Helveola
Figure 28-4. Massive hepatic necrosis secondary to mushroom poisoning. Section taken from an explanted liver, showing confluent lobular necrosis with collapse and extinction of most of the hepatic parenchyma (central area of photomicrograph) and broad areas of bridging necrosis. A few lobules of viable hepatocytes remain (left upper and right lower corners). Hematoxylin and eosin, original magnification 20¥. (Courtesy of Dr M.I. Fiel.)
which corresponds to three medium-sized mushrooms. There is an initial latent asymptomatic period of 6–24 hours; 12–24 hours of severe GI symptoms then ensue, with the patient often being misdiagnosed with viral gastroenteritis. A second latent phase follows, with improvement of GI symptoms but the development of abnormal liver chemistry tests. A hepatic phase occurs 48–96 hours after ingestion, with precipitous elevation of serum aminotransferases into the thousands, coagulopathy, and jaundice. Renal failure secondary to fulminant hepatic failure or to the direct nephrotoxicity of amatoxin also develops. Half of patients with Amanita poisoning have clinical or biochemical evidence of pancreatitis.68
Treatment Early consultation with a poison control center and liver transplant center is essential. The amount of mushroom ingested appears to be the main prognostic factor. Supportive care remains the mainstay of treatment. Induction of emesis can reduce the toxin load, but most patients present 6–8 hours post ingestion, making this of limited use. The administration of activated charcoal with a cathartic and gastric lavage is performed to remove any remaining toxin from the GI tract. As diarrhea can be severe, adequate intravenous fluid and electrolyte maintenance are essential. Although amatoxin and phalloidin are dialyzable, charcoal hemoperfusion and hemodialysis have not been effective in limiting hepatic injury. Although the precise mechanism of action is unknown, high-dose penicillin G (300 000–1 000 000 IU/kg/day) has been used in some patients, with resultant improvement of hepatic dysfunction. The hepatoprotective effects of penicillin G may be secondary to increased renal excretion of amatoxin, or via the inhibition of penetration of the toxin into hepatocytes. Silymarin, an extract of the milk thistle Silybum marianum, has been used widely in Europe in doses of 20–50 mg/kg/day, usually in combination with penicillin G. Its hepatoprotective effects may be related to inhibition of toxin binding to liver cells, prevention of the toxin’s penetration of hepa-
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The genus Lepiota can be easily mistaken for an edible variety of mushroom. It has a flat pink or brown ochre cap, is covered with fluffy scales, and bears white or cream-colored gills (Figure 28-3). Lepiota helveola contains high concentrations of amatoxin but no phalloidin. It has the same – if not a greater – hepatotoxic potential as Amanita species, and multiple poisonings resulting in FHF and the need for liver transplantation have been reported.67 There are many other types of mushroom that contain toxins with potential GI and neurological toxicities. Symptoms often begin within minutes to several hours of ingestion. Numerous fatalities have resulted from toxin ingestion, though none have been related to hepatic failure. Most toxic ingestions are self-limited, without related hepatotoxicity. As several types of mushroom may grow near each other or be consumed together, clinicians should always be aware of the possibility of the ingestion of both edible and potentially hepatotoxic and lethal varieties, especially if initial symptoms appear to begin after a 6-hour latent period. The presence of symptoms immediately after ingestion should not rule out the possibility of a more serious poisoning.
PYRROLIZIDINE ALKALOIDS Known as hepatotoxins since the 18th century, pyrrolizidine alkaloids (PAs) cause acute and chronic hepatic injury in experimental animals and in humans. Over 300 PAs have been identified in over 6000 plants, including approximately 3% of the world’s flowering plants. Some more common plants containing PA are shown in Table 28-6. The chief genera that produce the toxic alkaloids affecting livestock and humans are the Senecio, Heliotropium, Crotalaria, and Symphytum (comfrey) species. Large outbreaks of PA poisoning have occurred through contamination of wheat crops; significant amounts of PAs in concentrations capable of causing injury in experimental animals have also been found in milk and honey. A number of PAs are used as supplements or in traditional herbal medicines, with comfrey and coltsfoot as popular examples. Systemic bioavailability of PAs after dermal exposure is generally low, but toxicity can occur after use of PA-containing creams and shampoos as well. Liver injury seems to depend on the type of PA and the total dose ingested, along with the susceptibility of the person to the alkaloid.3,67
Mechanism of Injury Once the alkaloids are ingested they pass into the hepatocytes via the sinusoidal blood and are metabolized through the CYP3A to dehydro-PAs, N-oxides and pyrroles. Hydrolysis and N-oxide formation are detoxification reactions and usually do not cause harm to the cell; however, dehydro-PAs are the primary toxic metabolites that react with available nucleophiles within the hepatocyte. Pyrroles are alkylating agents that are highly reactive with proteins and nucleic acids, binding sulfur, nitrogen, and oxygen-containing groups on various macromolecules. They penetrate the nucleus and
Chapter 28 OCCUPATIONAL AND ENVIRONMENTAL HEPATOTOXICITY
Table 28-6. Plants Containing Pyrrolizidine Alkaloids Plant
Common names
Alkaloids contained
Geographic distribution
Armsinckia intermedia
Fiddleneck tarweed, firewood, yellow forget-me-nots Rattlebox, rattle pod Wild Lucerne Whiteback Streaked rattle pod Wedge-leaved rattle pod, earring plant Viper’s bugloss, Paterson’s curse, salvation Jane Common heliotrope, caterpillar weed, potato weed Heliotrope
Echiumine, lycopsamine, intermedin
USA
Dicrotaline Dicrotaline Intergerrimine Monocrotaline, retronecine, retusine Echiumine, echimidine
Many countries South Africa South Africa Jamaica Australia, South Africa USA, Australia
Crotolaria Crotolaria dura Crotolaria fulva Crotolaria mucronata Crotolaria reftsa Echium lycopsis Heliotropium europalum Heliotropium lasiocarpum Senecio jacoboae Senecio latifoolius Senecio reetrorus Senecio ridelli Senecio spartioides Senecio vulgaris Symphytum officinale
Common ragweed, stinking Willie Dan’s cabbage, groundsel, ragwort, Rhodesia ragwort Dan’s cabbage, Wooly groundsel Wooly groundsel, Riddels groundsel Brown groundsel Common groundsel
Heliotrine, lasiocarpine, europine, supinine Echimidine, echiumidine, lasiocarpine, heliotune Jacobine, jacodine, jacoline, jacovine Semicriphylline Retrorsine Retrorsine Seneccionine, senociphylline, spartioidine Retrorsine, senecione
Australia, USA Russia, Central Asia US, Jamaica, New Zealand South Africa South Africa USA USA UK, USA Japan
From Zimmerman HJ. Occupational hepatotoxicity. In: Zimmerman HJ. Hepatotoxicity: the adverse effects of drugs and other chemicals on the liver. Philadelphia: Lippincott Williams & Wilkins, 1999
react with DNA, causing cross-linking within DNA and between DNA and nucleoproteins. One of the mutations caused is in codon 249 of the p53 gene, the same target as for aflatoxin; however, this may only be specific for the PAs found in comfrey.68 The predominant feature of PA toxicity, veno-occlusive disease (VOD), is caused by pyrroles passing from the hepatocyte to the perisinusoidal space of Disse into the sinusoidal lumen, where they are able to react with the sinusoidal lining (endothelial cells) and the walls of small hepatic veins (Figure 28-5). They can also become bound on passing red blood cells and thus get carried to the lungs or heart, where they can induce pulmonary hypertension and right ventricular hypertrophy.69 Differences in susceptibility are at least partly explained by isoforms of the CYP3A subfamily. CYP3A catalyzes pyrrole formation as well as N-oxide formation, the latter being primarily responsible for the detoxification process. The differences in enzyme activity can vary by as much as 30-fold, leading to a large difference in the dose required to reach toxicity in different individuals. Older age has also been shown to lead to an increase in susceptibility to hepatotoxicity.3,68
Acute Effects Acute PA toxicity typically occurs through human consumption of contaminated grains with seed containing PA, or through herbal remedies. The syndrome of veno-occlusive disease, characterized by abdominal distention, hepatosplenomegaly, ascites, and peripheral edema, was first described in Jamaican children, and was related to their intake of bush teas made with Senecio and Crotalaria species as therapy for acute illnesses. In other parts of the world, serious outbreaks of PA poisoning have occurred in Afghanistan and central India, both after periods of drought when Heliotropium plants thrived in the region, leading to wheat crop contamination. The
Figure 28-5. Veno-occlusive disease. Liver needle biopsy showing fibroobliterative changes in a terminal hepatic venule consisting of fine fibrous tissue deposition along the wall (arrow) and within adjacent perisinusoidal spaces. The lumen is slightly diminished in caliber. Hematoxylin and eosin, original magnification 20¥. (Courtesy of Dr M.I. Fiel.)
most recent epidemic case occurred in 1992 in Tajikistan, where close to 4000 people developed PA hepatotoxicity. PA toxicity was also endemic in parts of South America in the second half of the 20th century, but with better education and proper identification of plants only sporadic cases are now reported.3,69 In acute PA hepatotoxicity, abdominal pain, jaundice and ascites typically develop within 3–6 weeks of ingestion, with centrilobular hepatic necrosis and sinusoidal dilatation seen on liver biopsy.
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Approximately half of the patients recover completely in a few weeks, but the course is rapidly fatal in approximately 20%. In the remainder of cases, 20% appear to recover clinically but then develop VOD and cirrhosis after several years. Others develop a subacute form of VOD which may eventually resolve or subsequently progress to chronic VOD and cirrhosis.3,69
P450 effect.70 Thus, identifying the concomitant use of ethanol or other medications may be important in predicting the chance of recovery from severe cocaine hepatotoxicity.71 Cocaine intoxication should be considered along with ischemic hepatitis, acetaminophen overdose, and Amanita mushroom poisoning in the differential diagnosis of any patient with acute hepatitis and extremely elevated serum aminotransferases that rise into the several thousand range.
Chronic Effects Chronic hepatotoxicity caused by PA exposure is associated with hepatocellular injury leading to cirrhosis. Owing to the production of persistent electrophilic metabolites that can be released from hepatocytes on an ongoing basis, there is ongoing hepatic injury in addition to VOD. The clinical picture is that of poor hepatic flow akin to the Budd–Chiari syndrome, with hepatosplenomegaly, ascites, and edema. Histologically, there is central fibrosis and bridging between central veins that leads to cirrhosis, similar to that seen in cardiac cirrhosis. Death is usually related either to complications of portal hypertension or to pulmonary hypertension leading to congestive heart failure.3,69
Cocaine Cocaine (benzoylmethylecgonine) is an alkaloid extracted from the leaf of Erythroxylon coa. For many years cocaine was presumed to be directly hepatotoxic to humans because of the frequent findings of jaundice and abnormal liver chemistry tests among cocaine abusers; however, in these persons there often is associated polysubstance abuse, alcohol intoxication, or viral hepatitis. In mice, cocaine is a potent hepatotoxin, causing steatosis and hepatocellular necrosis with high serum transaminase levels. Ten per cent of cocaine is converted in the liver by flavin adenine dinucleotide and cytochrome P450 to the active metabolite, the hepatotoxic free radical norcocaine nitroxide. There is further oxidation of norcocaine nitroxide to the nitrosonium ion, which is highly reactive with glutathione and results in its depletion.70 This ultimately leads to further free radical formation, covalent binding to hepatic proteins, and lipid peroxidation of hepatocyte cell membranes. Although hyperthermia and shock have been associated with cocaine intoxication, and the profound serum aminotransferase elevation found in some cocaine intoxications is similarly seen in patients with ischemic hepatitis, ischemia is not thought to play a major role in cocaine hepatotoxicity.67 Acute hepatotoxic injury from cocaine use is now well described in humans. Severe necroinflammatory hepatitis and liver failure, clinically similar to that seen with acetaminophen ingestion, can occur, with associated renal failure, disseminated intravascular coagulation, and rhabdomyolysis. Histologic changes include pericentral coagulative necrosis and micro- and macrovascular steatosis, with only mild inflammatory infiltration. The most severe histologic changes occur in zone 3 of the liver parenchyma, indistinguishable from changes seen in acetaminophen toxicity. Cocaine and acetaminophen are both metabolized by cytochrome P450. It appears that, as with acetaminophen toxicity, there can be enhancement of necrosis in cocaine hepatotoxicity via P450 induction or glutathione depletion. In mice, cimetidine effectively prevents cocaine-induced hepatic necrosis, whereas ethanol and barbiturates potentiate it, all presumably related to the cytochrome
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AFLATOXINS Aflatoxins were first isolated in the 1960s after an epidemic of acute hepatic necrosis and death in turkeys that were fed a peanut meal contaminated with Aspergillus flavus (turkey X disease).72 Since that time, aflatoxins have been subject to a great number of studies, confirming their hepatotoxic and hepatocarcinogenic potential in poultry and domestic animals, as well as in humans.73,74 Aflatoxininduced acute hepatotoxicity in humans has followed the ingestion of contaminated maize, soybeans, and cassava.
Epidemiology Aflatoxins are produced as secondary metabolites of Aspergillus flavus and Aspergillus parasiticus fungi in warm, humid temperatures. They are ubiquitous and contaminate a variety of food staples in tropical and subtropical areas, including rice, oats, wheat, corn, ground nuts, and spices. There is a wide (more than 100-fold) intraspecies variation in animal susceptibility to disease, and the predominant patterns of injury vary from species to species as well.74 Within species, older age and male gender appear to be protective factors. Choline-deficient diets and diets low in protein appear protective against acute toxicity, yet enhance the carcinogenic effects of aflatoxins.75,76
Acute Effects Upon ingestion, aflatoxins are rapidly metabolized to the reactive species by the microsomal enzymes of the liver. In binding to guanine residues on DNA these metabolites inhibit the synthesis of nuclear RNA. Acute hepatic injury with steatosis and/or hepatocellular necrosis is thought to result from injury to membranes (plasma, reticular, and mitochondrial) via either injury to ribonuclease or by direct toxic reactions of the metabolites with the membrane proteins. The clinical symptoms of acute aflatoxicosis are ascribed to hemorrhagic necrosis of the liver and bile duct proliferation, leading to hepatic failure.76,77
Chronic Effects The synergistic effects of aflatoxin and hepatitis B and C viruses in the development of hepatocellular carcinoma have been well described. Recent epidemiologic studies from Singapore and Shanghai,78,79 areas that have both seen tremendous economic improvement over the last two decades, have shown that the incidence of HCC has rapidly fallen over the same period. Clearly this is at least partly due to a decrease in aflatoxin contamination of the food supply; aflatoxins have significant late effects in the development of hepatocellular carcinoma. Geographic areas where aflatoxin contamination is prevalent show a strong correlation between the degree of contamination of common foodstuffs and the incidence of hepatocellular carcinoma.
Chapter 28 OCCUPATIONAL AND ENVIRONMENTAL HEPATOTOXICITY
High concentrations of aflatoxin B1 have been found in the livers of many patients developing hepatocellular carcinoma in these areas. It appears that aflatoxin contamination of food and coexisting chronic hepatitis B infection are associated with even higher rates of hepatocellular carcinoma in a given population (e.g. in Mozambique, which has both the highest average aflatoxin exposure and the highest incidence of hepatocellular carcinoma in the world). Thus, the effects of these two carcinogenic agents appear synergistic. Chronic feeding of aflatoxin to experimental animals leads reproducibly to the development of hepatocellular carcinoma. Evidence of a causative role for aflatoxin in the pathogenesis of human hepatocellular carcinoma has been further suggested by the description of a high frequency of a unique mutation in codon 249 of the p53 gene.80,81 The p53 gene influences the transcription of important cellular genes involved in regulation of the cell cycle. Mutations and allelic deletions of the p53 gene are the most common genetic alterations found in human tumors, with p53 abnormalities described in several hepatocellular carcinoma cell lines. Mutations at codon 249 of the p53 gene have been found in patients with hepatocellular carcinoma in China and southern Africa, areas of high aflatoxin contamination. The absence of similar mutations has been shown in geographic regions where hepatocellular carcinoma occurs frequently but where aflatoxin contamination is low. These mutations are consistent with those caused by aflatoxin B1 in experimental models of mutagenesis. The clustering of the codon 249 mutation in hepatocellular carcinomas occurring in areas with high aflatoxin contamination may thus be a clue to the carcinogenicity of an environmental toxin.80,81
CYANOBACTERIAL TOXINS Cyanobacteria (also known as blue-green algae) are an ancient group of photosynthetic organisms that grow in water, with habitats that range from hot springs to temporarily frozen ponds in Antarctica and include both fresh and seawater.82 Of the currently known 150 genera with about 2000 species, at least 40 are known to be toxicogenic, affecting primarily either the central nervous system or the liver. The hepatotoxins cylindrospermopsin, microcystins, and nodularin are all small cyclic peptides that are synthesized nonribosomally. The species that are capable of synthesizing these toxins must thus possess the peptide synthesis gene sequence that will be expressed under certain environmental conditions. The presence of cyanobacterial toxins in drinking water supplies poses a serious problem to water treatment facilities, as specific technical procedures that are not widely available are required to remove these toxins effectively.
Acute Effects The majority of reported outbreaks of acute cyanobacterial toxicity are cases of gastroenteritis with mild elevations of g-glutamyltransferase (GGTP). However, in 1996, improperly purified water with high levels of microcystins from the Tabocas reservoir was used at a dialysis center in Caruaru, Brazil, resulting in 101 of 124 exposed subjects developing acute liver injury, with 50 subsequent deaths.83 Clinically, patients developed primarily cholestatic jaundice with high bilirubin and alkaline phosphatase concentrations, as well as increases in hepatic enzymes (aspartate and alanine aminotrans-
ferase). On histopathology, panlobular hepatocyte necrosis with apoptosis was the predominant feature.
Chronic Effects Microcystin and nodularin have been shown to induce the expression of tumor necrosis factor-a and early response genes (c-jun, jun B, jun D, c-fos, fos B, fra-1) in rat liver and hepatocytes.84 In mice, mutations in the K-ras codon 12 as well as DNA fragmentations have been reported after an injection of cyanobacterial extract.85 In China the consumption of microcystin-contaminated drinking water from pond and ditch waters has been found to be associated with a 25-fold increase in incidence of HCC.85,86
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39. Shintani H, Nakamura A. Analysis of a carcinogen, 4,4 methylenedianiline, from thermosetting polyurethane during sterilization. J Anal Toxicol 1989;13:354–357. 40. Kanz MF, Gunasena GH, Kaphalia L, Hammond DK, Syed YA. A minimally toxic dose of methylene dianiline injures biliary epithelial cells in rats. Toxicol Appl Pharmacol 1998;150:414–426. 41. Cocco P, Kazerouni N, Zahm SH. Cancer mortality and environmental exposure to DDE in the United States. Environ Health Perspect 2000;1:1–4. 42. Battaller R, Bragulat E, Nogue S, et al. Prolonged cholestasis after acute paraquat poisoning through skin absorption. Am J Gastroenterol 2000;95:1340–1343. 43. Guzelian P. Comparative toxicology of chlordecone (kepone) in humans and experimental animals. Annu Rev Pharmacol 1982;22:89–113. 44. Tchounwou PB, Centeno JA, Patlolla AK. Arsenic toxicity, mutagenesis, and carcinogenesis – a health risk assessment and management approach. Mol Cell Biochem 2004;255:47–55. 45. Kitchin K. Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol Appl Pharmacol 2001;172:249–261. 46. Abernathy CO, Thomas DJ, Calderon RL. Health effects and risk assessment of arsenic. J Nutr 2003;133:1536S–1538S. 47. Tsuda T, Babazono A, Yamamoto E, et al. Ingested arsenic and internal cancer: a historical cohort study followed for 33 years. Am J Epidemiol 1995;141:198–209. 48. Yoshida T, Yamauchi H, Fan Sun G. Chronic health effects in people exposed to arsenic via the drinking water: dose-response relationships in review. Toxicol Appl Pharmacol 2004;198:243–252. 49. Chiu HF, Ho SC, Wang LY, et al. Does arsenic exposure increase the risk for liver cancer? J Toxicol Environ Health A 2004;67:1491–500. 50. Centeno JA, Mullick FG, Martinez L. Pathology related to chronic arsenic exposure. Environ Health Perspect 2002;110:883–886. 51. Ho SY, Tsai CC, Tsai YC, et al. Hepatic angiosarcoma presenting as hepatic rupture in a patient with long-term ingestion of arsenic. J Formos Med Assoc 2004;103:374–379. 52. Guo HR. The lack of a specific association between arsenic in drinking water and hepatocellular carcinoma. J Hepatol 2003;39:382–288. 53. Tenenbeim M. Hepatotoxicity in acute iron poisoning. J Toxicol Clin Toxicol 2001;39:721–726. 54. Henretig FM, Temple AR. Acute iron poisoning in children. Clin Lab Med 1984;3:575. 55. Kozaki K, Egawa H, Garcia-Kennedy R, et al. Hepatic failure due to massive iron ingestion successfully treated with liver transplantation. Clin Transplant 1995;9:85. 56. Pankit AN, Bhave SA. Copper metabolic defects and liver disease: Environmental aspects. J Gastroenterol Hepatol 2002;17:5403–5407. 57. Muller T, Langner C, Fuchsbichler A, et al. Immunohistochemical analysis of Mallory bodies in Wilsonian and non-Wilsonian hepatic copper toxicosis. Hepatology 2004;39:963–969. 58. Tanner MS. Role of copper in Indian childhood cirrhosis. Am J Clin Nutr 1998;67:1074S–1081S. 59. Muller T, Feichtinger H, Berger H, et al. Endemic Tyrolean infantile cirrhosis: an ecogenetic disorder. Lancet 1996;347:877–880. 60. Zietz BP, Dieter HH, Lakomek M, et al. Epidemiological investigation on chronic copper toxicity to children exposed via the public drinking water supply. Sci Total Environ 2003;302:127–144. 61. Lopez CM, Vallejo NE, Pineiro AE, et al. Alteration of biochemical parameters related with exposure to lead in heavy alcohol drinkers. Pharmacol Res 2002;45:47–50.
Chapter 28 OCCUPATIONAL AND ENVIRONMENTAL HEPATOTOXICITY
62. Chia SE, Yap E, Chia KS. Delta-aminolevulinic acid dehydratase (ALAD) polymorphism and susceptibility of workers exposed to inorganic lead and its effects on neurobehavioral functions. Neurotoxicology 2004;25:1041–1047. 63. Sanchez JA, de la Fuente JM, Castrillo JM, et al. Hepatotoxidad por plomo inorganico: resultados de 85 casos de saturnismo agudo. Gastroenterol y Hepatol 1985;8:246–250. 64. Ikeda M, Zhang ZW, Moon CS. Normal liver function in women in the general Japanese population subjected to environmental exposure to cadmium at various levels. Int Arch Occup Environ Health 2000;73:86–90. 65. Rikans LE, Yamano T. Mechanisms of cadmium-mediated acute hepatotoxicity. J Biochem Mol Toxicol 2000;14:110–117. 66. Velicia R, Sanz C, Martinez-Barredo F, et al. Hepatic disease in the Spanish toxic oil syndrome. J Hepatol 1986:3:59–65. 67. Schiano TD. Liver injury from herbs and other botanicals. Clin Liver Dis 1998;2:607–630. 68. Fu PP, Xia Q, Lin G, et al. Pyrrolizidine alkaloids: genotoxicity, metabolism enzymes, metabolic activation, and mechanisms. Drug Metab Rev 2004;36:1–55. 69. Chojkier M. Hepatic sinusoidal-obstruction syndrome: toxicity of pyrrolizidine alkaloids. J Hepatol 2003;39:437–446. 70. Selim K, Kaplowitz N. Hepatotoxicity of psychotropic drugs. Hepatology 1999;29:1347–1351. 71. Ponsoda X, Bort R, Jover R, Gomez-Lechon MJ, Castell JV. Increased toxicity of cocaine on human hepatocytes induced by ethanol: role of GSH. Biochem Pharmacol 1999;58: 1579–1585. 72. Swarbock O. Disease of turkey poults. Vet Rec 1960, 72:652. 73. Ross RK, Yuan JM, Yu MC, et al. Urinary aflatoxin biomarkers and risk of hepatocellular carcinoma. Lancet 1992;339:943– 946. 74. McGlynn KA, Hunter K, LeVoyer T, et al. Susceptibility to aflatoxin B1-related primary hepatocellular carcinoma in mice and humans. Cancer Res 2003;63:4594–4601. 75. Cullen JM, Newberne PM. Acute hepatotoxicity of aflatoxins. In: Eaton DL, Groopman JD, eds. The toxicology of aflatoxins: human health, veterinary, and agricultural significance. London: Academic Press, 1993: 1–26.
76. Williams JH, Phillips TD, Jolly PE, et al. Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. Am J Clin Nutr 2004;80:1106–1122. 77. Creppy EE. Update on survey, regulation and toxic effects of mycotoxins in Europe. Toxicol Lett 2002;127:19–28. 78. Ming L, Thorgerlsson SS, Gall MH, et al. Dominant role of hepatitis B virus and cofactor role of aflatoxin in hepatocarcinogenesis in Qidong, China. Hepatology 2002;36:1214–1220. 79. Wang LY, Hatch M, Chen CJ, et al. Aflatoxin exposure and risk of hepatocellular carcinoma in Taiwan. Int J Cancer 1996;67:620–625. 80. Yu MC, Yuan JM. Environmental factors and risk for hepatocellular carcinoma. Gastroenterology 2004;127:S72–S78. 81. Kensler TW, Egner PA, Wang JB, et al. Chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas. Gastroenterology 2004;127:S310–S318. 82. Hitzfeld BC, Hoger SJ, Dietrich DR. Cyanobacterial toxins: removal during drinking water treatment, and human risk assessment. Environ Health Perspect 2000;108:113–122. 83. Jochimsen EM, Carmichael WW, An JS, et al. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. N Engl J Med 1998;338:873–878. 84. Sueoka E, Sueoka N, Okabe S, et al. Expression of the tumor necrosis factor alpha gene and early response genes by nodularin, a liver tumor promoter, in primary cultured rat hepatocytes. J Cancer Res Clin Oncol 1997;123:413–419. 85. Rao P, Bhattacharya R, Parida MM, et al. Freshwater cyanobacterium Microcystis aeruginosa (UTEX 2385) induced DNA damage in vivo and in vitro. Environ Toxicol Pharmacol 1998;5:1–6. 86. Ueno Y, Nagata S, Tsutsumi T, et al. Detection of microcystins, a blue-green algal hepatotoxin, in drinking water sampled in Haimen and Fusui, endemic areas of primary liver cancer in China, by highly sensitive immunoassay. Carcinogenesis 1996;17:1317–1321.
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ALCOHOLIC LIVER DISEASE Stephen F. Stewart and Christopher P. Day Abbreviations ADH alcohol dehydrogenase AMPK adenosine monophosphate-activated protein kinase ALD alcoholic liver disease ALDHs aldehyde dehydrogenases ALT alanine transaminase AP-1 activator protein-1 apoB apolipoprotein B AST aspartate transaminase ATP adenosine triphosphate Bax Bcl-2-associated x protein Bid BH3-interacting domain death agonist COX-2 cyclooxygenase-2 CT computed tomography CTLA-4 cytotoxic T-lymphocyte antigen-4 CYP2E1 cytochrome P450 2E1 ER endoplasmic reticulum DF discriminant function DISC death-inducing signaling complex ECM extracellular matrix ELISA enzyme-linked immunosorbent assay FFA free fatty acids G3P glycerol-3-phosphate
GSH HCC HERs HRS HSC IgA IL-6 iNOS LBP LPS LSP MAA MARS MAT MDA MEOS MMPs mRNA MS MTP NaCNBH3 NO NK
mitochondrial glutathione hepatocellular cancer hydroxyethyl radicals hepatorenal syndrome hepatic stellate cells immunoglobulin A interleukin-6 inducible NO synthase LPS-binding protein lipopolysaccharide liver-specific membrane lipoprotein MDA-acetaldehyde molecular adsorbents recycling system methionine adenosyltransferase malondialdehyde microsomal ethanol-oxidizing system matrix metalloproteinases messenger RNA methionine synthase microsomal triglyceride transfer protein cyanoborohydride nitric oxide natural killer
INTRODUCTION Alcohol is consumed by a large percentage of the world’s population and is an effective anxiolytic and social lubricant. A small proportion of consumers become dependent, and a moderate proportion of these, and many who are not dependent, develop clinically significant liver disease. These problems are not new. Alcohol was recognized to be a cause of liver damage by the ancient Greeks, and is currently the commonest cause of liver disease in the western world. The magnitude and range of the health and socioeconomic problems attributable to alcohol abuse are enormous. Cirrhosis, predominantly alcoholic, is now the fourth commonest cause of death between the ages of 25 and 64 in the USA and alcohol may also make a significant contribution to cardiovascular-related mortality. The overall socioeconomic cost of alcohol abuse in the USA, in terms of health care, crime, and loss of work capacity, has been estimated at over $160 000 million per year. It is, therefore, a significant drain on limited health care resources. In common with all alcohol-related disease, abstinence is the cornerstone of management in patients with alcoholic liver disease (ALD). The development of specific therapies has, however, been hampered by a continued lack of a clear understanding of the mechanisms through which ethanol causes liver injury. Intense research efforts have now
NKT PAP PT PPAR-a
natural killer T phosphatidate phosphohydrolase prothrombin time peroxisome proliferator-activated receptor-a PUFA polyunsaturated fatty acid PTX pentoxifylline RA retinoic acid ROS reactive oxygen species SAH S-adenosylhomocysteine SAME S-adenosylmethionine SOD-2 superoxide dismutase SREBP-1c sterol response element-binding protein-1c TAG triacylglycerol TGF-b transforming growth factor-b TLR4 toll-like receptor 4 TNF-a tumor necrosis factor-a TNFR1 TNF-a receptor 1 TRAIL tumor necrosis factor-related apoptosisinducing ligand UPR unfolded protein response VLDL very-low-density-lipoproteins
highlighted several important metabolic and immunological consequences of excessive alcohol consumption that may contribute to disease pathogenesis and it is hoped that further defining these disease mechanisms may lead to the development of novel treatment strategies. It has also become increasingly clear that individuals are not “all equal” in their susceptibility to ALD. Although cumulative alcohol dose undoubtedly plays a role in determining disease risk, only a small proportion of heavy drinkers go on to develop the more advanced forms of ALD – hepatitis, fibrosis, and cirrhosis. Elucidating the genetic and environmental factors associated with disease progression would be a major step towards disease prevention. This chapter will focus on the pathogenetic mechanisms of ALD and the current treatments available.
EPIDEMIOLOGY Several independent studies have demonstrated a close correlation between deaths from cirrhosis and per capita alcohol consumption. Perhaps the best example of this is the effect of wine rationing in France during the Second World War; this was associated with an 80% reduction in cirrhosis deaths, followed by a return to pre-war levels when restrictions were removed.1 A similar effect was observed during Prohibition in the USA. Figure 29-1 shows the
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30
Second Third Fourth Fifth
Sixth Seventh Eighth
Ninth
Tenth
driven by the question of how alcohol leads to liver injury. With few exceptions they fail to address the fact that most individuals appear to be remarkably resistant to the deleterious effects of ethanol on the liver.
25
Rate per 100 000 population
PATHOGENESIS 20
15
10
5
Males Both sexes Females
0 1910
1920 1930
1940 1950
1960 1970 1980
1990
2000
Year Figure 29-1. Age-adjusted death rates from liver cirrhosis by sex. States with death registration 1910–1932, all United States 1933–2000. Reproduced with permission from National Institute on Alcohol Abuse and Alcoholism (www.niaaa.gov).
decline in cases after the act was passed in 1916 and a gradual increase following the repeal of the act in 1932. The worldwide increase in mortality from cirrhosis observed during the 1950s and 1960s was associated with a similar rise in alcohol consumption, attributed largely to the falling price of alcohol relative to income.2 Conversely, the reduction in per capita alcohol intake that has occurred in several countries since the late 1970s (including in the USA) has recently been reflected in some reduction of deaths due to cirrhosis. More recently, this reduction has stabilized and, once again, there has been a rise in ALD mortality rates in some countries.3 This may be associated with a rise in alcohol consumption, but may also be due, in part, to the increased prevalence of obesity, now recognized to be an important risk factor for the development of ALD.4 In 2001, liver cirrhosis was the 12th leading cause of death in the USA, and 44.4% of the cases of cirrhosis were alcohol-related. These figures translate to around 28 500 deaths from cirrhosis, of which 12 600 are alcohol-related.5 These deaths occur in an estimated total population of 2 million individuals with ALD of varying severities that represent approximately 1 in 7 of the estimated 14 million heavy drinkers in the USA. The potential environmental and genetic explanations for this clear interindividual variation in susceptibility to ALD will be discussed in the section on pathogenesis, below. First it is important to review the putative mechanisms through which this injury occurs in the next section of this chapter on pathogenesis. Most of the studies producing the data presented below were
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While there is good epidemiological evidence that heavy ethanol intake can result in liver disease in some individuals, there is still much debate about the main pathogenetic mechanisms through which this occurs. Several mechanisms have been proposed, and data from human and animal studies support the fact that more than one is likely to be important. The first and most direct is the effect of ethanol metabolism on liver biochemistry and the resulting steatosis and oxidative stress. The second is the indirect release of cytokines as a result of the increase in gut-derived endotoxin transported to the liver via the portal vein. The third is the liverdirected adaptive immune responses generated as a result of the development of new antigens formed by the reactive intermediates produced by the first two mechanisms. Many of these mechanisms have been elucidated using a variety of animal models. The intragastric ethanol-fed rat model designed by Tsukomoto and French has proven to be the most useful; however, there have also been mouse, guinea pig, hamster, and primate models, each producing its own challenges. As with all animal work, there is often difficulty in interpreting the data with regard to humans. Attempts to minimize this problem led to studies in the baboon, and, using this model, Rubin and Lieber described lesions resembling alcoholic hepatitis and cirrhosis.6 Working with animals so closely related to humans has obvious benefits; however, difficulty replicating the experiments and cost and ethical implications have somewhat limited the usefulness of this approach.7 Later sections of this chapter attempt to describe the putative mechanisms of ethanol-induced liver injury in detail and offer some theories as to how they may interact. First, however, there is a brief description of the absorption, distribution, metabolism, and elimination of alcohol. Alcohol metabolism will be discussed in terms of the fate of a unit of alcohol following ingestion. One unit is equivalent to 10 g or 12.5 ml of absolute alcohol which is present in approximately half a pint (284 ml) of beer and one standard measure of wine (114 ml) or liquor (24 ml).
ABSORPTION, DISTRIBUTION, AND EXCRETION The typical time course of blood alcohol concentration following the ingestion of 1 unit is shown in Figure 29-2. The peak level occurs approximately 20 min after ingestion and reaches between 10 and 15 mg/100 ml. The rate of rise and height of peak is a function of alcohol absorption and tissue distribution. In addition, it has been suggested that the peak value may be influenced by first-pass metabolism of alcohol by alcohol dehydrogenase (ADH) activity within the gastric mucosa; however, the biological importance of this effect is controversial. Alcohol is absorbed from the gastrointestinal tract by simple diffusion.8 Because of slow absorption of ethanol in the stomach, 50–80% of absorption occurs in the duodenum and upper jejunum. The rate of absorption is delayed following a meal and increases in proportion to the alcohol concentration of the drink consumed. Since absorption is more rapid from the intestine than the stomach, any pathological condition, drug, or surgical interven-
Chapter 29 ALCOHOLIC LIVER DISEASE
12 Blood alcohol (mg/100 ml)
On an empty stomach 10
Following a meal
8 6 (Km) of liver alcohol dehydrogenase activity
4 2 0 0
20
40
60
80
100
120
140
Time min Figure 29-2. The typical time course of blood alcohol concentration following the ingestion of 1 unit either following a meal (continuous line) or on an empty stomach (dotted line).
tion that delays or increases gastric emptying will also affect alcohol absorption accordingly. Following absorption, the tissue distribution of alcohol is principally determined by blood flow and water content. Thus, in organs with a rich vasculature such as brain, lungs, and liver, alcohol levels rapidly equilibrate with the blood. Alcohol is poorly soluble in lipids which will take up only 4% of the amount of ethanol that can be dissolved in a corresponding volume of water. As a result, tissues with a high fat/water ratio attain much lower levels than organs such as the kidney, where the high water content results in urinary alcohol levels 1.3 times higher than those in blood. The low lipid solubility of alcohol also explains why, following the ingestion of the same amount of alcohol per unit weight, an obese person attains a higher level of blood alcohol than a thin person. Furthermore, the higher fat content of female body composition compared to male has been invoked as part of the explanation for their higher alcohol levels following the ingestion of similar amounts of alcohol per unit weight.9 Over 90% of circulating alcohol is oxidatively metabolized, primarily in the liver, and excreted as carbon dioxide and water. The remainder is eliminated unchanged in the urine (15 y)
163g
177g
192g
227g
(144–210)
(160–224)
(197–275)
129
81
51
(average of minimum and maximum) (130–197)
No of cases
73
Cirrhosis of the liver Cirrhosis and potentially precirrhotic lesions (severe steatofibrosis with inflammatory reactions, chronic alcoholic hepatitis) Moderate-to-severe fatty infiltration Figure 29-7. Frequency of cirrhotic and precirrhotic liver lesions according to dose and duration of alcohol consumption in 334 drinkers. (Reproduced from Lelbach WK. Cirrhosis in the alcoholic and its relation to the volume of alcohol abuse. Ann NY Acad Sci 1975; 252:85–105, ©1975 with permission of New York Academy of Science.202)
Chapter 29 ALCOHOLIC LIVER DISEASE Figure 29-8. Alcoholic hepatitis.
during alcohol-induced injury, all of which have been shown to enhance collagen production by HSC.67,181,182 CYP2E1 may be particularly important in this regard given its inducibility by alcohol and a high-fat diet and its perivenular distribution. HSC grown in the presence of hepatocyte cell lines that overexpress CYP2E1 increase their production of collagen, an effect that is prevented by antioxidants or a CYP2E1 inhibitor.183 Hepatocyte apoptosis is a notable feature of alcoholic hepatitis (Figure 29-8),130 and apoptosing hepatocytes express Fas, that can promote stellate cell initiation through the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).184 Furthermore, apoptosing hepatocytes may also be ingested by Kupffer cells and HSC which subsequently release TGFb, capable of activating HSC.185,186
MECHANISMS OF HEPATOCELLULAR CANCER Epidemiological studies reveal that alcohol plays a major contributory role in the development of hepatocellular cancer (HCC); however the primary mechanisms through which this occurs are not clearly defined (Table 29-2). Cirrhosis itself is a precancerous condition, and alcohol-related HCC without pre-existing cirrhosis is rare. Nevertheless, three features indicate that alcohol may be a cocarcinogen. The first is that heavy alcohol consumption is associated with several extrahepatic cancers (discussed below). The second is that when the incidence of incidental HCCs in liver explants from patients with alcoholic cirrhosis is compared with that from other etiologies, it lies between that of immune-mediated liver disease and viral hepatitis.187 It appears, therefore, that the incidence of HCC
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Table 29-2. The primary mechanisms thought to be involved in ethanolinduced hepatic fibrosis and hepatocellular carcinoma Fibrosis Kupffer cell production of profibrotic cytokines Kupffer cell production of profibrotic reactive oxygen species Hepatocyte production of profibrotic reactive oxygen species Hepatocyte production of acetaldehyde Kupffer cell and hepatic stellate cell production of transforming growth factor-b after ingestion of apoptotic hepatocytes Hepatocellular carcinoma Lipid peroxidation and DNA mutagenesis Activation of carcinogenic xenobiotics Antiapoptotic effect of tumor necrosis factor-a DNA hypomethylation Immunosuppression
is above that of the “baseline” expected due to pure cirrhotic risk. The third is that there are several plausible mechanisms through which alcohol could promote carcinogenesis.
Lipid Peroxidation and DNA Mutagenesis MDA, an end-product of lipid peroxidation, can bind to DNA and form adducts as it does with other endogenous compounds.188 These adducts were found to be highly mutagenic in Escherichia coli189 and are repaired by nucleotide excision repair. They are also found at significant levels in healthy humans, and can induce cell cycle arrest.190 This latter property results in an increase in the number of hepatic progenitor cells (oval cells)191 which are more resistant to oxidative stress than fully differentiated hepatocytes. It has been suggested that this may promote HCC as the oval cells survive through oxidative damage but remain susceptible to mutagenesis.191
Activation of Xenobiotics Another mechanism proposed for the increased rate of HCC seen in alcoholic cirrhosis is the increased production of carcinogenic metabolites from other environmental carcinogens (xenobiotics) that are metabolized through the MEOS and other metabolic pathways up-regulated in heavy drinkers. This mechanism has been suggested to explain the increased cancer risk seen with tobacco smoking,192 aflatoxin,193 and other chemicals.194 In addition, CYP2E1 is responsible for the metabolism of retinoic acid (RA) in the liver.195 Up-regulation of CYP2E1 by ethanol therefore synergizes with its inhibition of RA synthesis and results in reduced RA levels, increased expression of the activator protein-1 (AP-1) (c-Jun and c-Fos) transcriptional complex, and increased hepatocyte proliferation.196 Supplementation with RA reverses this effect.195
TNF-a-induced survival factors As discussed above, TNF-a has both pro- and antiapoptotic properties and the balance of these appears to depend on the local microenvironment and the disease. While apoptosis may reduce the risk of HCC, increased cell survival through TNF-a-induced NFkB activation could have the opposite effect, particularly in combination with the mutagenic effects of lipid peroxidation products.
594
Reduced DNA methylation DNA methylation is an important negative regulator of gene expression and hypomethylation of oncogenes has been shown in human and rat HCC.197 Chronic ethanol consumption results in reduced concentrations of SAME, the main methyl donor (as discussed above), and dietary depletion of SAME increases the risk of HCC in rats.198
Immunosuppression Malnutrition, vitamin deficiencies, and acute ethanol per se can all result in reduced immunosurveillance. Of particular relevance is the effect on NK cells, thought to be central to tumor surveillance. While this is debated, the primary functional effect appears to be one of suppression.137,199 In addition, there are other, more widespread effects on the innate and adaptive immune responses that could all have knock-on effects on tumor surveillance (discussed below). A reduction in immunosurveillance and a subsequent increase in viral replication may also be the mechanism through which alcohol leads to an increased rate of HCC in hepatitis C cirrhosis.200
SUSCEPTIBILITY TO ALCOHOLIC LIVER DISEASE While the majority of heavy drinkers will develop some degree of steatosis (fatty liver), only around a third go on to develop alcoholic hepatitis and only between 1 in 4 and 1 in 12 ever progress to cirrhosis.201 This leads to the obvious question: what factors determine whether or not a heavy drinker develops advanced ALD?
DOSE OF ETHANOL The observation that only a minority of heavy drinkers develop ALD was first reported 30 years ago by Lelbach. This author showed that, although the risk of disease increased in proportion to the duration of intake, only 20% of consumers of more than 200 g of ethanol (around 20 standard “drinks”) per day develop cirrhosis after 13 years and less than 50% after 20 years.202 Further work from around this time showed that women appeared to develop ALD at lower doses of alcohol consumption than men.203 More detailed studies examining the precise dose–response relationship between alcohol intake and risk of ALD, the gender effect, and the risk threshold have been reported in the last 10 years. A large cohort study from Italy involving 6917 subjects between the ages of 12 and 65 reported that the risk of developing ALD begins at 30 g/day of ethanol.201 However, only 5.5% of the individuals drinking this much showed signs of liver disease. The risk increased according to daily dose, reaching 10% at 60 g/day. The study also reported that risk is higher among the over-50s if alcohol is drunk outside mealtimes or consumed in a variety of different beverages rather than one “tipple of choice.” Interestingly, this study showed no gender effect. Further evidence for a dose–response relationship and a risk threshold came from an even larger study from Copenhagen involving 13 285 subjects between the ages of 30 and 79.204 A self-administered questionnaire assessed intake, and incidence of disease was taken from death certificates and hospital
Chapter 29 ALCOHOLIC LIVER DISEASE
medical records. This study revealed a dose-dependent increase in risk, with women having a significant risk above 7–13 units per week, and men 14–27 units per week. This group updated their data analysis recently by looking at type of alcohol consumed.205 Their results suggest that the highest risk is seen in drinkers who do not include wine in their drinking repertoire. Furthermore the relative risk of cirrhosis fell as the proportion of wine increased. This association between wine intake and ALD risk may be confounded by other factors associated with wine drinking such as a lower prevalence of obesity compared to beer and spirit drinkers. While all these studies have their flaws, with data collection being the most obvious, they do allow a number of conclusions to be drawn. No dose of alcohol confers a guarantee of developing cirrhosis regardless of the period it is consumed for, and relatively low doses can cause problems.
DIET The data from ethanol-fed rats linking a diet high in polyunsaturated fats with an increased risk of alcoholic liver injury, discussed above, are supplemented by an epidemiological study linking cirrhosis mortality with pork (high in linoleic acid) consumption and dietary intake of unsaturated fats.206 A further case–control study from France has reported that the risk of cirrhosis is increased by diets high in fat and alcohol and low in carbohydrate.207 A more obvious role for diet in ALD risk has been suggested by two studies showing that obesity and associated hyperglycemia increase the incidence of all stages of ALD in heavy drinkers.4,208 While these studies have provided evidence that dose, pattern, and type of alcohol consumption and dietary (and presumably exercise-related) factors play a role in determining ALD risk, they have also demonstrated that other endogenous factors are likely to be equally, if not more, important.
GENDER AND RISK OF ALD The most obvious endogenous or “genetic” factor determining ALD risk is female gender. It has long been appreciated that women develop ALD at a lower intake of alcohol than men. The traditional explanation has been that women develop higher blood alcohol concentrations per unit of alcohol consumed due to their lower volume of distribution for alcohol. This, in turn, is attributed to their lower body mass index and to fat constituting a higher percentage of their body mass than in men. More recent evidence has, however, suggested an explanation based on disease mechanisms. Enomoto and colleagues have demonstrated in the rat model that estrogen increases gut permeability to endotoxin and accordingly up-regulates endotoxin receptors on Kupffer cells, leading to an increased production of tumor necrosis factor in response to endotoxin.209 These exciting data suggest several new directions for research into human gender-specific susceptibility to ALD.
NON-GENDER-LINKED GENETIC FACTORS AND RISK OF ALD Evidence for non-gender-linked genetic susceptibility to ALD comes principally from a twin study showing that the concordance rate for alcoholic cirrhosis was three times higher in monozygotic than in dizygotic twin pairs.210 This difference in concordance rates was not
entirely explained by the difference in concordance rates for alcoholism per se. Further indirect evidence of a genetic component to disease risk comes from the observation that the death rate from ALD is subject to wide interethnic variation that is not entirely explained by variations in the prevalence of alcohol abuse.211,212 Hispanics appear to be at particularly high risk, for example. Difficulties in performing family linkage studies in ALD have resulted in almost all of the relevant information thus far coming from classical case–control, candidate gene, allele association studies. Accordingly these studies are subject to all the common pitfalls of this type of study design and must be interpreted with caution.213 Many early reports of positive associations are likely to be subject to type I errors (chance findings), while negative reports may be subject to type II errors (false negatives) attributed to small underpowered studies. Given that the most likely mechanisms of hepatocyte injury in excessive drinkers are related to fat accumulation, oxidative stress, endotoxin-mediated release of proinflammatory cytokines, and immunological damage, the majority of studies reported thus far have focused on genes encoding proteins involved in these various pathways.
Genes Influencing the Severity of Steatosis Recognition of the role played by steatosis in the pathogenesis of more advanced liver disease60 suggests that factors determining its severity may play a key role in determining the risk of cirrhosis. Clearly genetic and environmental factors determining the degree of obesity would fall into this category, as would functional polymorphisms of genes encoding enzymes involved in hepatic lipid metabolism. Of interest in this respect is a preliminary report that a “low-activity” promoter polymorphism in the gene encoding MTP, the principal protein responsible for the export of fat from the liver, is associated with an increased risk of advanced ALD.214
Genes Influencing Oxidative Stress and Risk of ALD The principal class of genes that influences the oxidant load in heavy drinkers are those genes encoding enzymes involved in alcohol metabolism. Polymorphisms have been identified in two of the seven genes encoding ADHs (ADH2 and ADH3), in the promoter region of the CYP2E1 gene and in the coding region of the gene encoding the mitochondrial form of ALDH (ALDH2). The genes encoding ADH2 and ALDH2 undoubtedly play a role in determining the risk of alcoholism and, to a lesser extent, ALD in oriental populations.215–217 Previously reported associations with ADH3 probably reflect linkage disequilibrium with ADH2.218 In caucasians, results from studies reported to date support a role for the ADH2 polymorphism in determining the risk of alcoholism, but not ALD.219 Several studies have looked for an association between the c2 promoter (Rsa I) polymorphism of the CYP2E1 gene and ALD with no consistent results emerging in any population, although one study did report that the cumulative lifetime alcohol intake of patients with ALD heterozygous for the c2 (more transcriptionally active) allele was almost half that of patients with ALD homozygous for the c1, wild-type allele.220 The HFE gene is another obvious candidate gene for ALD, since liver iron promotes oxidative stress and iron deposition is common in ALD. Unfortunately, a case–control study
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of over 400 patients and controls found no evidence of an association between ALD and either of the HFE mutations associated with hemochromatosis.221 This lack of association was explained by the observation that hepatic iron content did not differ between patients with and without the mutations. The lack of any striking associations between polymorphisms in genes encoding proteins involved in the generation of ROS and ALD has recently turned attention towards polymorphisms in genes encoding proteins involved in the body’s antioxidant defenses. Manganese-dependent SOD-2 is the most important mitochondrial antioxidant enzyme and a polymorphism altering its mitochondrial targeting sequence has been associated with ALD in a small French study,222 although not confirmed in a larger study from the UK.223 This and other polymorphisms affecting the function of antioxidant defense systems are clearly worthy of further study.
Endotoxin Receptor and Cytokine Genes and Risk of ALD Evidence supporting a role for endotoxin-mediated cytokine release in the pathogenesis of ALD, together with the identification of promoter polymorphisms in genes encoding endotoxin receptors, cytokines, and cytokine receptors, has recently suggested an alternative set of candidates to explain genetic susceptibility to ALD. CD14, an LPS receptor on monocytes, macrophages, and neutrophils, has no intracellular domain but enhances signaling through another LPS receptor, TLR4. A C/T polymorphism is present at position -159 in the CD14 promoter, with the TT genotype associated with increased levels of soluble and membrane CD14.224 A study from Finland has recently reported an association between possession of the TT CD14 genotype and advanced ALD;125 however, this has not been observed in a larger study in north-east England.225 This latter study also showed no association between ALD and possession of the Asp299Gly polymorphism in the TLR4 gene, previously reported to be linked to hyporesponsiveness to LPS.226 With respect to polymorphisms in the cytokine genes, the first such association was reported between alcoholic hepatitis and a polymorphism at position -238 in the TNF-a promoter region.227 The functional significance of this polymorphism is, however, unclear and the association may well be either spurious or due to linkage disequilibrium with another true “disease-associated polymorphism” on chromosome 6. An association with ALD has also been reported for a promoter polymorphism in IL-10. IL-10 is the classical anti-inflammatory cytokine which inhibits: (1) the activation of CD4+ T-helper cells; (2) the function of cytotoxic CD8+T cells and macrophages; (3) class II human leukocyte antigen/B7 expression on antigen-presenting cells; and (4) HSC collagen synthesis. A variant CÆA substitution at position -627 in the IL-10 promoter has been associated with decreased reporter gene transcription, decreased IL-10 secretion by peripheral blood monocytes, and an increased response to a-interferon in patients with chronic hepatitis C – all consistent with the polymorphism being associated with lower IL-10 production. A strong association between possession of the A allele and ALD has been reported from a study of over 500 heavy drinkers with and without advanced liver disease.228 This is consistent with low IL-10 favoring inflammatory and
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immune-mediated mechanisms of disease as well as HSC collagen production.
Immune Response Genes and Risk of ALD In view of the immunoregulatory functions of IL-10, the association between ALD and a low-activity promoter polymorphism in IL-10 may be considered as further evidence that immune mechanisms are involved in the pathogenesis of ALD. Further evidence supporting a role for immune mechanisms in determining individual susceptibility to ALD has come from a recent study showing that, compared to drinkers with no evidence of ALD, patients with ALD are more likely to have high titers of autoantibodies against CYP2E1173 and to have T-cell responses against oxidative stress-derived adducts.176 Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a T-cell surface molecule that normally acts to “damp down” the immune response to antigens either directly, by competing with CD28 on the surface of CD4+ Th cells for the antigen-presenting cell co-stimulatory molecule B7, or indirectly, by activating T-regulatory cells which act to inhibit CD4+ Th cell function.229 CTLA-4 knockout mice develop lethal autoreactive lymphoproliferative disease and an AÆG polymorphism in exon 1 leading to a ThrÆAla substitution has recently been associated with autoimmune liver diseases, insulin-dependent diabetes, and autoimmune thyroid disease. These associations strongly suggest that this polymorphism is associated with impaired CTLA-4 function, although recent data suggest that other tightly linked CTLA-4 polymorphisms may be responsible for the functional effect.230 Although the exon 1 polymorphism has been associated with the titer of anti-CYP2E1 antibodies in one study173 and with ALD in another,231 this has yet to be confirmed as an ALDsusceptibility allele in large studies examining the full CTLA-4 gene haplotype. In future, the choice of candidate genes for detailed study as ALD risk factors is likely to be guided by: (1) genome and proteome expression studies in tissue from patients with various stages of ALD; (2) whole-genome single nucleotide polymorphism scans of cases and controls; and (3) mouse mutagenesis studies. Most importantly, however, as with other so-called complex diseases, establishing reliable genetic associations is critically dependent on the collection of large numbers of well-phenotyped cases and controls, which almost certainly require national and multinational collaborations. Only then are we likely to come up with associations that are robust enough to guide targeted treatment and prevention strategies.
CLINICAL FEATURES DIAGNOSIS Chronic alcohol abuse produces a wide range of morphological changes in the liver, the most frequent being fatty liver (steatosis), alcoholic hepatitis, and cirrhosis.232,233 For ease of presentation the three principal lesions will be discussed separately in terms of their pathology, clinical features, and prognosis, but it is important to appreciate that alcohol-related liver damage is a spectrum, with the various lesions occurring more commonly in combination than in isolation. Significantly, the clinical manifestations of each of these histological lesions are extremely variable, ranging from completely
Chapter 29 ALCOHOLIC LIVER DISEASE
asymptomatic forms to a first presentation with severe hepatic failure. Patients with none or minimal symptoms are, however, more likely to have the earlier, more reversible, forms of liver disease and therefore the early recognition of these patients is critical to allow intervention at a stage when it is likely to be of most benefit. Patients most commonly present with symptoms unrelated to the liver, typically non-specific digestive symptoms or vague psychiatric complaints. The patient may seek advice concerning the social effects of alcohol abuse on family life or work performance. Often, physical examination will be normal, other than occasional plethora, suffused conjunctivae, tremulousness, and aggressive behavior. Up to 30% of patients with ALD have no symptoms related to excessive alcohol intake and may present following the chance finding of hepatomegaly or abnormal blood tests at routine medical examination. The key to the early recognition of patients with alcoholrelated disease is a high index of suspicion. Once the diagnosis is suspected it is usually easy to confirm by direct questioning for alcohol history and alcohol-related symptoms, careful clinical examination, and supportive laboratory investigations.
History Features in the history important for both the confirmation of alcohol abuse and to aid in its subsequent management include the amount and duration of alcohol intake, the pattern of intake, precipitating factors of drinking bouts, and evidence of physical dependence such as early-morning tremor, blackouts, and morning drinking. Confirmation of the history should be sought from a family member or close associate. Specific liver-related symptoms, such as jaundice and hematemesis, should be sought but are uncommon, even in patients with established disease. In addition, since all alcoholics with liver disease do not necessarily have disease of alcoholic etiology,234 enquiries should be made concerning other risk factors for liver disease, including a history of foreign travel, blood transfusions, or intravenous drug use.
Clinical Examination Important features to note on examination are the signs of chronic liver disease, including hepatomegaly, and signs indicative of alcoholrelated pathology in other organs, such as hypertension, atrial fibrillation, and a cushingoid appearance. It is important to understand that many of the classical signs of chronic liver disease, including spider nevi, Dupuytren’s contractures, palmar erythema, and parotid swelling, can occur in alcoholics in the absence of cirrhosis. Clinical signs and history cannot be relied upon to distinguish the various histological subtypes of ALD, since patients with cirrhosis can be asymptomatic, while patients with hepatocellular failure may have only severe fatty change.235
Laboratory Investigations Biochemical and hematological tests can confirm the presence of alcohol abuse and indicate the presence of liver damage, but are not useful in determining the severity of the histological lesion. Blood alcohol estimations are an often-underused method of confirming a suspicion of excess drinking, with levels >100 mg/100 ml at a morning clinic or levels >150 mg/100 ml without obvious intoxication strongly suggestive of alcohol abuse. Elevation of g-glutamyl-
transferase has been reported in up to 90% of patients abusing alcohol.236 The rise is mainly due to hepatic microsomal induction and is independent of the presence of liver disease, although hepatocellular necrosis and cholestasis may contribute. It is not specific for alcohol abuse and is raised in other forms of liver injury and in patients taking other enzyme-inducing drugs.237 Its main clinical use is probably in monitoring a period of supposed abstinence, since it falls within a week of cessation of drinking. Other biochemical markers of alcohol abuse rather than liver disease include elevated serum uric acid,238 hypertriglyceridemia, and desialylated transferrin.239 The classical hematological marker of alcohol abuse is a raised mean corpuscular volume, which has been reported to occur in between 80% and 100% of alcoholics with and without liver disease240 and may be more common in alcoholic women. It is due to a direct toxic effect of alcohol on the marrow, although nutritional folate and vitamin B12 deficiencies may contribute in some patients. With regard to biochemical markers of alcohol-related liver damage, a rise in serum aspartate transaminase (AST) activity of up to five times normal is common in patients abusing alcohol and reflects the presence, but not the severity, of liver damage.241 However, unlike non-ALD, alanine transaminase (ALT) activity is raised less often than AST, and the AST/ALT ratio has been suggested as a means of distinguishing liver disease of alcoholic and nonalcoholic etiology.242 Recently, however, it has been appreciated that an AST greater than the ALT can also be a marker of severe nonALD.243 Biochemical markers of the stage of liver disease have so far proved elusive. Possible exceptions include plasma IgA, which is twice normal in less than 30% of alcoholics with early disease and greater than three times normal in 60% of patients with cirrhosis,244 and the procollagen peptides. Levels of procollagen III in particular have been shown to distinguish advanced from early ALD.245
Liver Biopsy Liver biopsy is a mandatory investigation in all patients chronically abusing alcohol who have hepatomegaly and/or abnormal liver blood tests. First, it is used to establish the diagnosis of alcohol-related liver disease. This is important since it has been shown that up to 20% of liver disease in alcoholics with abnormal liver function is due to an alternative etiology.234 Second, it is the only way of accurately staging the disease, which cannot be achieved by any combination of clinical or laboratory data246 Without knowledge of the histological severity, no prognostic information can be given to the patient and no rational treatment plan can be devised.
ALCOHOL-INDUCED FATTY LIVER Pathology Fatty liver is the earliest lesion seen in ALD. The classical appearance is of a single large fat droplet displacing the nucleus occurring predominantly in perivenular hepatocytes (macrovesicular steatosis). Very rarely, the steatosis is panacinar and may be associated with severe cholestasis, cholangiolitis, and clinical presentation with hepatic failure.235 Inflammation is rare in simple fatty liver although occasional lipogranulomata may be seen as a response to the extrusion of cellular lipid. Mild fibrosis may occur in response to lipogranulomata and is usually considered reversible; however, the presence of marked perivenular fibrosis in an otherwise uncompli-
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cated fatty liver may be a marker of high risk of progression to cirrhosis.247 Microvesicular steatosis, in the form of finely dispersed lipid droplets, may also occur in some patients (alcoholic foamy degeneration) and is associated with bilirubinostasis and focal liver necrosis.248 This lesion resolves with abstention. Alcoholic fatty liver is histologically indistinguishable from non-alcoholic fatty liver associated with the metabolic syndrome (hyperlipidemia, hypertension, type II diabetes, and obesity).
Clinical Features Patients with fatty liver are usually asymptomatic or present with non-specific digestive symptoms. Rarely, fatty liver may be associated with hyperlipidemia, hemolytic anemia, and jaundice (Zieve’s syndrome, discussed below) or hepatic failure. Smooth non-tender hepatomegaly is usually the only clinical finding, although signs of portal hypertension may be observed if perivenular fibrosis (central hyaline sclerosis) is present. All, or none, of the laboratory investigations discussed may be abnormal; most commonly, the g-glutamyltransferase, AST, and mean corpuscular volume are mildly raised.
Prognosis It is widely considered that fatty liver is an entirely benign lesion reversible with abstention from alcohol. Fat starts to accumulate in the liver after as little as one weekend of heavy drinking in nonalcoholic human volunteers.249 Fortunately the reverse seems to hold true, and in the majority of patients with fatty liver who stop drinking the laboratory abnormalities quickly return to normal250 and the histological abnormality rapidly regresses.251 Accordingly, no treatment options have been evaluated in patients with fatty liver other than abstention and a well-balanced diet. However, there are reports that alcoholic fatty liver per se is not always benign, with occasional mortality due to hepatic failure, fat emboli, and hypoglycemia. Furthermore, fatty liver may be a precursor of alcoholic cirrhosis. In a study by Sorensen and colleagues,61 it was found that the extent of fatty liver on initial liver biopsy was a better predictor of subsequent progression to cirrhosis 10 years later than alcohol history. Furthermore, a more recent study revealed that even “pure” fatty liver can progress to fibrosis and cirrhosis in a proportion of patients.62 In this study, the presence of mixed macro- and microvesicular fat and giant mitochondria were associated with disease progression. This suggests, as discussed previously, that fatty liver may be causative in the development of cirrhosis rather than simply an epiphenomenon of alcohol abuse.
ALCOHOLIC HEPATITIS Pathology Alcoholic hepatitis consists of a constellation of histological abnormalities. The features obligatory for diagnosis are:232 1. liver cell damage, typified by ballooning degeneration progressing to necrosis ± Mallory bodies. Ballooning degeneration is characterized by hepatocyte swelling, a pale granular cytoplasm, and a small hyperchromatic nucleus. Mallory bodies are intracytoplasmic inclusions staining purplish-red with hematoxylin and eosin and consisting of aggregates of inter-
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mediate filament proteins, reflecting impaired function of the microtubular system 2. inflammatory cell infiltrate, predominantly neutrophils. These are typically arranged round necrotic hepatocytes that contain Mallory bodies (“satellitosis”) 3. pericellular fibrosis, producing a “chicken-wire” appearance. In addition, there is often fibrous thickening around the hepatic vein radicals and eventual obliteration of the veins, a process referred to as central hyaline sclerosis 4. perivenular distribution, unless cirrhosis is present, when lesions occur at the periphery of nodules. As the severity increases the damage extends to involve the whole lobule Other features, which are often present but are not obligatory for diagnosis, include: fatty change, bridging necrosis, bile duct proliferation, apoptotic bodies, cholestasis, and giant mitochondria. Histological features considered to indicate a high risk of progression to cirrhosis are the extent and degree of fibrosis (central hyaline sclerosis is the worst sign), a panlobular distribution, and widespread Mallory body formation. It is important to highlight that this pattern of lesions can also occur in other conditions, including: diabetes mellitus, obesity, jejunal-ileal bypass, total parenteral nutrition, and following treatment with various drugs, when it is referred to as non-alcoholic steatohepatitis.
Clinical features There is no good correlation between the severity of the histological lesion and the clinical presentation, which can range from asymptomatic to life-threatening hepatic decompensation.252 However, patients with the milder histology are more likely to present with non-specific symptoms, incidental hepatomegaly, or raised transaminases, while patients with severe histology usually present with symptoms specifically related to hepatocellular failure, such as jaundice, ascites, and encephalopathy or variceal bleeding. The episode of decompensation leading to clinical presentation may be precipitated by vomiting, diarrhea, anorexia, increased alcohol intake, or intercurrent infection. The majority of patients have tender, smooth hepatomegaly, with an arterial bruit in severe cases. Signs of chronic liver disease may be present, even without coexisting cirrhosis, and the more advanced cases may also have signs of portal hypertension and encephalopathy. Non-liver signs commonly present include pyrexia, signs of associated vitamin deficiency and malnutrition, a hyperdynamic circulation, and cyanosis due to intrapulmonary arteriovenous shunting. Abnormalities of liver-related blood tests are always present and include decreased albumin and increased gglutamyltransferase, AST, bilirubin, alkaline phosphatase, and prothrombin time (PT). In addition, blood urea and serum sodium and potassium are all low, unless hepatorenal syndrome (HRS) supervenes, and hypoglycemia may be present. Macrocytic anemia, neutrophil leukocytosis, and thrombocytopenia are present in all but the mildest cases. A peculiar clinical feature of patients with severe alcoholic hepatitis is that they often rapidly deteriorate in the days immediately following hospital admission.253 This has been observed in up to 40% of patients and varies from deteriorating blood tests to increasing encephalopathy or variceal bleeding. The pathophysiological basis of this is not clear but suggestions have included the nutritional implications of withdrawing an alcoholic from his/her
Chapter 29 ALCOHOLIC LIVER DISEASE Figure 29-9. Alcohol-induced hepatic fibrosis.
principal source of calorific intake and a reduction in hepatic blood flow consequent upon a reduction in levels of acetaldehyde, which, via conversion to adenosine, has vasodilatory actions.
Prognosis The short-term outcome in patients with alcoholic hepatitis depends largely on the severity of the initial histological lesion. Thus in the Veterans Administration Cooperative study 30-day mortality was 1% in those with mild alcoholic hepatitis, 12% in those with moderately severe histology, and 34% in patients with severe disease.254 One of the management problems is that many patients will have prolongation of the PT that precludes transabdominal liver biopsy. It is these patients who are likely to have severe disease and in whom
aggressive and experimental therapy might be justified. While transjugular liver biopsy is an alternative, it is not always locally available. In view of this difficulty, many clinical and laboratory variables have been suggested as indicators of histological severity and therefore of potential use in predicting short-term mortality in alcoholic hepatitis. Based on these variables there have been three main attempts at creating prognostic indices. First is the modified Child’s criteria which combines the presence of encephalopathy and ascites with serum albumin, bilirubin, and PT.255 Second is a more complex system combining 12 different variables to derive a combined clinical and laboratory index,256 and third is the discriminant function (DF) of Maddrey and colleagues, which is based on PT and bilirubin only.257 This has been confirmed as a useful predictor of mor-
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tality prospectively and, in view of its simplicity, is probably the most clinically useful index at present. Perhaps surprisingly, none of these indices includes the presence of renal failure, which is not uncommon in the most severely ill patients. This may reflect the fact that the occurrence and outcome of the HRS are entirely dependent on the severity of the hepatocellular dysfunction, which is better indicated by other clinical and laboratory variables. Alternatively, renal function may be an important prognostic indicator that has not been utilized in the indices to date. A fourth scoring system, the Glasgow Alcoholic Hepatitis Score, now incorporates this parameter. While this scoring system needs validation in large numbers prospectively, initial work suggests that it is more sensitive and specific than the Maddrey score at determining prognosis.258 If the patient survives to hospital discharge, then the long-term prognosis is determined by the initial histology, the progression to cirrhosis, and the subsequent drinking behavior. Thus, the 5-year survival falls from 70% to 50% in patients with severe compared to mild alcoholic hepatitis,259 and in the Veterans Administration study, patients with mild hepatitis who developed cirrhosis had a 71% 2year survival compared to 81% in those who did not.260 In addition, the 7-year survival has been reported to fall from 80% to 50% in patients who continue to drink compared with abstainers,259 which is presumably due, at least in part, to the influence of intake on the risk of progression to cirrhosis. In men with mild histology, drinking behavior is the major factor determining progression to cirrhosis, while in women and men with severe histology, progression can occur independently of drinking behavior.261
CIRRHOSIS Pathology With progressive injury the features of cirrhosis, namely fibrous septa linking hepatic and portal veins, and regenerative nodules eventually appear. The cirrhosis is usually micronodular, possibly reflecting the inhibition of regenerative activity by alcohol, and frequently reverts to a macronodular cirrhosis with abstention.262 The coexistence of steatosis and hepatitis is common and usually indicates continued consumption. In contrast, alcohol withdrawal at the cirrhotic stage can make the histological determination of etiology almost impossible.
Prognosis The survival of patients with alcoholic cirrhosis is determined by the clinical and histological severity of the disease at presentation and their subsequent drinking behavior. It has also been shown in some studies that gender263 and ethnicity264 may influence survival. Several studies have shown that patients who present with decompensated disease do significantly worse than those presenting with compensated disease.265,266 The influence of drinking behavior on this trend is best illustrated by the seminal study of Powell and Klatskin.267 They showed that in patients with compensated disease, continued drinking reduced the 5-year survival from 89% to 68%. Abstaining patients with ascites or jaundice had lower survival rates than compensated patients, but higher survival rates than patients with ascites or jaundice who continued to drink. The lowest survival was seen in patients with variceal bleeding and alcohol habits had no effect on their mortality. The presence of coexisting alcoholic hepatitis on initial biopsy also adversely affects prognosis.268 HCC develops in 5–15% of patients with alcoholic cirrhosis.269 It is most common in abstaining men and the majority of patients die within a few months of diagnosis.266,270
ASSOCIATED CONDITIONS AND EXTRAHEPATIC MANIFESTATIONS INTRODUCTION The range of health problems associated with excess alcohol consumption extends beyond the liver, with virtually every system in the body affected (Figure 29-10). As with ALD, the pathogenetic pathways are not always clear, but are more complex than the direct effect of ethanol per se. This section will give an overview of these problems.
GASTROINTESTINAL EFFECTS As the first site of exposure after ethanol ingestion, the gastrointestinal system is a prime candidate for toxicity. As well as the liver, alcohol can affect most parts of the gastrointestinal system, as summarized in Table 29-3 and discussed below.
Salivary Glands and Oropharynx Clinical Features As with other forms of cirrhosis the clinical presentation of alcoholic cirrhosis can range from asymptomatic hepatomegaly to hepatic failure and the complications of portal hypertension such as ascites or variceal bleeding. Presentation with severe hepatic decompensation usually implies the presence of continued drinking and superimposed alcoholic hepatitis, but may signal the development of HCC or portal vein thrombosis. The clinical findings will depend on the presence of portal hypertension or encephalopathy and do not differ significantly from those observed in other forms of cirrhosis. Patients with compensated cirrhosis, particularly if abstinent from alcohol, can have completely normal laboratory investigations, while patients with continued intake will have a similar range of abnormal laboratory investigations to those seen in patients with alcoholic hepatitis. In addition, a raised a-fetoprotein suggests the presence of HCC and indicates the need for further investigations.
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Parotid enlargement is frequently observed in heavy drinkers with and without chronic liver disease. A histological study at necropsy demonstrated an increase in adipose tissue at the expense of acinar tissue in the salivary glands of patients with alcoholic cirrhosis compared to controls.271 It may be this which contributes to the reduction in both basal and stimulated parotid gland salivary flow reported in these patients.272 Whether the reduced secretion and altered gland structure in patients with alcoholic cirrhosis is primarily associated with the liver disease or the effects of prolonged alcohol consumption per se is not entirely clear; however, reports of increased resting salivary flow in alcoholics without liver disease would suggest that the development of liver disease is the important factor leading to reduced secretion. The prevalence of glossitis and stomatitis is higher in alcoholics than controls,273 presumably reflecting their poor nutritional status, which includes deficiencies in B-vitamins and iron. In addition,
Chapter 29 ALCOHOLIC LIVER DISEASE Figure 29-10. Extrahepatic complications of excess alcohol consumption.
Wernike-Korsakoff sydrome Cerebellar disease Brain stem disease Cerebral atrophy Parotid enlargement Oropharyngeal tumours
Arrhythmias, hypertension cardiomyopathy, ischemic heart disease Gastritis Acute/chronic pancreatitis Malabsorption Hematological complications Peripheral neuropathy myopathy
Diarrhoea Sexual dysfunction reduced fertility
Osteoporosis
heavy drinkers have a very significant increase in the incidence of oropharyngeal tumors. Tobacco and alcohol are the principal etiological factors associated with the development of head and neck malignancies and appear to act in synergy. One study has reported a history of alcohol and tobacco use in more than 75% of patients with tumors of the oropharynx.274 As these tumors are more common in Asians carrying the null ALDH2 gene, it has been suggested that acetaldehyde, which has been shown to accumulate in the saliva of these individuals, plays a role in the pathogenesis.275 A history of alcoholism or alcohol-related disease is also associated with a worse prognosis in patients with head and neck malignancy.
Esophagus Acute alcohol ingestion alters esophageal motor function by reducing lower esophageal sphincter pressure, and inhibiting the primary peristaltic movement of the distal esophageal body.276 This reduces esophageal clearance and increases gastroesophageal reflux. Chronic ethanol consumption also results in reduced esophageal clearance, but lower sphincter pressures are increased unless the patient has concomitant autonomic neuropathy. While ethanol is associated with heartburn, there is no good evidence that drinkers are more prone to esophagitis. Nausea and vomiting are frequent in chronic alcoholics and may induce Mallory–Weiss tears.277 Esophageal cancer is the sixth commonest cancer in the world, and alcohol has been identified as a major risk factor since 1962. This association is dose-related, and there is no dose below which there is no increased risk. Smoking is an important cofactor. Alcohol-associated nutritional deficiencies and the enhanced bioactivation of dietary mycotoxins and nitrosamines and tobaccorelated carcinogens may be important cofactors.278
Stomach The effects of ethanol on gastric motility have been inconclusive, but tend towards an inhibitory effect on gastric emptying. Gastric acid secretion is greatly increased by beer and less so by wine. Most spirits do not lead to an increase in gastric acid secretion, leading to the suggestion that it is other products of fermentation that have this effect.279 Acute alcohol consumption causes an acute erosive hemorrhagic form of gastritis, with loss of surface epithelial cells and neutrophil infiltration. This peaks at 60 min and lasts at least 24 h. Both non-steroidal anti-inflammatory drugs and portal hypertensive gastropathy are risk factors. Chronic heavy drinkers are more likely to develop a superficial or atrophic type of gastritis. Whether alcohol abuse per se induces this classical chronic gastritis with a mononuclear cell infiltrate and glandular atrophy is unclear. This may be due to the increased incidence of Helicobacter pylori infection in the gastrointestinal tract of heavy drinkers.280 While there is some debate over whether chronic ethanol consumption increases the risk of duodenal ulcers,281 there is no evidence that the incidence of gastric ulceration is higher than in the general population.
Small Intestine Alcohol is one of the main causes of malnutrition in the western world. It can be severe and is associated with neurological problems, skin abnormalities, and glossitis, and may also contribute to increased susceptibility to infection and malignancy. Malnutrition in an alcoholic can be both primary, due to inadequate nutrient intake, and secondary due to malabsorption or maldigestion resulting from gastrointestinal complications. While pancreatic and hepatic dysfunction can play a role, particularly in fat malabsorption, the most
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Table 29-3. Important extrahepatic gastrointestinal conditions associated with excess alcohol consumption Salivary glands and oropharynx
Esophagus
Small bowel
Colon
Pancreas
Parotid enlargement Glossitis/stomatitis Oropharyngeal malignancy
Gastroesophageal reflux disease Mallory–Weiss tears Esophageal malignancy
High-transit diarrhea Malabsorption
High-transit diarrhea
Acute and chronic pancreatitis Exocrine and endocrine pancreatic insufficiency
important cause of malabsorption is probably altered small-bowel function. Several factors contribute to alcohol-related intestinal dysfunction, including the effects of alcohol on gut motility, cellular structure, and function and blood flow. Significant increases in motility have been reported, the most obvious clinical manifestation of which is reduced transit time and diarrhea.282 Cellular changes observed in the jejunoileal epithelium of alcoholics include abnormal mitochondria, dilated ER, altered membrane fluidity, and focal cytoplasmic degradation.283 These changes are manifest macroscopically by a decrease in villous height,284 biochemically by a decrease in the activity of mucosal disaccharidases,285 and functionally by an increased permeability to water and solutes.286 Intraluminal ethanol also causes regional changes in blood flow within the jejunal mucosa.287 Together, these various effects of alcohol intake impair the absorption of a variety of nutrients and minerals, including glucose, amino acids, trace elements, and vitamins such as thiamine, B12, B6, and folic acid. This intestinal malabsorption can lead to overall weight loss and multiple deficiencies of micronutrients. The role of oxidative stress in the pathogenesis of many alcohol-related diseases highlights the importance of micronutrient deficiencies in antioxidant vitamins and trace elements such as zinc, manganese, and selenium.288
Colon Alcohol has been shown to have direct effects on colonic motility, with alcohol increasing propulsive activity and contributing to alcohol-induced diarrhea. This effect can be observed following alcohol withdrawal when colorectal transit time increases significantly from approximately 25 h to 33 h.289 There is no confirmed impact of alcohol on the incidence of colorectal cancer.
Pancreas Alcohol can cause a chronic, recurrent, calcifying pancreatitis, typically after a period of at least 6 years of heavy consumption. There is also an established association between excessive alcohol intake and acute pancreatitis. In practice, the first clinical episode of acute pancreatitis will occur after the histological changes of chronic pancreatitis have been well established. With time, attacks often become less severe as the features of pancreatic insufficiency set in. The precise mechanisms of alcohol-related pancreatic damage are unclear, though alcohol per se does not seem to be directly toxic.290 As in alcohol-induced liver disease, oxidative stress may play a role.291 Oxidative injury can lead to a block in exocytosis, leading to the shunting of secretions into the interstitium. The resulting inflammatory response leads initially to acute pancreatitis and, if the insult (excess alcohol intake) persists, eventually to chronic pancreatitis as the acini dedifferentiates into tubular structures, losing their secre-
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tory capacity and fibrosing. The fibrosis is particularly prominent in areas of fat necrosis,292 presumably reflecting the direct fibrogenic effect of lipid peroxidation products. If alcohol abuse continues after the first episode of pancreatitis, the majority of patients will suffer from recurrent attacks of pain, occurring at intervals of weeks or months. Eventually, with progressive loss of acinar tissue, patients develop clinical features of chronic pancreatitis. These include diabetes mellitus, reflecting impaired endocrine function, and malabsorption associated with steatorrhea due to impaired exocrine function. It remains controversial whether or not alcohol abuse is a risk factor for pancreatic cancer.
CARDIOVASCULAR SYSTEM Acute and chronic alcohol ingestion leads to a variety of beneficial as well as deleterious effects on the heart and cardiovascular system. There now seems little doubt that moderate alcohol intake is associated with a decreased risk of ischemic heart disease293 while excessive alcohol intake can lead to hypertension and disordered cardiac rhythm, including sudden cardiac death, cardiomyopathy, and cerebrovascular accidents. This dual effect of alcohol on the cardiovascular system is largely responsible for the well-known U- or J-shaped curve describing the relationship between alcohol intake and total as well as cardiovascular-related mortality.294 This shows that mortality amongst light (1–9 drinks per week) and moderate (10–34 drinks per week) drinkers is lower than in abstainers and heavy drinkers. The left-hand part of the curve is due to an inverse relationship between death from coronary artery disease and alcohol intake, while the right-hand portion is attributable to a greater risk of non-ischemic cardiovascular and non-cardiovascular deaths (accidents, suicide, cancer, liver disease) in heavy drinkers. Importantly, contrary to popular belief, almost 50% of the excess deaths occurring in heavy drinkers are attributable to circulatory diseases rather than to liver disease.295
Hypertension A number of epidemiological studies, controlling for variables such as diet and smoking, have established a dose–response relationship between blood pressure and alcohol consumption.296–298 It has been estimated that 30% of all cases of hypertension may be attributable to alcohol with females apparently less susceptible.299 The threshold for alcohol-associated hypertension appears to be around three standard drinks per day, with some studies showing a dose–response relationship with higher levels of intake.300 Findings from short-term studies have suggested that cessation of alcohol consumption in hypertensive patients results in a decrease in blood pressure.299 Whether alcohol-induced hypertension remains reversible in the
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long term is unknown. The mechanisms underlying the association between alcohol and hypertension are unclear.
Coronary Artery Disease As discussed, in recent years a number of epidemiological studies have demonstrated a negative correlation between moderate consumption of alcohol and fatal coronary artery disease.301 Case–control studies have also shown a lower incidence of myocardial infarction in moderate drinkers compared to abstainers.302 In these studies “moderate” drinking was no more than two drinks per day in men and one drink per day in women. Supportive evidence for a protective effect of alcohol on ischemic heart disease is provided by its biological plausibility.303 Moderate alcohol consumption increases the plasma levels of the protective high-density lipoprotein cholesterol by as much as 33%.304 The mechanism is likely to be a result of altered hepatic synthesis and secretion of lipoproteins. Alcohol intake is also associated with impaired platelet aggregation305,306 and lower levels of fibrinogen, thereby reducing the risk of thrombo-occlusive events.
Cerebrovascular Disease All types of strokes have been associated with alcohol consumption. This is perhaps not surprising in view of the association between alcohol and most of the established stroke risk factors, including hypertension, cardiomyopathy, arrhythmias, diabetes, and cigarette smoking.307 In view of its negative association with coronary heart disease, it might be expected that moderate consumption would be associated with a reduced risk of ischemic stroke. The consumption of one drink per day has been associated with a reduced risk of ischemic stroke in one study308 but this has not been confirmed in other similar studies, with some reporting a positive association between heavy alcohol intake and cerebral infarction in young men following alcohol “binges.”309 This may be attributed either to dehydration or to the occurrence of alcohol-related supraventricular arrhythmias with resulting embolic events. The expected positive association with hemorrhagic strokes has been reported310 but it remains unclear whether this association is independent of alcohol’s effect on other risk factors, particularly hypertension.
Cardiomyopathy It has been recognized since the early 1960s that long-term, heavy alcohol consumption is the main cause of a non-ischemic, dilated cardiomyopathy. Postmortem and endomyocardial biopsy studies performed in chronic alcoholics both with and without cardiac symptoms have shown dilation of the atria and ventricles, increased myocardial mass, interstitial fibrosis, and small-vessel coronary artery disease.311 While subclinical alcoholic cardiomyopathy, characterized by left ventricular hypertrophy and mild systolic and diastolic dysfunction, appears to be relatively common in heavy drinkers, clinical presentation is relatively uncommon and appears to require at least 5 years of > 90 g ethanol per day. Interestingly, the duration of drinking seems to be more important than the dose over this threshold, with the mean duration in symptomatic patients being 25 years of heavy drinking compared to 16 years in asymptomatic patients.312 The onset is usually insidious, with non-specific fatigue and chest pain associated with palpitations, most commonly
due to atrial fibrillation. As the disease progresses, features of biventricular failure develop. With continued drinking, death from cardiac failure or arrhythmias usually occurs within 4 years of presentation, although in the early stages of disease dramatic recovery can occur with abstention.313
Arrhythmias and Sudden Cardiac Death Heavy drinking increases the risk of cardiac arrhythmias whether or not heart disease is present. This evidence has come from clinical observations, retrospective case–control studies, controlled studies of consecutive admissions for supraventricular tachyarrhythmias, and prospective epidemiological studies.314 The association is best established for atrial fibrillation, although in one study individuals drinking more than six drinks per day had a higher risk of all supraventricular tachyarrhythmias than those drinking less than one drink per day when matched for age, sex, and smoking.315 The tendency of these arrhythmias to present following weekend or holiday “binges” has led to the term “holiday heart syndrome.” Alcohol has also been shown to promote the onset of ventricular tachyarrhythmias316 and this presumably explains the increased incidence of sudden cardiac death observed in heavy compared to occasional or light drinkers.317 The mechanism of alcohol-related arrhythmogenesis is almost certainly multifactorial. Factors that may play a role include subclinical cardiomyopathy producing conduction delays, potassium and magnesium depletion, the hyperadrenergic state accompanying alcohol withdrawal, autonomic neuropathy, and a direct effect of ethanol on cardiac conduction.316 The mechanism of ventricular tachyarrhythmias is most likely early after-depolarizations provoked by catecholamine release and potassium depletion during withdrawal in the presence of a prolonged action potential due to the autonomic neuropathy. In support, patients with a prolonged action potential, manifest on the surface electrocardiogram as QT interval prolongation, have been shown to be at risk of sudden cardiac death.318
EFFECTS ON THE NERVOUS SYSTEM Acute and chronic alcohol intake is associated with a wide range of effects on the nervous system. The depressant effect of alcohol means that acute heavy consumption can lead to blackouts and even coma. After a sudden reduction in alcohol consumption, tremulousness and agitation are common, while the full-blown syndrome of delirium tremens, including hallucinations and seizures, is less frequently seen, and more serious. Alcohol and its metabolite acetaldehyde are almost certainly directly neurotoxic, but associated nutritional deficiencies undoubtedly contribute to the pathogenesis of some, if not all, alcohol-related neurological diseases.319
The Wernicke–Korsakoff Syndrome The Wernicke–Korsakoff syndrome is a nutritional disorder caused by thiamine deficiency, and is predominantly observed in alcoholics. Wernicke’s encephalopathy represents its acute phase, while Korsakoff ’s psychosis represents the chronic continuation of the disease. The major pathologic changes of this syndrome are predominantly in the paraventricular parts of the thalamus and hypothalamus, the mammillary bodies, the periaqueductal gray matter,
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and the floor of the fourth ventricle. An abrupt onset and the triad of oculomotor disturbances, cerebellar ataxia, and mental confusion characterize classical Wernicke’s encephalopathy. The most common ocular abnormality is nystagmus (vertical or horizontal), but bilateral sixth-nerve palsy, palsies of conjugate gaze, and complete ophthalmoplegia are also seen. Ptosis and pupillary abnormalities may also occur. Mental inattention is characterized by disorientation, inattention, and unresponsiveness, which progresses to coma if untreated. Treatment is with parenteral thiamine. The disease can be aggravated by giving intravenous dextrose before thiamine supplementation is administered. Patients either recover within 48– 72 h or progress to Korsakoff ’s psychosis. Korsakoff ’s psychosis is characterized by various degrees of both retrograde and anterograde amnesia, with relative preservation of other intellectual functions. The Korsakoff state is potentially reversible by early intervention with thiamine and prompt treatment of Wernicke’s encephalopathy. Unfortunately, recovery is incomplete in more than 50% of cases and individuals may be left with devastating chronic memory deficits.
Bordeaux, France, revealed that consumption of between 250 and 500 ml of wine per day resulted in a significant reduction in the risk of dementia and Alzheimer’s disease in later life.321 Computed tomography (CT) studies have shown a substantially higher incidence of cerebral cortical atrophy in alcoholics, and brain weights in chronic alcoholics at autopsy are less than half that of agematched controls. However, no correlation has been demonstrated between either the CT or histological changes and the neuropsychological impairment frequently seen in chronic alcoholics. For example, ventricular and sulcal enlargements are often seen on CT in alcoholics with no clinical evidence of cerebral dysfunction. Furthermore, there is little firm evidence of any histological abnormality in the brains of alcoholics other than that related to the complications of alcoholism such as Wernicke’s encephalopathy, post-traumatic changes, and chronic hepatocerebral degeneration. Thus, cerebral atrophy is common in alcoholics, and dementia may occur as a result of the direct toxic effect of ethanol on the brain, but there is no defined clinicopathological entity that constitutes “alcoholic dementia” and the mental disturbances are more likely to be related to other established complications of alcohol abuse.
Cerebellar Disease Alcoholic cerebellar degeneration is characterized clinically by an ataxic gait and truncal ataxia, while typically the upper limbs are unaffected.320 Pathologically there is degeneration of the cerebellar cortex, predominantly of the anterior and superior vermis and anterior lobes. In most cases the syndrome evolves over a period of several weeks or months, after which it remains unchanged for years. Acute cerebellar degeneration may respond to large doses of thiamine and abstinence, but patients usually present long after the onset of their symptoms. At this stage the likelihood of improvement is small, and probably occurs as a result of an improvement in the peripheral neuropathy which is present in around half the patients.
Brainstem Disease Central pontine myelinolysis is a rare demyelinating disease characterized by neuronal dysfunction centered on the pons. It is encountered predominantly in malnourished alcoholics with disordered electrolytes. Cerebral edema associated with either severe hyponatremia or the rapid correction of hyponatraemia during electrolyte replacement may play a role in the pathogenesis. Clinical features include the subacute onset of a progressive quadriparesis, pseudobulbar palsy affecting speech and swallowing, and paralysis of horizontal eye movements. More extensive brainstem dysfunction may result in pupillary abnormalities, decerebrate posturing, altered conscious level, and respiratory paralysis. Not surprisingly, the prognosis of this condition is poor, with the diagnosis often only made at postmortem. Central pontine myelinolysis may be associated with Marchiafava–Bignani syndrome which is a rare demyelinating disease of the corpus callosum also occurring predominantly in alcoholics. This presents with acute bilateral hemispheric dysfunction and has a poor prognosis.
Neuropathies Peripheral neuropathy is another common nutritional complication in alcoholics. The precise mechanism is unclear but histology reveals a non-inflammatory degeneration of myelin sheaths and axon cylinders, which is more intense in distal segments. In advanced cases, degeneration may also be observed in the anterior and posterior roots of the spinal cord. Patients with electrophysiological evidence of peripheral neuropathy can be asymptomatic, or, more typically, present with pain and paresthesia initially affecting the lower limbs. In severe cases weakness and atrophy may be seen. With continued drinking the symptoms progress relentlessly, so that in advanced cases significant distal motor deficits with atrophy may be seen. Treatment consists of abstinence and nutritional supplementation, particularly with B-vitamins. Recovery is slow and often incomplete. An association between alcoholism and autonomic neuropathy was first reported in 1980.322 The subsequent observation that it was more common in alcoholics with liver disease than those without suggested that the liver disease rather than alcohol per se might be the primary cause.323 This hypothesis was supported by a report that the incidence of autonomic neuropathy was similar (45%) in patients with alcohol and non-alcohol-related liver disease.324 More recently, evidence for a reversible metabolic effect of liver disease on autonomic function has been provided by a study demonstrating an improvement in autonomic function 3 months after successful liver transplantation.325 Importantly, autonomic neuropathy is associated with an adverse prognosis in patients with liver disease, attributed either to an impaired response to stresses or to the associated QT interval prolongation and subsequent risk of ventricular arrhythmias.318 As many as 50% of patients with liver disease experience typical symptoms of autonomic neuropathy, including postural dizziness, abnormal sweating, and impotence.324
Alcoholic Dementia While some studies suggest that a high level of alcohol consumption may be a contributing factor in some of cases of dementia, this is an area of great controversy. One study of moderate drinkers in
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ALCOHOLIC MYOPATHY Alcoholic myopathy can occur in an acute form with variable severity. In the mild form it may represent a mild rise in muscle enzymes,
Chapter 29 ALCOHOLIC LIVER DISEASE
while at its most severe there may be rhabdomyolysis. In the severe form the patient presents, often after a heavy bout of drinking, with muscle pain and weakness. Muscle enzymes are markedly raised and electromyography abnormal. Occasionally the rhabdomyolysis can be severe enough to cause myoglobinuria and acute renal impairment. The condition improves in most cases over days. Alcoholic myopathy also has a more common, chronic form, which presents with progressive, painless weakness and wasting of the proximal muscle groups. The condition is associated with chronic alcohol consumption, and histology reveals a loss of type IIb muscle fibers. While ethanol may partly induce myopathy through the wellcharacterized neuropathy, there appears to be an additional impact of ethanol and its metabolism on muscle per se. These mechanisms remain unclear, but are likely to involve the generation of oxidative stress, mirroring other end-organ damage.
THE FETAL ALCOHOL SYNDROME A conservative estimate for the incidence of fetal alcohol syndrome has been put at 0.33 per 1000 live births, with many more children suffering from various alcohol-related effects not amounting to the full syndrome.326 A similar per capita frequency is likely to occur in other industrialized countries, but few data are available on the magnitude of this problem in the developing or third world. Fetal alcohol syndrome is caused by excessive alcohol consumption during pregnancy which results in a variety of abnormalities in the fetus, thought to be due to a direct effect of alcohol and its metabolite acetaldehyde, rather than to associated nutritional deficiencies or other drugs. The severity of the syndrome depends on both the timing and severity of maternal alcohol consumption during gestation. The diagnostic criteria include features of growth retardation and developmental delay, central nervous system involvement, and characteristic facial dysmorphology in the presence of a maternal alcohol consumption of more than two drinks per day. The central nervous system involvement typically presents as behavioral dysfunction and mental retardation. The characteristic facial features include short palpebral fissures, an elongated mid-face, an indistinct philtrum, a thin vermilion, and a foreshortened maxilla.
ALCOHOL AND CANCER Results from several large epidemiological studies have firmly established that alcohol is associated with a higher cancer incidence and mortality.327 Alcohol consumption is most strongly associated with cancers of the esophagus (as discussed above), oropharynx and larynx, with the increased risk particularly prominent in smokers (Table 29-4). The controversy over the association between alcohol Table 29-4. Cancers confirmed to be associated with excess alcohol consumption Alcohol and cancer Mouth Pharynx Larynx Esophagus Breast Liver
and breast cancer has been resolved by two large meta-analyses. The first was a meta-analysis of six prospective cohort studies.328 This has clearly demonstrated that, for intakes less than 60 g/day, breast cancer risk increases linearly with intake. A daily intake of 30–60 g was associated with a relative risk of 1.41 (1.18–1.69) when compared to non-drinkers and this risk was independent of other known risk factors. The second, more recent study had very similar findings.329 The mechanisms underlying alcohol-related cancers are unclear but several factors have been suggested to play a role. Alcohol may be important in the initiation of cancer, either by increasing the expression of certain oncogenes, or by impairing the cell’s ability to repair DNA, thereby increasing the likelihood that oncogenic mutations will occur. Alcohol may act as a co-carcinogen by enhancing the effect of direct carcinogens such as those found in tobacco and the diet. This effect of alcohol may be, at least in part, via induction of the cytochrome P450 family of enzymes that are found in the liver, lung, and intestine and are capable of metabolizing various tobacco and dietary constituents into cancer-promoting free radicals. Since reduced levels of iron, zinc, and vitamins A, B, and E have been experimentally associated with some cancers, the nutritional deficiencies associated with chronic alcohol intake may also play a role in alcohol-related cancers, possibly by increasing the magnitude of free radical-related oxidative stress. Finally, alcoholism is associated with immunosuppresion, which makes chronic alcoholics more susceptible to infection and theoretically reduces immune surveillance of early tumors.
HEMATOLOGICAL COMPLICATIONS Heavy alcohol consumption, with or without liver disease, can have profound effects on the hematological system. While the earliest and most obvious effects are on erythrocytes, derangements in production, function, and consumption of leukocytes, platelets, and coagulation can also have important consequences.
Erythrocytes It has long been recognized that many alcoholics have increased mean corpuscular volume. This simple macrocytosis can occur in the absence of vitamin deficiency, and is thought to be a direct effect of alcohol, or the products of its metabolism, on the development of the red cell. In line with this, the corpuscular volume returns to normal several weeks after abstinence. In true folate deficiency, there may also be a macrocytic, megaloblastic anemia. This is quite common in alcoholics, who may also suffer from vitamin B12 deficiency, which can cause a similar picture. Alcohol can also promote a sideroblastic anemia in which heme synthesis is impaired. In this condition serum ferritin is raised and the red cells are hypochromic in the peripheral blood, and have ring sideroblasts in the marrow. Heavy drinkers also suffer from the anemias associated with some degree of alcohol-induced liver injury. Zieve’s syndrome is a condition described in 1958,330 typically found in middle-aged male heavy drinkers with alcoholic fatty liver and severe hyperlipidemia. It is rare, and improves with abstinence. This is in contrast to the more concerning spur-cell anemia, which tends to be associated with advanced alcoholic cirrhosis, though it can occasionally occur in
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other types of cirrhosis. Spur cells, or acanthocytes, are caused by the equilibration of the outer lipid layer of the cell membrane with the cholesterol-rich abnormal lipoproteins in the plasma. The problem is compounded by the reduced fluidity of the red-cell membrane seen in cirrhosis due to a reduction in the proportion of polyunsaturated versus saturated fatty acids. The acute development of spur-cell anemia is a poor prognostic indicator in alcoholic cirrhosis, and has been considered an indication for transplantation, which may be curative. Finally, red-cell consumption can occur as a result of congestive hypersplenism resulting from liver disease and portal hypertension.
Leukocytes Chronic alcoholics are more prone to a variety of infections331 and malignancies.332 This sensitivity has often been attributed to defects in innate and acquired immunity due to ALD and ethanol consumption per se. While the effects of ethanol on neutrophil333,334 and macrophage function128,335,336 have been well investigated, revealing in particular a disruption in phagocytosis337 and antigenprocessing and presentation,338 studies into the effects on lymphocyte functioning have been less conclusive. A uniform finding, however, in both chronic alcoholics and chronically ethanol-fed animals is a reduction in circulating T-cell numbers, and, in mice, a reduction in spleen and lymph node size.339,340 Whether this reduction in numbers is primarily due to a failure of proliferation or an increase in apoptotic rates is, however, unclear. It appears therefore that chronic, or even acute, ethanol consumption may alter the host’s ability to mount an appropriate-magnitude immune response.341
Platelets While an acute single dose of ethanol may not affect platelet number or function, chronic heavy drinking does. The changes that occur can do so in the absence of folate deficiency or hypersplenism, although these problems can compound the condition. The thrombocytopenia seen as a consequence of alcohol ingestion appears to be a direct myelosuppressive effect of ethanol on bone marrow megakaryocytes, and is usually mild, and rarely of clinical consequence.342 In addition, ethanol can also affect platelet function, even in the absence of thrombocytopenia. Chronic heavy drinkers have been found to have prolonged bleeding times and platelets that are significantly less responsive to standard platelet aggregation tests and have decreased thromboxane A2 release. When these patients are followed up during an in-hospital period of abstinence, these abnormalities return to normal during 2–3 weeks of alcohol withdrawal.
Coagulation One of the difficult problems in patients with ALD is the derangement in coagulation which can compound acute episodes of gastrointestinal bleeding. These disturbances are common and complex. Liver synthesis of clotting factors can be impaired by hepatocellular dysfunction or inadequate absorption of vitamin K, which is required for the synthesis of factors II, VII, IX, and X. These abnormalities present with an abnormal PT. Treatment requires replacement of the factors plus vitamin K. In many cases of cirrhosis there will also be a reduced fibrinogen level.
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EFFECTS ON THE ENDOCRINE SYSTEM The pathogenesis of decreased libido and impotence in heavy drinkers is not fully understood. There is evidence from human and rat studies that chronic alcohol consumption reduces testosterone synthesis. While alcoholics may develop the resulting hypoandrogenization, there is not the expected rise in gonadotrophins that normal accompanies this end-organ failure.343 This in turn suggests a problem with the hypothalamic and pituitary feedback mechanisms. These problems are compounded by the hyperestrogenization seen in liver disease and manifested by spider nevi and gynecomastia. Heavy alcohol consumption may also induce changes in peripheral testosterone and estrogen metabolism as well as changes in estrogen receptors. Even in patients without liver disease, alcohol can affect fertility. In men, abnormal spermatogenesis is more frequent, with decreased numbers and motility of sperm. In women, amenorrhea, anovulation, and accelerated onset of menopause have all been associated with alcohol intake. Alcohol-induced pseudo-Cushing’s syndrome has the same characteristics as classical Cushing’s, namely moon face, central obesity, muscle wasting, abdominal striae, fatigue, easy bruising, and hypertension.344 This syndrome can be indistinguishable from true Cushing’s syndrome, except for the fact that it resolves with abstinence and may recur when heavy drinking is resumed. Whether pseudo-Cushing’s is a true identity or simply a syndrome combining several of the clinical features of alcohol abuse is presently in debate. In addition to the endocrinological associations of heavy drinking described above, there are also more subtle effects resulting in a reduction in growth hormone and a rise in prolactin. The first of these has no direct impact apart from enhancing hypoglycemia, described below. The second can exacerbate the effects of hypogonadism and hyperestrogenism.
Hypoglycaemia and ketoacidosis Inhibition of hepatic gluconeogenesis, depleted hepatic glycogen stores, and deranged glucocorticoid secretion may all contribute to the presentation of the alcoholic with severe hypoglycemia. These often malnourished patients are prone to episodes of ketoacidosis which, when compounded with starvation and vomiting, can be lifethreatening.
HYPERURICAEMIA Hyperuricemia is caused by a decrease in the excretion of uric acid, secondary to hyperlactacidemia. Lactate competitively inhibits uric acid clearance by the proximal renal tubule, and consequently reduces its excretion. This situation is exacerbated by the alcoholinduced increase in urate synthesis secondary to accelerated degradation of adenine nucleotides.
OSTEOPOROSIS Even in the absence of liver disease, alcohol can cause osteopenia,345 possibly through a direct toxic effect on osteoblasts and bone remodeling. This loss of bone can result in an increased incidence of fractures in alcoholics. While the pathogenesis of this problem may involve the influence of endocrine factors, such as pseudo-
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Cushing’s or hypogonadism, nutritional deficiencies associated with alcoholism, low levels of osteocalcin, which rapidly rises on abstinence, point to a more direct effect of alcohol itself on bone formation.346
TREATMENT The age-adjusted death rate from all-cause cirrhosis, the greatest percentage of which is alcohol-related, fell by 45.5% in the USA between 1970 and 2001. This greatly exceeded the age-adjusted allcause mortality, which fell by 28.7%. The total annual per capita ethanol consumption rose steadily from 9 liters (2 gallons) to 12.6 liters (2.8 gallons) between 1955 and 1980 and then dropped back to around 9.9 liters (2.2 gallons) in the following 20 years. In crude terms, it appears, therefore, that the improvement in alcoholic cirrhosis mortality exceeds the reduction in ethanol consumption. This may be due to the fact that there are fewer individuals drinking heavily and more drinking within healthy levels. It may also reflect improvements in the management of patients with ALD. Clearly, any improvement in the treatment of the complications of cirrhosis will have a beneficial effect on the management of patients with end-stage ALD. However, this section focuses on treatment strategies that have been specifically directed at mechanisms involved in the pathogenesis of alcohol-related liver injury and considers their current and future role in the management of patients with the various stages of ALD.
ACHIEVING ABSTINENCE Since cessation or a marked reduction in alcohol intake has been shown to improve the histology and/or survival of patients with all stages of ALD,259,261,267 measures aimed at establishing and maintaining abstinence are critical in the management of patients with ALD. This is best achieved by close liaison between liver physicians and addiction psychiatrists with support from specialist alcohol nurses and trained counselors.347 Available treatments for alcoholdependent patients can be divided into psychological and pharmacological. So-called “brief interventions” are the simplest form of psychological therapy and can be implemented by non-psychiatric staff. This involves educating and informing the patients about the nature of their problem and providing them with advice on how to go about changing their behavior. In spite of the apparent simplicity of this form of management, brief interventions have been shown significantly to increase the chances of heavy drinkers moderating their drinking at 6 and 12 months in an outpatient setting.348 With only minimal training, medical and nursing staff can also deliver a variety of manual-guided psychosocial treatments, including cognitive-behavioral therapy and motivational enhancement therapy, both of which have been shown to reduce drinking in dependent patients in a randomized controlled trial.349 As an alternative, or an addition, to psychological therapies, some patients may derive benefit from pharmacological therapy (Table 295). Both acamprosate and naltrexone have been shown to reduce drinking days and increase abstinence rates in more than one randomized controlled trial and a recent meta-analysis.350–352 Acamprosate is derived from taurine and its beneficial effect is thought to be via binding to the g-aminobutyric acid receptor with a reduc-
Table 29-5. Non-pharmacological and pharmacological therapies to obtain and maintain abstinence Non-pharmacological • Brief intervention • Cognitive therapy • Motivational enhancement therapy • Psychotherapy Pharmacological Acamprosate Naltrexone Disulfiram
• • •
tion in the neuronal excitation that is normally observed during alcohol craving. Importantly for patients with ALD, acamprosate, unlike naltrexone, is well tolerated in all but patients with Child–Pugh C cirrhosis353 and its benefit seems to persist for at least 1 year after treatment withdrawal. Disulfiram, an inhibitor of acetaldehyde dehydrogenase, has been used for many years in the management of alcohol-dependent patients. As discussed previously, it induces an acetaldehyde-mediated adverse reaction to alcohol intake characterized by nausea and flushing. Trials of effectiveness, however, have given conflicting results.354,355 The drug also requires compliance and its potential for hepatotoxicity has limited its use in patients with established ALD.356 Importantly, there have been no formal trials of either psychological or pharmacological therapies in drinkers with ALD. However, previous evidence that the severity of alcohol dependence in ALD patients is less than that observed in an unselected group of alcoholdependent patients357 suggests that these treatments may be even more beneficial in the ALD population. Consistent with the low level of dependency, up to 50% of ALD patients will either abstain completely or achieve a significant reduction in intake after being given simple advice by physicians during their initial presentation, with a significant improvement in survival compared to continued heavy drinkers.358
ALCOHOLIC HEPATITIS Alcoholic hepatitis covers a spectrum of disease from subclinical to a severe, life-threatening disorder. Independent predictors of survival in these patients are serum bilirubin, PT, and the presence of hepatic encephalopathy. As discussed, the two laboratory indices have been combined to derive a DF (bilirubin (mg/dl) + 4.6 (PT prolongation)), and a value of 32 or greater has been shown to predict a high short-term mortality in several prospective studies.140,257,359,360 Accordingly, almost all treatment trials in patients with alcoholic hepatitis have been short-term (usually 1 month) and restricted to patients with a DF > 32 and/or encephalopathy. Patients with less severe disease appear to have a good short-term prognosis even when jaundiced.260 Accordingly, in these patients and the severe patients surviving their initial presentation, treatment is focused on achieving abstinence, which has been convincingly shown to improve long-term outcome.259,261,267 Reports that some patients with alcoholic hepatitis can progress to cirrhosis even with abstention,261 and that patients with coexisting alcoholic hepatitis and cirrhosis have a worse long-term survival than patients with cirrhosis
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only,268 suggest the need for longer-term treatment trials in patients with alcoholic hepatitis. Progress in developing specific treatments for acute alcoholic hepatitis has been hampered by a poor understanding of disease pathogenesis. Reflecting this paucity of information, many treatment modalities have been tried in patients with alcoholic hepatitis; however, none has been consistently shown to have a beneficial effect and, accordingly, none has achieved consensus status among practicing hepatologists.
Despite 13 randomized controlled trials and six meta-analyses, the debate over the use of steroids continues. It appears that they are probably beneficial in patients with severe disease; however, mortality on treatment remains high, particularly when renal impairment is present, and treatment is relatively contraindicated in the large number of patients with concomitant infection and gastrointestinal bleeding. It is because of these limitations that alternative therapeutic strategies have been sought.
Pentoxifylline Corticosteroids Of all the treatments available for patients with severe alcoholic hepatitis, corticosteroids are the most intensively studied, and probably the most effective. Steroids are aimed at suppressing or “switching off ” the hepatic inflammatory response seen in liver biopsies from patients with severe alcoholic hepatitis. The mechanism of this effect is, at least in part, through the inhibition of NFk-B transcriptional activity.361 The transcription of many inflammatory cytokines, chemokines, and adhesion molecules is dependent on the NFk-B signaling cascade.362 Two important side effects of steroids used in medium dose include poor wound-healing and susceptibility to infection, both of which can lead to life-threatening complications in this group of patients. Concern over these adverse effects coupled with a continued uncertainty over efficacy has contributed to the reluctance of many clinicians to prescribe steroids for patients with alcoholic hepatitis. Patients with alcoholic hepatitis form a heterogeneous population, both in severity and probably in disease pathogenesis. Without a liver biopsy it is difficult to differentiate a patient with severe acute inflammatory alcoholic hepatitis from one with alcohol or nonalcohol-induced cirrhosis that has decompensated while drinking. Many initial trials of steroids were poorly designed and included patients with a variety of disease severities and almost certainly patients without alcoholic hepatitis. Most of these trials showed no treatment benefit. However, two randomized controlled trials focused only on patients who had the worst prognosis, defined by a DF of ≥ 32 and/or encephalopathy, and both showed a survival benefit in the steroid-treated patients.257,360 Several meta-analyses have attempted to resolve the controversy, and although most have shown a survival benefit,363–365 this has not been a universal finding.366 Rather than performing a further conventional meta-analysis, the authors of the last three large randomized controlled trials have pooled their individual patient data, only including patients with encephalopathy and/or a DF > 32.367 This study showed that steroids improved survival versus placebo (85 versus 65%), with placebo treatment, increasing age, and creatinine independent predictors of mortality on multivariate analysis. A weakness of this study is that two of the three original trials included gastrointestinal bleeding as a contraindication, while one did not and only one trial required a liver biopsy for diagnosis. Nonetheless, the large numbers (102 on placebo, 113 on steroids) make this the most robust meta-analysis to date. The same group has now published evidence to support the withdrawal of steroids if the bilirubin has not fallen by the seventh day of steroid treatment. This simple clinical observation significantly reduces the length of treatment in nonresponders.368
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As discussed in the section on pathogenesis, there is good evidence from animal models that TNF-a plays an important role in acute alcohol-mediated liver injury. Pentoxifylline (PTX) is a non-selective phosphodiesterase inhibitor that is approved for use in claudication at a dose of 400 mg three times a day due to its effect on red blood cell deformability. In the late 1980s, PTX was observed to have an anticytokine effect, later attributed to a reduction in TNF-a gene transcription369 and, accordingly, to reduced levels of important downstream TNF-a effectors, including other proinflammatory cytokines, chemokines, and adhesion molecules. The first randomized controlled trial of PTX in 101 patients with alcoholic hepatitis was reported in 2000.370 The effective claudication dose was given for 28 days to patients with a DF > 32 and led to a 40% reduction in mortality compared to placebo. The secondary end-point of HRS was reduced in the treated population by 65%. Importantly, almost all of the improvement in survival was due to a fall in mortality from HRS, suggesting that PTX may have a specific beneficial effect in alcoholic hepatitis patients developing this ominous complication.371 Clearly, further trials are needed to determine whether PTX should become standard treatment for patients with alcoholic hepatitis. In particular, comparisons should be made with steroids and placebo (in patients in whom steroids are contraindicated), and trials of PTX in combination with steroids should be performed.
Nutritional Supplementation Trials investigating the role of nutritional supplementation have been prompted by the degree of protein–calorie malnutrition seen in patients presenting with acute alcoholic hepatitis and the correlation between the severity of malnutrition and mortality.372 Initial trials with parenteral amino acid therapy yielded conflicting results;373,374 however, more consistent and promising results have been reported from two randomized controlled trials of enteral tube-feeding. The first compared enteral tube feeding of an energydense formula supplying 2115 kcal/day with an isocaloric standard oral diet.375 The enteral feed contained whole protein plus branchedchain amino acids, medium- and long-chain triglycerides, and maltodextrin. Thirty-five severely malnourished cirrhotics were randomized and in-hospital mortality was 12% in the tube-fed group compared to 47% in the oral group. This prompted a further study 10 years later comparing enteral feeding to steroids in 71 patients with acute severe alcoholic hepatitis. In this trial, whilst there was no difference in mortality between the groups during the 28-day treatment period, deaths occurred earlier in the steroid-treated patients and the mortality rate was lower in the enterally fed group in the year following treatment. The overall mortality rate at 1 year was 61% and 38%, in steroid- and enteral-treated groups, respec-
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tively. While this difference did not reach significance (P = 0.26), it must be appreciated that this treatment was being compared with what is currently considered to be the best available treatment. Furthermore, the trial is ongoing,376 with a further 69 patients expected to be recruited. A further pilot study in a small group of patients has shown promising results for enteral nutrition combined with a shortened course of steroids. Further, larger studies are required to confirm this benefit. In summary, nutritional supplementation may have a role in improving medium- to long-term survival in patients with severe alcoholic hepatitis. Which patients benefit most and the mechanisms by which they derive benefit are, as yet, unclear. There is no doubt, however, that this form of treatment deserves further investigation.
Antioxidants Interest in the potential value of antioxidant therapy in the treatment of alcoholic hepatitis has arisen as a result of the growing body of evidence, discussed above, implicating oxidative stress as a key mechanism in alcohol-mediated hepatotoxicity. These considerations have recently led to three trials investigating the effect of antioxidant supplementation in patients with severe alcoholic hepatitis. In the first study, 56 patients were randomized to receive vitamin E, selenium, and zinc supplementation or placebo.377 Whilst treated patients had an in-hospital mortality of 6.5% compared with 40% in the placebo group, the entry criteria and patient details were not clear. The second trial compared steroids with an antioxidant cocktail (vitamins A, C, E, selenium, allopurinol, desferrioxamine, and N-acetylcysteine) and was stopped after an interim assessment found steroid treatment to be associated with a significantly higher survival.378 This trial did not examine whether antioxidants conferred any benefit in patients in whom steroids were contraindicated, or in combination with steroids. The most recent study investigated the role of antioxidants in patients with severe alcoholic hepatitis stratified by gender and steroid treatment. The active group received a loading dose of N-acetylcysteine of 150 mg/kg followed by 100 mg/kg per day for 1 week, and vitamins A–E, biotin, selenium, zinc, manganese, copper, magnesium, folic acid, and coenzyme Q daily for 6 months. The decision to treat with steroids was made by the supervising clinician according to conventional criteria.92 While white blood cell count and bilirubin at trial entry were both associated with increased mortality, antioxidant therapy showed no benefit either alone or in combination with steroids. In summary, on the basis of the data available thus far, high-dose antioxidant therapy confers no survival benefit in patients with severe alcoholic hepatitis.
Hepatic Mitogens The observation that survival in patients with severe alcoholic hepatitis correlates with the intensity of hepatocyte staining for proliferating cell nuclear antigen379 implies that the liver’s capacity for regeneration is an important determinant of outcome and suggests that therapy directed at enhancing proliferation might be beneficial. An infusion of insulin and glucagon has been shown to improve liver regeneration in a rat partial hepatectomy model,380 and to improve survival in a mouse model of fulminant hepatitis.381 These observations were the stimulus to several trials investigating the role of
insulin and glucagon therapy in the treatment of patients with severe alcoholic hepatitis. While the first trial showed a significant reduction in mortality in the treated group,382 two subsequent larger studies showed no benefit, with one reporting a high incidence of hypoglycemia.383,384 At present, therefore, this form of therapy cannot be recommended. Anabolic steroids have also been shown to promote hepatocyte regeneration; however, three large randomized controlled trials with either testosterone or oxandrolone in males with alcoholic hepatitis have reported no treatment benefit.385
Propylthiouracil Centrilobular hypoxia is a feature of animal models of ALD and has been postulated to play a role in the liver injury, which is characteristically most severe in the centrilobular acinar zone 3.386 The hypoxia has been attributed to the hypermetabolic state induced by ethanol which is similar to the hypermetabolic state associated with hyperthyroidism, and can be attenuated in the rodent model of ALD by the antithyroid drug, propylthiouracil.386 Two trials have evaluated the role of this drug in improving short-term mortality in patients with alcoholic hepatitis. Although the first trial reported a more rapid improvement in clinical and laboratory indices, neither trial showed any survival benefit.387,388
Experimental Therapies In addition to the treatments described above, several novel therapies for acute alcoholic hepatitis are currently undergoing investigation. The one that initially showed the most promise was the anti-TNF-a antibody, infliximab. This chimeric human/mouse monoclonal antibody binds to TNF-a and blocks its biological effects. Its potential use in alcoholic hepatitis has been suggested by its reported benefit in several other inflammatory conditions, the putative role of TNF-a in the pathogenesis of ALD, and a report that anti-TNF-a antibodies ameliorate the liver injury in a mouse model of ALD.389 Two initial reports demonstrated an improvement in biochemistry and a satisfactory safety profile when used alone390 or in combination with steroids.391 A further pilot study using a “sister” molecule, etanercept, also showed a satisfactory safety profile, though only 7 of 13 patients had a DF > 32.392 The safety aspect is important since experience with infliximab in other diseases has raised concerns over the risk of infection.393 This could potentially limit the number of patients with AH suitable for treatment and was the rationale for excluding patients with severe disease (DF > 55) from one of the initial trials.391 The beneficial role of TNF-a in promoting liver regeneration is another potential problem for antiTNF-a treatments in patients with alcoholic hepatitis, for the reasons discussed above.394 To date, one clinical trial has examined the role of infliximab in combination with steroids in patients with acute severe alcoholic hepatitis. Infliximab was used at twice the dose given for Crohn’s disease and at 0, 2, and 4 weeks. The study was stopped after 36 patients were randomized because of the high mortality in the treated group. Most of the deaths were infectionrelated, and the Maddrey scores were not found to be different in the treated group after a mean of 2 months.395 While this study may put a halt to further trials of infliximab in this group of patients, its role has not yet been fully assessed. There may be benefit at lower doses, instead of steroids, or for those patients who cannot have steroids because of bleeding. Randomized controlled studies are still
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required to determine which patients might benefit most from treatment, the important adverse effects, and the optimal duration of therapy. A further experimental therapy that may benefit patients with alcoholic hepatitis is the molecular adsorbents recycling system (MARS). The primary aim of this treatment is to support impaired liver function while the liver recovers or the patient undergoes liver transplantation. It may, therefore, have a role in patients with alcoholic hepatitis either alone or in combination with other pharmacological therapies. The principal of the MARS procedure is to dialyze blood against an albumin solution aimed at removing albumin-bound toxins including bilirubin and bile salts. To date, only a small series of patients with alcoholic hepatitis have been treated with this procedure;396 however, clinical improvement has been reported in these cases. In addition, a rapid fall in portal pressure has been observed within hours of starting the dialysis, which may in itself may have therapeutic potential.397 Further randomized trials are awaited with interest.
Treatment of HRS in Alcoholic Hepatitis As alluded to above, in patients with severe AH, the development of renal failure is associated with a survival of less than 10% even with intensive management and renal support.371 Perhaps the most significant advance in the management of patients with advanced liver disease over the past decade has been the introduction of albumin infusions combined with splanchnic vasoconstrictor agents for patients with HRS. This combination appears significantly to improve the survival of patients with cirrhosis who have this lifethreatening complication.398–400 Although no randomized trials have specifically examined this form of therapy in patients with alcoholic hepatitis, the previously reported high mortality in alcoholic hepatitis patients with HRS suggests that it will have a significant and beneficial impact on patient survival.
ALCOHOLIC CIRRHOSIS While the high mortality of severe alcoholic hepatitis, coupled with the young age of many of the patients, makes it an important area for therapeutic trials, the vast majority of patients with ALD in clinical practice have advanced fibrosis or cirrhosis. These patients may be asymptomatic or present with symptoms related to portal hypertension, advanced liver failure, or the development of HCC. As discussed above, the most important therapy is achieving and maintaining abstinence, since this has been shown to improve survival in both well-compensated and decompensated patients.267 Unfortunately, as with alcoholic hepatitis, no adjunctive pharmacotherapies have been consistently shown to improve survival in more than one randomized controlled trial, although some have shown promise and will be reviewed below. Potential reasons for the lack of progress thus far include: (1) a lack of a clear understanding of disease pathogenesis; (2) problems with compliance in long-term treatment trials; and (3) the confounding effect of drinking behavior during the duration of the trial. As a result, at present the management of patients with advanced fibrotic ALD is directed primarily at preventing and treating the complications of portal hypertension, liver failure, and HCC, and deciding if and when to consider patients for orthotopic liver transplantation.
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Pharmacological Therapy Propylthouracil. In contrast to its lack of effect in patients with acute alcoholic hepatitis, propylthiouracil may improve the longterm survival of patients with alcoholic cirrhosis. There has, however, been only one trial reported thus far.401 In this study, the investigators went to great lengths to assess drinking behavior and compliance by checking daily urine samples for alcohol and a drug biomarker. Treatment for 2 years improved mortality in the patient group as a whole, particularly in patients who continued to drink moderately during the trial. No improvement was seen in abstinent patients, who had an excellent prognosis on drug or placebo or in continued heavy drinkers who had a universally bad prognosis. Although the patient numbers were high,310 a large percentage of patients were either non-compliant or dropped out of the study. For this reason and the lack of any confirmatory studies, propylthiouracil has not been widely adopted by the liver community. In view of the promising results, however, it does seem surprising that no centers have attempted to repeat the study, which remains an excellent model of how to perform a randomized controlled trial in this potentially difficult group of patients. Colchicine. This anti-inflammatory drug has been evaluated in the treatment of patients with alcohol and non-alcohol-related cirrhosis because of its antifibrotic effect in vitro.402,403 To date, clinical results have been conflicting. The most convincing evidence supporting the use of colchicine comes from a study including 100 patients followed up for up to 14 years. Survival was 75% and 34% in treated and placebo groups, respectively. Some patients appeared to have a resolution of their cirrhosis to either minimal fibrosis or normal histology.404 Three further trials, however, with median follow-ups of 1,405 6,406 and 40407 months have all shown no benefit. A recent metaanalysis has also reviewed 14 randomized controlled trials and found no benefit of colchicine treatment on mortality or liver histology.408 This has been confirmed by a further large randomized study.409 Antioxidants. In addition to trials in patients with alcoholic hepatitis the accumulating evidence that oxidant stress is involved in the pathogenesis of ALD has prompted trials of antioxidants in patients with chronic disease. Two trials have evaluated the drug silymarin, which is the active component of the herb milk-thistle and has potent antioxidant properties in vitro410 and in vivo.411,412 The first trial in 170 patients with cirrhosis (92 had ALD) followed up for between 2 and 6 years reported a beneficial effect on survival.413 In contrast, a later, larger study of 200 patients with cirrhotic ALD, followed up for 5 years, showed no benefit.414 SAME, which acts as both an antioxidant by replenishing GSH and a methyl donor maintaining cell membrane fluidity, has also been evaluated in patients with alcoholic cirrhosis. Using death or liver transplantation as a combined end-point, Mato and colleagues reported a significant beneficial effect of SAME treatment in patients with Child’s A and B cirrhosis.415 Clearly further trials with this agent are awaited with interest. Phosphatidylcholine. Phosphatidylcholine is an essential component of all cell membranes and is vulnerable to attack by lipid peroxidation. Through mechanisms that are, as yet, unclear, dietary supplementation with phosphatidylcholine has been shown to attenuate ethanol-induced fibrosis in baboons.416 Potential mechanisms of
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action include stimulation of collagenase417 and acting as a “sink” for free radicals.418 A long-term trial in patients with alcoholic cirrhosis has just been completed in the USA. While there was a trend to improvement in transaminases and bilirubin in the phosphatidylcholine group in certain patient subgroups (heavy drinkers and those with hepatitis C), overall there was no improvement in liver histology as determined by liver biopsies 24 months apart. The potential benefits of the drug may not have been evaluated appropriately because of the dramatic reduction in drinking seen in the treated and placebo groups of patients that were followed up to completion.419
Liver Transplantation for Advanced ALD Since the initial report of its success in 1988,420 ALD has become an increasingly common indication for orthotopic liver transplantation in both Europe421 and North America.422 However, transplantation for ALD remains controversial, principally due to concerns over the risk of post-transplant recidivism and its effect on outcome and public opinion at a time of increasing donor shortage. This issue, coupled with a perception that these patients are more likely to have contraindications to transplantation due either to extrahepatic complications of excessive alcohol abuse or to an associated lack of selfcare, has contributed to a continued reluctance of many centers to offer transplantation to patients with ALD. An accumulating number of reports of transplantation in patients with ALD have now provided a firm evidence base from which these issues can be addressed. Outcome of liver transplantation for ALD. Several studies have convincingly demonstrated that the survival of patients transplanted for cirrhotic ALD is comparable to patients with cirrhosis of alternative etiologies, with 5- and 10-year survivals lying somewhere between those of patients transplanted for cholestatic and viral hepatitis-related liver disease.423 Furthermore, there is no evidence that patients with ALD have a higher frequency of postoperative complications or resource utilization compared to patients transplanted for other indications, despite being transplanted at a more advanced stage of disease.424 The improvement in quality of life following transplantation also compares favorably with other indications in the short term,425,426 although not after 3 years follow-up.424 The reason for this decline is unclear, but does not seem to be related to a return to problem drinking. As for other indications, the decision to offer transplantation to a patient with ALD is based on their expected survival with and without transplantation. Without transplantation, survival depends on the severity of their liver disease and their subsequent drinking behavior. Patients with Child’s C cirrhosis have a 1-year survival of 50–85% compared to 75–95% in patients with Child’s B.427 This suggests that, in the absence of other predictors of high mortality, such as a history of spontaneous bacterial peritonitis, recurrent variceal hemorrhage, or the development of HCC, transplantation should be restricted to patients with Child’s C cirrhosis. In support of this policy, Poynard and colleagues recently demonstrated that ALD patients whose disease severity approximated to a Child–Pugh score of 11 or higher had a significantly improved 2-year survival compared to matched controls,428 while in the UK, the 1- and 2-year survival rates of 87% and 82% compare well with the 41% and 30%
survival predicted from the Beclere prognostic model.428 The potential effect of abstinence on prognosis of these patients has led most units to adopt a policy of offering transplantation to patients whose Child–Pugh score remains high after a period of abstinence. Post-transplant recidivism. Perhaps the greatest concern when considering transplantation for patients with ALD is the risk of recidivism and its effect on outcome and public opinion. With respect to the frequency of recidivism, this depends critically on its definition. Studies that have considered any alcohol use posttransplantation as a “relapse” have reported recidivism rates as high as 49%,429–431 whilst those that have restricted the definition to heavy or problem drinking have reported lower rates of 10–15%.425,432–436 With respect to the influence of recidivism on outcome, thus far, there are few data on which to base firm conclusions. From the information available, the incidence of graft dysfunction related to recidivism ranges from 0 to 17% and mortality ranges from 0 to 5%.431,432,434,435 In a study from 2001, the recidivism rates were 30%, with many showing evidence of recurrent disease on their protocol biopsies. Neither recidivism nor histology affected 84-month survival rates.437 In spite of this apparently reassuring report there is now evidence that if the follow-up is prolonged to 10 years, mortality in recidivists is significantly higher than in abstainers.438 It is therefore imperative that patients are monitored carefully for relapse following transplantation, with relapsers offered appropriate counseling. It is important that this is done for all ALD patients since, at present, there are few factors have been identified that reliably predict the risk of post-transplant recidivism prior to transplantation. Efforts to minimize the risk of post-transplant recidivism are important not only for the individual patient, but also to avoid the likely adverse effect this has on the organ-donating public. Public opinion. With organ shortage as a significant problem and while the decision to be an organ donor remains voluntary in most of the western world, it is imperative that the public are convinced that donated livers are being given to the most deserving patients. A recent UK study clearly demonstrated that, when compared to other patient groups, the general public, primary care physicians, and gastroenterologists all place patients with ALD well down their list of patients most deserving a liver transplant.439 The perception that patients with ALD have played a significant role in their disease and the widely held belief that “once a drinker, always a drinker” seem likely to be the most important factors contributing to this negative view of ALD patients. It is therefore vital that the public are made aware that patients are only offered transplantation if they fail to recover after a period of abstinence and that the incidence of significant post-transplant recidivism is low. Comorbidity. Excessive alcohol consumption can, and often does, affect many organ systems apart from the liver and this can potentially give rise to contraindications to surgery. An increased risk of pancreatitis, cardiomyopathy, osteoporosis, cerebrovascular disease, dementia, and malnutrition might all be expected to limit the numbers of patients fit for surgery. In practice, however, although most transplant units routinely screen ALD patients for cardiac and cerebral complications of excessive alcohol intake, this results in the
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exclusion of very few patients.436 Similarly, despite the increased risk of psychiatric comorbidity in heavy drinkers, this rarely, if ever, leads to the exclusion of patients at the stage of transplant assessment.436 This seems likely to be attributable to patients with significant physical or psychiatric comorbidities not being referred for formal transplant assessment and/or to the tendency of many alcohol-related morbidities to improve during the period of abstinence required by most units prior to assessment. Preoperative abstinence. In light of the above considerations, it is perhaps not surprising that most centers require patients to have been abstinent for a period of time prior to assessment. This is primarily to give the liver a chance to recover spontaneously; however, it also allows time for other alcohol-related morbidities to recover, thereby improving the patient’s fitness for surgery and, importantly, satisfies public opinion. During this period, the patient can also be put in contact with alcohol treatment services for support both before and after transplantation. Whilst there is broad consensus on the need for a period of pretransplant abstinence, there is far less agreement on the requirement for a minimum duration. Many units have previously insisted on a 6-month period of abstinence, possibly attributable to early reports that this was a positive predictor of post-transplant abstinence.425 While a recent study confirms a 6month period of abstinence to be associated with reduced rates of recidivism at 1 and 2 years post-transplant,440 other studies have shown this period of abstinence to have little if any predictive power for subsequent drinking habits.435 This is obviously a very important question as the price of insisting on a fixed abstinence period may be death in some patients. A recent study demonstrating that the chance of recovery in patients with decompensated ALD can be predicted as early as 3 months441 has led some observers to suggest that, if required at all, the minimum period of abstinence could safely be reduced to 3 months rather than 6 months.442 Currently, it appears that, in practice, most centers do not adhere strictly to a fixed period of abstinence, instead preferring to assess each case on an individual basis and listing the patient when it is considered that recovery is unlikely.423
Transplantation for Alcoholic Hepatitis Clearly, many of these issues are pertinent when it comes to considering the possibility of transplanting patients with severe acute alcoholic hepatitis. A reasonable period of abstinence is not possible to assess the liver’s potential for spontaneous recovery, significant comorbidities are almost universal, and formal psychiatric assessment and pretransplant counseling are often precluded by the severity of the illness. Accordingly, although these patients undoubtedly have a poor prognosis without transplantation, most clinical centers do not consider these patients for liver transplantation. Nonetheless, there have been isolated reports of survival following transplantation of these patients371,443 and a recent report that the presence of histological alcoholic hepatitis in the explanted liver of patients transplanted for apparently chronic stable ALD is not associated with a worse prognosis or an increased risk of recidivism.444 Clearly, some patients with alcoholic hepatitis can benefit from transplantation; however, more data are required before any firm recommendations can be given on which patients (if any) are likely to derive the most benefit.
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Gut permeability
Alcohol metabolism ADH
CYP2E1/Fe Oxidative stress
Endotoxin
Acetaldehyde
4
1
Immunological damage (adducts)
Lipid peroxidation KC
4
MDA
2 Cytokines
5
Necroinflammation and/or apoptosis 3
Figure 29-11. Putative mechanisms of hepatocyte injury in alcoholic hepatitis with potential targets for therapy: (1) antiendotoxin therapy; antibiotics, probiotics, enteral nutrition; (2) anticytokine therapy; corticosteroids, pentoxifylline, Infliximab; (3) antiapoptotic therapy; caspase inhibitors; (4) antioxidants; (5) immune-based therapy; corticosteroids. CYP2E1/Fe, ADH, MDA, KC.
CONCLUSIONS The short-term social and psychological benefits of alcohol have meant that its use and abuse have been widespread in many societies. End-stage liver disease is the result of prolonged heavy alcohol intake among a small proportion of users. Nevertheless, ALD still accounts for around half the total number of deaths from cirrhosis in the USA, and a great many more patients with fibrosis and alcoholic hepatitis. It therefore makes up a significant proportion of the workload of most liver units. The interaction between the physical and psychological dependence of the drug and the complexity of disease susceptibility makes this patient population a fascinating group. The multiple potential mechanisms of pathogenesis make it an intriguing disease to study, made all the more interesting considering the gray areas around the relative importance of each mechanism (Figure 29-11). Animal work is slowly improving this knowledge. This is filtering down into human trials of novel treatments, but improvements are slow considering the high mortality of acute alcoholic hepatitis, and liver transplantation remains the mainstay of treatment for advanced cirrhosis. Further research to understand the basics of hepatocyte injury are required to fuel further clinical trials and it must be expected that the next 10 years will lead to significant improvements in patient survival.
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385. Rambaldi A, Iaquinto G, Gluud C. Anabolic-androgenic steroids for alcoholic liver disease: a Cochrane review. Am J Gastroenterol 2002; 97:1674–1681. 386. Israel Y, Kalant H, Orrego H, et al. Experimental alcoholinduced hepatic necrosis: suppression by propylthiouracil. Proc Natl Acad Sci USA 1975; 72:1137–1141. 387. Orrego H, Kalant H, Israel Y, et al. Effect of short-term therapy with propylthiouracil in patients with alcoholic liver disease. Gastroenterology 1979; 76:105–115. 388. Halle P, Pare P, Kaptein E, et al. Double-blind, controlled trial of propylthiouracil in patients with severe acute alcoholic hepatitis. Gastroenterology 1982; 82:925–931. 389. Iimuro Y, Gallucci RM, Luster MI, et al. Antibodies to tumor necrosis factor alfa attenuate hepatic necrosis and inflammation caused by chronic exposure to ethanol in the rat. Hepatology 1997; 26:1530–1537. 390. Jalan R, Williams R, Kaser R, et al. Clinical and cytokine response to anti-TNF antibody therapy in severe alcoholic hepatitis. Hepatology 2001; 34:441A. 391. Spahr L, Rubbia-Brandt L, Frossard JL, et al. Combination of steroids with infliximab or placebo in severe alcoholic hepatitis: a randomized controlled pilot study. J Hepatol 2002; 37:448–455. 392. Menon KV, Stadheim L, Kamath PS, et al. A pilot study of the safety and tolerability of etanercept in patients with alcoholic hepatitis. Am J Gastroenterol 2004; 99:255–260. 393. Keane J, Gershon S, Wise RP, et al. Tuberculosis associated with infliximab, a tumor necrosis factor alpha-neutralizing agent. N Engl J Med 2001; 345:1098–1104. 394. Akerman P, Cote P, Yang SQ, et al. Antibodies to tumor necrosis factor-alpha inhibit liver regeneration after partial hepatectomy. Am J Physiol 1992; 263:G579–G585. 395. Naveau S, Chollet-Martin S, Dharancy S, et al. A double-blind randomized controlled trial of infliximab associated with prednisolone in acute alcoholic hepatitis [see comment]. Hepatology 2004; 39:1390–1397. 396. Jalan R, Sen S, Steiner C, et al. Extracorporeal liver support with molecular adsorbents recirculating system in patients with severe acute alcoholic hepatitis [see comment]. J Hepatol 2003; 38:24–31. 397. Sen S, Mookerjee RP, Cheshire LM, et al. Albumin dialysis reduces portal pressure acutely in patients with severe alcoholic hepatitis. J Hepatol 2005; 43:42–48. 398. Ortega R, Gines P, Uriz J, et al. Terlipressin therapy with and without albumin for patients with hepatorenal syndrome: results of a prospective, nonrandomized study. Hepatology 2002; 36:941–948. 399. Uriz J, Gines P, Cardenas A, et al. Terlipressin plus albumin infusion: an effective and safe therapy of hepatorenal syndrome. J Hepatol 2000; 33:43–48. 400. Guevara M, Gines P, Fernandez-Esparrach G, et al. Reversibility of hepatorenal syndrome by prolonged administration of ornipressin and plasma volume expansion. Hepatology 1998; 27:35–41. 401. Orrego H, Blake JE, Blendis LM, et al. Long-term treatment of alcoholic liver disease with propylthiouracil. N Engl J Med 1987; 317:1421–1427. 402. Ehrlich HP, Ross R, Bornstein P. Effects of antimicrotubular agents on the secretion of collagen. A biochemical and morphological study. J Cell Biol 1974; 62:390–405. 403. Diegelmann RF, Peterkofsky B. Inhibition of collagen secretion from bone and cultured fibroblasts by microtubular disruptive drugs. Proc Natl Acad Sci USA 1972; 69:892–896. 404. Kershenobich D, Uribe M, Suarez GI, et al. Treatment of cirrhosis with colchicine. A double-blind randomized trial. Gastroenterology 1979; 77:532–536. 405. Akriviadis EA, Steindel H, Pinto PC, et al. Failure of colchicine to improve short-term survival in patients with alcoholic hepatitis. Gastroenterology 1990; 99:811–818.
622
406. Trinchet JC, Beaugrand M, Callard P, et al. Treatment of alcoholic hepatitis with colchicine. Results of a randomized double blind trial. Gastroenterol Clin Biol 1989; 13:551– 555. 407. Cortez-Pinto H, Alexandrino P, Camilo ME, et al. Lack of effect of colchicine in alcoholic cirrhosis: final results of a double blind randomized trial. Eur J Gastroenterol Hepatol 2002; 14:377–381. 408. Rambaldi A, Gluud C. Colchicine for alcoholic and nonalcoholic liver fibrosis and cirrhosis. Cochrane Database of Systematic Reviews 2001; 3:002148. 409. Morgan TR, Weiss DG, Nemchausky B, et al. Colchicine treatment of alcoholic cirrhosis: a randomized, placebocontrolled clinical trial of patient survival. Gastroenterology 2005; 128:882–890. 410. Carini R, Comoglio A, Albano E, Poli G. Lipid peroxidation and irreversible damage in the rat hepatocyte model. Protection by the silybin–phospholipid complex IdB 1016. Biochem Pharmacol 1992; 43:2111–2115. 411. Pietrangelo A, Borella F, Casalgrandi G, et al. Antioxidant activity of silybin in vivo during long-term iron overload in rats. Gastroenterology 1995; 109:1941–1949. 412. Masini A, Ceccarelli D, Giovannini F, et al. Iron-induced oxidant stress leads to irreversible mitochondrial dysfunctions and fibrosis in the liver of chronic iron-dosed gerbils. The effect of silybin. J Bioenergetics Biomembranes 2000; 32:175–182. 413. Ferenci P, Dragosics B, Dittrich H, et al. Randomized controlled trial of silymarin treatment in patients with cirrhosis of the liver. J Hepatol 1989; 9:105–113. 414. Pares A, Planas R, Torres M, et al. Effects of silymarin in alcoholic patients with cirrhosis of the liver: results of a controlled, double-blind, randomized and multicenter trial. J Hepatol 1998; 28:615–621. 415. Mato JM, Camara J, Fernandez de Paz J, et al. S-adenosylmethionine in alcoholic liver cirrhosis: a randomized, placebo-controlled, double-blind, multicenter clinical trial. J Hepatol 1999; 30:1081–1089. 416. Lieber CS, Robins SJ, Li J, et al. Phosphatidylcholine protects against fibrosis and cirrhosis in the baboon. Gastroenterology 1994; 106:152–159. 417. Li J, Kim CI, Leo MA, et al. Polyunsaturated lecithin prevents acetaldehyde-mediated hepatic collagen accumulation by stimulating collagenase activity in cultured lipocytes. Hepatology 1992; 15:373–381. 418. Lieber CS. Alcoholic liver disease: new insights in pathogenesis lead to new treatments. J Hepatol 2000; 32 (suppl):113–128. 419. Lieber CS, Weiss DG, Groszmann R, et al. II. Veterans Affairs cooperative study of polyenylphosphatidylcholine in alcoholic liver disease. Alcoholism Clin Exp Res 2003; 27:1765–1772. 420. Starzl TE, Van Thiel D, Tzakis AG, et al. Orthotopic liver transplantation for alcoholic cirrhosis. JAMA 1988; 260:2542–2544. 421. European Liver Transplant Registry. www.ELTR.org. 2001. 422. Belle SH, Beringer KC, Detre KM. Liver transplantation for alcoholic liver disease in the United States: 1988 to 1995. Liver Transplant Surg 1997; 3:212–219. 423. Neuberger J, Schulz KH, Day C, et al. Transplantation for alcoholic liver disease. J Hepatol 2002; 36:130–137. 424. Wiesner RH, Lombardero M, Lake JR, et al. Liver transplantation for end-stage alcoholic liver disease: an assessment of outcomes. Liver Transplant Surg 1997; 3:231–239. 425. Bird GL, O’Grady JG, Harvey FA, et al. Liver transplantation in patients with alcoholic cirrhosis: selection criteria and rates of survival and relapse. Br Med J 1990; 301:15–17. 426. Burra P, De Bona M, Canova D, et al. Longitudinal prospective study on quality of life and psychological distress before and one
Chapter 29 ALCOHOLIC LIVER DISEASE
427.
428.
429.
430.
431.
432.
433.
434.
435.
year after liver transplantation. Acta Gastroenterol Belg 2005; 68:19–25. Propst A, Propst T, Zangerl G, et al. Prognosis and life expectancy in chronic liver disease. Digest Dis Sci 1995; 40:1805–1815. Poynard T, Barthelemy P, Fratte S, et al. Evaluation of efficacy of liver transplantation in alcoholic cirrhosis by a case-control study and simulated controls. Lancet 1994; 344:502–507. Lucey MR, Carr K, Beresford TP, et al. Alcohol use after liver transplantation in alcoholics: a clinical cohort follow-up study. Hepatology 1997; 25:1223–1227. Gerhardt TC, Goldstein RM, Urschel HC, et al. Alcohol use following liver transplantation for alcoholic cirrhosis. Transplant Proc 1996; 62:1060–1063. Berlakovich GA, Steininger R, Herst M, et al. Efficacy of liver transplantation for alcoholic cirrhosis with respect to recidivism and compliance. Transplant Proc 1994; 58:560–565. Kumar S, Stauber RE, Gavaler JS, et al. Orthotopic liver transplantation for alcoholic liver disease. Hepatology 1990; 11:159–164. Osorio RW, Ascher NL, Avery M, et al. Predicting recidivism after orthotopic liver transplantation for alcoholic liver disease. Hepatology 1994; 20:105–110. Pageaux GP, Michel J, Coste V, et al. Alcoholic cirrhosis is a good indication for liver transplantation, even for cases of recidivism [see comment]. Gut 1999; 45:421–426. Foster PF, Fabrega F, Karademir S, et al. Prediction of abstinence from ethanol in alcoholic recipients following liver transplantation. Hepatology 1997; 25:1469–1477.
436. Anand AC, Ferraz-Neto BH, Nightingale P, et al. Liver transplantation for alcoholic liver disease: evaluation of a selection protocol. Hepatology 1997; 25:1478–1484. 437. Burra P, Mioni D, Cecchetto A, et al. Histological features after liver transplantation in alcoholic cirrhotics. J Hepatol 2001; 34:716–722. 438. Cuadrado A, Fabrega E, Casafront F, Pons-Romero F. Alcohol recidivism impairs long-term patient survival after orthotopic liver transplantation for alcoholic liver disease. Liver Transplant 2005; 11:420–426. 439. Neuberger J, Adams D, MacMaster P, et al. Assessing priorities for allocation of donor liver grafts: survey of public and clinicians. Br Med J 1998; 317:172–175. 440. Miguet M, Monnet E, Vanlemmens C, et al. Predictive factors of alcohol relapse after orthotopic liver transplantation for alcoholic liver disease. Gastroenterol Clin Biol 2004; 28:845–851. 441. Veldt BJ, Laine F, Guillygomar’h A, et al. Indication of liver transplantation in severe alcoholic liver cirrhosis: quantitative evaluation and optimal timing. J Hepatol 2002; 36:93–98. 442. Lucey MR. Is liver transplantation an appropriate treatment for acute alcoholic hepatitis? J Hepatol 2002; 36:829–831. 443. Shakil AO, Pinna A, Demetris J, et al. Survival and quality of life after liver transplantation for acute alcoholic hepatitis. Liver Transplant Surg 1997; 3:240–244. 444. Tome S, Martinez-Rey C, Gonzalez-Quintela A, et al. Influence of superimposed alcoholic hepatitis on the outcome of liver transplantation for end-stage alcoholic liver disease [see comment]. J Hepatol 2002; 36:793–798.
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30
HEPATITIS A Maria H. Sjogren Abbreviations 5¢ NTR 5¢ non-translated anti-HAV antibodies against HAV FHF fulminant hepatic failure
HAV HBV
hepatitis A virus hepatitis B virus
INTRODUCTION Experimental work in humans begun in the 1940s led to the clinical recognition of viruses as etiological agents of hepatitis A (“infectious hepatitis”) and hepatitis B (“serum hepatitis”).1,2 These initial observations were later amplified demonstrating the existence of two hepatitis viruses: hepatitis A virus (HAV) and hepatitis B virus (HBV).3 The hepatitis A viral agent was first characterized in 1973, when scientists discovered the virus in human stools from volunteers who were infected with HAV.4 The subsequent development of sensitive and specific serological techniques for the diagnosis of HAV infection and the isolation of HAV in cell culture5 were important advances which permitted understanding of the epidemiology of HAV infection and ultimately protection against infection by the development of effective vaccines.
VIROLOGY Initially, HAV was classified as an enterovirus type 72 belonging to the picornaviridae family. Subsequent sequencing of HAV nucleotides and amino acid sequences questioned this classification and the new genus Hepatovirus was created for HAV.6 HAV is a non-enveloped virus that measures 27–28 nm in diameter, has a buoyant density of 1.33–1.34 g/cm3 in CsCl, and a sedimentation coefficient of 156–160 S by ultracentrifugation. Hepatitis A survives exposure to ether and an acid environment at pH 3. It also survives heat exposure at 60oC for 60 min, but is inactivated at 85oC for 1 min. Only one serotype of HAV is known which has no antigenic crossreactivity with the hepatitis B, C, D, E, or G agents. The HAV genome consists of a positive-sense RNA, 7.48 kb long, singlestranded, and linear. The HAV RNA has a sedimentation coefficient of 32–33 S and a molecular weight of 2.8 ¥ 104. The HAV RNA has a long open reading frame (6681 nucleotides) and is covalently linked to a 5¢ terminal protein and a 3¢ terminal poly-A tract. Onset of HAV replication in cell culture systems takes a long time, usually months. Primate cells are favored for HAV in-vitro cultivation, among them African green-monkey kidney cells, primary human fibroblasts, human diploid cells (MRC-5), and fetal rhesus kidney cells. The virus is not cytopathic and persistent infection of cell cultures is the rule. Two conditions control the outcome of HAV replication in cell culture.7 First, the genetic make-up of the virus is important, as HAV strains mutate in distinct regions of
IgM PCR
immunoglobulin M polymerase chain reaction
the viral genome, as they become cell-culture-adapted. Another important condition is the metabolic activity of the host cell at the time of infection. Cells in culture, although infected simultaneously, initiate HAV replication in an asynchronous manner, probably due to differences in the metabolic activity of individual cells, but there is no definitive evidence of cell cycle dependence of HAV replication.8 An initial step in the life cycle of a virus is its attachment to a cell surface receptor. Location and function of these receptors determine tissue tropism. Little is known about the mechanism of cell entry of HAV. Scientific work has suggested that HAV could infect cells by a surrogate receptor-binding mechanism (a non-specified serum protein). HAV infectivity in tissue culture was shown to require calcium and to be inhibited by treatment of the cells with trypsin, phospholipases, and b-galactosidase.9 A surface glycoprotein on African green-monkey kidney cells has been identified as a receptor for HAV. The receptor was named HAVcr-1. Blocking of HAVcr-1 with specific monoclonal antibodies prevented infection of otherwise susceptible cells. Whatever the entry mechanism, once HAV enters a cell, the viral RNA is uncoated, cell host ribosomes bind to viral RNA, and polysomes are formed. HAV is translated into a large polyprotein of 2227 amino acids. This polyprotein is organized into three regions: P1, P2, and P3. The P1 region encodes for structural proteins VP1, VP2, VP3, and a putative VP4. The P2 and P3 regions encode for non-structural proteins associated with viral replication. The viral RNA polymerase copies the plus RNA strand (genome) that in turn is used for translation into proteins and for assembly into mature virions. It appears that down-regulation of HAV RNA synthesis occurs as defective HAV particles appear.10 In addition a group of specific RNA-binding proteins have been observed during persistent infection.11 The origin and nature of these proteins are unknown, but they exert activity in the RNA template and are believed to play a regulatory role in the replication of HAV.12 Numerous strains of hepatitis A exist, with considerable nucleotide sequence variability (15–25% difference within the P1 region of the genome). Human HAV strains can be grouped in four different genotypes (I, II, III, and VII), while simian strains belong to genotypes IV, V, and VI.13 Despite the nucleotide sequence heterogeneity, the antigenic structure of human HAV is highly conserved among strains. A recent report suggested that the hepatitis A VP1/2A and 2C genes are responsible for virulence. Investigators studied the ability of recombinant HAV virus to cause acute hepatitis in experimental
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Figure 30-1. The reported and estimated cases of acute hepatitis A have declined over the last 10 years, particularly after hepatitis A virus vaccine became commercially available in the USA.16
70000 Vaccine licensed 60000
Cases
50000 40000 30000 20000 10000 0 1966
1970
1974
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1982
1986
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animals by constructing 14 chimeric virus genomes from two infectious cDNA clones encoding a virulent and an attenuated HAV isolate (HM175 strain). Comparisons of the genotype and phenotype of each virus supported their findings.14 Among the many strains of HAV, the HM175 and CR326 human HAV strains are important because they were used for the production of commercially available vaccines; strain HM175 was isolated in 1978 from human feces from Australian patients who were members of a small hepatitis A outbreak. CR326 was isolated from Costa Rican patients infected with the HAV. The nucleotide and amino acid sequence show 95% identity between these two strains. Vaccines prepared with these strains are expected to provide protection against all relevant human strains. To study a possible viral contribution to the development of fulminant hepatic failure (FHF) during acute infection with HAV, the 5¢ non-translated (5¢ NTR) region of the HAV genome in 84 serum samples, including 12 FHF patients, were sequenced.15 The investigators observed relatively fewer nucleotide substitutions in FHF compared to non-FHF subjects with acute HAV infection (P < 0.001). This finding was most prominent between nucleotides 200 and 500, suggesting that this nucleotide variation in the central portion of the 5¢ NTR of HAV may influence the clinical severity of the infection.
EPIDEMIOLOGY Acute hepatitis A is a reportable infectious disease in the USA, with a rate of infection of 4/100 000.16 In the USA, the number of reported and estimated subjects infected with HAV has been decreasing, as shown in Figure 30-1.16 In the year 2003, 7653 HAV infections were reported; however, taking into consideration the underreporting of cases and asymptomatic infections, the true number of infections was calculated to be 61 000.16 The highest rate of reported disease has been among children 5–14 years of age and 25% of reported cases are among persons aged 20 years or less;17 however, HAV infection can occur in any age group. A recent communication from the Centers for Diseases Control and Prevention reported the current epidemiological risk factors for the US population, as shown in Figure 30-2.18
628
Sexual or household contact 12% International travel 9% Unknown 57%
MSM 8% IVDA 5% Day care 5%
Outbreak 1% Contact with patient 4% Figure 30-2. The epidemiological risks for acute hepatitis A virus infection have not changed significantly over the years. Data depicted in the graph were obtained from reported cases in the USA in 2002.18 MSM, men who have had sex with men; IVDA, intravenous drug abuse.
Hepatitis A infection generally follows one of three epidemiological patterns.19 First, in countries with poor sanitary conditions, most children are infected at an early age. Although earlier seroepidemiological studies routinely showed that 100% of preschool children had detectable anti-HAV, presumably reflecting previous subclinical infection, recent studies show that the average age of infection is rapidly increasing to 5 years and above, when symptomatic infection is more likely. As such, 82% of 1393 Bolivian school children were shown to have detectable anti-HAV in sera; however, when stratified in two groups according to family income, there was a
Chapter 30 HEPATITIS A
significant difference between the groups: 56% of children from high-income families had detectable anti-HAV versus 95% of children from low-income families.20 The second epidemiological pattern is seen in industrialized countries where there is a low prevalence of HAV infection among children and young adults. Thus, in the USA the prevalence of antiHAV is approximately 10% in children, whereas 37% of adults have detectable anti-HAV.21 The third epidemiological pattern is observed in closed or semi-closed communities, in which HAV is capable (through epidemics) of infecting the entire population, which thus becomes immune. Thereafter, newborns remain susceptible but free of anti-HAV until the virus is reintroduced into the community. The primary route of human transmission is fecal–oral by either person-to-person contact or ingestion of contaminated food or water. Although rare, transmission by parenteral route has been documented following blood transfusion22,23 or the use of blood products.24 Cyclic outbreaks among users of injecting and non-injecting drugs and among men who have sex with men (up to 10% in outbreak years) have been reported.25 In the USA the rate of antibody to HAV increases with age:25 9% of adolescents are found to have detectable antibody, while 75% of individuals over 70 years of age have evidence of remote infection with HAV.
nificant problem in adults and older children. Approximately 11–22% of patients25 with acute hepatitis A require hospitalization, with an average cost of $6914 per patient.28 In one outbreak involving 43 persons, the cost was approximately $800 000.25 On average 27 workdays are lost per adult case with a total loss of 829 000 workdays per year.25,28 Combined direct and indirect costs associated with hepatitis A infection in the USA totaled more than $200 million in 1989 and approximately $488.8 millions in 1997.15,29 The clinical characteristics of hepatitis A cases reported in 2002 were similar to previous years with a preponderance of cases among men rather than women in all ages, 72% of cases manifesting jaundice, 25% requiring hospitalization and 0.5% resulting in death.18 Mortality and the need for hospitalization increase with age (Table 30-1), and subjects older than 60 years of age are at increased risk.18 Hepatitis A infection usually presents in one of five different clinical patterns:
PATHOGENESIS
Young children (less than 2 years of age) are usually asymptomatic; only 20% develop jaundice, while most 5-year-old children or older (80%) develop symptoms. This high rate of symptoms is maintained in adulthood. HAV infection with prolonged cholestasis is rare but could lead to invasive procedures, as the diagnosis of acute hepatitis may not be readily accepted in patients with a history of jaundice for several months even in the presence of detectable immunoglobulin M (IgM) anti-HAV.30 Relapsing hepatitis A is observed in approximately 10% of patients with acute hepatitis A; it is a benign syndrome and resolves by itself.31 Cholestasis and relapsing hepatitis do not have an increased mortality and treatment is symptomatic. Shedding of HAV has been documented during the relapse phase.31 Acute hepatitis A, unlike hepatitis E, has not been shown to have an increased mortality in pregnant women.
Once the HAV is ingested and survives the gastric acid, it traverses the small-intestine mucosa and the virus reaches the liver through the portal vein. The putative cellular receptor for the virus has been identified as a surface glycoprotein on an experimental model in African green-monkey kidney cells.26 Once the virus reaches the liver cell, it starts replicating in the hepatocyte cytoplasm where it has been shown as a fine granular pattern, but it is not present in the nucleus. The distribution of HAV is throughout the liver. Although HAV antigen has been detected in other organs (lymph nodes, spleen, kidney), the virus appears to replicate exclusively in liver cells. Once the virus is mature, it reaches the systemic circulation through hepatic sinusoids and is released into the biliary tree through bile canaliculi and the small intestine and during acute hepatitis it is eventually excreted in feces. The pathogenesis of HAV-associated cell injury is not completely defined. The lack of cell injury in cell culture systems does not support the hypothesis of a cytopathic virus. Immunologically mediated cell damage is greatly favored. The hypothesis is that the emergence of antibody to HAV could result in hepatic necrosis during immunologically mediated elimination of the virus.27
1. asymptomatic (not jaundiced) 2. symptomatic (jaundiced): self-limited to approximately 8 weeks 3. cholestatic, in which jaundice lasts 10 weeks or more 4. relapsing, consisting of two or more bouts of acute HAV infection occurring over a 6–10-week period 5. FHF
FULMINANT HEPATIC FAILURE Fulminant hepatic failure (FHF) due to hepatitis A infection is rarely seen in young people. When hepatic failure is due to hepatitis A, it usually becomes manifest in the first week in about 55% of patients and in 90% during the first 4 weeks following the onset of disease; it is rarely seen after this interval.32 There are reports of increased
CLINICAL FEATURES AND DIAGNOSIS
Table 30-1. Age-specific mortality due to hepatitis A virus
Chronic infection with the HAV does not occur, it only manifests as an acute bout of hepatitis, and rarely acute hepatitis A can have prolonged clinical manifestations such as relapsing hepatitis or cholestasis. Commonly, the incubation period is 2–4 weeks, rarely up to 6 weeks. Mortality is low in previously healthy individuals. The impact of the disease is in the morbidity it causes, usually a sig-
49 Total
Age group (years)
Case fatality (per 1000) 3.0 1.6 1.6 3.8 17.5 4.1
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contribution of HAV to acute liver failure cases in populations classified as hyperendemic for hepatitis A. As such, in a recent communication from India where 276 patients with FHF were seen between 1994 and 1997, 10.6% of the cases among adults were due to HAV infection. HAV had been responsible for only 3.5% of ALF among 206 patients seen in the same community during the years 1978–1981.33 In recent years, it has been appreciated that certain populations are at high risk of developing increased morbidity and acute liver failure due to hepatitis A infection, among them the elderly34 and individuals with chronic liver disease. A 1998 scientific report described the clinical outcome of 256 individuals hospitalized with acute hepatitis A in the state of Tennessee between January 1994 and December 1995.35 All patients had a serological diagnosis of acute hepatitis A based on a detectable IgM antibody to hepatitis A. Figure 30-3 depicts the clinical outcome of these patients. The age of the individuals played a major role when rates of complications were analyzed. Twenty-five percent of patients aged 40 or above and 11% of patients younger than 40 years of age had at least one complication (P = 0.014). Although two recent reports described the further decline of acute viral hepatitis as a cause of fulminant failure,36,37 this decline
100
89%
80
Percentage of patients
60
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40
26% 20
5% 0 Died
Serious Prolonged PT complications
Nausea vomiting
Figure 30-3. Hospitalized patients due to acute hepatitis A have significant disease.35 PT, prothrombin time.
630
is principally due to the control of hepatitis B, while the contribution of HAV infection to FHF has remained unchanged over the last three decades, despite the availability of highly efficacious vaccines.
CLINICAL SYMPTOMS OF ACUTE HAV INFECTION Prodromal symptoms include fatigue and weakness, anorexia, nausea and vomiting, and abdominal pain. Less common symptoms include fever, headache, arthralgias, myalgias, and diarrhea. Usually dark urine precedes other symptoms in approximately 90% of infected individuals; this sign occurs within 1–2 weeks of the onset of prodromal symptoms. Symptoms may last a few days, up to 2 weeks, usually decreasing with the onset of clinical jaundice. Tenderness and mild liver enlargement are present in 85% of individuals, splenomegaly in 15%, and cervical lymphadenopathy in 15%. Complete clinical recovery is achieved in 60% of individuals within a period of 2 months and in almost everybody by 6 months. Overall the prognosis of acute hepatitis A infection is excellent in otherwise healthy adults. Some subjects develop complications that can be fatal (FHF).
DIAGNOSIS, BIOCHEMICAL ABNORMALITIES, AND SEROLOGY Acute hepatitis A is clinically indistinguishable from other forms of viral hepatitis. The diagnosis of infection is based on the determination of antibodies against HAV (anti-HAV) in serum. A diagnosis of acute hepatitis A requires demonstration of IgM anti-HAV in serum. This test is positive from the very onset of symptoms29 and usually remains positive for approximately 4 months.38 However, certain patients have been shown to have low levels of detectable IgM anti-HAV for more than a year after the initial infection.38 IgG anti-HAV is also positive at the onset of the disease, remains detectable usually for life, and is interpreted as a marker of previous infection. The diagnosis of acute HAV should be differentiated from any other acute viral hepatitis, autoimmune hepatitis, and others by means of appropriate serological testing. However, some cases may be difficult because individuals may harbor more than one viral infection, such as patients with chronic hepatitis B or chronic hepatitis C with superimposed acute HAV. Testing for HAV RNA is limited to research laboratories. HAV RNA has been detected in serum, stool, and liver tissue. Amplification of viral RNA by polymerase chain reaction (PCR) is available. Using this test, HAV RNA has been documented in human sera for up to 21 days after onset of illness.39 Briefly, the principles of PCR are: HAV RNA is reverse-transcribed (RT-PCR) into complementary cDNA before proceeding to amplify the viral product. Viral amplification is obtained in the presence of specific oligonucleotide primers, which are described in the scientific literature.40 In a recent description of 76 French patients with acute HAV infection, seen between January 1987 and April 2000, 19 were diagnosed as having FHF.41 Ten patients experienced liver death and required orthotopic transplantation and one patient died while awaiting liver transplantation. The article describes the HAV RNA status in 39 of the 50 patients whose sera and clinical data were available, including the 19 with FHF. Of interest, HAV RNA was detected in 36/50 (72%) cases. The likelihood of having an undetectable HAV RNA was more
Chapter 30 HEPATITIS A
pronounced in fulminant hepatitis cases than in non-fulminant cases (P < 0.02). When HAV RNA was detectable, titers were lower in encephalopathic patients than in non-fulminant cases (3.6 log versus 4.4 log, P = 0.02). These data suggest that the finding of a detectable IgM antibody against hepatitis A and undetectable or low-titer HAV RNA in patients with acute liver failure may signal an ominous prognosis and warrant consideration of early referral for liver transplantation. As with other studies, HAV genotypes did not seem to play a role in the clinical manifestations.42
PREVENTION AND TREATMENT Hepatitis A is still an infectious disease of potentially serious consequences due to morbidity and mortality. Recommendations concerning HAV immunoprophylaxis by the Advisory Committee on Immunization Practices were published in December 1999.25 The overall strategy is to protect individuals from disease and to lower the incidence of HAV infection in the USA. The vaccine is not licensed for use in children less than one year of age. At the present time, high-risk populations are targeted for immunization. Table 302 depicts these populations. Since childhood vaccination in high-risk areas was recommended, the overall hepatitis A rate has declined steadily, and in 2002, it was the lowest yet recorded (3.1/100 000). The decline in rates has been greater among children and in states where routine childhood vaccination is recommended, suggesting a positive impact of childhood vaccination. As such, hepatitis rates declined 20-fold during the years 1997–2001 among American Indian and Alaska Native children where routine hepatitis A vaccine was implemented.43 However, a 2003 Centers for Disease Control analysis of hepatitis A vaccination coverage for children aged 24–35 months who reside in the 11 states where the HAV vaccine is routinely recommended showed that immunization ranged from 6.4% to 72.7%,
Table 30-2. High-risk patient groups25a, 25b ∑ All children 12–23 months old ∑ Healthy individuals who travel to endemic areas, work in occupations where likelihood of exposure is high, are family members of infected patients, or adopt infants or children from endemic areas ∑ Persons with liver disease, including chronic hepatitis B or C or those undergoing liver transplantation ∑ HIV-positive patients ∑ Men who have sex with men ∑ Users of injecting and non-injecting illicit drugs ∑ Persons with clotting-factor disorders ∑ Laboratory workers who handle live hepatitis A virus ∑ Persons who live in communities with high or intermediate rates of hepatitis A ∑ Routine vaccination is recommended for children living in 11 states where rates of hepatitis A are at least twice the national average (≥20 cases per 100 000 population). These states are: Alaska, Arizona, California, Idaho, Nevada, New Mexico, Oklahoma, Oregon, South Dakota, Utah, and Washington. In addition, vaccination should be considered for children who live in Arkansas, Colorado, Missouri, Montana, Texas, and Wyoming because the average annual incidence of infection with HAV is 10–20 cases per 100 000 inhabitants HIV, human immunodeficiency virus; HAV, hepatitis A virus.
with an average of 50.9%; while immunization among children of the same age residing in the six states where HAV vaccination should be considered averaged 25% (range 0.6–32.3%). The analysis concluded that HAV immunization rates for children aged 24–35 months are lower than overall rates for other child vaccines.44 It is likely that universal immunization was not recommended in the USA because communities were considered to have high, intermediate, and low rates of hepatitis A and US government surveillance data demonstrated that communities with high and intermediate rates were primarily responsible for an average of 50% of reported HAV cases each year.25 Hence the recommendation was based on the concept that reducing hepatitis A incidence in states with high (Table 30-2) or intermediate average annual incidence of hepatitis A through routine vaccination of children would substantially reduce the national disease incidence. However, recent outbreaks in Georgia, Tennessee, and Pennsylvania, where more than 600 symptomatic cases and 3 deaths were reported, and thousands of exposed individuals needed immediate passive immunization,45 seem to contradict the recommendation for immunization for high or intermediate rates of endemic HAV and it is likely that immunization directed to specific groups would not control infection as efficiently as universal immunization would. There are no specific medications to treat acute hepatitis A; symptomatic treatment is recommended. Sanitary measures and administration of serum Ig were the principal means of prevention against HAV infection in years past. However, the availability of excellent hepatitis A vaccines has rendered the use of Ig for pre-exposure prophylaxis unnecessary. When immune serum globulin is used for postexposure prophylaxis, it should be given within 2 weeks of exposure. In these cases, the recommended dose of Ig is 0.02 ml/kg by intramuscular injection. Although considered safe, it can cause fever, myalgias, and pain at injection sites. Post-exposure prophylaxis with Ig can be safely accompanied by active immunization against hepatitis A.46 Figure 30-4 depicts the magnitude of anti-HAV titer following administration of one dose of Ig or two doses of hepatitis A vaccine. Hepatitis A vaccine was licensed in the USA in 1995; two inactivated hepatitis A vaccines are commercially available in the country. Their extensive use during clinical trials and postmarketing surveillance support the safety and efficacy of these products. Havrix is manufactured by SmithKline Biologicals, Rixensart, Belgium, and Vaqta by Merck Sharp & Dohme, West Point, Pennsylvania. Both vaccines have been prepared with HAV grown in cell culture. The final products are purified and formalin-inactivated and contain alum as an adjuvant. The basic difference between the two commercially available vaccines is the HAV strain used for preparation. Havrix was prepared with the HM175 strain, while Vaqta was prepared with the CR326 strain,47,48 a difference of little or no practical importance since both vaccines are safe and immunogenic. Doses and schedule of immunization are given in Table 30-2. Persistence of anti-HAV serum level is estimated to be detectable for approximately 20 years following immunization with Havrix.49 During clinical trials it was observed that the vaccine was well tolerated and the frequency of adverse events decreased with successive doses. Rare cases of anaphylaxis have been reported and repeated doses should not be given if individuals experience hypersensitivity reactions to the original dose. An incidence of 1–10% has been reported for local reactions at the injection site (induration,
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Section V. Liver Diseases Due to Infectious Agents
GMT=geometric mean titer 1000 IG 900
Figure 30-4. The magnitude of antibody to hepatitis A virus following immunoglobulin (Ig) administration is usually not detectable by commercial assays and is short-lived. GMT, geometric mean titer.
10000 Vaccine
800 1000 700
500
GMTs
GMTs
600 100
400 300
10
200 100 1
0 Dose of IG
15
30
Primary dose
redness, swelling), fatigue, mild fever, malaise, anorexia, and nausea. An incidence of less than 1% was reported for hematoma at injection site, pruritus, rash, upper respiratory tract infections, abdominal pain, diarrhea, vomiting, arthralgias, myalgias, lymphadenopathy, insomnia, photophobia, and vertigo.50 Post-marketing reports have shown similar events, plus rare side effects such as syncope, jaundice, hepatitis, dyspnea, Guillain–Barré syndrome, multiple sclerosis, and others. Whether these events are related to vaccine administration is unclear. In recent years a combination of hepatitis A and B vaccines has become available (Twinrix), with an excellent record of efficacy and safety.51 The dose schedule is shown in Table 30-3.
IMMUNIZATION AGAINST HEPATITIS A IN SUBJECTS WITH CHRONIC LIVER DISEASE Individuals with chronic liver disease are susceptible to infection with HAV, and increased HAV-related mortality has been observed in these individuals. It is logical to recommend pre-exposure prophylaxis with hepatitis A vaccine for patients with chronic liver disease who are susceptible to infection.52 This recommendation should be extended to pre- or post-liver-transplant individuals, although the efficacy of HAV vaccines is lessened in these individuals.53 Figure 30-5 depicts the seroconversion rate observed in patients with compensated chronic liver disease.52 An acute hepatitis episode in patients with underlying chronic liver disease is fraught with possible exacerbation of symptoms, and increased morbidity and mortality. Although the current guidelines recommend immunization against HAV of all patients with chronic liver disease,25 cost-effective analysis has found controversial results. A report published in 2000 found that it would cost 23 million Canadian dollars to save one life if patients with HCV infection were immunized.54 However, some of the assumptions on this report were challenged and remain unsolved.55 Two other studies on patients with chronic hepatitis C decidedly show an advantage of immunizing these patients against HAV.56,57 The methods used in these studies are dissimilar and some may be insensitive to the inci-
632
180 Days
Seroconversion at mos (%)
100
210 Booster at 6 months
98.2
97.7
94.3
95.2
80 60 40 20 0 Healthy adults Chronic HBV Chronic HCV
Other CLD
Figure 30-5. The seroconversion rate to anti-hepatitis A virus was similar in healthy controls and in subjects with compensated chronic liver disease (CLD); however the anti-hepatitis A virus titer was lower in chronically ill individuals compared to controls.55 HBV, hepatitis B virus; HCV, hepatitis C virus. (Reproduced from Keeffe EB. Vaccination against hepatitis A and B in chronic liver disease. Viral Hepatol 1999; 5:77–88, ©1999, with permission of Blackwell Publishing.)
Table 30-3. Recommended doses of hepatitis A vaccinesa (HAV) Vaccine
Age (years)
Dose
Volume
Dosing schedule
Havrix > 18 Vaqta > 18 Twinrix ≥ 18
2–18
720 EL.U 1440 EL.U 25 U 50 U 720 EL.U HAV 20 mg HBV
0.5 ml 1.0 ml 0.5 ml 1.0 ml 1.0 ml
0, 6–12 months 0, 6–12 months 0, 6–18 months 0, 6–18 months 0, 1, 6 months
a
2–18
Vaccines are injected intramuscularly in the deltoid area. EL.U, ELISA units; HBV, hepatitis B vaccine.
Chapter 30 HEPATITIS A
dence of HAV, underestimation of cases, and economic and societal costs of even one case of FHF. Universal immunization during childhood before the establishment of any chronic liver disease promises excellent prevention of HAV infection.58
REFERENCES 1. MacCallum FO, McFarlan AM, Miles JAR, et al. (eds) Infective hepatitis: studies in East Anglia during the period 1943–1947. Medical Research Council special report no. 273. London: HMSO; 1951:1–144. 2. Havens WP, Ward R, Drill VA et al. Experimental production of hepatitis by feeding icterogenic materials. Soc Exp Biol Med 1944; 57:206. 3. Krugman S, Ward R, Giles JP, et al. Infectious hepatitis: detection of virus during the incubation period and in clinically inapparent infections. N Engl J Med 1959; 261:729–734. 4. Feinstone SM, Kapikian AZ, Purcell RH. Hepatitis A: detection by immune electron microscopy of a viruslike antigen associated with acute illness. Science 1973; 182:1026–1028. 5. Provost PJ, Hilleman MR. An inactivated hepatitis A virus vaccine prepared from infected marmoset liver. Proc Soc Exp Biol Med 1978; 159:201–203. 6. Minor PD. Picornaviridae. Classification and nomenclature of viruses. Fifth report of the International Committee on Taxonomy of Viruses. Arch Virol 1991; 2(suppl):320–326. 7. Siegl G. Replication of hepatitis A virus and processing of proteins. Vaccine 1992; 10:S32–S35. 8. Harmon SA, Summers DF, Ehrenfeld E. Detection of hepatitis A virus RNA and capsid antigen in individual cells. Virus Res 1989; 12;361–369. 9. Seganti L, Superti F, Orsi N, et al. Study of the chemical nature of Frp/3 cell recognition units for hepatitis A virus. Med Microbiol Immunol 1987; 176:21–26. 10. Siegl G, Nüesch JPF, de Chastonay J. DI-particles of hepatitis A virus in cell culture and clinical specimens. In: Brinton MA, Heinz FX, eds. New aspects of positive strand RNA viruses. Washington: American Society for Microbiology; 1990: 102–107. 11. Nüesch JPF, Weitz M, Siegl G. Proteins specifically binding to the 3¢ untranslated region of hepatitis A virus RNA in persistently infected cells. Arch Virol 1993; 128:65–79. 12. Robertson BH, Jansen RW, Khanna B, et al. Genetic relatedness of hepatitis A virus strains recovered from different geographic regions. J Gen Virol 1992; 73:1365–1377. 13. Mathiesen LR, Feinstone SM, Purcell RH, Wagner JA. Detection of hepatitis A antigen by immunofluorescence. Infect Immun 1977; 18:524–530. 14. Emerson SU, Huang YK, Nguyen H, et al. identification of VP1/2A and 2C as a virulence genes of hepatitis A virus and demonstration of genetic instability of 2C. J Virol 2002; 76:8551–8559. 15. Fujiwara K, Yokosuka O, Ehata T, et al. Association between severity of type A hepatitis and nucleotide variations in the 5¢ non-translated region of hepatitis A virus RNA: strains from fulminant hepatitis have fewer nucleotide substitutions. Gut 2002; 51:82–88. 16. Disease burden from viral hepatitis A, B and C in the United States. www.cdc.gov/hepatitis. 17. Centers for Diseases Control and Prevention. Guidelines for viral hepatitis surveillance and case management. Atlanta, GA: CDC; 2005:1–42. 18. Centers for Diseases Control and Prevention. Hepatitis surveillance report no. 59. Atlanta, GA: CDC; 2004:1–60. 19. Gust ID. Epidemiological patterns of hepatitis A in different parts of the world. Vaccine 1992; 10:S56–S58.
20. Gandolfo GM, Ferri GM, Conti L, et al. Prevalence of infections by hepatitis A, B, C and E viruses in two different socioeconomic groups of children from Santa Cruz, Bolivia. Med Clin (Barc) 2003; 120:725–727. 21. Centers for Disease Control and Prevention. Prevention of hepatitis A through active or passive immunization: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 1996; 45:1–30. 22. Skidmore SJ, Boxall EH, Ala F. A case report of post-transfusion hepatitis A. J Med Virol 1982; 10:223. 23. Hollinger FB, Khan NC, Oefinger PE, et al. Posttransfusion hepatitis type A. JAMA 1983; 250:2313–2317. 24. Mannucci PM, Gdovin S, Gringeri A, et al. Transmission of hepatitis A to patients with hemophilia by factor VIII concentrates treated with organic solvent and detergent to inactivate viruses. Ann Intern Med 1994; 120:1–7. 25. Prevention of hepatitis A through active or passive immunization: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 1999; 48:1–37. 25a Hepatitis A vaccine: what you need to know www.cdc.gov/hepatitis 25b. Immunization Action Coalition www.immunize.org 26. Kaplan G, Totsuka A, Thompson P, et al. Identification of a surface glycoprotein on African green monkey kidney cells as a receptor for hepatitis A virus. EMBO J 1996; 15:4282–4296. 27. Kurane I, Binn LN, Bancroft WH, Ennis FA. Human lymphocyte responses to hepatitis A virus-infected cells: interferon production and lysis of infected cells. J Immunol 1985; 135:2140–2144. 28. Berge JJ, Drennan D, Jacobs J, et al. The cost of hepatitis A infections in American adolescents and adults in 1997. Hepatology 2000; 31:469–473. 29. Liaw YF, Yang CY Chu CM, Huang MJ. Appearance and persistence of hepatitis A IgM antibody in acute clinical hepatitis A observed in an outbreak. Infection 1986; 14:156–158. 30. Gordon SC, Reddy KR, Schiff ER. Prolonged intrahepatic cholestasis secondary to acute hepatitis A. Ann Intern Med 1984; 101:635–637. 31. Sjogren MH, Tanno H, Fay O, et al. Hepatitis A virus in stool during clinical relapse. Ann Intern Med 1987; 106:221–226. 32. William R. Classification, etiology and considerations of outcome in acute liver failure. Semin Liver Dis 1996; 16:343–348. 33. Chadha MS, Walimbe AM, Chobe LP, Arankalle VA. Comparison of etiology of sporadic acute and fulminant viral hepatitis in hospitalized patients in Pune, India during 1978–81 and 1994–97. Ind J Gastroenterol 2003; 22:11–15. 34. Brown GR, Persley K. Hepatitis A epidemic in the elderly. South Med J 2002; 95:826–833. 35. Willner IR, Mark DU, Howard SC, et al. Serious hepatitis A: an analysis of patients hospitalized during an urban epidemic in the United States. Ann Intern Med 1998; 128:111–114. 36. Ostapowicz G, Fontana R, Schiedt FV, et al. Results of a prospective study of acute liver failure at 17 tertiary care centers in the United States. Ann Intern Med 2002; 137:947–954. 37. Schiodt FV, Atillasoy E, Shakill AO, et al. Etiology and outcome for 295 patients with acute liver failure in the United States. Liver Transplant Surg 1999; 5:29–34. 38. Kao HW, Ashcavai M, Redeker AG. The persistence of hepatitis A IgM antibody after acute clinical hepatitis A. Hepatology 1984; 4:933–936. 39. Yotsuyanagi H, Iino S, Koike K, et al. Duration of viremia in human hepatitis A viral infection as determined by polymerase chain reaction. J Med Virol 1993; 40:35–38. 40. Mannucci PM, Gdovin S, Gringeri A, et al. Transmission of hepatitis A to patients with hemophilia by factor VIII concentrates treated with organic solvent and detergent to inactivate viruses. Ann Intern Med 1994; 120:1–7.
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41. Rezende G, Roque-Alsonso M, Samuel D, et al. Viral and clinical factors associated with fulminant course of hepatitis A infection. Hepatology 2003; 38:613–618. 42. Fujiwara K, Yokosuka O, Imazeki F, et al. Analysis of the genotype-determining region of hepatitis A viral RNA in relation to disease severities. Hepatol Res 2003; 25:124–134. 43. Bialek SR, Thoroughman D, Hu D, et al. Hepatitis A incidence and hepatitis A vaccination among Anerican Indians and Alaska Natives, 1990–2001. Am J Public Health 2004; 94:996–1001. 44. Hepatitis A vaccination coverage among children aged 24–35 months – United States. MMWR 2005; 54:141–144. 45. Sjogren MH. The clinical profile of acute hepatitis A infection: is it really so severe? Hepatology 2004; 39:572–573. 46. Leentvaar-Kuijpers A, Coutinho RA, Brulein V, Safary A. Simultaneous passive and active immunization against hepatitis A. Vaccine 1992; 10:S138–S141. 47. Andre FE, D’Hondt E, Delem A, Safary A. Clinical assessment of the safety and efficacy of an inactivated hepatitis A vaccine. Vaccine 1992; 10(suppl 1):S160–S168. 48. Provost PJ, Hughes JN, Miller WJ, et al. An inactivated hepatitis A viral vaccine of cell culture origin. J Med Virol 1986; 19:23–31. 49. Van Damme P, Thoelen S, Cramm K, et al. Inactivated hepatitis A vaccine: reactogenicity, immunogenicity, and long-term antibody persistence. J Med Virol 1994; 44:446–451.
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50. Just M, Berger R. Reactogenicity and immunogenicity of inactivated hepatitis A vaccines. Vaccine 1992; 10(suppl 1):S110–S113. 51. FDA approval for a combined hepatitis A and B vaccine. MMWR 2001; 50:806–807. 52. Reiss G, Keefe EB. Review article: hepatitis vaccination in patients with chronic liver disease. Aliment Pharmacol Ther 2004; 19:715–727. 53. Aeslan M, Wiesner RH, Poterucha JJ, Zein NN. Safety and efficacy of hepatitis A vaccination in liver transplantation recipients. Transplantation 2001; 72:272–276. 54. Myers RP, Gregor JC, Marotta P. The cost-effectiveness of hepatitis A vaccination in patients with chronic hepatitis C. Hepatology 2000; 31:834–839. 55. Jacobs RJ, Koff RS. Cost-effectiveness of hepatitis A vaccination in patients with chronic hepatitis C. Hepatology 2000; 32:873–874. 56. Jacobs RJ, Koff RS, Meyerhoff AS. The cost-effectiveness of vaccinating chronic hepatitis C patients against hepatitis A. Am J Gastroenterol 2002; 97:427–434. 57. Arguedas MR, Heudebert GR, Fallon MB, Stinnett AA. The cost-effectiveness of hepatitis A vaccination in patients with chronic hepatitis C viral infection in the United States. Am J Gastroenterol 2002;97:721–728. 58. Rosenthal P. Cost-effectiveness of hepatitis A vaccination in children, adolescents and adults. Hepatology 2003; 37:44–51.
Section V: Liver Diseases Due to Infectious Agents
31
HEPATITIS B Robert Perrillo, Satheesh Nair Abbreviations AFP a-fetoprotein ALT alanine aminotransferase anti-HBc antibody to HBcAg anti-HBe antibody to HBeAg anti-HBs antibody to HBsAg anti-HDV antibody to hepatitis delta virus AST aspartate aminotransferase BCP basal core promoter cccDNA covalently closed circular DNA CTLs cytotoxic T lymphocytes
HAV HBcAg HBeAg HBIG HBsAg HBV HBV DNA HBX HCC HDAg
hepatitis A virus hepatitis B core antigen hepatitis B e antigen hepatititis B immune globulin hepatitis B surface antigen hepatitis B virus hepatitis B virus deoxyribonucleic acid hepatitis B X antigen hepatocellular carcinoma hepatitis delta antigen
HEPATITIS B: A GLOBAL HEALTH ISSUE There are more than 350 million carriers of hepatitis B virus (HBV) in the world today, of whom 75% reside in Asia and the western Pacific. Effective vaccines have been available for more than 20 years, but perinatal and early life exposure continues to be a major source of infection in high-prevalence areas. Moreover, high-risk behaviors, such as promiscuous heterosexual contact and injecting drug use, account for many new cases in young adults in other parts of the world. Fulminant acute hepatitis B accounts for several hundred deaths per year in the United States, and chronic HBV infection accounts for one million deaths worldwide each year due to complications of end-stage liver disease, including hepatocellular carcinoma. Although significant progress has been made in the area of antiviral therapy, many patients cannot be successfully managed. Thus, universal hepatitis B vaccination is likely to have the greatest impact on liver disease-related mortality in future generations.1
EPIDEMIOLOGY OF HBV INFECTION GEOGRAPHICAL DISTRIBUTION AND SOURCES OF EXPOSURE The prevalence of hepatitis B varies markedly around the world. In highly endemic regions, such as Southeast Asia (excluding Japan), China, and much of Africa, 8% or more of the population are chronic HBV carriers, and the lifetime risk of infection varies between 60% and 80%. In these areas perinatal transmission and horizontal spread among children are the major sources of infection. Nearly half of the HBV carriers in the world reside in these highly endemic areas.2 Areas of intermediate risk include parts of southern and eastern Europe, the Middle East, Japan, the Indian subcontinent, much of the former Soviet Union, and northern Africa. In intermediate-risk
HDV HIV HLA IFN MCSF MHC PCR Si RNA Th1 YMDD
hepatitis delta virus human immunodeficiency virus histocompatability locus antigen interferon macrophage colony-stimulating factor major histocompatibility complex polymerase chain reaction small interfering deoxyribonucleic acid T helper 1 tyrosine-methionine-aspartate-aspartate
areas the lifetime risk of infection is between 20% and 60%. Individuals of all age groups are infected, but as with high-risk areas most infections occur during infancy or early childhood. Regions of low prevalence include North America, western Europe, certain parts of South America, and Australia. Here the lifetime risk of HBV infection is less than 20% and transmission is primarily horizontal (i.e. between young adults). Sexual transmission is the main mode in Europe and North America, with injecting drug use making a major contribution to new cases as well.3 Transmission of infection from an HBV carrier mother to the neonate accounts for the majority of new infections in the world today: 60–90% of hepatitis B surface antigen (HBsAg)-positive mothers who are also hepatitis B e antigen positive will transmit the disease to their offspring, whereas mothers who are positive for antibody to HBeAg (anti-HBe) do so less frequently (15–20%). Other less frequent sources of infection include household contact with an HBV carrier, hemodialysis, exposure to infected healthcare workers, tattooing, body piercing, artificial insemination, and receipt of blood products or organs. Since routine screening of the blood supply was implemented in the early 1970s, transfusion-associated hepatitis B has become rare in the US. Hepatitis B can be transmitted by HBsAg-negative but anti-HBc-positive blood owing to the presence of a small amount of circulating HBV DNA that is only detectable by polymerase chain reaction (PCR) in 10–20% of cases.4 In addition, liver donors who are positive for anti-HBc alone can transmit hepatitis B.5 HBV is efficiently transmitted by percutaneous and mucous membrane exposure to infectious body fluids. The virus is 100 times more infectious than human immunodeficiency virus (HIV) and 10 times more infectious than hepatitis C virus. The presence of HBeAg positivity indicates a higher risk of transmission from mother to child, after needlestick exposure, and in the setting of household contact. HBV DNA has been detected by sensitive testing such as PCR in most body fluids. Although HBV replicates primarily in hepatocytes, the presence of replicative intermediates and virally
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Section V. Liver Diseases Due to Infectious Agents
Decline among MSM & HCWs
Decline among injecting drug users
16
Cases per 100 000 population
14 12
Vaccine licensed
Universal HBsAg screening of pregnant women Infant immunization
10 8 6
Figure 31-1. Rates of acute hepatitis B infection 1966–2000, and vaccine milestones. Rates of infection have been declining but are most notable in healthcare workers and neonates.
HBsAg screening of high risk pregnant women
OSHA rule Adolescent immunization
4 2
Immunization all children
0 1967 1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000 Years
encoded proteins in other sites, such as adrenal glands, testis, colon, nerve ganglia, and skin, suggests that there is a vast extrahepatic reservoir for infectious virus.6 Small amounts of HBV DNA have been demonstrated in peripheral mononuclear cells and liver tissue years after apparent resolution of chronic infection.7,8 Extrahepatic localization of low levels of replicating virus explain the relatively high rate of transmission of infection from anti-HBc positive organ donors.9
Other* 5% None identified 16% Heterosexual 42%
RATES OF INFECTION IN THE UNITED STATES Hepatitis B virus has infected an estimated 150 000–450 000 persons in the US each year during the past two decades.10 There has been a 75% reduction in incidence of acute hepatitis B between the years 1987 and 1998 owing to vaccination programs, changes in sexual lifestyle, refinements in blood screening procedures, and the availability of virus-inactivated blood components (Figure 31-1). Most striking has been the decrease among children and healthcare workers, groups with the highest rates of vaccination. Nonetheless, an estimated 78 000 cases of new HBV infections occurred in 2001, with the highest incidence of disease among sexually active young adults (20–29 years old), and higher rates among black and Hispanic people than in white persons.11 Between 1991 and 2001, approximately 40% of cases of acute hepatitis B reported to the Centers for Disease Control were caused by intimate contact among heterosexuals, approximately 18% were due to injecting drug use, and 19% occurred in men who have sex with other men (Figure 31-2). Between 1994 and 1998, more than half of all patients (56%) reported treatment for a sexually transmitted disease or imprisonment prior to their illness, suggesting that these cases might have been prevented by routine immunization in sexually transmitted disease clinics and correctional health care programs.10 According to the third National Health and Nutrition Examination Survey (1988–1994), one or more serologic markers of HBV infection were demonstrated in 4.9% of the US population, and the
636
MSM 19%
Injecting drug use 18% Figure 31-2. Reported risk factors for acute hepatitis B in adults, United States, 1991–2001. Source: Sentinel Counties Study of Viral Hepatitis, CDC, courtesy of Dr Eric Mast. *Other includes household contact, institutionalization, hemodialysis, blood transfusion, and occupational exposure.
prevalence of chronic infection was 0.2%.12 Traditional estimates based on the results of blood donation screening in the late 1970s also indicated a prevalence rate for chronic infection of 0.2–0.4% in the US population. Although it has traditionally been estimated that there are between 1.25 and 1.5 million HBV carriers in the US, this is likely to be a serious underestimate because of changing immigration patterns and under-representation of certain minorities, such as Asians and Pacific Islanders, in field surveys, ethnic groups in which chronic infection rates of 10% are routinely seen.
Chapter 31 HEPATITIS B
CLINICAL OUTCOME OF HBV INFECTION DEFINITIONS In common usage, the term ‘carrier’ has often been used to refer to persistently infected individuals with normal serum aminotransferase levels (sometimes inappropriately referred to as healthy HBV carriers). Because of the potentially confusing nomenclature, it has been proposed that the carrier state be categorized as inactive or active, with the former referring to patients who have evidence for HBV replication by polymerase chain reaction (PCR)-based assay only and normal or only mildly abnormal serum aminotransferase levels.13 Long-term follow-up of inactive carriers suggests that the majority of patients do not have progressive liver disease and do not develop complications. However, some of these patients will ultimately have one or more episodes of reactivated hepatitis in which there an increase in viremia and elevated serum aminotransferase activity. Also, some patients with the inactive carrier state may develop hepatocellular carcinoma. Active carriers, on the other hand, have evidence for HBV replication by non-PCR-based assays for HBV DNA, intermittently or persistently abnormal serum aminotransferase levels, and liver biopsy evidence for chronic hepatitis.
CLINICAL SEQUELAE OF ACUTE HBV INFECTION The age at which an individual becomes infected with HBV determines the clinical outcome. HBV infection in adults with an intact immune system is more likely to cause clinically apparent acute hepatitis B, with only 1–5% of cases becoming chronically infected.3 By contrast, as many as 95% of infected neonates become chronic HBV carriers owing to immunologic tolerance to the virus. In adults, fulminant liver failure due to acute hepatitis B occurs in less than 1% of cases, but this still accounts for 5% of all cases of acute liver failure and approximately 400 deaths annually in the US. Spontaneous survival in acute liver failure due to hepatitis B is approximately 20%. Liver transplantation has resulted in a 50–60% survival rate. Recurrent disease in the allograft is now infrequent owing to the administration of hepatitis B immunoglobulin and antiviral therapy. Rapid viral elimination may result in HBsAg clearance by the time of initial presentation. In these cases, the accurate diagnosis of fulminant liver failure due to hepatitis B may require testing with IgM antibody to hepatitis B core antigen (see Section on Serologic Markers of Infection).
CLINICAL SEQUELAE OF CHRONIC HBV INFECTION Between one-third and one-quarter of people chronically infected with HBV are expected to develop progressive liver disease (including cirrhosis and primary liver cancer). An estimated 15–25% of patients over the age of 40 with chronic HBV infection will die of liver-related causes, including hepatocellular carcinoma. The presence of active viral replication and long-standing necroinflammatory liver disease due to HBV strongly influences the rate
of progression to cirrhosis. The major determinant of survival for patients with chronic hepatitis B is the extent of the liver disease when they first come to medical attention.14 Cirrhosis is associated with decreased survival and a higher frequency of hepatocellular carcinoma. Five- and 20-year survival rates of 55% and 25%, respectively, have been reported in cirrhotic patients, whereas rates of 97% and 63% have been reported for those with mild disease.15,16 The most dramatic difference in survival seems to be between patients with compensated versus decompensated cirrhosis. An 84% 5-year survival was reported for compensated HBV-related cirrhosis. In the same study, a 14% 5-year survival was found for patients with ascites, jaundice, encephalopathy and/or a history of variceal bleeding.17 Multivariate analyses in several large cohort studies have identified that age, ascites, hyperbilirubinemia, and other features of advanced liver disease correlate independently with survival in cirrhotics. Interferon-induced clearance of HBeAg has been associated with longer survival without complications or the need for transplantation.18
MOLECULAR BIOLOGY OF HBV HBV is a small (3.2 kb) DNA virus that belongs to the family Hepadnaviridae. Other members of this family include human HBV-like agents that infect the woodchuck, the ground and tree squirrels, the woolly monkey, crane, heron, Ross goose, and the Pekin duck. HBV has a DNA genome that is in a relaxed, circular, partially doublestranded configuration (Figure 31-3). The genome is composed of four open reading frames in which several genes overlap and use the same DNA to encode viral proteins. The four viral genes are core, surface, X, and polymerase. The core gene encodes the core nucleocapsid protein that is important in viral packaging and HBeAg. The surface gene encodes the pre-S1, pre-S2, and S proteins (large [L], middle [M], and small [S] surface proteins, respectively). The X gene encodes the X protein, which has trans-activating properties and may be important in hepatic carcinogenesis. The polymerase gene has a large open reading frame (approximately 800 amino acids) and overlaps the entire length of the surface open reading frame. It encodes a large protein with functions critical for packaging and DNA replication (including priming, RNA- and DNA-dependent DNA polymerase, and Rnase H activities). Although HBV is a DNA virus, replication is through an RNA intermediate requiring an active viral reverse transcriptase/polymerase enzyme. The reverse transcriptase lacks a proofreading function, and this results in a higher mutation rate than other DNA viruses (estimated to be 1010–11 point mutations per day).19 Complete HBV genomic sequencing has identified a large number of mutations within the HBV genome, many of which are silent or do not alter the amino acid sequence of encoded proteins. Because of genomic overlap, however, some of the silent mutations in one open reading frame (for example the polymerase gene) may result in an amino acid substitution in an overlapping reading frame (HBsAg gene). The clinical implications of this are currently uncertain. Figure 31-4 illustrates the life cycle of HBV. The initial phase of hepadnaviral infection involves the attachment of mature virions to host cell membranes. The human receptor for HBV remains
637
Section V. Liver Diseases Due to Infectious Agents
k 2.4
2.1
NA bR
kb
Figure 31-3. Overlapping HBV genome and major transcripts. The hepatitis B genome is partially double-stranded with four overlapping open reading frames (ORF) or genes. The S gene encodes the viral surface envelope proteins (HBsAg) and is composed of the pre-S1, pre-S2 and S regions. The core gene consists of the precore and core regions, which give rise to the HBeAg and the core protein, respectively. The polymerase gene overlaps the entire S gene and mutations here may give rise to changes in the HBsAg protein. The fourth gene codes for an incompletely understood protein, HBX. Two 11 base-pair direct repeats (DR1 and DR2) are required for strand-specific HBV DNA synthesis during viral replication.
RN A
Pre-S 2 e Pr
1 -S
nd 3.5 kb RNA
+St r an d
-S tr a
-P
-S
RF
F OR
O
OR
F-C
5' DR 1 5'
DR2
Pre -C AA A AA A AA A AA A
ORF X
0.7 kb R
NA
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Excess HBsAg
Assembly ER Uncoating
HBsAg cccDNA
Positive Strand Synthesis
Removal of pregenome
Translation Repair Transcription 2.4 / 2.1 kb RNA 3.5 kb RNA
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Figure 31-4. Life cycle of hepatitis B virus. The receptor for viral entry has not been identified. Once inside the cell, the virus undergoes uncoating and nuclear entry of the HBV genome occurs, followed by repair of the single-stranded DNA strand and formation of the covalently closed (ccc) DNA template. Viral transcripts are formed for the HBsAg, DNA polymerase, X protein, and the RNA pregenome; the pregenome and polymerase are incorporated into the maturing nucleocapsid. The surface protein enveloping process occurs in the endoplasmic reticulum. Some of the nonenveloped nucleocapsid recirculates back to the nucleus and the cycle begins again. Excess tubular and spherical forms of HBsAg are secreted in great abundance. (Adapted from Perrillo R, Nair S. Hepatitis B and D. In: Feldman M, Friedman L, eds. Sleisenger and Fordtran’s gastrointestinal and liver disease, 8th edn. Philadelphia: Elsevier, 2005, ©2005, with permission from Elsevier.)
Chapter 31 HEPATITIS B
unknown. Entry of the virus results from fusion of the viral and host membranes as the nucleocapsid is released into the cytoplasm. Mechanisms of intracellular transport of viral genome into the nucleus are poorly understood, but the first step in genomic replication involves conversion of the relaxed circular form of HBV DNA into a double-stranded covalently closed circular form (cccDNA). These cccDNA molecules serve as the template for viral transcription and are the major form of viral DNA in the nucleus of the infected hepatocytes. Subgenomic (0.7–2.4 kb) and pregenomic (3.5 kb) RNA molecules are transcribed from this template. The L protein is translated from the 2.4 kb RNA, the M and S proteins from the 2.1 kb message, and the HBX protein from the 0.7 kb transcript. The pregenomic RNA serves as the template for reverse transcription as well as the mRNA for core and polymerase; the precore RNA codes for the precore gene product. HBV replication begins with encapsidation of the pregenome RNA through complex interactions between host and viral proteins. HBV DNA polymerase reverse transcribes the pregenomic RNA into a negative-strand HBV DNA, which in turn serves as the template for positive-strand synthesis to form a partially doublestranded genome. Concurrent with HBV DNA synthesis, the nucleocapsid undergoes maturation and, through a yet incompletely understood mechanism, interaction occurs with the S protein to initiate viral assembly in the endoplasmic reticulum. S protein is synthesized in the endoplasmic reticulum, where monomer aggregates that exclude host membrane proteins subsequently bud into the lumen as subviral particles. Once formed, the HBsAg undergoes glycosylation in the endoplasmic reticulum and the Golgi apparatus. Non-infectious subviral particles (spherical and filamentous forms of HBsAg) are secreted in great abundance compared to mature virions.
GENOMIC VARIATION HBV Genotypes A genetic classification based on the comparison of complete genomes has demonstrated eight genotypes of HBV (A–H).20 The different genotypes are based on an intergroup divergence of 8% or more in the complete nucleotide sequence. During the last few years there has been a rapid increase in information regarding the epidemiology, molecular characteristics, clinical impact and treatment response of different HBV genotypes (Table 31-1). In the next several years, new extrapolations of the importance of HBV genotypes should emerge because several methods are now available for their detection, including a commercially available line probe assay (INNO-LiPA, Innogenetics, Ghent, Belgium). HBV genotype A is found mainly in Caucasians in northwestern Europe and the USA. Genotypes B and C are highly prevalent in Asia. Recent changes in immigration patterns have resulted in an influx of Asian HBsAg carriers with these genotypes into the US.21 Genotype D has been found in all continents, with the highest prevalence in the Mediterranean region and the Middle East. Genotype E is almost entirely restricted to West Africa and is genetically very similar to genotype D. The most divergent genotype F is found in South and Central America. It is believed that this is the original genotype of the New World.22 Cases of genotype G have been reported in the US and France. A newly defined genotype, H, has been described in Mexico, Nicaragua, and California.
Table 31-1. Hepatitis B Genotypes and Their Possible Clinical Associations Eight well characterized genotypes (A–H) Different geographic distributions A Northwestern Europe, North America, Central Africa B Southeast Asia, including China, Japan and Taiwan (increasing prevalence in North America) C Southeast Asia (increasing prevalence in North America) D Southern Europe, Middle East, India E West Africa F Central and South America, American natives, Polynesia G USA, France H Central and South America: Proposed clinical associations Time to HBeAg seroconversion and probability of HBsAg loss (B shorter than C) Response to treatment with interferon-a (A>B>C>D) Precore/core promoter mutant frequency (precore not selected with genotypes A and F) Liver disease activity and risk of progression (B purpuric rash Papular acrodermatitis of childhood (Gianotti–Crosti syndrome) Articular manifestations Polyarthralgia, polyarthritis, tenosynovitis Manifestations of chronic HBV infection Nephritic Membranous and membranoproliferative glomerulonephritis Immunoglobulin A nephropathy Polyarteritis nodosa Cutaneous Purpuric rash > urticaria and maculopapular rash Rare manifestations* Neurologic Guillain–Barré syndrome Peripheral mononeuropathy, mononeuropathy multiplex, seizures Myopathies Polymyositis, dermatomyositis, myocarditis Cutaneous manifestations Lichen planus, pyoderma gangrenosum, erythema nodosum Dermatomyositis-like syndrome, porphyria cutanea tarda Rheumatologic Rheumatoid arthritis, polymyalgia rheumatica, systemic lupus erythematosus Essential mixed cryoglobulinemia (type II, III) Hematologic Aplastic anemia * Data based on isolated case reports and small case series.
The Gianotti–Crosti syndrome, also known as papular acrodermatitis of childhood, is a rare skin disorder associated with acute HBV infection in children aged 2–6 years. Typically, eruptions of flat-topped small papules are observed on the face and extremities in association with generalized lymphadenopathy.
Arthropathy As with skin manifestations, synovial symptoms may present either alone or as part of the serum sickness-like syndrome prior to the onset of hepatic symptoms. The arthropathy can be difficult to distinguish from acute rheumatoid arthritis, as it may afflict the same joints and be associated with morning stiffness. A minority of patients may experience polyarthritis or tenosynovitis. Typically, symptoms begin abruptly 6–20 weeks after exposure to HBV and 2–3 weeks before other systemic symptoms. The joint symptoms almost always resolve before the development of jaundice, should that occur, and do not lead to joint deformity. In some cases, polyarthropathy may persist for several months.
Polyarteritis Nodosa Polyarteritis nodosa (PAN) occurs sporadically or as a serious complication of chronic HBV infection, conferring a mortality rate of 30–50%. The process affects small to medium-sized arteries and arterioles. There is a strong relationship between PAN and HBV. Positive HBV serology has been reported in 36–70% of patients with PAN.50 Conversely, only two in 500 HBV infections result in PAN. PAN typically presents with a non-specific systemic prodrome that includes fever, malaise, myalgias, and arthralgias. Organ-specific complications subsequently develop owing to end-organ damage from fibrinoid necrosis and perivascular inflammation, which cause microaneurysm formation, stenosis, and vessel occlusion. Neurologic manifestations are very common and include polyneuropathy, mononeuropathy, and CNS involvement leading to confusion, memory loss, seizures, or intracranial hemorrhage. Renal failure and hypertension are common. Cutaneous manifestations include tender subcutaneous nodules mostly on the lower extremities, livedo reticularis, urticaria, ulceration and angioedema. Other complications include eosinophilia, polyarthralgia, myalgia, and cardiomyopathy. Whereas PAN is generally associated with the same range of complications for both the sporadic and the HBV-associated forms, some manifestations, such as renal infarction, malignant hypertension, orchiepididymitis and mesenteric ischemia, are seen more commonly with HBV infection. The disease is characterized by circulating immune complexes that contain HBsAg; for this reason plasmapheresis may be indicated. Good therapeutic responses have also been observed with antivirals, either given alone or in combination with plasmapheresis. There is no apparent relationship between the severity of the vasculitis and the severity of the hepatic disease, and often the hepatic disease is relatively mild although high-level viral replication is frequently present. The course of the disease is variable, but the prognosis is gravest for those with significant proteinuria (>1 g/day), renal insufficiency, gastrointestinal involvement, cardiomyopathy, and CNS involvement.
Glomerulonephritis Several types of glomerular lesion have been described in chronic HBV infection, with membranous glomerulonephritis and
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membranoproliferative glomerulonephritis being the most common. Renal biopsies have found immune complex deposition and cytoplasmic inclusions in the glomerular basement membrane. These complexes activate complement and cytokine production, with a subsequent inflammatory response. Nephrotic syndrome is the most frequent presentation of HBV-associated glomerulonephritis. During childhood, significant renal failure at presentation is infrequent and a prior history of clinical liver disease is uncommon. Nevertheless, liver biopsies almost always demonstrate varying degrees of chronic viral hepatitis. The diagnosis of HBV-associated glomerulonephropathy is usually established by serologic evidence of HBV antigens or antibodies, the presence of immune complex glomerulonephritis on renal biopsy, and by the demonstration of glomerular deposits of one or more HBV antigens such as HBsAg, HBcAg, or HBeAg by immunohistochemistry. Most patients have detectable HBeAg in serum, and in addition will demonstrate activation of the classic complement cascade, with low serum C3 and occasionally C4 levels. The renal disease often resolves over months to several years, especially in children. Often this resolution occurs with seroconversion of HBeAg. Rarely, however, renal failure may ensue. The natural history of HBV-related glomerulonephritis in adults has not been well defined, but several reports suggest that glomerular disease is often slowly and relentlessly progressive.51 Treatment has been successfully accomplished with interferon-a and has been linked to successful long-term control of HBV replication.52 Nucleoside analogue therapy has resulted in improved renal function and diminished proteinuria.
Pathology of Chronic Hepatitis B Chronic hepatitis B infection is characterized by mononuclear cell infiltration in the portal triads. Periportal inflammation often leads to the disruption of the limiting plate of hepatocytes (referred to as
interface hepatitis), and inflammatory cells can often be seen at the interface between collagenous extensions from the portal tract and liver parenchyma (referred to as active septae). During reactivated hepatitis B there is more intense lobular inflammation that is reminiscent of the features of acute viral hepatitis. Unlike hepatitis C, steatosis is not a feature of chronic hepatitis B. The only histologic feature on routine light microscopy that is specific for chronic hepatitis B is the presence of ground-glass hepatocytes (Figure 31-6). The morphological findings are due to HBsAg particles (20–30 nm in diameter) that accumulate in the dilated endoplasmic reticulum. Owing to the presence of high levels of cystine in HBsAg, ground-glass cells have a high affinity for certain dyes, such as orcein, Victoria blue and aldehyde fuchsin. Groundglass hepatocytes may also be seen in HBsAg carriers, where they may be detected in up to 5% of cells. When present in abundance, they are often indicative of a state of active viral replication. Immunofluorescence and electron microscopic studies have shown HB core antigen inside the hepatocyte nuclei of affected cells. During periods of intense hepatitis activity, cytoplasmic core antigen staining is generally easily observed. With successful treatment with nucleoside analogs the cytoplasmic core antigen staining often disappears, but nuclear core antigen staining may remain, indicating persistence of the cccHBV DNA template.
ACUTE FLARES IN CHRONIC HEPATITIS B Chronic hepatitis B is often punctuated by sudden flares of disease activity that are reflected by an increase in serum aminotransferase levels. Although a uniform definition of a flare is lacking, they have frequently been described as increments that are at least equal to
Figure 31-6. Ground-glass hepatocytes indicated by arrows. The cellular inclusions represent large amounts of HBsAg in the endoplasmic reticulum of infected hepatocytes. This finding is essentially diagnostic of hepatitis B infection in a patient with chronic hepatitis. (Hematoxylin and eosin; ¥630.) Photo courtesy of Dr Gist Farr, Department of Pathology, Ochsner Clinic Foundation.
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Chapter 31 HEPATITIS B
Table 31-3. Hepatitis Flares in Chronic Hepatitis B Virus Infection Cause
Comment
Spontaneous
Precipitating factors for antecedent viral replication unclear Often observed during withdrawal; requires pre-emptive antiviral therapy
Immunosuppressive therapy
adulthood owing to a breakdown of immunotolerance to HBV. Multiple episodes of reactivation and remission have been shown to accelerate the progression of chronic hepatitis B, and are particularly likely to occur in patients with the precore mutant form.42
IMMUNOSUPPRESSIVE THERAPY
twice the baseline values. These spontaneous flares are an important part of the natural history of hepatitis B, as when repeated they cause histologic progression. Acute flares in chronic hepatitis B occur in association with a number of circumstances and clinical situations (Table 31-3). Most of these flares are due to a change in the balance between immunologic response to HBV and the extent of viral proliferation. Acute flares in chronic hepatitis B that are not explainable by infection with other hepatotropic viruses often occur as a secondary response to increased levels of replicating wild-type or mutant virus, or as a result of therapeutic intervention with immunologic modifiers such as interferon, corticosteroids, or cancer chemotherapy. In most instances, the initiating events for the acute exacerbations in chronic hepatitis B may not be readily identifiable, and these flares are considered to be spontaneous in nature.
Reactivation of HBV replication is a well-recognized complication in patients with chronic HBV infection who receive cytotoxic or immunosuppressive therapy.54 Suppression of the normal immunological responses to HBV leads to enhanced viral replication and is thought to result in widespread infection of hepatocytes. Upon discontinuation of immunosuppressive medications such as cancer chemotherapy, antirejection drugs, or corticosteroids, immune competence is restored and infected hepatocytes are rapidly destroyed. The more potent the immunosuppression, the greater the level of viral replication, and thus the greater the potential for serious clinical consequences of sudden withdrawal and restoration of immunologic competence. Postmortem study of liver tissue in cases of severe liver injury has documented sparse staining of viral antigens, suggesting that patients were in an active state of immune clearance.55 The vast majority of patients have been HBsAg positive before treatment, but some studies have emphasized the reappearance of this marker in patients who were initially positive for anti-HBc alone.56 Reactivated hepatitis in HBsAg-negative patients with antiHBc is explainable by the possible latency of HBV in liver and mononuclear cells and the large extrahepatic reservoir of HBV. Chemotherapy given to cancer patients who are chronic hepatitis B carriers has been shown to be associated with an increased risk of liver-related morbidity and mortality.57 Acute flares of hepatitis B due to cancer chemotherapy and other immunosuppressive drugs are often detected relatively late. The use of antiviral treatment after major biochemical abnormalities have been detected should be anticipated to have relatively little effect on reducing liver injury, because much of the immunologic response to HBV and viral elimination has already occurred. Instead, the key to management of this situation lies in anticipating its occurrence and early antiviral treatment. Current treatment guidelines suggest that nucleoside analog therapy should commence at the onset of cancer chemotherapy or a finite course of immunosuppressive therapy and be maintained for 6 months after completion of the immune-modifiying therapy.
SPONTANEOUS FLARES
ANTIVIRAL THERAPY-INDUCED FLARES
Antiviral therapy Interferon Lamivudine On treatment YMDD mutant Withdrawal*
HIV treatment
Genotypic variation Precore and core promoter mutants Superinfection with other hepatitis viruses
Often during the second to third month; may herald virologic response No more common than placebo Can have severe consequences in patients with advanced disease Caused by rapid re-emergence of wild-type HBV; can have severe consequences in patients with advanced disease As above when using lamivudine; can also occur with immune reconstitution or secondary to antiretroviral drug hepatotoxicity Fluctuations in ALT common with precore mutant May be associated with suppression of HBV replication
*Has also been reported with other nucleoside analogs.
Spontaneous exascerbations are often due to reactivated infection, and it has been shown that an increase in serum HBV DNA often precedes an increase in serum aminotransferase level. Histologic evidence for acute lobular hepatitis superimposed on the changes of chronic viral hepatitis is frequently observed during these flares. Immunoglobulin M (IgM) antibody to hepatitis B core antigen (HBcAg), a marker which is often diagnostic of acute viral hepatitis, also may appear at this time. The reasons for reactivated infection are unknown, but are probably explained by subtle changes in the immunologic control of viral replication. Reactivation seems to occur more commonly in individuals who are infected with HIV-1.53 In individuals who acquire their infection early in life, flares become more common during
Antiviral treatment of chronic hepatitis B can be associated with flares of hepatitis in several ways. Flares may occur during interferon therapy, after withdrawal from nucleoside analogs, following withdrawal of corticosteroids, and in association with lamivudineresistant mutants.
Interferon Interferon-induced flares occur in approximately a third of treated patients and are explainable by the immunostimulatory properties of the drug. Flares generally occur during the second to third month of treatment with the conventional preparations of interferon. It is currently unknown whether or not they occur as commonly with
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the newer long-acting (pegylated) forms of interferon. ALT flares have been shown to be a predictor of sustained virologic response, particularly in patients with high-level viremia.58 Flares tend to be particularly common in patients who have decompensated liver disease, having occurred in 50% of patients in one series.59 They are frequently associated with clinical deterioration in these patients.
Nucleoside Analogs ALT flares occur in approximately 20–25% of patients after withdrawal of nucleoside analogs such as lamivudine and adefovir. These are probably due to rapid resurgence of wild-type virus, and although generally well tolerated they have been associated with serious clinical exacerbations in patients with advanced liver disease.60 Reinstitution of therapy is often associated with a decline in HBV DNA and aminotransferase levels. Flares have been seen to follow the emergence of YMDD mutant HBV during lamivudine therapy.61 Initial reports emphasized the temporal occurrence of these flares at the time of or shortly after the detection of lamivudine resistance. However, further follow-up of patients with lamivudine-resistant HBV mutants indicates that the incidence of moderate to severe ALT flares (defined as >5 and >10 times the upper limit of normal) increases with time after detection of lamivudine resistance. In one long-term follow-up study the cumulative incidence of ALT flares was as follows: 4 years (61%).62
Corticosteroid Withdrawal ALT levels increase, often with an inverse decline in HBsAg concentration and serum HBV DNA, following withdrawal of corticosteroids. Clinical trials have been carried out in which a short course of corticosteroids was used prior to conventional antiviral therapy and suggest that this may enhance virologic response rates.63,64 The immune rebound following withdrawal from a 4–8-week course of corticosteroids may be due to increased activation of lymphocytes that promote Th1 cytokine responses at a time when there is increased viral antigen expression. Serious hepatic decompensation has been reported in patients with advanced disease, and this approach is no longer being used.
Antiretroviral Therapy ALT flares occur in HIV–HBV co-infected patients receiving highly active antiretroviral therapy (HAART) owing to a number of potential causes.65 Lamivudine resistance and withdrawal may be associated with ALT flares, and HBV infection clearly increases the risk of toxicity from antiretroviral medication, usually within the first 6 months of starting treatment. Immune reconstitution due to HAART may also be associated with ALT flares. These patients may also be particularly susceptible to flares due to infection with other hepatitis viruses.
Genotypic Variation Chronic infection with precore mutant HBV is often associated with multiple flares of liver cell necrosis interspersed with periods of asymptomatic HBV carriage.42 Approximately 45% of patients have episodic ALT flares with normal levels in between episodes, and 20% have flares superimposed on persistently abnormal ALT.66 These
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flares have been attributed to increases in the concentration of precore mutants and changes in the proportion of precore to wildtype HBV.67 Mutations at the basal core promoter (BCP) region of the HBV genome are associated with decreased HBeAg synthesis, active liver histology, and increased viral replication. Multiple exacerbations of hepatitis due to reactivated HBV infection have been described in patients with BCP mutation, either alone or in association with precore mutation.68 HBeAg-negative patients who harbor both the precore and the core promoter mutants may be particularly predisposed to severe reactivation episodes following chemotherapy for malignancies.69
Infection with Other Viruses Patients with chronic HBV infection may exhibit severe flares in serum aminotransferases and even frank liver failure when superinfected with other hepatotropic viruses, such as hepatitis A virus (HAV), hepatitis C virus (HCV) and hepatitis delta virus (HDV). Increased mortality has been reported when delta virus superinfection is superimposed on chronic hepatitis B, and chronic hepatitis delta virus infection is often associated with multiple fluctuations in serum aminotransferase levels (see section on Hepatitis Delta Virus). Acute hepatitis C superimposed on chronic hepatitis B has been reported to be as severe as delta superinfection and has been associated with a high rate of liver failure (34%) and death (10%).70 Acute hepatitis C often leads to chronic infection, and the subsequent course also may be characterized by frequent fluctuations in serum aminotransferase levels. Patients with chronic hepatitis B who become infected with other hepatotropic viruses may become HBeAg negative and serum HBV DNA negative by non-PCR-based assays owing to a process of viral interference. This has been described with superimposition of HAV, HCV, and HDV infections and combined infections with HCV and HDV.
SEROLOGIC DIAGNOSIS OF ACUTE AND CHRONIC HEPATITIS B SEROLOGIC MARKERS OF ACTIVE OR PAST INFECTION Hepatitis B surface antigen (HBsAg) appears in serum 2–10 weeks after exposure to the virus and before the onset of symptoms or elevation of serum aminotransferase levels. HBsAg usually becomes undetectable after 4–6 months in self-limited acute hepatitis. Persistence for more than 6 months implies progression to chronic HBV infection. Figure 31-7A indicates the typical time course for acute hepatitis B with complete immunologic recovery. The disappearance of HBsAg is followed several weeks later by the appearance of hepatitis B antibody (anti-HBs). In most patients anti-HBs persists for life and provides long-term immunity. In some cases anti-HBs may not become detectable, but these patients do not appear to be susceptible to recurrent infection. Anti-HBs may not be detectable during a window period of weeks to months after the disappearance of HBsAg. During this period, HBV infection is diagnosed by the detection of IgM antibodies against hepatitis B core antigen.
Chapter 31 HEPATITIS B
HBsAg
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HBeAg Symptoms 1000
1000 800
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Figure 31-7. A Typical acute hepatitis B. There is a brief period of viremia during which HBV DNA and HBeAg is detectable in serum. These events precede the onset of ALT abnormality. Both HBeAg and HBV DNA are generally no longer detectable after 12 weeks from the onset of illness. B Typical HBeAg-positive chronic hepatitis B. During the replicative phase of infection there is variable expression of ALT abnormality and persistence of circulating HBeAg. C Chronic Hepatitis B with transition to inactive carrier state. During the inactive HBV carrier state low levels of HBV DNA are detectable in serum by PCR only. Generally, HBV DNA values are 2 log10 or less. Continued
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Section V. Liver Diseases Due to Infectious Agents
HBV DNA
Figure 31-7, cont’d. D Chronic hepatitis B: Evolution of HBeAg-negative mutant. In some patients HBeAg-negative mutants of HBV (precore and core promoter mutants) are selected after the loss of HBeAg. Such patients often have fluctuations in serum HBV DNA and ALT, as indicated in the right hand side of the graph.
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Coexistence of HBsAg and anti-HBs has been reported in approximately 10 to 25% of HBsAg-positive individuals, and this occurs more commonly in those with chronic hepatitis B.71 In most instances the antibodies are low level, non-neutralizing, and heterotypic, i.e. directed against a different subtype of HBsAg than that present in the infected patient. The mechanisms behind this finding are not clear, but it may be due to antibody formed against minor variants of the HBsAg protein. The presence of these heterotypic antibodies is not associated with specific risk factors or change in clinical course, and may occur in the setting of active liver disease and viral replication.72 Hepatitis B core antibody (anti-HBc) is detectable in acute and chronic HBV infection. During acute infection, anti-HBc is predominantly of the IgM class and is usually detectable for 4–6 months after an acute episode of hepatitis, and may rarely persist for up to 2 years. IgM anti-HBc may become detectable during exacerbations of chronic hepatitis B. Anti-HBc persists in those who recover from acute hepatitis B, and also in association with HBsAg in those who progress to chronic infection. In areas with low HBV endemicity the occurrence of isolated presence of anti-HBc has been found in 1–4% of the general population. Isolated reactivity for anti-HBc may occur in a number of situations. It may occur during the window period of acute hepatitis B, when it is predominantly of the IgM class; many years after recovery from acute hepatitis B, when anti-HBs has fallen to undetectable levels; as a false positive serologic test; after many years of chronic infection when the HBsAg titer has decreased below the level of detection; in individuals who are co-infected with hepatitis C, and rarely, as a result of variable sensitivity of HBsAg assays.73 Evidence for co-infection with hepatitis C virus has been demonstrated in up to 60% of individuals with anti-HBc only.74 The results of PCR testing of sera have shown that 0–30% of patients with isolated anti-HBc have HBV DNA in serum. Usually, the HBV DNA is detectable at a low level and not by standard hybridization assays. The presence of low-level viremia in these HBsAg-negative subjects has clinical implications with regard to potential infectivity. It has been shown that anti-HBc testing of
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HBV DNA (million copies/ml)
900
blood donors prevents some cases of post-transfusion hepatitis B. Also, the risk of transmission of HBV infection from a liver donor with isolated anti-HBc to a liver transplant recipient has been found to be as high as 50–70% in some series, with the risk of transmission depending partly on the HBV serological status of the recipient.75,76 Low-level viral replication also has implications with regard to the possibility of underlying liver disease. HBV DNA in serum and liver tissue has been confirmed by PCR in HBsAg-negative patients with cirrhosis and hepatocellular carcinoma, and PCR has confirmed an association in some cases of fulminant non-A–C hepatitis.77,78 Hepatitis B e antigen (HBeAg) is a soluble viral protein that is found in serum early during acute infection. HBeAg reactivity usually disappears at or soon after the peak in serum enzymes, and persistent detection 3 or more months after the onset of illness indicates a high likelihood of transition to chronic infection. The finding of HBeAg in the serum of an HBsAg carrier indicates greater infectivity and a high level of viral replication that may require antiviral therapy. Using a commercially available PCR assay, nearly 90% of cases with HBeAg positive chronic hepatitis B were found to have serum HBV DNA levels that were persistently above 105 copies/ml, and the mean value was 8.37 log10 copies.79 In contrast, anti-HBepositive patients had much lower serum HBV DNA levels, with the values being higher in those with persistently or intermittently abnormal ALT (mean of 5.1 log10 copies/ml) than patients with persistently normal ALT (3.10 log10 copies/ml). Most HBV carriers who are HBeAg-positive have active liver disease, the exception being HBeAg-positive children and young adults with perinatally acquired HBV infection, who usually have normal ALT levels and minimal inflammation in the liver.12 Figure 31-7B indicates the serologic features of a typical case of HBeAgpositive chronic hepatitis B. In general, seroconversion from HBeAg to anti-HBe is associated with a mean 3 log10 or greater reduction in serum HBV DNA and remission of liver disease. Patients who have normal ALT and detection of small amounts of HBV DNA (103 copies or less) after HBeAg seroconversion are considered to be inactive carriers (Figure 31-7C). Some patients, however,
Chapter 31 HEPATITIS B
Digene corp.
Roche molecular systems
Bayer corp.
1010 109 108
HBV DNA IU/ml
107
Figure 31-8. Commercially available HBV DNA assays. The dynamic ranges of quantification of the available HBV DNA assay vary considerably and none detects the full range of HBV values that can be observed. The expression of results in IU/ml is preferred when trying to equate the results of one assay with another. (Adapted from Standring DN, Bridges EG, Placidi L, et al. Antiviral B-L-nucleosides specific for hepatitis B virus infection. Antivir Chem Chemoter 2001; 12:119–129.)
106 105 104 103 102 10 1 HBV digene HBV digene Ultra- Amplicor Cobas Cobas hybridhybrid- sensitive HBV amplicor taqman capture I capture II digene monitor HBV 48 HBV hybridmonitor capture II
continue to have active liver disease and detectable HBV DNA in serum, due to either low levels of wild-type virus or the selection of precore or core promoter mutations that impairs HBe Ag secretion (Figure 31-7D). HBV DNA can be measured in serum using qualitative or quantitative assays. The clinical utility of testing for serum HBV DNA has been hampered by the absence of a licensed test in the US, as well as an accepted international reference standard. A number of non-PCR-based assays are available, with varying levels of sensitivity from 103 to 105 genomic copies per milliliter of serum (Figure 31-8). Although less sensitive than PCR, the results of the non-PCRbased assays correlate with a clinical response to antiviral therapy, and several of the currently available antiviral therapies were licensed based on clinical trials that used these assays. There are several shortcomings, however, to the use of these less sensitive assays, and as a result most clinical laboratories use one of several commercially available PCR assays with enhanced sensitivity (102 genomic copies/ml or less). The measurement of serum HBV DNA by quantitative PCR is potentially useful in a number of clinical circumstances, perhaps one of the most common of which is in distinguishing HBeAg-negative chronic hepatitis B from the inactive HBV carrier state with another source of liver test abnormalities, such as alcohol or obesity. Quantitative serum HBV DNA testing is also useful in evaluating candidacy for antiviral therapy, as well as to monitor response during treatment. Patients with high serum HBV DNA have been found to respond less frequently to conventional interferon therapy.80 When using solution hybridization testing, a baseline level of 200 pg/ml or
Versant Versant HBV HBV DNA 1.0 DNA 3.0
more (roughly equivalent to 56 million copies by PCR assay) has been found to be associated with a very low rate of virologic response. By contrast, quantitative PCR testing of baseline serum HBV DNA has not been shown to correlate with a response to nucleoside analog therapy owing to more potent inhibition of viral replication. Monitoring the HBV DNA level at key intervals during treatment allows one to assess the likelihood of HBeAg clearance. Several studies have found that the level of serum HBV DNA at 12–24 weeks of nucleoside analog treatment may distinguish those individuals more likely to go on to HBeAg seroconversion.81,82 Other studies have suggested that baseline HBV DNA level during treatment can be used to evaluate the likelihood of relapse after treatment discontinuation, as well as the chance of developing resistance to lamivudine.83,84 Reappearance of HBV DNA, or a 1 log increase in serum HBV DNA during treatment, suggests that drug resistance has occurred.85Finally, high pretreatment levels of serum HBV DNA have been shown to correlate with a higher rate of recurrent infection in liver transplant recipients treated with lamivudine.86,87 Qualitative PCR is an even more sensitive method of detecting HBV DNA than quantitative PCR. Qualitative PCR has altered traditional concepts of clearance of HBV DNA in acute and chronic infection. HBV DNA can be detected in serum and peripheral mononuclear cells years after recovery from acute viral hepatitis.46 The disappearance of HBsAg is followed by the loss of HBV DNA from serum in chronic hepatitis B. Even then, HBV DNA persists in small amounts in liver tissue and peripheral mononuclear cells years after the infection has subsided.8 Detection of HBV DNA before liver transplantation may identify patients who are at a higher
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risk of apparent de novo hepatitis after transplantation, and this also may allow the identification of HBV as the cause of liver disease in HBsAg-negative patients.88,89 Finally, detection of minute amounts of HBV DNA may be particularly important in patients with fulminant hepatitis B, who frequently have cleared HBsAg by the time they obtain medical attention.90
ANTIVIRAL THERAPY OF CHRONIC HBV INFECTION VIROLOGIC ENDPOINTS AND DEFINITIONS OF RESPONSE The primary goal of treatment for chronic hepatitis B is durable suppression of HBV DNA to levels below that associated with liver disease. This can be accomplished with either interferon-a or nucleoside analogs. The level at which this occurs is generally considered to be 2 ¥ ULN
+ or -
+
Cirrhosis
+ or -
-
Cirrhosis
Duration of therapy IFN-a, 16 weeks Lamivudine, minimum of 1 year Continue for 3–6 months after HBeAg seroconversion Adefovir, minimum of 1 year IFN-a non-responders/contraindications to IFN-a Æ lamivudine or adefovir Lamivudine resistanceÆadefovir IFN-a, lamivudine, or adefovir may be used as initial therapy, but IFN-a or adefovir preferred owing to need for long-term therapy End-points of treatment—sustained normalization of ALT and undetectable HBV DNA by PCR assay Duration of therapy IFN-a, 1 year Lamivudine, >1 year Adefovir, >1 year IFN-a non-responders/contraindications to IFN-aÆ lamivudine or adefovir Lamivudine resistanceÆadefovir £2 ¥ ULN No treatment required Compensated: lamivudine or adefovir Decompensated: lamivudine or adefovir, refer for liver transplantation; IFN-a contraindicated Compensated: observe Decompensated: refer for liver transplantation
Adapted from reference 136. * Treatment recommendations for compensated hepatitis B are intended primarily for those with moderate to severe hepatitis. ** HBV DNA >105 copies/mL.
of normal (Table 31-6). This recommendation is based on the observation of very low rates of sustained virologic response with either interferon or nucleoside analogs in patients with minimal pretreatment ALT elevation.96All guidelines indicate that decisions should ideally be made in the context of liver histology, and treatment should be preferentially directed to individuals with moderate to severe hepatitis. Some experts feel that the guidelines are too restrictive, however, and that patients with advanced fibrosis may benefit from treatment even when serum HBV DNA is below 105 copies.97 Also, the exclusion from treatment of patients with ALT levels that are normal or less than twice the upper limit of normal is controversial, because such individuals may occasionally have significant fibrosis and necroinflammatory disease.97 Liver biopsy is key in making the most appropriate treatment decision in this situation.
CURRENTLY AVAILABLE ANTIVIRAL AGENTS Interferon-a Interferon is effective after a relatively short course of treatment (6 months to 1 year), and unlike the nucleoside analogs has not been associated with drug resistance. Also, in contrast to nucleoside analogs, interferon has direct immunomodulatory properties. Interferon enhances human leukocyte antigen (HLA) class I antigen expression on the surface of infected hepatocytes and augments CD8+ cytotoxic T-lymphocyte activity. This could be operatively important in reducing the amount of the covalently closed circular (ccc) form of HBV DNA (the genomic template for viral transcrip-
tion), which may explain the loss of HBsAg that occurs in approximately 5–8% of interferon-treated patients. The major disadvantages of interferon are related to its poor acceptance in comparison to nucleoside analogs, its lower level of HBV DNA suppression, and the greater cost during a year of treatment. Flares of ALT have been described during interferon-a therapy, and although these are potentially important in achieving a virologic response, their unpredictability leads to inconsistent antiviral efficacy. The magnitude of an ALT flare has been shown to predict the likelihood of sustained virologic response in patients with high-level viremia, suggesting that vigorous cell-mediated immune responses are often required to overcome high levels of viral replication.58 Pegylated interferon has been found to be more effective than conventional interferon in the treatment of HBV infection.98 Doses of 1.0 mg/kg body weight of pegylated interferon-a2b and 180 mg of pegylated interferon-a2a given once weekly have been studied in clinical trials.99–101 No data are yet available for judging whether the increased effectiveness of pegylated interferon is primarily a function of a more pronounced effect on viral replication or of greater immunomodulatory action.
Impact of Genotype on Response Viral genotype appears to effect the response to interferon. Early studies to recognize the potential importance of genotype in interferon-treated patients were limited by small sample sizes.102 A relationship between virological response and genotype, however, has been recently reaffirmed in a large multicenter study of pegylated
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Section V. Liver Diseases Due to Infectious Agents
LAM ADV ETV IdT
5V 20 M
23
6T
4V N
20
41 M
20
1 M
G
02 S2
84
V A1
81 A1
80
L
M
FTC
L1
Nucleoside analogs have excellent oral bioavailability, a good safety record, and antiviral efficacy comparable to that observed with interferon-a2b. They are also considerably less expensive than interferon when given for 48–52 weeks, as recommended in the prescribing information. These drugs have proved to be particularly useful in the management of decompensated cirrhosis, a clinical situation in which even small doses of interferon can lead to worsening liver failure and severe infections. Table 31-7 lists the available agents and those in late-stage development. Nucleoside analogs replace natural nucleosides during the synthesis of the first or second strand (or both) of HBV DNA. They thus serve as competitive inhibitors of the viral reverse transcriptase and DNA polymerase. Because nucleoside analogs partially and reversibly suppress viral replication, they have to be given for more than 1 year in most cases to achieve maximal efficacy. Unfortunately, drug resistance occurs with prolonged monotherapy (Table 31-7). Salvage therapy with another nucleoside or nucleotide analog may be possible if the two drugs do not share similar resistance sites (Figure 31-9). Nucleoside analogs have several other limitations as well. With these agents, demonstrating the clearance of the covalently closed circular form of HBV DNA (cccDNA) has been difficult, and unlike with interferon HBsAg clearance rarely occurs after 1 year of treatment. These problems may be partly due to the fact that nucleoside analogs, unlike interferon, do not have a direct enhancing effect on the immunologic response to HBV.56 Also, as indicated above, post-withdrawal ALT flares have been seen in approximately 25% of cases after discontinuation of treatment.
Lamivudine has the distinction of being the first nucleoside analog to be specifically licensed for the treatment of hepatitis B. The drug has been shown to be a relatively potent inhibitor of viral replication, convenient to administer, and free of severe adverse effects. Clinical trials demonstrated that a 1-year course of lamivudine resulted in the suppression of viral replication and histologic improvement.103 In one study, HBeAg seroconversion and HBeAg loss occurred in 17% and 32% of patients, respectively.104 A 2-year course of lamivudine proved to be more effective, resulting in an increase of HBeAg seroconversion from 17% at 1 year to 27% at 2 years.105 Prolongation of treatment beyond 1 year, however, has been associated with incremental changes in viral resistance (approximately 40% at 2 years), and the longer treatment is continued the more frequently resistance is seen (65% at year 5).62 Resistance is even more commonly encountered (90% at 4 years) in patients co-
73
Nucleoside Analogs
Lamivudine
V1
interferon-a2b. In this study, HBeAg-positive patients with genotype A responded more frequently than those with genotypes B, C and D (47%, 44%, 28%, and 25%, respectively).99 These results confirm and extend the finding of earlier studies in HBeAg-positive and HBeAg-negative patients suggesting that patients with genotype A respond more frequently than those with genotype D. The impact that genotype exerts on response to interferon is particularly relevant in the selection of therapy in the growing population of AsianAmericans, who often carry the less responsive genotype C.21
Figure 31-9. HBV drug resistance sites. Reported HBV polymerase mutations by treatment. With the exception of adefovir, dipivoxil and tenofovir (not shown), the mutational patterns of the L-nucleoside analogs overlap with that for lamivudine at nucleotide site 204, suggesting that the selection of lamivudine-resistant mutants affects future treatment options. LAM, lamivudine; ADV, adefovir; ETV, entecavir; LdT, telbivudine; FTC, emtricitabine. Adapted from 85 with permission.
Table 31-7. Available Antiviral Agents and Those Under Late-Stage Development Drug
Antiviral potency at weeks 48–52 (log10 decline HBV DNA)*
Rate of resistance**
Lamivudine Emtricitabine Adefovir
–4 to –4.5 log –3 log –3 to – 3.5 log
Tenofovir Entecavir Telbivudine Pegylated interferon†
–5 to –6 log –5 to –6 log –5 to –6 log –3.5 to –4 log
High rate of resistance (15–20% at year 1, 60–70% at year 4) Similar resistance pattern to lamivudine Effective against lamivudine resistant and wild-type HBV; resistance 3% at year 2; 6% at year 3, 15% at year 4, and 28% at year 5. Not known but presumably similar to adefovir owing to close chemical similarity Uncommon ( 0.05). Taken from 147 with permission.
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Section V. Liver Diseases Due to Infectious Agents
adefovir may not add to the greater antiviral activity of lamivudine. The possibility exists that nucleoside analogs such as telbivudine and lamivudine taken together sterically inhibit binding to the HBV DNA polymerase or compete for phosphorylation enzymes (kinases) that are required for drug activation. Thus, not only is no benefit seen for a combination of these two drugs, but combination therapy might actually be detrimental compared to either agent alone. Recently, a combination of emtricitabine and adefovir was associated with more rapid and greater HBV DNA suppression, and this was associated with enhanced T-cell responses.149 More studies need to be carried out with combination nucleoside analog therapy before definitive conclusions regarding benefits and costs of combination therapy can be drawn.
Combination Interferon and Nucleoside Analog Therapy From a conceptual standpoint, the combination of interferon with a nucleoside analog might prove to be more effective than either drug alone because these agents have different mechanisms of action. This might also allow for a shorter course of nucleoside analog therapy, thereby reducing the risk of viral resistance. A number of studies in the woodchuck and several clinical trials in humans have provided support for additive or synergistic effects when interferon is used in conjunction with a nucleoside analog. Three large multicenter studies have evaluated the combination of pegylated interferon and lamivudine. In one study, HBeAg-positive patients received pegylated interferon-a2b with either lamivudine or placebo for 1 year.99 Despite a 3 log10 greater decline in serum HBV DNA at the end of treatment in the combination therapy group, the frequency of loss of HBeAg at the end of follow-up was nearly equivalent (35% and 36%, respectively). It is possible that the low dose of pegylated interferon used in this study (100 mg weekly for 8 months, followed by 50 mg weekly until the end of treatment) may have influenced the high relapse rate in the combination therapy group. In a second study, 814 HBeAg-positive patients were treated with either 48 weeks of pegylated interferon-a2a in a dose of 180 mg once weekly or pegylated interferon in the same dose given concurrently with lamivudine; a third arm received lamivudine alone.100 Patients in the combination therapy arm had a greater degree of HBV DNA suppression (-7.2 log10 copies vs -5.8 for lamivudine and -4.5 for interferon monotherapy). At the end of a 6-month follow-up period, however, HBeAg seroconversion had occurred in 32% of the pegylated interferon monotherapy group, 27% in the combination group, and 19% of the lamivudine-only patients. Between 9 and 15% of patients in this study had previously been treated with lamivudine, which may have influenced the findings. In the third study, patients with HBeAg-negative chronic hepatitis B were treated with 180 mg of pegylated interferon-a2a given weekly for 1 year in combination with either placebo or lamivudine, and these two treatment groups were compared against lamivudine monotherapy.101 Both virologic response and ALT normalization were significantly more common at completion of follow-up in patients treated with the interferon-containing regimens, but as with the study in HBeAg-positive patients those receiving combination therapy did not demonstrate a higher rate of sustained virologic response than those receiving pegylated interferon alone. There was, however, a more rapid decline in serum
656
HBV DNA and an approximate 1 log10 greater reduction in serum HBV DNA at the end of treatment in patients who received combination therapy. Thus, at present a combination of lamivudine and pegylated interferon does not appear to offer any durable offtreatment therapeutic advantages over pegylated interferon alone. Unlike the situation with combination nucleoside analog therapy, however, all three studies cited above provide proof of concept that pegylated interferon and lamivudine have additive antiviral effects on therapy, and further studies using pegylated interferon-a and a nucleoside analog in different treatment schedules may be warranted.
FUTURE ISSUES AND AREAS OF NEED Many advances in the treatment of chronic hepatitis B have been made over the past decade, but many unresolved issues remain. Time-limited treatments that induce a durable virologic response while remaining both safe and easily affordable have not been developed. The treatment of patients with normal or near-normal ALT levels, individuals who are immunosuppressed or HIV co-infected, and those with HBeAg-negative chronic hepatitis B remain the greatest clinical challenges. Significant developments with nucleoside analog therapy over the past decade have overshadowed the importance of the host immune response in achieving therapeutic end-points and many authorities have relegated interferon to consideration as second-line therapy. However, data demonstrating the enhanced potency of pegylated interferon now provide reason to re-evaluate this issue. As nucleoside or nucleotide analogs have to be given for extended periods this encourages the selection of drug-resistant HBV mutants. This occurs to some extent with all of these agents when used as monotherapy. Furthermore, recognition that sequential nucleoside analog therapy can result in the selection of mutant HBV that is resistant to more than one drug may ultimately lead to the use of a combination of nucleos(t)ide analogs as first-line therapy in some patients. This strategy is likely to be most useful in patients who have features that correlate with a high probability of drug resistance (for example high serum HBV DNA level at baseline). Immunologic modifiers that are better accepted and more predictably effective than interferon would be a great step forward for this field, and could provide an opportunity for durable viral suppression with much shorter courses of nucleos(t)ide analog therapy. The same could potentially be said of other drugs that reduce serum HBV DNA levels independently of inhibition of viral DNA polymerase.
IMMUNIZATION FOR HEPATITIS B Immunization against HBV can be achieved by vaccination against HBs Ag, which confers active immunity, or by immunoglobulin, which confers passive immunization. Active immunization gives long-term immunity, whereas passive immunization confers only immediate protection (2–4 months).
HEPATITIS B IMMUNOGLOBULINS Hepatitis B immunoglobulin (HBIG) contains high titers of antiHBs antibody. Several clinical trials have established the efficacy of HBIG in preventing HBV in postexposure prophylaxis settings.150–153
Chapter 31 HEPATITIS B
HBIG licensed in the United States has an anti-HBs antibody titer of 1:100 000. In Europe, several preparations of HBIG with different concentrations and pharmacokinetics are available. HBIG is safe, but anaphylactic reactions can rarely occur, particularly when given intravenously. Myalgias, skin rash and arthralgias have been reported, and these are believed to be due to antigen–antibody complexes that can occur in HBsAg positive-patients who have mistakenly been given HBIG.
HEPATITIS B VACCINES At present there are two vaccines that are specifically licensed for hepatitis B prevention in the US: Recombivax HB (Merck, licensed in 1986) and Engerix-B (SmithKline Beecham, licensed in 1989). The two vaccines are similar in efficacy. Aluminum hydroxide is added as an adjuvant and thiomersal as a preservative. Because of concerns about the mercury content in thiomersal, preservative-free vaccines are available. For optimal response, the hepatitis vaccine is administered intramuscularly in the deltoid area of adults and in the anterolateral thigh in infants or neonates. HBV vaccines make use of DNA recombinant technology by introducing HBs antigen gene (S gene) into yeast, Saccharomyces cerevisiae. These vaccines induce HBsAg-specific T-helper cells and T cell-dependent B cells to produce neutralizing HBs antibodies against the epitope a (aa124-148) of HBsAg as early as 2 weeks after the first immunization.154 HBV vaccines are highly efficacious in preventing HBV infection. Recipients of HBV vaccine develop only antibody to HBsAg (antiHBs). The detection of antibody to hepatitis B core antigen implies infection, which is frequently subclinical. Anti-HBs titers greater than 100 MIU/ml confer 100% protection against hepatitis B. Most recipients achieve such a high anti-HBs level. The antibody titer may wane over the years, but even at lower titers there is excellent protection against HBV. A number of factors have been identified (smoking, obesity, chronic liver disease, and age over 50) that affect the antibody response, leading to lower levels of anti-HBs.155,156 These ‘hyporesponders’ may benefit from higher doses of vaccine. The response rate is also lower in immunocompromised patients, such as transplant recipients and patients receiving chemotherapy. Injection of vaccine into the buttocks elicits a lower rate of response than injection in the deltoid or anterolateral thigh. HBV vaccination elicits a lower response in hemodialysis patients, with only 50–60% responding adequately. The factors associated with a poor response are old age, presence of DR3, DR7 DQ2 and absence of A2 alleles.157 Hence patients with chronic renal insufficiency should be vaccinated early before renal disease progresses, so that a better response can be achieved.158 In a recent report, repeated dosing with intradermal vaccination (5 mg every 2 weeks, aiming for a titer of >1000 IU/l) achieved a response rate of 97.6%.159 Approximately 5–10% of vaccine recipients do not achieve detectable antibody levels (non-responders). Recent reports, mostly in animals, indicate that intradermal injection may produce a stronger humoral and cellular immunity than conventional intramuscular doses.160,161 Intradermal injection, by recruiting dendritic cells, stimulates primary MHC class I as well as class II restricted T-cell responses. In one study, all but one of nine non-responders to
conventional intramuscular dosing responded to two to three doses of vaccine given intradermally.161 Despite these interesting observations, recommendations for use of intradermal vaccination are lacking at present owing to limited data with regard to the lasting nature of antibody responses and inherent problems with standardization of intradermal delivery. Because vaccination results in a strong immunologic memory capable of preventing infection even in patients with very low or undetectable antibody titers, there is no role for booster doses in immunocompetent adults and children.162 Current recommendations for a booster dose only include patients undergoing hemodialysis. In these patients anti-HBs titer should be tested annually and a booster dose given if the titer is less than 10 mIU/ml.163 There are no serious side effects to vaccine. An increased incidence of neurologic disease, such as aseptic meningitis or Guillain– Barré syndrome, has not been observed.
High-Risk Groups Table 31-9 indicates the high-risk groups for whom vaccination is recommended. Targeted vaccination has not achieved its objective in certain high-risk groups, such as injecting drug users, but has been moderately successful in healthcare workers. Failure of strategies to vaccinate high-risk adults has led to policies of universal vaccination of newborns and preadolescent children in the US.
Vaccination Schedule The dosage of HBV vaccine and the schedule are listed in Table 3110. The typical schedule is 0, 1, and 6 months. The first two doses are important in the recruitment of responders and the third acts as a booster to achieve the highest anti-HBs titer. In immunocompromised patients and those on hemodialysis four doses are recommended, with the fourth intended to provide the highest possible titer. If a vaccination series is interrupted, the second dose should be administered as soon as possible.163 If the third dose is interrupted it should be given when convenient. The second and third doses should be separated at least by 2 months.163
Table 31-9. High-Risk Groups Requiring Consideration for HBV Vaccine Healthcare workers Public safety workers with likelihood of exposure to blood Staff and clients of institutions for developmentally disabled Hemodialysis patients Patients who are likely to require multiple transfusions with blood or blood products Household contacts and sex partners of HBV carriers or patients with acute hepatitis B International travelers to endemic areas who may have intimate contact with local population or who may take part in medical activities in endemic areas Injecting drug users Sexually active bisexual and homosexual men Sexually active heterosexual men and women if they have more than one partner Inmates of correctional facilities Patients with chronic liver disease Potential organ recipients
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Section V. Liver Diseases Due to Infectious Agents
Table 31-10. Recommended Dosing for the Currently Available HBV Vaccines*
Infants* and children < 11 yrs Children/adolescents (11–19 yrs) Adults (≥20 yrs) Hemodialysis patients Immunocompromised patients
Recombivax HB (10 mg/ml)
Engerix-B (20 mg/ml)
2.5 mg 5 mg 10 mg 40 mg (1.0 ml)¶ 40 mg (1.0 ml)¶
10 mg 20 mg 20 mg 40 mg (2.0 ml)# 40 mg (2.0 ml)#
* The standard schedule is 0, 1, and 6 months. ** Infants born to HBs-negative mothers. ¶ Special formulation. # Two 1.0 ml doses administered at one site in four-dose schedule (0, 1, 2, 6 months).
Table 31-11. Hepatitis B Prophylaxis for Infants Born to an HbsagPositive Mother Vaccine
First dose Second dose Third dose¶
Recombivax HB 5 mg (0.5 ml)
HBIG or
Engerix B 10 mg (0.5 ml)
Age of the infant 0.5 ml IM*
5 mg (0.5 ml)
10 mg (0.5 ml)
None
Within 12 hours of birth 1 month
5 mg (0.5 ml)
10 mg (0.5 ml)
None
6 months
* HBIG should be administered at a site different from that used for vaccine. ¶ If four doses of vaccine are administered, the third dose is given at 2 months and the fourth at 12–18 months.
HBV vaccine is currently administered to all infants and children as a part of a universal immunization program in the US and in many countries worldwide. In Taiwan universal vaccination of newborns was implemented in 1984, and this has led to a decline in hepatitis B carrier rates among children, from 10% to less than 1%.164 Prevention by vaccination is also the best way to control the complications of chronic infection, such as hepatocellular carcinoma. In Taiwan this has proved to be effective in reducing the incidence of cancer to a quarter to a third of that in children born prior to 1984.165 Unfortunately, as of the year 2000 only 116 of 215 countries had implemented a universal vaccine program, owing to lack of resources and poor education as to its strategic importance.166 Combination vaccine, HBV with DPT and Hib (DTPw-HB/Hib, the current vaccine for immunization of infants) does not affect the immunogenicity of any of the components.167 Adolescents should also be vaccinated if they are at high risk. Table 31-10 lists the highrisk adult groups who are targeted for HBV vaccine.
Postexposure/Perinatal Prophylaxis Table 31-11 outlines the recommendations for prevention of perinatal transmission. Postexposure vaccination should be considered for any percutaneous, ocular or mucous membrane exposure. The type of immunoprophylaxis is determined by the HBsAg status of the source and the vaccination-response status of the exposed person. Table 31-12 outlines the postexposure prophylaxis for exposure to a known HBsAg-positive source.
658
Table 31-12. Postexposure Prophylaxis: if Source is HBsAg positive Vaccination status of exposed person
Immune prophylaxis
Unvaccinated
HBIG (0.06 ml/kg) and initiate HB vaccination series
Previously vaccinated Known responder* Known non-responder Antibody response unknown
No treatment HBIG ¥ 2 doses or HBIG ¥ 1 dose and initiate revaccination Test for anti-HBs If adequate* : no treatment If inadequate**: HBIG ¥ 1 dose and give vaccine booster
* Anti-HBs titer >10 mIU/ml. ** Anti-HBs titer 20 years is predicted to increase substantially before peaking in 2015.8 Those born between 1940 and 1965 appear to have the highest lifetime risk of HCV infection.8 These are precisely the individuals who now, at age 40–65 years, are seeking medical care for HCV and as their liver disease progresses, are being referred for evaluation for liver transplantation. Recent results from the CDC suggest that the prevalence of HCV infections has not greatly changed since the National Health and Nutrition Examination Survey (NHANES) III study conducted a decade ago.4,9 A recent survey conducted during 1999–2002 of 15 079 non-institutionalized persons has shown that the current prevalence of anti-HCV in the USA is 1.6%, corresponding to 3.8 million individuals. Prevalence is higher among blacks (3.0%) than whites (1.5%) or Mexican Americans (1.3%) and higher in males (2.1%) than females (1.1%). The prevalence of anti-HCV peaked in 45–49-year-olds in the ongoing NHANES study, compared with a peak in 35–39-year-olds in NHANES III conducted a decade earlier. These data support an aging cohort of HCV-infected individuals in our society. The prevalence in those aged 45–49 years is 7.1% among men and 2.3% among women. Of all anti-HCVpositive persons, 69.9% are between the ages of 35 and 54 years, an age group that may also be at risk for non-hepatic comorbid conditions.10 Eighty-eight percent of those who are anti-HCV-positive are also HCV RNA-positive. Among black men in this age group, 17.9% tested anti-HCV-positive.9 Consistent with results from NHANES III, the ongoing NHANES study found that the prevalence of anti-HCV depends on a number of demographic and risk factors. Highest prevalence is seen in individuals who have ever injected drugs (57.3%) compared with a prevalence of 3.5% in those who have used non-injection drugs and 0.7% in those who denied any drug use.9 Prevalence increased with increasing numbers of lifetime sexual partners and was higher among those with a history of blood transfusion before 1990 (4.2%) than among those without (1.4%). This recent survey suggests that, while the overall prevalence of HCV infection has not changed in the past 10 years, the peak in age-specific prevalence has shifted to older age groups, resulting in a cohort of people, now aged 40–59 years, with a high prevalence of infection. With prolonged infection comes risk of hepatic
666
complications. The incidence of HCC, a highly lethal complication of long-standing infection, has increased twofold in the USA from 1.3 per 100 000 during 1978–1980 to 3.0 per 100 000 during 1996–1998.11,12 This measured rise in incidence of HCC is consistent with the rise in complications of HCV predicted by the CDC.13 There also appears to be a shift towards the occurrence of HCC in younger individuals.11 The incidence of HCC among those over the age of 65 years continues to rise from 14.2 per 100 000 in 1993 to 18.1 per 100 000 in 1999. While much of this rising incidence is due to HCV, HBV-related HCC also appears to be increasing.14 This change in the incidence of the long-term consequences of HCV infection is of concern. Particularly worrisome is the observation that, once HCC is diagnosed, median survival is only 7–8 months and survival has increased minimally in the past 25 years despite increased screening for HCC and HCC interventions such as radiofrequency ablation and chemoembolization.15 These poor outcomes in patients with advanced HCV-related liver disease underscore the importance of evaluating individuals with hepatitis C for therapy, overcoming barriers to treatment if they exist, and improving outcomes of treatment by maximizing adherence.
MODES OF TRANSMISSION Transmission of HCV infection is highly linked to parenteral exposures such as occurs with injection drug use, from contaminated needles in the health care setting, and through exposure to infected blood products. Risk factors for HCV infection include injection drug use or intranasal cocaine drug use, clotting factors transfused before 1987, blood or blood products transfused before 1992, highrisk sexual activity, mother-to-infant (vertical) transmission, occupational exposure, and tattoos or body-piercing with contaminated needles1 (Table 32-1). Potential exposures that have been evaluated in case-control studies and have been found to be weakly associated, if at all, with HCV infection, include medical, surgical, and dental procedures, tattooing, acupuncture, ear-piercing, incarceration, military service, and foreign travel.16 Prevalence of HCV infection in health care workers is low, although HCV has clearly been transmitted in hospital settings, and outbreaks of HCV have been described in dialysis units and surgical centers.16 Despite the low risk (0.5%) after needlestick exposure from an HCV source patient,
Table 32-1. Sources of infection for hepatitis C
Injection drug use Sexual Transfusion Occupational Unknown Othera a
Previously acquired (before 1990s)
Newly acquired (1995–2000)
60% 15% 10% 4% 10% 1%
68% 18% ~0% 4% 9% 1%
Other includes nosocomial, iatrogenic, perinatal. Data from the Centers for Disease Control and Prevention, Atlanta, GA.
Chapter 32 HEPATITIS C
acquisition of HCV infection in health care workers has been described.16 HCV transmission from infected health care worker to patient has also been described, although this is typically secondary to a breakdown in routine infection control practices.16 HCV is not transmitted through casual household contact, although sharing of toothbrushes and razors with infected household members should be avoided. Serological tests were introduced to screen units of donated blood in 1990 and were improved in 1992. More recently, in the USA, nucleic acid testing that identifies low-level viremia has further improved the safety of the blood supply such that now the risk of acquiring HCV by contaminated blood is less than 1:200 000 000 units transfused. Approximately 6 million people donate blood each year, and 0.6% of these are seropositive for HCV, accounting for 36 000 newly identified infections through this mechanism alone.16 Risk factors in seropositive volunteer blood donors include previous blood transfusion, intranasal cocaine and injection drug use, multiple heterosexual partners, and ear-piercing among men.17 Recommendations for testing for anti-HCV in different at-risk groups are summarized in Table 32-2. While public health measures have led to a marked decline in incident infections, they remain large reservoirs of infection, largely in those with prior or ongoing injection drug use. HCV antibodies are identified in 88.7% of injection drug users and the prevalence of anti-HCV antibodies increased directly with the duration of drug use.18 Injection drug use accounts for two-thirds of new infections in the USA. The prevalence of HCV is increased in injection drug users who are black, infected with HIV, who inject frequently, and who use cocaine. More than 80% of injection drug users acquired HCV within 2 years of initiating drug use, a finding that has been used to estimate the time of initial infection and calculate the duration of infection in many studies.18,19
HCV is transmitted sexually, although the efficiency is low.20 High-risk heterosexual behavior (typically defined as more than 20 or in some studies more than 50 lifetime sexual partners) is independently associated with risk for HCV infection. In sexual partners of HCV-infected persons, prevalence of infection is 2–3%, which is similar to that in the general US population. However, incidence is a little higher (0.4–1.8% per year) in those who have multiple heterosexual partners, sex workers, and men who have sex with men than in those in long-term monogamous partnerships (0–0.6% per year).20 Prevalence of antibody-positive, genotypeconcordant couples varies in different studies from around the world, from 2.8 to 11% in South-East Asia, 0 to 6.3% in northern Europe, to 2.7% in the USA.20 HIV co-infection appears to increase the risk of HCV sexual transmission. In addition, studies from sexually transmitted disease clinics and studies of men who have sex with men suggest that sexual practices associated with trauma are more common in HCV-positive than HCV-negative individuals.20 Whether HCV sexual transmission differs depending on the gender of the infected partner is unclear.20 Current recommendations for preventing HCV transmission differ for those in long-term monogamous versus short-term relationships (Table 32-3).20 Mother-to-infant transmission of HCV is well documented, although the risk to the infant of acquiring infection from a seropositive mother is less than 5%. This risk is higher in mothers with high levels of virus (greater than 106 copies/ml) and those with HCV/HIV co-infection (22% versus 4% in mothers with and without HIV respectively).21 The rate of transmission from viremic mothers to their infants is 3–7%.21 There are no specific recommendations to reduce the risk of perinatal infection. Cesarean section and breast-feeding do not appear to alter the risk.21 Part of the difficulty in evaluating rates of transmission and risk factors for transmission across different studies relates to different definitions of infection and different durations of follow-up from study to study. Infants are frequently anti-HCV-positive from passive trans-
Table 32-2. Recommendations for hepatitis C testing History
Risk for infection and prevalence
Testing recommended
Ever injected drugs Received clotting factors prior to 1987 Persistently abnormal LFTs Chronic hemodialysis Received blood or organs prior to 1992 Born to infected mother Health care workers
High, 60–90% High, 70–90%
Yes Yes
Intermediate, 15% Intermediate, 10% Intermediate, 6%
Yes Yes Yes
Intermediate, 6% Low, 1–2%
Sex with infected partner
Low, 2%
Sex with multiple partners Tattooed Body-piercing Snorted cocaine
Low, 2–5% Low, 500 000 IU/ml to 1 470 000 IU/ml. The Cobas Amplicor HCV Monitor v2.0 is an automated version of the Amplicor test and has a dynamic range of 600–500 000 IU/ml. Samples above the upper limit should be retested after dilution, particularly in those with high levels of virus prior to therapy, in whom EVR and RVR are going to be measured. There are two commercially available assays for determining HCV genotype, assays based on PCR amplification of the 5¢ non-coding region. With these assays, the six genotypes can be readily identified, although tests are less accurate in measuring HCV subtypes, with errors occurring in 10–25% of cases because of variations in the target 5¢ NC region.
SELECTION FOR ANTIVIRAL THERAPY AND PRETREATMENT ASSESSMENTS All patients with confirmed, chronic HCV infection should be evaluated for antiviral therapy. Because of limitations in efficacy and the potential for toxicity, each patient needs careful assessment of the relative risks and benefits of beginning therapy immediately, delaying therapy until a later time, or deferring treatment indefinitely. Pretreatment evaluations include the following: (1) clinical assessments; (2) laboratory tests; and (3) liver biopsy.
CLINICAL ASSESSMENTS A complete medical evaluation is essential in order to rule out medical comorbid conditions that might contraindicate or might worsen with treatment. Particular attention should be paid to coronary artery disease, since ribavirin-associated anemia may lead to worsening of cardiac ischemia on therapy. Other potentially lifedetermining conditions such as non-skin cancer and chronic obstructive pulmonary disease should be identified since life expectancy may be limited by these comorbid conditions rather than HCVrelated liver disease. In such cases, patients may elect not to be treated. All patients should be evaluated for psychiatric disorders, particularly depression and suicide risk. Uncontrolled depression is an absolute contraindication to interferon-based therapies. Patients with psychiatric disorders in remission or stabilized with antidepressants may receive antiviral therapy, with the involvement of mental health professionals throughout the course of therapy. All patients should be evaluated for current substance use, including alcohol and drug use. Current heavy alcohol use, binge alcohol use (more than four drinks per occasion at least once a month), or active current injection drug use requires referral to an addiction specialist prior to treatment initiation. Since illicit non-injection drug use may affect treatment adherence, each case should be evaluated individually. Establishing abstinence from drugs and alcohol prior to initiating treatment is recommended. Patients should be evaluated for autoimmune disorders since these may worsen on interferon therapy. Controlled autoimmune thyroid disease (on replacement therapy if necessary), and con-
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trolled diabetes (with normal or near-normal serum glycosylated HbA1C) are not contraindications to HCV antiviral therapy but these patients should be monitored closely for signs of worsening disease. Other medical disorders such as psoriasis, rheumatoid arthritis, and Crohn’s disease might worsen on interferon therapy, so that these diseases must be medically controlled prior to treatment and they should be managed by a specialist familiar with these disorders. Patient adherence to treatment should also be assessed. Evidence of prior non-adherence to medical, psychiatric, or addiction therapies provides indirect evidence of likely non-adherence to HCV therapies and should be considered before a final decision to treat HCV disease is made. A baseline ophthalmic exam should be performed in patients with risk factors for retinal disease (such as hypertension or diabetes) to identify any disease that might be worsened by interferon and to provide a reference point should any symptomatic ophthalmic symptoms occur on therapy.
LABORATORY TESTS Patients should have adequate platelet counts (>75 ¥ 109/l), neutrophils (an absolute neutrophil count of >1.5 ¥ 109/l), and hemoglobin (>13 g/dl for men and >12 g/dl for women) in order to tolerate therapy. Patients with neutrophil and platelet counts below these recommended levels can begin therapy but may require dose reductions. African-Americans with constitutional neutropenia 75 kg) HCV genotypes 2/3: 800 mg In combination with pegylated interferon alpha-2b: 800 mg, 105 kg/day). In order to minimize side effects as well as costs, the “early stopping rule” may be applied to genotype 1 patients who fail to achieve an EVR. This means that quantitative HCV RNA measurement should be performed before and after the initial 12 weeks of treatment. If there is a less than 2 log decline in viral load, treatment should be stopped since further treatment is unlikely to lead to an SVR. Side effects with treatment may be significant and occur in the vast majority of patients (Table 32-7). For interferon these are arthralgias, muscle pain, flu-like symptoms, fever, and others. Concerning laboratory tests, leukopenia, granulocytopenia, and thrombocytopenia are most important and need to be monitored. For ribavirin, anemia is the most prominent adverse event leading to a significant drop in hemoglobin within the first 4 weeks of treatment. In patients with coronary artery disease a careful evaluation of the risk–benefit ratio of this potentially dangerous treatment is essential. In individual patients, interferon-related adverse events may occur that require specific management and, in some cases, termination of therapy. Among the most important side effects are depression, autoimmune thyroid disease, alopecia, and diabetes (Table 32-7). Patients and doctors must be aware of these adverse events and need to react appropriately. Reduction of peginterferon or ribavirin may become necessary in up to 20% of patients due to side effects. Retrospective analysis has shown that it is particularly important for genotype 1 patients to be compliant and take more than 80% of both drugs for more than 80% of the time in order to optimize response rates.83 Treatment of patients with persistently normal ALT is safe and responses are comparable to those with abnormal ALT.84 Thus in most current guide-
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lines, elevated ALT is not a requirement for therapy.22 In patients with normal ALT a biopsy should be performed and treatment initiated if a fibrosis score of at least 1 (Ishak or Metavir) is present.
NON-RESPONDERS TO PREVIOUS TREATMENT In most countries the present standard treatment with the combination of peginterferon plus ribavirin is only approved and reimbursed for treatment-naive or those previously treated with interferon monotherapy. Unfortunately, a growing number of patients are non-responders to current therapies (more than 50% of genotype 1 patients, 20% of genotype 2 and 3 patients, and 40% of genotype 4 patients). These patients are highly motivated and are often waiting for new approaches. These patients are primary candidates for the new therapies described below. In addition, there have been numerous trials attempting to improve treatment outcomes with the drugs available today. High-dose induction regimens with either peginterferon or ribavirin are under investigation. Several studies have also explored retreatment with low-dose peginterferon for prolonged durations, in order to slow disease progression by exploiting the antifibrogenic and antiproliferative effects of interferons. Long-term trials of this approach are in progress, including Hepatitis C Antiviral Long-term Treatment against Cirrhosis (HALT-C), that is exploring low-dose PEG-IFN alpha-2a,85 Evaluation of Peg-Intron in Control of Hepatitis C Cirrhosis (EPIC-3), that is investigating PEG-IFN alpha-2b86 compared to placebo, and Colchicine versus Peg-Intron Long Term (COPILOT), that is evaluating low-dose PEG-IFN alpha-2b compared to colchicine.87 However, significant progress in treatment will only be achieved using innovative new drugs, as described below.
SELECT PATIENT POPULATIONS HCV infection is prevalent in certain populations in the USA. These include 300 000 injection drug users,88 175 000 homeless,89 100 000 children,90 280 000 veterans,91 300 000 with HIV infection,92 310 000 of those who are incarcerated, and 940 000 living below the
Chapter 32 HEPATITIS C
poverty level.4 Many of these patient groups have been excluded from the registration trials of peginterferon plus ribavirin therapy.
AFRICAN-AMERICANS African-Americans have the highest prevalence of HCV infection of any racial group in the USA. African-Americans also have a high prevalence of HCV genotype 1 infection.93 Unfortunately, treatment is particularly problematic in this group. African-Americans have lower rates of spontaneous viral clearance and lower rates of response to peginterferon and ribavirin (SVRs in African-Americans were 18–26%).94,95 In genotype 1 patients, blacks have a slower and quantitatively smaller decline in HCV RNA levels with interferon with or without ribavirin compared with whites.96 In one study of genotype 1 patients, PEG-IFN alpha-2a 180 mg/week plus ribavirin 1000 or 1200 mg/day was administered in a prospective trial for 48 weeks to 78 blacks and 28 whites. End-of-treatment responses were 39 and 52% and SVRs were 26 and 39% in the African-Americans and Caucasians respectively. Histological improvement (defined as an improvement of >2 in histological activity index) was seen in 66% of African-Americans compared with 75% of Caucasians. Treatment was tolerated equally well, except for a higher incidence of severe neutropenia (defined as absolute neutrophil count less than 500/mm3) in the African-American group that was not associated with increased risk of infection. A second trial of PEG-IFN alpha2b (1.5 mg/kg per week) plus ribavirin (1000 mg/day for 12 weeks and 800 mg for an additional 36 weeks) further underscored reduced treatment responses. End-of-treatment responses were 20% and 58% and SVRs were 19% and 52% in African-Americans and Caucasians respectively.95 Reasons for resistance to HCV antiviral therapy are under investigation, but low response rates are not simply explained by the high prevalence of genotype 1 infection seen in this group. As with other patient populations, failure to achieve an early virological response at 12 weeks is strongly predictive of a low likelihood of achieving an SVR with 48 weeks of peginterferon plus ribavirin. Whether prolonging therapy beyond 48 weeks is beneficial is currently unknown.
HEPATITIS C/HIV CO-INFECTION Highly active antiretroviral therapy (HAART) has markedly changed the natural history of HIV infection, such that life expectancy is now comparable to those who are HIV-negative.97 Because of overlapping risk factors for HCV and HIV infection, co-infection with the two viruses is common, particularly in those with HIV who have acquired infection through injection drug use. Of the approximately 1 000 000 HIV-infected individuals in the USA, 30% are estimated to have HCV/HIV co-infection. Similar proportions of HIVinfected patients in Europe seem to be HCV/HIV co-infected, although the proportion is even higher in certain countries (such as Spain, with a prevalence of 50%). The prevalence of HCV depends on the risk factor for HIV infection (85% in injection drug users, 14% heterosexual and 10% with homosexual contact as their risk for HIV98). The CDC and the American Association for the Study of Liver Diseases have recommended that all HIV-infected patients be tested for HCV and that all HIV-positive patients with liver injury of unknown cause should be tested for HCV RNA, even if the anti-HCV test is negative.22 Guidelines have recommended consideration of HCV antiviral therapy in HCV/HIV co-infected
patients, although response to peginterferon plus ribavirin therapy is lower than observed in those with HCV infection alone.22 These guidelines are supported by results of randomized controlled clinical trials.99–102 The need to treat patients with HCV/HIV co-infection is driven in part by concerns that liver disease progression is accelerated in those with co-infection, compared to the natural history in those with HCV monoinfection.44 A meta-analysis of eight separate studies has shown that HCV/HIV-infected patients have a twofold risk of cirrhosis diagnosed on liver biopsy and a sixfold risk of decompensated liver disease with clinical complications when compared to HCV monoinfected patients.103 A 10-year incidence of cirrhosis has been estimated to be 14.9 and 2.6% respectively in HCV-infected patients with and without HIV infection.104 Risk of liver disease progression appears to be particularly true for patients with HIV-related immune compromise.105 Currently only one therapy, PEG-IFN alpha-2a (180 mg/week) plus ribavirin, is FDA-approved for HCV treatment of HCV/HIV co-infection. While the FDA-approved dose of ribavirin is 800 mg/day, and this was the dose of ribavirin included in the phase III trials of peginterferon plus ribavirin,100 several guidelines have advocated the use of higher doses of ribavirin (1000/1200 mg/day based on a 75-kg body weight) in order to give these patients an improved likelihood of treatment response.22,102 Current recommendations are to consider treatment of patients with baseline CD4 counts of >200 cells/ml, or CD4 counts of >100 but 6) has been described.106 The primary goal of treatment in patients with co-infection is viral clearance (achieving an SVR), although secondary goals include delay of histological and clinical liver disease, as for those with HCV monoinfection. Unfortunately overall response to peginterferon plus ribavirin therapy is reduced in patients with HCV/HIV, in part because of a high prevalence, as variables were shown to be associated with nonresponse in HCV monoinfected patients (genotype 1 infection, high HCV RNA levels, advanced liver disease, and African-American race). Pretreatment HCV RNA levels in an international trial of PEG-IFN alpha-2a plus ribavirin was 14 000 000 copies/ml, more than twofold higher than baseline levels in monoinfected treatment trials.100 There have been three recent published studies of PEG-IFN plus ribavirin in co-infected patients demonstrating superiority to interferon plus ribavirin.99–101 In the AIDS Clinical Trials Group (ACTG) 5071 study, 133 HIV/HCV-co-infected adults were randomized to receive either standard interferon alpha-2a subcutaneously at 3 million international units (MIU) three times a week (tiw) or PEGIFN alpha-2a subcutaneously at 180 mg/week, each combined with increasing doses of ribavirin escalated from 600 to 1000 mg daily. Virologic tests at both 48 weeks end of treatment response (ETR) and 72 weeks (SVR) showed a greater response in the PEG-IFN arm when compared to the standard interferon arm (overall SVR of 27%, and SVR of 14 and 73% in genotype 1 and non-1 in the peginterferon group compared to overall SVR of 12%, and SVR of 6 and 33% in genotype 1 and non-1 in the interferon plus ribavirin arm). Overall tolerability of antiviral therapy was lower than in HCV monoinfected patients with 12% of patients in both arms discon-
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Section V. Liver Diseases Due to Infectious Agents
tinuing treatment. In the AIDS PEGASYS Ribavirin International Coinfection Trial (APRICOT),100 860 HIV/HCV-co-infected adults were randomized to one of three treatment arms: (1) standard interferon alpha-2b at 3 MIU tiw plus ribavirin at 800 mg/day; (2) PEGIFN alpha-2a at 180 mg/week plus ribavirin 800 mg/day; or (3) PEG-IFN alpha-2a at 180 mg/week plus placebo. Overall SVR in the peginterferon plus ribavirin arm demonstrated superiority to standard interferon plus ribavirin (49 and 14% respectively) with an intermediate response in patients receiving peginterferon monotherapy (SVR of 33%). SVR in genotype 1 and non-1 patients receiving peginterferon plus ribavirin were 29 and 62% respectively, based on an intent-to-treat analysis. Peginterferon also appeared to have an anti-HIV effect, since HIV RNA levels decreased by 0.9 logs. The third multicenter trial (RIBAVIC study) of peginterferon plus ribavirin for co-infection, included 412 patients randomized to either PEG-IFN alpha-2b at 1.5 mg/kg per week compared to standard interferon alpha-2b at 3 MIU tiw, with both arms receiving ribavirin 800 mg/day. As for the PEG-IFN alpha-2a trials, SVR rates in the PEG-IFN arm were superior to the standard interferon arm (27 and 15% respectively). Response to peginterferon plus ribavirin in genotype 1 and non-1 patients were 15 and 46% respectively.99 While all three studies show the superiority of peginterferon plus ribavirin to standard interferon plus ribavirin, the SVR rates reported appear to vary. Several factors may account for the lower overall sustained virologic response rates in the ACTG 5071 trial and the RIBAVIC Study. In the ACTG 5071 trial, the initial starting dose of ribavirin was lower than the other two studies at a total dose of 600 mg/day, and ribavirin dose seems particularly important for response in genotype 1-infected patients. In addition, about 33% of patients in the ACTG 5071 trial were African-American, a population that has consistently been shown to be resistant to HCV therapy. Treatment discontinuations were also high in these trials (42% in RIBAVIC), with severe side effects occurring in 31% of patients. Many patients (80%) had a history of intravenous drug use that may have contributed to non-adherence. Thus, differences in response to PEG-IFN plus ribavirin therapy between trials appear to be multifactorial, in part due to differences in study design, but also due to differences in characteristics of the study populations. Given the lower response rates in HCV/HIV co-infected patients, recent guidelines have recommended ribavirin dosing of 1000/ 1200 mg/day,22,102 although ribavirin-associated hemolytic anemia would be predicted to be significant and may necessitate the use of adjunctive therapy with epoietin, to improve tolerability.107 Recently, the first European Consensus Conference on coinfected patients (1stEEC) published its recommendations.107a
PATIENTS WITH COMPENSATED CIRRHOSIS Patients with cirrhosis have a substantial need for therapy yet have a somewhat lower response to peginterferon plus ribavirin than patients with earlier-stage disease. Goals of treatment include eradication of HCV infection, delay in cirrhosis progression and decompensation and prevention of HCC. Risk of developing HCC is 1.5% per year in a cirrhotic patient, a risk that may be reduced by HCV antiviral therapy.108,109 Data on treating patients with compensated cirrhosis are largely derived from subgroup analyses of clinical trials110; few prospective studies have focused on this population alone. One prospective trial of PEG-IFN alpha-2a in patients with
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bridging fibrosis and/or cirrhosis demonstrated that viral eradication was achievable in 30% of patients.111 As with all HCV trials, SVR was lower in patients with cirrhosis and genotype 1 infection (SVR in patients with low viral load and high viral load of 16 and 10% respectively). 111 Histological response (defined as >2-point reduction in histological activity index) was seen in 54% of patients. No prospective study has been conducted of peginterferon plus ribavirin in patients with cirrhosis, but subset analysis of patients with stage III and IV fibrosis from the registration trials of peginterferon provides guidance as to expected response. PEG-IFN alpha-2a (180 mg/week) plus ribavirin (1000/1200 mg/day) yielded SVR of 43 and 52% in two separate studies,80,81 while PEG-IFN alpha-2b (1.5 mg/kg per week) plus ribavirin (800 mg/day) yielded a response of 33%.79 Long-term goals of preventing complications of HCV cirrhosis with low-dose peginterferon therapy are under evaluation.
PATIENTS WITH COMPLICATIONS OF CIRRHOSIS HCV-associated end-stage liver disease with or without HCC has become the leading diagnosis in patients undergoing liver transplantation, affecting approximately half of all patients who are potential candidates (Chapter 49). Liver transplantation remains a life-saving option in patients with decompensated HCV cirrhosis, including those with HCV-related HCC, although recurrent HCV infection of the graft is a major cause of morbidity and mortality following liver transplantation (Chapter 52). Thus there is a great unmet medical need to treat HCV infection in patients with complications of cirrhosis. Goals of such treatment include eradication of infection prior to liver transplantation, in order to prevent post-transplantation reinfection, as well as slowing of HCV disease progression with the hope of obviating the need for liver transplantation. Unfortunately, peginterferon plus ribavirin is poorly tolerated in patients with complications of cirrhosis and, as such, these drugs are largely contraindicated. Major concerns about therapy are that leukopenia and thrombocytopenia will be worsened by interferon, making patients at risk for life-threatening infections and/or bleeding complications. Select patients with HCV may be candidates for low doses of interferon and ribavirin therapy provided that therapy is administered under close supervision of providers with experience in the management of end-stage liver disease. Outcomes of a low accelerating-dose regimen of interferon and ribavirin in a case series of 111 patients have recently been reported.69 Sixty-three percent of patients had clinical complications of HCV cirrhosis. While many patients were Child’s A cirrhotics, 45 were Child’s B and 23 were Child’s C. Mean Child–Pugh score of the group was 7 and model for end-stage liver disease (MELD) score was 11. Forty-six percent of patients lost HCV RNA at the end of treatment and 24% were HCV RNA-negative at follow-up. SVR was highly dependent on genotype (13 and 50% in patients with genotype 1 and non-1 infection respectively). Predictors of achieving an SVR included non-1 genotype, Child’s A cirrhosis (for genotype 1 only), treatment duration, and the ability to tolerate full dose therapy. Of the 15 patients who were HCV RNA-negative at the time of liver transplantation, 12 remained virus-free post-transplantation.69 Prevention of posttransplantation reinfection of the graft is a highly desirable goal, given the progressive nature of post-transplantation liver disease.39 These encouraging results support the approach of treating patients with clinical complications of cirrhosis with low doses of interferon
Chapter 32 HEPATITIS C
plus ribavirin. Tolerability of peginterferon plus ribavirin is less clear since cytopenias would be predicted to be worse with peginterferon than with standard interferon preparations, although hematological growth factors may be able to ameliorate some of these side effects.69 While interferon plus ribavirin may provide benefit to select patients with complications of cirrhosis, the medical need for new non-interferon-based options is high.
is common in patients following liver transplantation. Consequently use of ribavirin for treatment of recurrent HCV disease is associated with a high likelihood of treatment-associated hemolysis.119 Difficulties in administering ribavirin in patients following liver transplantation may also contribute to reduced treatment responses in this group (Chapter 52).
PATIENTS WITH RENAL DISEASE
FUTURE THERAPEUTIC APPROACHES AND NOVEL THERAPIES (Figure 32-3, Table 32-8)
Renal disease can be an extrahepatic manifestation of HCV infection with cryoglobulinemia and MPGN112 (see above). Management of HCV-related MPGN is problematic. While improvement in cryoglobulin levels and serum creatinine may occur, benefits appear to be transient, with relapse frequent when treatment is stopped.22 Potential benefits of improvement in renal function and decrease in proteinuria from long-term viral suppression with antiviral treatment have been inadequately studied. There are several other interactions between HCV-associated liver disease and renal dysfunction. The prevalence of HCV antibodies in patients on chronic hemodialysis is 8.6%,113 although prevalence in certain US dialysis centers may be higher, and prevalence from other parts of the world ranges from 5 to 50%.22 HCV infection is an independent risk factor for death in dialysis patients.114 Reasons for the high prevalence of HCV infection include transfusions before the availability of effective screening tests as well as nosocomial transmission of HCV infection in dialysis units. Screening and careful attention to infection control practices are essential to prevent HCV transmission. In patients on hemodialysis, ALT values are typically lower than in non-uremic patients but normal ALT levels do not exclude histologically significant liver disease. The goals of antiviral therapy in patients on dialysis are to slow liver disease progression and to eradicate HCV infection in those who might subsequently undergo kidney transplantation. Eradication of HCV is particularly important since chronic HCV disease adversely affects patient and graft survival after renal transplantation, and HCV antiviral therapy is not possible following kidney transplantation because treatment carries an unacceptable risk of allograft rejection.115 Ribavirin is also contraindicated in patients on dialysis and PEG-IFN alpha-2b should be used with caution in patients with creatinine clearance of less than 50 ml/min. Specific recommendations for dosing of PEG-IFN alpha2a in dialysis patients are available (135 mg/week).22 There is a general sense that treatment responses are greater with interferon monotherapy in dialysis patients than in those with normal renal function, possibly because reduced clearance of interferon effectively increases the administered dose. However, information about treatment outcomes with interferon/peginterferon monotherapy is limited and largely derived from case series. Overall SVRs from these case series are 33% (range 14–71%) with SVRs in genotype 1 patients of 26%.22,116,117 Virological relapse and side effects are common and patients should be monitored closely for toxicity. HCV antiviral therapy is also problematic in HCV-infected patients with lesser degrees of renal dysfunction.118 Since ribavirin is contraindicated in patients with a creatinine clearance of less than 50 ml/min, treatment outcomes are reduced in patients who can only receive peginterferon monotherapy. Because of nephrotoxicities associated with immunosuppressive therapies, renal dysfunction
UNMET NEEDS IN THE TREATMENT OF HEPATITIS C Impressive progress has been made in the treatment of hepatitis C since the discovery of the virus in 1989. Acute hepatitis C has become a treatable disease and chronicity can be prevented with therapy in 90% of cases. Genotype 2 and 3 patients, particularly those with genotype 2 infection, can be cured in around 90%. However effective therapy is still not available for a significant proportion of patients. Fifty percent of genotype 1 patients are nonresponders to standard treatment. These patients are highly motivated and, since many have advanced liver disease, are in substantial need of treatment. In addition, present treatments are
Table 32-8. Future therapeutic approaches and novel hepatitis C therapies Optimization of the use of interferon and ribavirin Optimizing dose and duration Additional agents _ Amantadine _ Histamine hydrochloride _ Thymosin-alpha New interferon delivery systems New generations of interferons, interferon inducers, other cytokines, and growth factors Consensus interferon Omega-interferon Albumin-bound interferon IL-28 A, IL-28 B, IL-29 ANA 245 – oral interferon inducers: TLR 7 and 9 agonists Erythropoietin Thrombopoietin Ribavirin analogs Viramidine Inosine monophosphate dehydrogenase (IMPDH) inhibitors – VX497 Small molecules with direct antiviral modes of action Specific hepatitis C viral enzyme inhibitors • Protease inhibitors – BILN 2061 – VX 950 – SCH 503034 • Polymerase inhibitors – NM 283/valopicitabine • Helicase inhibitors Antisense oligonucleotides Therapeutic vaccines E2 protein vaccine IC41 polypeptide vaccine HCl, hydrochloric acid; IL, interleukin; ANA, antinuclear antibody; IMPDH,
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Section V. Liver Diseases Due to Infectious Agents
On Market
Ribavirin
IFN & PEG IFN
Phase III
Viramidine Thymosin
Other IFNs Gamma IFN
STAGE
Phase II
antisense
IMPDH inhibitors
Ribozymes
Phase I
Polymerase inhibitors
E2 Vaccine
Histamine HCl
IL 10 & IL 12 others
Figure 32-3. Hepatitis C drug development. Various drugs at various stages of development. Some of these compounds such as levovirin and ribozymes are no longer in clinical development. Polymerase inhibitors are now in both phase I and phase II development. IFN, recombinant interferon alpha; PEG-IFN, pegylated recombinant interferon-alpha; HCl, hydrochloric acid; IMPDH, inosine monophosphate dehydrogenase; IL, interleukin; HCV, hepatitis C virus; siRNA, small interfering RNA. (Modified according to McHutchinson, Falk Liver Week, Freiburg, Germany 2004).
Protease Inhib Levovirin Apoptosis Inhibitors
HCV Immunotherapy
siRNA
Preclinical HCV Vaccines
Research
Many others including Antisense Antifibrotics Immune stimulants Gene therapy
long-term, costly, and associated with significant side effects. Furthermore our knowledge on the treatment of HCV genotypes other than 1, 2, and 3 is very limited. Only recently have we seen reliable data on HCV genotype 4, the most frequent genotype in the Arab world, where the prevalence of HCV in the general population reaches 20%. At present we stratify our treatment according to infecting genotype, although in the future, early viral kinetics may be more important than genotype in guiding treatment regimens. Regardless of evolving treatment strategies, it is likely new innovative drugs will be necessary in order to improve treatment outcomes substantially. The development of innovative drugs has been problematic due to unpredicted adverse events as well as to the early development of resistance. More drugs are failing each year than are successfully entering clinical development. Therefore there is still a strong rationale for optimizing the use of the currently available drugs (peginterferon and ribavirin) and/or for developing next generations of both compounds. Approaches for future therapies are as follows: (1) optimization of the use of interferon and ribavirin; (2) development of new generations of type 1 interferons, interferon inducers, other cytokines, and growth factors; (3) development of ribavirin analogs with an improved safety profile; (4) development of small molecule inhibitors with direct antiviral modes of action; and finally, (5) development of therapeutic vaccines. Each approach will be discussed individually.
OPTIMIZATION OF THE USE OF INTERFERON AND RIBAVIRIN An important question is whether a proportion of patients with genotype 1 infection would benefit from prolonged treatment with peginterferon plus ribavirin. Patients who have cleared the virus
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from their blood by week 12 have a high chance of becoming a sustained responder with 48 weeks of treatment. Those who clear virus between week 12 and week 24, the so-called “late” or “slow” responders, have a high relapse rate after 48 weeks. The likelihood of achieving an SVR in those with a 2-log drop by week 12, but who still have detectable virus at this time point, is intermediate for achieving an SVR with 48 weeks of treatment. Treatment duration may need to be adjusted according to individual viral kinetics. Initial studies of PEG-IFN alpha-2a plus ribavirin have shown that, while the overall population of genotype 1 patients does not benefit from prolonging treatment to 72 weeks, a subgroup of patients with a slow response may do so.120 In addition, patients known to be difficult to treat, such as those with high body mass index (>25) and those with high viral load (>6.5 log HCV RNA in serum) may also show a better SVR with prolonged therapy.121 However, extending treatment beyond 1 year may also lead to greater treatment discontinuations. Several recent studies have shown that 24 weeks may be more than adequate for certain patients with genotype 2 or 3 infection. The initial study evaluating the combination of PEG-IFN alpha and ribavirin79 included an arm with a lower dose of PEG-IFN alpha-2b (0.5 mg/kg per week) after 1.5 mg/kg per week had been given for the first month. Treatment responses for genotype 2 and 3 patients were equally good or better than the current FDA-approved dose of 1.5 mg/kg per week (80% SVR). Therefore it is logical to evaluate lower doses of peginterferon as well as shorter duration for these easy-to-treat patients. An RVR after 4 weeks of therapy seems to be the crucial factor for the success of a shorter duration of therapy. Patients with this RVR may only be treated for 16 weeks,122 14 weeks,123 or even 12 weeks.124 While in Europe and North America genotype 2 and 3 patients make up to 30% of all HCV patients, in many parts of Asia (India, Thailand, and more), genotype 2 and 3
Chapter 32 HEPATITIS C
patients are the predominant genotype and make up to 80% of all infections. Cost-effective approaches to the treatment of genotype 2/3 infection are particularly important in the developing world.
treat patient populations such as those with HIV co-infection or those prior to or following liver transplantation.
RIBAVIRIN ANALOGS NEW GENERATIONS OF TYPE 1 INTERFERONS, INTERFERON INDUCERS, OTHER CYTOKINES, AND GROWTH FACTORS There are a number of other type 1 interferons on the market or under clinical development. In addition conventional interferon alpha will soon be available as a generic, which will be particularly relevant in parts of the world where patients have to pay for their drugs and/or where social security systems are inadequate for drug reimbursement. Consensus interferon is approved in many countries, although a pegylated formulation is not available. Therefore daily injections of consensus interferon may be necessary to achieve optimum response. Many other cytokines have been tried with limited or no success, among them IL-10 and IL-12. Thymosin alpha is still under evaluation in combination with peginterferon. At present interferon alpha linked to albumin (albuferon) is starting phase III development. This interferon only requires once-monthly dosing.125 A different approach is the application of orally applied interferon inducers, but none has proven successful thus far. Anemia is a major problem in peginterferon and ribavirin combination therapies. Reduction of ribavirin does impair treatment outcomes, particularly for genotype 1 patients. The application of recombinant erythropoietin pre-emptively or when anemia develops has been studied and may offer the advantage of preventing ribavirin dose reductions.126 However, this growth factor adds significantly to the cost of treatment, and epoietin has not been shown to improve SVR. Erythropoietin use is probably most justified in difficult-to-
Adding ribavirin to interferon alpha clearly has been a substantial advance in the treatment of hepatitis C. Non-immune hemolytic anemia is the most important dose-limiting side effect. For treatment of genotype 1 infection in particular, compliance with full dose of ribavirin seems to be an important determinant of treatment response. Therefore several attempts have been made to develop ribavirin analogs with less anemia but comparable or possibly even better antiviral effects as ribavirin. At present viramidine, a “livertargeted” prodrug of ribavirin, is in phase III clinical development. Preliminary analysis of phase II results has suggested that anemia is less severe and less frequent than with ribavirin.127 Whether SVR results will be the same with viramidine as with ribavirin in combination with peginterferon is currently unknown. Results of the phase III trials are awaited. Approaches such as viramidine may be particularly useful for special patient groups such as those with recurrent hepatitis C after liver transplantation and those with renal disease.
SMALL MOLECULES WITH DIRECT ANTIVIRAL MODES OF ACTION (POLYMERASE, HELICASE, AND PROTEASE INHIBITORS) After unraveling the crystal structures of all three major HCV enzymes involved in replication and after the discovery of an HCV replicon system128 that facilitated in vitro testing of novel antiviral agents, there has been a concerted effort in drug discovery for innovative hepatitis drugs (Figure 32-4). Numerous patents have been Figure 32-4. The hepatitis C virus (HCV)-replicon system: milestone for the development of anti-HCV compounds.128 IFN, interferon.
R2884G HCV 5¢
EMCV Neo
IRES
3
4A
4B
5A
5B
3¢
IRES
subgenomic replicon
G418 IFN-a HuH-7 cells with HCV-replicon
IFN-a/G418 resistant cell clones 19 cell clones
expanded cell clones Preparation of total-RNA
2/16
G418-resistant HuH-7 cell clones 16 cell clones
Transfection in naive HuH-7 NUR G418!!!
IFN-a IC50
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filed but only a small number of these compounds have so far entered clinical development. The proof concept supporting this approach was established by Boehringer Ingelheim with BILN 2061 that demonstrated a 4-log decrease in viral load after 48 h of oral therapy (Figure 32-5).129,130 These drugs work in treatment-naive as well as in non-responders to prior peginterferon and ribavirin, but show 100-fold less activity against genotype 2 and 3 compared with genotype 1 infection.131 This selectivity for certain genotypes is likely a function of the interaction between small-molecule inhibitors and the HCV protease, since the protease demonstrates sequence differences between genotypes. The in vitro replicon system also suggested that these novel drugs would face problems of drug resistance in vivo. Due to side effects from long-term use in animals, BILN 2061 was withdrawn from further clinical development. Recently, VX 950 became a second protease inhibitor that has also shown a 4.4-log decrease
of HCV viral load after 2 weeks of treatment. Phase II studies are planned to explore this drug in combination with peginterferon.132 Polymerase inhibitors are a different group of drugs with less potent antiviral activity. In monotherapy NM283, or valopicitabine, achieves a 1–2-log decrease in HCV RNA levels133 after 4 weeks. Viral suppression is more potent when this drug is combined with peginterferon, reaching a 3-log reduction in viral load after 24 weeks.134 Addition of interferon seems to increase antiviral efficacy and may prevent resistance development. Apart from gastrointestinal side effects, valopicitabine was well tolerated. Phase II studies are under way. Due to either resistance development and/or to insufficient antiviral activity, all these small molecules acting directly on viral replication will likely need to be administered in combination with drugs such as peginterferon with or without ribavirin. Another antiviral approach has been to cleave the hepatitis C genome in a region that is crucial for the virus. Ribozymes are such
10 000 000
1.GT1b 2.GT1b 3.GT1a 4.GT1a
Virus load: copies RNA/ml
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5.GT1b 6.GT1a 7.GT1b 1000 8.GT1b 9.GT1b 10.GT1b Upper end of linear range
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X
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Hours Figure 32-5. Proof of principle: the BILN 2061 hepatitis C virus (HCV) protease inhibitor. Drop in HCV viral load over time in patients with chronic hepatitis C, genotype 1, naive and non-responders to previous therapy.130
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“molecular scissors” that cleave the HCV internal ribosome entry site.135 This concept works in vitro but ribozymes have not been demonstrated to be beneficial in patients. Further concepts are antisense molecules (ISIS-14803)136 and gene silencing using small interfering RNAs (SiRNA).137
THERAPEUTIC VACCINES Patients with chronic HCV infection have a diminished T-lymphocyte reactivity against the various T-cell epitopes compared to patients resolving acute HCV infection. Furthermore T lymphocytes from patients with chronic HCV have less capability to produce cytokines, such that their cellular immune functions are impaired.64 Therefore it is logical to explore whether stimulation of the patients’ immune system with therapeutic vaccines can overcome these deficiencies. Two such therapeutic vaccines are currently in clinical development and data are in the public domain. First, the E2 protein vaccine by Innogenetics has demonstrated reduced liver fibrogenesis despite a lack of viral suppression in a phase I/II study.138 A second vaccine from Intercell in Vienna includes five peptides spanning all the major T-cell epitopes together with an innovative adjuvant. Although CD4 and CD8 T-cell responses could be demonstrated, the associated reduction in viral load has thus far been unimpressive.139 The potential therapeutic value of these vaccines should perhaps be evaluated in easy-to-treat patients in order to prevent relapse, rather than in the difficult-to-treat non-responder population. Only clinical studies will reveal whether HCV therapeutic vaccines will have a place in the management of patients with chronic HCV.
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HEPATITIS D Mario Rizzetto Abbreviations ALT alanine aminotransferase anti-HD antibody to the HD Ag HBIg hyperimmune serum against HBsAg HBsAg hepatitis B surface antigen HBV hepatitis B virus HDV hepatitis delta virus
IFN IgM IgM antiHD l-HD Ag LKM
interferon immunoglobulin M antibody to the HD Ag large HD Ag liver–kidney microsomal
INTRODUCTION The hepatitis delta virus (HDV) is a unique transmissible human pathogen with an RNA genome which shares similarities with RNA pathogens of higher plants. The HDV is defective and requires the hepatitis B virus (HBV) for transmission; it therefore can only infect individuals with simultaneous HBV infection. It causes both acute and chronic hepatitis D; these are distinct medical entities usually more severe than the disease caused by HBV alone. The pathobiology of hepatitis D is complex, requiring an appreciation of the virologic and pathogenic interactions of two distinct agents within a common host.1
VIROLOGY The HDV is a defective RNA pathogen dependent for infection on obligatory helper functions provided by the HBV; it is the only member of the genus Deltavirus.2 Other hepadnaviruses can support HDV and its infection was experimentally established in the eastern woodchuck carrying the woodchuck HBV. The hepadnavirus provides the hepatitis B surface antigen (HBsAg) coat that is necessary in HDV for binding to the hepatocytes and for virion assembly.3 In vitro, intracellular replication of HDV has been established in mice by the intrahepatic injection of naked viral HDV DNA or RNA sequences4 and by the engraftment of human hepatocytes into heterochimeric severe combined immunodeficiency (SCID) mice.5 The virion is an approximately 36 nm particle containing within an HBsAg coat a 1.7 kilobase single-stranded circular RNA genome and two related structural phosphoproteins sharing a common antigenic reactivity (the hepatitis delta antigen = HD Ag).6 The genome contains only about 1700 nucleotides and is therefore the smallest among animal viruses, resembling viroid RNAs of plants. In analogy with viroids and components of eukaryotic cells, HDV RNA can act as a ribozyme, i.e., it encloses a segment that can self-cleave and self-ligate the genome. The HDV replicates by a rolling-circle mechanism similar to that involved in the replication of viroids; using the circular genomic RNA as a template, linear oligomeric forms of antigenomic polarity
Mu PCR SCID s-HD Ag THF g-2
million units polymerase chain reaction severe combined immunodeficiency small HD Ag thymic humoral factor g-2
are generated, which are then cleaved and ligated to circular monomers by the autocatalytic ribozyme. Replication occurs through RNA-directed RNA synthesis performed by a host RNA polymerase II which is normally DNA-dependent but is redirected by HDV to transcribe its RNA genome. The two forms of HD Ag produced by HDV, the small HD Ag (s-HD Ag) and the large HD Ag (l-HD Ag), differ in their C-terminal 19 amino acids. Their synthesis is regulated through the editing of the antigenomic RNA at position 1012 by cellular double-stranded RNA adenosine deaminase;7 the editing process changes a UAG stop codon to a UGG tryptophan codon that allows transcription to proceed for 19 further amino acid residues, leading to the synthesis of l-HD Ag. The sHDAg is required for viral replication; l-HD Ag inhibits replication of HDV and is required for its assembly.8 Replication of the antigenomic RNA strand requires multiple post-translational modifications, including the phosphorylation and methylation of HDAg.9 In vitro virion assembly is critically dependent on prenyl lipid modification (prenylation) of l-HD Ag.8,10 The HDV has evolved in at least three major genotypes, designated I, II, III, that differ by as much as 40% over the entire genome and have different geographic prevalences; two subgroups have been identified within genotype I and II, named genotype I A and I B and genotype II A and II B.11,12
TRANSMISSION The HDV is transmitted by the same routes as the helper HBV, i.e., by the parenteral route, either overtly or covertly. The highest rates of hepatitis D were reported in parenteral drug addicts; in contrast, vertical transmission from mother to newborn is rare.13 Transmission can occur by sexual contacts, in particular mercenary heterosexual contact, as attested by the prevalence of HDV in prostitutes and sexual partners of HDV-infected persons; however the spread of HDV has not been evident among homosexual man. Household transmission was important in 1970–1980 in endemic areas in southern Europe and cohabitation with an HDV carrier was identified as a major risk factor for the acquisition of the virus;14 molecular studies have confirmed sexual and household spread of HDV.15
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The efficiency of HDV transmission is primarily determined by whether the person exposed to HDV is or is not a carrier of HBsAg. In normal (HBsHg-negative) persons, HDV cannot be transmitted unless HBV infection has previously been established; therefore HDV is acquired simultaneously with HBV, i.e., by HBV–HDV coinfection (Figure 33-1A), and transmission efficiency depends on the infectious titer of co-infecting HBV. In the HBsAg carrier the pre-existing HBV state acts as a selective magnet to activate HDV and this infection is therefore rapidly established and amplified – i.e. there is HDV superinfection on prior HBV infection (Figure 33-1B). In the chimpanzee, titers of HDV as low as 10–11 serum dilutions were sufficient to establish infection in animals carrying the HBsAg. As the HBsAg state provides the HDV with indefinite biological support, superinfected HBsAg carriers often also become chronic carriers of HDV. Therefore HBsAg carriers are the selective victims of HDV, and the main epidemiologic reservoir and source of the virus.
EPIDEMIOLOGY Hepatitis D was endemic in the 1980s in many areas of the world but its distribution and ratio relative to the local prevalence of HBV varied widely. Overall prevalence rates were higher in tropical and
ALT
IgM anti-HD
IgM anti-HBc
IgG anti-HD
HDV HBV
HDV HBV Months A
Months
Weeks Course of coinfection with Hepatitis B virus and Hepatitis D virus ALT
IgM anti-HD
IgG anti-HD
HDV HBV
HDV HBV B
Course of Hepatitis D viral superinfection
Figure 33-1. (A) Course of co-infection with hepatitis B virus (HBV) and hepatitis D virus (HDV). (B) Course of hepatitis D viral superinfection. ALT, alanine aminotransferase; IgM, immunoglobulin M.
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subtropical areas with a higher prevalence of HBV than in north America and northern Europe, where the prevalence of HBV was low. In developing areas hepatitis D occurred as a secondary event in the context of high HBV endemicity; in settings of overcrowding and poor hygienic condition HDV infection mostly occurred in children. In most areas of the developed world, where the prevalence of HBV was low, HDV infection was confined to intravenous drug addicts.16 The pattern of infection was composite in areas with intermediate HBV prevalence such as southern Europe and Taiwan, resulting from an endemic pattern in the general population and an epidemic pattern within drug addicts.17 In Italy virus spread occurred primarily in overcrowded south Italian families and the age of acquisition of infection was adolescence–early adulthood; at-risk groups were also institutionalized patients, prisoners, hemophiliacs, and hemodialysis patients. Genetic analysis of HDV worldwide has shown significant geographic variations.18,19 Genotype I predominates in the USA, Europe,20 Turkey,21 the Middle East, and Mongolia;22 genotype I A predominates in Asia, I B in the USA, and both are common in the Mediterranean basin. Genotype II was found in Japan, Taiwan, and in Yakutia in Russia.23 Genotype III has been identified in northern South America.24 In the late 1970s to early 1980s the prevalence of HDV infection was relatively high in southern Europe but important epidemiologic changes have occurred in this area in the last decade. Changes were best documented in Italy. In a baseline study performed in the years 1978–1981 the prevalence of HDV infection, as determined from the prevalence of the antibody to the HD Ag (anti-HD) in HBsAg carriers with liver disease, was 24%. The prevalence remained stable up to the second half of the 1980s, but by the early 1990s it had diminished to 14% and in a nationwide survey performed in the late 1990s it has further declined to 8%.25 A decline in HDV infection was noted in Spain,26 where the rate of anti-HD in HBsAg carriers with chronic hepatitis dropped from 15% in 1975–1985 to 7.1% in 1986–1992 and a significant decrease in the prevalence of HDV superinfection as the cause of hepatitis flares in HBsAg carriers was also noted in Taiwan.27 The decrease of HDV infection throughout southern Europe is related to the control of HBV achieved in recent years through better public health standards, universal HBV vaccination and the measures introduced to contain the spread of HIV, which is transmitted in the same way as HBV/HDV; in Italy the reduction in family size brought about by social and economic changes had a major additional impact in containing hepatitis D. Sparse information was made available in the last years from other countries.28 A decrease from 47.6 to 15.4% in the prevalence of antiHD in patients with HBsAg-positive cirrhosis was noted in Belarus from 1991 to 1997. No marker of HDV infection was found in Indian children in the reservation of Xinger in central Brazil, a region adjacent to the Amazon jungle where the highest level of HDV endemicity was reported in the 1980s and where outbreaks of hepatitis D have continued to occur (in the Peruvian sector). Hyperendemic foci were reported from west Greenland, Tunisia, and Japan and markers of HDV were found in a consistent proportion
Chapter 33 HEPATITIS D
(18.35%) of HBsAg-positive pregnant women in Moldova as well as in 13.15% of hepatitis B patients in the Shandong province in China.28 In contrast, hepatitis D is rare in Poland29 and no infection marker was detected in rural areas of the high Andean plateau and in Nigeria; the rate of infection was also low, 6.5 and 4% respectively, in chronic HBsAg-positive liver disease in sub-Saharan Africa and in HBsAg carriers in north-west Mexico. In India HDV was implicated as a major risk factor of fulminant hepatitis but was rarely found in a series of hospital patients and mixed clinical HBsAgpositive populations in this country.28 Only a few studies comparing the prevalence of HDV infection over the years in drug addicts are available. In Taiwan, the prevalence of HDV in HBsAg-positive drug addicts has declined from 79% in 1985 to 14% in 2004;30 in this population the average rate of decrease in the prevalence of HDV infection was 4.7% per year.31 No HDV case was found in intravenous drug users in Rio de Janeiro, Brazil, despite a 7.8% prevalence of serum HBsAg in this community; low prevalence rates were found in a community in Gran Canaria, and a 14.7% prevalence rate was found in drug users in Jeddah, Saudi Arabia.28 It is unlikely that the pattern of HDV infection has changed in many endemic areas of the developing world, as the factors determining transmission and circulation of the virus were not modified in the last decade whereas reduction of HBV infection and the consequent shrinkage of the HBsAg pool within drug abusers has diminished the impact of HDV infection in these communities.
DIAGNOSIS Detection of indirect antibody markers is the first step in diagnosing HDV infection.13,19 These are the immunoglobulin M (IgM) antibody to the HD Ag (IgM anti-HD) and total antibody to the HD Ag (anti-HD) which predominantly detects the IgG antibody. The IgM antibody is also composed of monomeric 7S IgM molecules. The antibodies to HDV are not protective; anti-HD may persist at low titer as a serological scar to past HDV infection in both HBsAgpositive and negative individuals. Detection of intrahepatic HD Ag by direct immunohistochemistry was initially the gold standard for the diagnosis of HDV infection, as its finding was diagnostic of viral replication and ongoing infection.13 However, intrahepatic HD Ag is not detectable in as many as 50% of patients with current HDV infection and its expression decreases with evolution to advanced fibrotic disease.32 The detection of HDV RNA in serum by the polymerase chain reaction (PCR) is presently the most specific and sensitive diagnostic method.33 This assay has overcome the limitations of the detection of HD Ag in serum by enzyme or radioimmunoassay, due to antigen sequestration in immune complexes with circulating antibodies; it can detect 10–100 copies of the viral genome. The highest level of diagnostic efficacy is obtained by amplification of the most conserved region, the C-terminal half of the HD Ag gene. The HDV genotype may be determined in serum by restriction fragment length polymorphism analysis of PCR amplification products or by sequencing;20,33 HDV genotypes can also be determined in liver tissue by immunohistochemistry.34
NATURAL HISTORY Hepatitis D comes about through co-infection with the HBV or through superinfection of a HBsAg carrier (Figure 33-1). Individuals protected from HBV by antibodies to the HBsAg (anti-HBs) in serum are also protected from HDV.13 In normal individuals who become co-infected with HBV and HDV, the activation of HDV is dependent on the prior activation of HBV. Expression of HDV may vary from massive to abortive; accordingly, co-infection with hepatitis D presents clinically as an acute hepatitis of variable severity. As the underlying HBV infection is usually self-limiting and the defective HDV cannot survive the clearance of its helper virus, co-infection with hepatitis D usually resolves; fewer than 2% of cases were reported to progress to chronicity.19 In contrast, since the HBV state will indefinitely support HDV, superinfected HBsAg carriers run a high risk of developing chronic hepatitis D; nevertheless, HDV superinfection can occasionally terminate the carriage of HBsAg with clearance of both HBV and HDV, and the rate of HBsAg clearance is increased over the years in chronic HDV patients compared to ordinary HBsAg carriers.35 Triple HBV–HDV–HCV infection can be observed in drug addicts. In studies in Spain36 and France,37 HDV acted as the dominant virus and cirrhosis was more frequent in patients with multiple infections; in a study of triple infections in Taiwan, HCV acted as the dominant virus.38 Superinfection presents clinically as an icteric hepatitis which often runs a severe course, and HDV superinfection was recognized worldwide as an important factor leading to fulminant liver disease.39 Surveys in the Mediterranean area in the 1980s have shown that chronic hepatitis D ran an indolent non-progressive or slowly progressive course in about 10–15% of cases while in the remaining patients the disease was progressive, advancing to cirrhosis within 2–6 years in 40% of cases. Infection with HDV increases the risk for hepatocellular carcinoma threefold and for mortality twofold in patients with HBsAg-positive cirrhosis;40 in cirrhotic patients impressive splenomegalies may develop. Because of the young age at HDV acquisition and the accelerated course of the disease, chronic hepatitis D was recognized as a major cause of juvenile cirrhosis in endemic areas. Evolution to cirrhosis occurs in three phases: (1) an early one characterized by florid disease and infection with significantly elevated alanine aminotransferase (ALT), active HDV replication, and suppressed HBV; (2) a second phase characterized by diminished ALT and HDV synthesis and occasionally by reactivation of HBV; and (3) a late cirrhotic phase characterized by marked reduction of the synthesis of both HBV and HDV.32 In children the course of the disease is similar to that seen in adults. In co-infections the serological pattern includes the markers of acute HBV infection (the IgM antibody to HB core antigen and HBV DNA) with superimposed makers of HDV. Hepatitis D antigenemia occurs early, often is not detectable and, when initially present, becomes rapidly undetectable for sequestration of the serum HD Ag by the homologous antibody. The viremic phase is best detected by HDV-RNA PCR assaying. The IgM antibody to HD rises days to
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a few weeks after onset of disease while total (IgG) anti-HD rises a few weeks after. In superinfected carriers, both the IgM and IgG anti-HD rise rapidly to high titers and persist as hepatitis D becomes chronic. The HDV RNA is usually detectable at onset of disease and persists, while the IgM antibody to the HB core antigen is usually negative except if the carrier of HBsAg had chronic liver disease related to active HBV infection. In patients whose acute or chronic disease resolves spontaneously or following interferon (IFN) therapy, the IgM antibody disappears in a few months; in most of these patients IgG anti-HD also disappears but occasionally it may persist at low titers.
CLINICAL COURSE Acute and chronic hepatitis D do not exhibit clinical or histologic features that are specific or distinctive from ordinary hepatitis B.16,18,19 Acute co-infection with hepatitis D may run a biphasic course with two peaks of ALT several weeks apart and expression of HDV in the second peak. In asymptomatic carriers of HBsAg who are unaware of their state, superinfection with hepatitis D may appear as acute hepatitis B; however, in contrast to acute hepatitis B, the IgM antibody to the HB core antigen is usually negative. In carriers with prior chronic hepatitis B, superinfection with hepatitis D may be mistaken for a recrudescence of the underlying HBV infection or it may decompensate a pre-existing stable HBV disease. The concomitant HBV infection is most often inactive (HBV DNA negative by conventional hybridization assays or positive at low titer by PCR; HBe Ag negative and anti-HBe positive in serum). A proportion of patients, usually with early and florid disease, generate a variety of autoantibodies. The most frequent is a liver–kidney microsomal (LKM) antibody which displays a pattern of tissue immunofluorescence similar to that displayed by the LKM1 antibody of autoimmune type 2 hepatitis; it is directed against uridine diphosphate glucuronyltransferase 1 instead of the cytochrome superfamily and is therefore distinguished as LKM3.41 The pathogenicity of HDV appears to be modulated by the degree of replication of the helper HBV; disease is usually most severe in patients whose HDV and HBV infections are both active.42 Variations in pathogenicity may be related to the genotype of HDV; genotype III was associated with outbreaks of fulminant hepatitis in South America24 and Japanese patients with genotype II B showed greater progression of chronic hepatitis to cirrhosis than those with genotype II B.12 The striking changes of the last 15 years in the epidemiology of HBV/HDV in the Mediterranean basin have modified the clinical impact and features of hepatitis D in this area. While most of the HDV patients collected in Italy in the 1980s had a florid chronic active hepatitis, and inactive cirrhosis with residual burnt-out inflammation was seen in fewer than 20% of cases, the proportion of cirrhotic patients has increased in Italy to 70% in the years 1996–2000; these patients represent the survivors to the epidemic of hepatitis D that ravaged HBsAg carriers in the 1970s–1980s.43 Thus the contemporary medical scenario of hepatitis D in areas such as Italy where the circulation of HDV has much diminished in
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recent years is predominantly made up of advanced fibrotic disease unresponsive to IFN, which is unlikely to achieve benefit from any medical treatment, and for which only liver transplantation offers adequate therapy.
PREVENTION AND THERAPY Successful vaccination against HBV provides protection against HDV. No effective HDV vaccine has been developed to protect the HBsAg carrier. In clinical trials evaluating the long-term administration of IFN-a in chronic hepatitis D, the rate of response (normalization of the serum ALT level and clearance of serum HDV RNA) has varied, occurring at different times from the beginning of treatment.44 It was proportional to the dose of IFN. In a study45, patients given 9 million units (Mu) thrice weekly responded better than patients given lower dosages and, at the end of treatment, the normalization of ALT in responders corresponded to histologic improvement and clearance of HDV RNA from blood; however, most experienced a biochemical and virological relapse shortly after stopping therapy and in the five patients who exhibited normal ALT for up to 4 years, the effect on HDV was not sustained. Nevertheless, after 14 years of followup complete fibrosis regression has been reported in some of the patients who had a persistent biochemical response and lost IgM anti-HD, all of whom had an initial diagnosis of active cirrhosis at the end of therapy.46 A virological and biochemical relapse was also common when IFN was reduced to 3 MU/m2 after a 4-month course with 5 MU/m2.44 Sustained responses are often followed by loss of HBsAg and seroconversion to anti-HBs47 and resolution of chronic hepatitis D has occurred up to 12 years after continuous IFN therapy.48 Responses have occurred in immunodeficiency virus-positive drug abusers with HDV in whom immunologic competence was preserved.49 In HDV patients with concomitant HBV infection, therapy may abrogate HBV replication. Several such patients, both adult and children, cleared HBV DNA and hepatitis B e antigen from serum and seroconverted to anti-HBe during interferon therapy; however the control of HBV infection had no impact on HDV infection or on disease activity. The severity of side effects is proportional to the IFN dose and to the age of the patient. Four patients were reported to experience a severe exacerbation of hepatitis during therapy; one of them had LKM antibodies. Suramin, aciclovir in combination with IFN, ribavirin, and Thymic Humoral Factor g-2 (THF g-2) a synthetic thymus-derived octapeptide, have not produced beneficial therapeutic effects. As HBsAg is required by HDV to perpetuate infection, repression of the helper HBV and of HBsAg production might prevent the spread of virus and thus help to eradicate HDV infection. On this rationale famciclovir and lamivudine, which are inactive on HDV replication but capable of inhibiting HBV, have been tested in hepatitis D. However, results were discouraging. None of 15 patients with chronic hepatitis D treated with famciclovir 500 mg three times a day for 6 months and then followed up for 6 months responded.50
Chapter 33 HEPATITIS D
In a study five patients received lamivudine 100 mg orally daily for 12 months.51 Though HBV DNA became undetectable in four patients and decreased in the other, ALT remained abnormal, HDV RNA detectable, and HBsAg positive in all. Liver biopsy showed no improvement in inflammatory or fibrotic score. A second study52 compared a 12-month with a 24-month course of lamivudine 100 mg/day. Thirty-one patients were randomized to treatment, 11 to placebo, and 20 to lamivudine for 12 months; thereafter all were given lamivudine on an open-label basis for 12 months and followed up for further 16 weeks. At the end of treatment HDV RNA was negative and ALT normal in three patients but only two patients remained virus-free at the end of follow-up. The effects of the combination of IFN with lamivudine were also investigated.53 Eight patients with chronic hepatitis D were treated with lamivudine for at least 24 weeks. Lamivudine was then combined with high-dose IFN followed by 9 MU IFN thrice weekly; the patients were followed up for 12 weeks post-therapy. The HBsAg concentration in serum decreased in two patients. There was no significant reduction of HDV RNA in plasma from baseline during treatment. At the end of treatment ALT normalized in one patient and decreased in three other patients, but three of these four patients showed a biochemical rebound after withdrawal of therapy. Available data indicate that IFN is the only potential – albeit limited – therapy for chronic hepatitis D. To achieve a response, high doses of conventional IFN are required (9–10 MU thrice weekly for 1 year or longer); although there is no information yet on pegylated IFNs, they appear nevertheless to represent a logical therapeutic option for the long-term treatment required for chronic hepatitis D. Parameters predictive of response are still unidentified. As response can take up to 10 months, IFN should be given for at least a year before a patient is regarded as a non-responder. Treated patients can lose HDV markers from serum while on therapy but may relapse when treatment is discontinued if they remain HBsAg-positive; therefore therapy should not be discontinued prematurely on the basis of the clearance of HDV. Loss of HBsAg is a reliable marker of resolution of hepatitis D. Preclinical data in a mouse-based model of HDV infection capable of yelding viremia support a potential role of prenylation inhibitors at clearing viremia through interference within the cxxx box within the 19 amino acids unique to l-HD Ag, which is the substrate for the prenyltransferases required to prenylate the antigen.54
TRANSPLANTATION Liver transplantation provides a valid treatment option for end-stage HDV liver disease. The spontaneous risk of reinfection is lower for HDV than for HBV and the clinical course of recurrent hepatitis is milder than for HBV. The prospect of an uneventful clinical course after transplantation was significantly improved by the long-term administration of hyperimmune serum against HBsAg (HBIg); the 5-year survival rate of 76 transplant recipients in Paris for terminal HDV cirrhosis given HBIg long-term was 88%, with reappearance of HBsAg in only 9%.55 None of 62 HDV patients transplanted in the last years in Turin experienced a viral recurrence; 48 were given HBIg and 14 were given lamivudine before transplantation and lamivudine together with HBIg indefinitely post-transplantation.56
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22. Takahashi M, Nishizawa T, Gotanda Y, et al. High prevalence of antibodies to hepatitis A and E viruses and viremia of hepatitis B, C, and D viruses among apparently healthy populations in Mongolia. Clin Diagn Lab Immunol 2004; 11:392–398. 23. Ivaniushina V, Radjef N, Alexeeva M, et al. Hepatitis delta virus genotypes I and II cocirculate in an endemic area of Yakutia, Russia. J Gen Virol 2001; 82:2709–2718. 24. Casey JL, Brown TL, Colan EJ, et al. A genotype of hepatitis D virus that occurs in northern South America. Proc Natl Acad Sci USA 1993; 90:9016–9020. 25. Gaeta GB, Stroffolini T, Chiaramonte M, et al. Chronic hepatitis D: a vanishing disease? An Italian multicenter study. Hepatology 2000; 32:824–827. 26. Navascues CA, Rodriguez M, Sotorrio NG, et al. Epidemiology of hepatitis D virus infection: changes in the last 14 years. Am J Gastroenterol 1995; 90:1981–1984. 27. Huo TI, Wu JC, Lin RY, et al. Decreasing hepatitis D virus infection in Taiwan: an analysis of contributory factors. Gastroenterol Hepatol 1997; 12:745–746. 28. Ciancio A, Rizzetto M. Clinical patterns, epidemiology and disease burden of hepatitis D virus chronic liver disease. Margolis H, Alter M, Liang T, et al, eds. 10th International Symposium on Viral Hepatitis and Liver Disease. London: International Medical Press; 2002:271–275. 29. Chlabicz S, Grzeszczuk A, Lapinski TW, et al. Search for hepatitis delta virus (HDV) infection in hepatitis C patients in north-eastern Poland. Comparison with anti-HDV prevalence in chronic hepatitis B. Eur J Epidemiol 2003; 18:559–561. 30. Huo TI, Wu JC, Wu SI, et al. Changing seroepidemiology of hepatitis B, C, and D virus infections in high-risk populations. J Med Virol 2004; 72:41–45. 31. Kao JH, Chen PJ, Lai MY, Chen DS. Hepatitis D virus genotypes in intravenous drug users in Taiwan: decreasing prevalence and lack of correlation with hepatitis B virus genotypes. J Clin Microbiol 2002; 40:3047–3049. 32. Wu JC, Chen TZ, Huang YS, et al. Natural history of hepatitis D viral superinfection: significance of viremia detected by polymerase chain reaction. Gastroenterology 1995; 108:796–802. 33. Smedile A, Niro MG, Rizzetto M. Detection of serum HDVRNA by RT-PCR. In: Hamatake RK, Lay JYN, eds. Methods in molecular medicine; hepatitis B and D protocols. Totowa, NJ: Humana Press; 2005:85–94. 34. Hsu SC, Syu WJ, Ting LT, Wu JC. Immunohistochemical differentiation of hepatitis D virus genotypes. Hepatology 2000; 32:1111–1116. 35. Niro GA, Gravinese E, Martini E, et al. Clearance of hepatitis B surface antigen in chronic carriers of hepatitis delta antibodies. Liver 2001; 21:254–259. 36. Jardi R, Rodriguez F, Buti M, et al. Role of hepatitis B, C, and D viruses in dual and triple infection: influence of viral genotypes and hepatitis B precore and basal core promoter mutations on viral replicative interference. Hepatology 2001; 34:404–410. 37. Mathurin P, Thibault V, Kadidja K, et al. Replication status and histological features of patients with triple (B, C, D) and dual (B, C) hepatic infections. J Viral Hepatol 2000; 7:15–22.
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38. Lu SN, Chen TM, Lee CM, et al. Molecular epidemiological and clinical aspects of hepatitis D virus in a unique triple hepatitis viruses (B, C, D) endemic community in Taiwan. J Med Virol 2003; 70:74–80. 39. Rizzetto M. Hepatitis delta virus disease: an overview. Prog Clin Biol Res 1993; 382:425–430. 40. Fattovich G, Giustina G, Christensen E, et al. Influence of hepatitis delta virus infection of morbidity and mortality in compensated cirrhosis type B. The European Concerted Action on Viral Hepatitis (Eurohep). Gut 2000; 46:420–426. 41. Philip T, Durazzo M, Trautwein C, et al. Recognition of uridine disphosphate glucuronosyl transferases by LKM-3 antibodies in chronic hepatitis D. Lancet 1994; 344:578–581. 42. Smedile A, Rosina F, Saracco G, et al. Hepatitis B virus replication modulates pathogenesis of hepatitis D virus in chronic hepatitis D. Hepatology 1991; 13:413–416. 43. Rosina F, Conoscitore P, Cuppone R, et al. Changing pattern of chronic hepatitis D in southern Europe. Gastroenterology 1999; 117:161–166. 44. Niro GA, Rosina F, Rizzetto M. Clinical update: treatment of hepatitis D. J Viral Hepatitis 2005; 12:2–9. 45. Farci P, Mandas A, Coiana A, et al. Treatment of chronic hepatitis D with interferon alfa-2a. N Engl J Med 1994; 330:88–94. 46. Farci P, Roskams T, Chessa L, et al. Long term benefit of interferon alpha therapy of chronic hepatitis D: regression of advanced hepatic fibrosis. Gastroenterology 2004; 126:1740–1745. 47. Lau JY, King R, Tibbs CJ, et al. Loss of HBsAg with interferonalpha therapy in chronic hepatitis D virus infection. J Med Virol 1993; 39:292–296. 48. Lau DT, Kleiner DE, Park Y, et al. Resolution of chronic delta hepatitis after 12 years of interferon alfa therapy. Gastroenterology 1999; 117:1229–1233. 49. Puoti M, Rossi S, Forleo MA, et al. Treatment of chronic hepatitis D with interferon alpha-2b in patients with human immunodeficiency virus infection. J Hepatol 1998; 29:45–52. 50. Yurdaydin C, Bozkaya H, Gurel S, et al. Famciclovir treatment of chronic delta hepatitis. J Hepatol 2002; 37:266–271. 51. Lau DT, Doo E, Park Y, et al. Lamivudine for chronic delta hepatitis. Hepatology 1999; 30:546–549. 52. Niro GA, Ciancio A, Tillman HL, et al. Lamivudine therapy in chronic delta hepatitis: a multicentre randomized-controlled pilot study. Aliment Pharmacol Ther 2005;22:227–232. 53. Wolters LM, van Nunen AB, Honkoop P, et al. Lamivudine-high dose interferon combination therapy for chronic hepatitis B patients co-infected with the hepatitis D virus. J Viral Hepatitis 2000; 7:428–434. 54. Bordier BB, Ohkanda J, Liu P, et al. In vivo antiviral efficacy of prenylation inhibitors against hepatitis delta virus. J Clin Invest 2003; 112:319–321. 55. Samuel D, Zignego AL, Reynes M, et al. Long term clinical and virological outcome after liver transplantation for cirrhosis caused by chronic delta hepatitis. Hepatology 1995; 21:333–339. 56. Marzano A, Gaia S, Ghisetti V, et al. Viral load at the time of liver transplantation and risk of hepatitis B recurrence. Liver Transplant 2005;11:402-409.
Section V: Liver Diseases Due to Infectious Agents
34
HEPATITIS E S.K. Sarin and Manoj Kumar Abbreviations ALT alanine aminotransferase cDNA complementary DNA EIA enzyme immunoassays ET-NANBH enterically transmitted non-A, non-B hepatitis FHF fulminant hepatic failure
HAV HEAg HEV HIV IEM IFM
hepatitis A virus hepatitis E antigen hepatitis E virus immunodeficiency virus immune electron microscopy immune fluorescent microscopy
INTRODUCTION Hepatitis E virus (HEV) is an RNA virus that causes an acute, selflimiting hepatitis. This unclassified virus is enterically transmitted, although other routes of transmission may exist. Infection with HEV may be asymptomatic or may cause hepatitis varying in degree of severity from mild to fulminant disease. Hepatitis E is the commonest form of acute hepatitis in adults in highly endemic regions of Asia, but the disease is rarely diagnosed in industrialized countries. Fulminant hepatitis E has been reported with increased frequency in pregnant women. Evidence for an enterically transmitted virus different from the hepatitis A virus (HAV) came from serologic studies of waterborne epidemics of acute hepatitis in India in the late 1970s. Khuroo1 and Wong et al.2 demonstrated that patients involved in such epidemics of hepatitis in the Kashmir region and in Delhi, India, respectively, lacked serologic evidence of recent HAV infection and only showed evidence of past infection. Therefore, they concluded that another agent must have caused the acute hepatitis. Balayan et al.3 in 1983 provided the first proof of the existence of a newly identified form of acute viral hepatitis by transmitting hepatitis to a volunteer from a patient involved in an outbreak of enterically transmitted non-A, non-B hepatitis in central Asia. The volunteer, who had pre-existing antibody to HAV, developed a severe hepatitis, shed 27–30-nm virus-like particles in his feces detected by immune electron microscopy (IEM), and developed antibodies to the virus-like particles during convalescence. The researchers also inoculated cynomolgus monkeys with the new virus; again, the monkeys developed hepatitis, shed virus-like particles, and developed an immune response to the particles. In 1990, Reyes et al. succeeded in cloning and sequencing a part of the genome of the virus.4 The new form of non-A, non-B hepatitis came to be known as epidemic non-A, non-B hepatitis or enterically transmitted non-A, non-B hepatitis (ET-NANBH), and later, the name of the disease was changed to hepatitis E to conform with the accepted nomenclature for the other types of viral hepatitis.5,6 Ironically, hepatitis occurring in previous centuries and the early 20th century, and known variously as “campaign jaundice” and “infective hepatitis,” was probably not due to HAV but instead was
IG MAbs pORF2 RT-PCR VLPs
immunoglobulin monoclonal antibodies polyprotein open reading frame 2 strand-specific reverse transcriptase polymerase chain reaction virus-like particles
due to HEV because the epidemiological descriptions of such disease resemble those of hepatitis E, not hepatitis A.6
VIROLOGY CLASSIFICATION As per the physicochemical properties, HEV was initially grouped into the Calicivirus family.7 HEV closely resembles the sequence of rubella virus, a member of the virus family Togaviridae, as well as the sequence of beet necrotic yellow vein virus, a plant furovirus.8 The sequences of the putative RNA polymerase and helicase of HEV resemble those of superfamily 3 viruses (rubella, and other alphaviruses) rather than those of superfamily 1 (caliciviruses, picornaviruses, and others).9 The Caliciviridae Study Group recently submitted a proposal to the International Committee on Taxonomy of Viruses to remove HEV from the family Caliciviridae and place it into an “unclassified” status. The decision was based primarily on the lack of phylogenetic relatedness between the HEV and caliciviruses.10,11 as well as profound differences in the types of putative replicative enzymes used by HEV, as described by Koonim and Dolja.12 The final taxonomic status of HEV remains to be determined.13
STRUCTURE Physiochemical Characteristics The buoyant density of HEV is 1.35–1.40 g/cm3 in CsCl.3,14 Its sedimentation coefficient is 183 S.15 The virus is relatively stable to environmental and chemical agents.16 HEV contains an RNA genome enclosed within a capsid that is composed of one or possibly two proteins, but direct analysis of purified virions has not been possible to date.17
Morphology HEV is a spherical, non-enveloped particle that is approximately 27–34 nm in diameter and has an icosahedral symmetry.18,19 It has an indefinite surface structure that is intermediate between that of the Norwalk agent (a member of the Caliciviridae family) and that of HAV (a member of the Picornaviridae family).20 Since HEV is
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refractory to growth in continous cell cultures and is not present in large amounts in clinical material, there has been very little characterization of authentic viral particles. However, the expression of truncated polyprotein open reading frame 2 (pORF2: aa 112–660) in the baculovirus system leads to the formation of HEV virus-like particles (VLPs).21,22 At around 27 nm, these VLPs are smaller than the intact virus particle. Xing et al. used cryoelectron microscopy to study the HEV structure.23 Their analysis suggested that HEV VLPs are assembled as a T=1 icosahedral particle containing 30 dimeric subunits of 50-kDa pORF2, with the potential to form an intact virion of the correct size with a T=3 arrangement of 90 dimeric subunits.23 pORF2 dimerization appears to be due to non-covalent interactions in the C-terminal part of the protein and may contribute to the assembly of the immunodominant ORF2.1 epitope. It is not known whether the pORF2 protein is truncated in viral particles (as it is in VLPs), and further studies are needed to enhance the understanding of HEV structure.
GENOME ORGANIZATION Early studies postulated that ET-NANBH was caused by an RNA virus. Therefore Reyes et al. attempted to construct a complementary DNA (cDNA) library. Virus-enriched gallbladder bile from cynomolgus macaques infected with the second passage Burma isolate was used as a source to construct a cDNA library in gglutamyltransferase 10. One clone, designated ET1.1, contained a 1.3-kb cDNA exogenous to non-infected human genomes. Using a sequence-independent single-primer amplification procedure, cDNA from the bile of an infected cynomolgus macaque was amplified and hybridized with the clone ET1.1. Subsequently similar hybridization analyses of human fecal materials collected from outbreaks of ET-NANBH in geographically separated locations indi-
cated that a common pathogen was responsible for the majority of ET-NANBH seen worldwide. A partial nucleotide sequence of the ET1.1 clone was published in 19904 and the entire viral genome was reported in 1991. The genome of HEV is a single-stranded positive-sense RNA molecule approximately 7.2 kilobases in length followed by a polyA tail.9 The HEV has a genome that encodes structural and non-structural proteins through the use of discontinuous and partially overlapping ORFs. The genome consists of a short 5¢ non-translated region, three ORFs, each in a different coding frame, and a short 3¢ nontranslated region that is terminated by a stretch of adenosine residues, organized as 5¢-ORF1–ORF3–ORF2-3¢, with ORF3 and ORF2 largely overlapping (Figure 34-1). The 5¢ and 3¢ untranslated regions are highly conserved and are likely to play roles in RNA replication and encapsidation. Recently it has been shown that the 5¢ end of the genome has a 7-methylguanosine cap.24 ORF1, the largest ORF, begins at the 5¢ end of the viral genome after a 27-bp non-coding sequence and extends 5079 bp to the 3¢ end (in the Burmese prototype strain), and encodes about 1693 amino acids encompassing non-structural, enzymatically active proteins probably involved in viral replication and protein processing. Based on the identification of characteristic amino acid motifs,25 the following genetic elements have been identified, in order, from the 5¢ to the 3¢ end of the ORF: (1) a methyl transferase, presumably involved in capping the 5¢ end of the viral genome; (2) the “Y” domain, a sequence of unknown function that is found in certain other viruses, including rubella virus; (3) a papain-like cysteine protease, a type of protease found predominantly in alphaviruses and rubella virus26 ; (4) a proline-rich “hinge” that may provide flexibility and that contains a region of hypervariable sequence;27,28 (5) an “X” domain of unknown function that has been found adjacent to
Genomic organization of HEV 5' UTR
3' UTR poly (A) tail RNA
Viral
Translation
MT
PORF 1 N
PP
Hel
RdRp C PORF 2 N PORF 3 Poly(cys)
N
C C Poly(pro)
Figure 34-1. Genomic organization of hepatitis E virus. The hepatitis E virus (HEV) genome is a ~7.5kb polyadenylated RNA. At its 5¢ and 3¢ termini, the viral RNA carries two short untranslated regions (UTRs). It is positive-sense and includes three open reading frames (ORFs. The three ORFs are organised as 5¢-ORF1-ORF3ORF2–3¢ and encode the viral proteins. ORF1 encodes a putative nonstructural polyprotein (pORF1) that includes domains found in viral methyltransferases (MT), papain-like cysteine proteases (PP), viral RNA helicases (Hel) and viral RNA-dependent RNA polymerases (RdRp). ORF2 encodes the major viral capsid protein (pORF2). ORF3 encodes a small protein (pORF3) with two hydrophobic domains in its N-terminal half, which includes a polycysteine [Poly(Cys)] region, and prolinerich [Poly(Pro)] sequences in its C-terminal half. pORF3 is phosphorylated at a single (Ser80) amino acid by the cellular mitogen-activated protein kinase (MAPK), associates with the cytoskeleton through its N-terminal end and with proteins carrying the src-homology 3 (SH3) motifs through its C-terminal end. pORF, polyprotein open reading frame.
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papain-like protease domains in the polyproteins of other positivestrand RNA viruses;29 (6) a domain containing helicase-like motifs similar to those found in viruses containing type I (superfamily 3) helicases;30 and (7) an RNA-dependent polymerase, with motifs most closely related to those found in viruses containing an RNA polymerase of superfamily 3.31 Expression of pORF1 alone in HepG2 cells or in an in-vitro translation system failed to demonstrate any proteolytic processing into mature products, suggesting that other cofactors may be required for correct processing.32 ORF2, approximately 2000 nucleotides in length, begins approximately 40 nucleotides after the termination of ORF1 and consists of a 5¢ signal sequence, a 300-nucleotide region rich in codons for arginine, probably representing an RNA-binding site, and three potential glycosylation sites.33 ORF2 encodes a 660-amino-acid protein, most likely representing one or more structural or capsid protein(s) of HEV. The pORF2 is an 80-kDa glycoprotein with a potential endoplasmic reticulum directing signal at its N-terminus (a region containing high concentrations of arginine and lysine). It is synthesized as a precursor, then processed through signal sequence cleavage into the mature protein, and probably glycosylated at three potential glycosylation sites, which is common for the surface proteins of the envelope but not for the non-enveloped viruses. In-vitro assays have suggested that the protein is cotranslationally translocated across the endoplasmic reticulum and is expressed intracellularly as well as on the cell surface of the hepatocyte.34 When pORF2 is expressed in mammalian cells, a large proportion of the nascent protein is modified by N-glycosylation.35 However, this glycosylated form of the protein is highly unstable,36 and it remains to be clarified whether the authentic viral particle contains glycosylated capsid proteins. When pORF2 is expressed in insect cells, it is cleaved at a site between amino acids 111 and 112 and at various other sites within the C-terminus of the protein. Some of these truncated forms of the pORF2 have the ability to self-assemble into VLPs or subviral particles.37–39 However, studies on native virus particles are awaited, to confirm the biological relevance of these subviral particles. Recently, by using the yeast two-hybrid system and in-vitro immobilization experiments, it has been shown that full-length and N-terminal deletion fragment of the ORF2 protein can dimerize. This dimerization property may not be amino-acid sequencedependent but instead is a complex formation of a specific tertiary structure that imparts to pORF2 its property to self-associate.40 The ORF2 protein also contains RNA-binding activity and specifically binds to the 5¢ end of the HEV genome. A 76-nucleotode region at the 5¢ end of the HEV genome was responsible for binding the ORF2 protein. This interaction may be responsible for bringing the genomic RNA into the capsid during assembly, thus playing a role in viral encapsidation.41 ORF3, less than 400 nucleotides in length, overlaps ORF1 by one nucleotide at its 5¢ end and overlaps ORF2 by over 300 nucleotides at its 3¢ end. It codes for a 123-amino-acid, 13.5-kDa nonglycosylated protein (pORF3), which is a very basic protein (pI 12.5), and is the most variable protein among the HEV strains. The function of pORF3 is unknown, but it has been reported to be a phosphoprotein. When expressed in animal cells, pORF3 is phosphorylated at a single serine residue (Ser80) in its 123-amino-acid primary sequence, by a mitogen-activated protein kinase, and does not seem to undergo other major post-translational modifications.42
This phosphoprotein, which possesses two very strong hydrophobic regions in the N-terminal half of the molecule (possibly transmembrane a-helices with the potential to be associated with cellular membranes) was found to associate with the hepatocellular cytoskeleton, suggesting a possible role as a cytoskeletal anchor site for the assembly of virus particles.42 Recently a possible role of pORF3 in the modulation of cellular signal transduction pathways has been suggested. By using in-vitro binding assays, pORF3 has been shown to bind to a number of SH3-containing cellular signal transduction pathway proteins, including the protein tyrosine kinases Src, Hck, and Fyn, the p85a regulatory subunit of phosphatidylinositol 3-kinase, phospholipase C, and the adapter protein Grb2.43 Although ORF3 maps in the structural region of the HEV genome, it may have some regulatory functions. Also, as ORF3 interacts with ORF2, the possibility of a non-structural function for the ORF3 protein exists. By using fluorescence-based colocalization in yeast two-hybrid experiments, transiently transfected COS-1 cell (a cell line of saccharomyces cerevisiae) co-immunoprecipitation, and cellfree coupled transcription–translation techniques, it has been shown that ORF3 protein interacts with the ORF2 protein, the interaction being dependent on the phosphorylation at Ser80 of ORF3. Such an interaction suggests a possible well-regulated role for ORF3 in HEV structural assembly.44 The observations that pORF3 can homodimerize in vivo and the overlap of the dimerization domain with the SH3-binding and phosphorylation domains, suggests that pORF3 may have a dimerization-dependent regulatory role to play in the signal transduction pathway.45
GENOTYPES The initial two isolates of HEV from Burma4 and Mexico46 were only about 76% similar in overall genomic organization. In the years immediately following identification of the Burmese and Mexican prototype strains, a number of additional isolates were identified from Pakistan, India, and China. All of these isolates were closely related to the original Burmese isolate, having greater than 93% nucleotide identity across the genome. At that time, the genotypic distribution of HEV consisted of a group of Burmese-like isolates and the lone Mexican isolate.47 Additional genetic diversity was reported in an acute hepatitis patient from the USA.48–50 A number of additional isolates of HEV were subsequently identified from patients from Italy and Greece.51–53 Genetic diversity is seen not only in HEV from different regions, but also from isolates from the same region. Isolates from Argentina and Austria were found to be distinct from each other as well as all previously identified isolates.54–56 Also, the two isolates from patients from Spain were most closely related to one of the Greek isolates.57 Extensive diversity has also been reported between HEV strains from a number of patients from China and Taiwan that are distinct from the original Chinese isolates (genotype IV), and are closely related to the Burmese isolate.58–60 In Japan multiple HEV strains of genotype III or IV have been isolated from patients with acute hepatitis of non-ABC etiology who had never been abroad.61–65 Cocirculating strains within the same country have also been reported from Pakistan and Namibia, South Africa. The 1987 Pakistan-Sar55 isolate clustered with other Asian isolates in subgenotype I-1a, while the 1988 Pakistan-Abb2B isolate clustered with Burmese isolates in subgenotype I-1b.66 Despite the fact that the
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1983 and the 1995 Namibian outbreaks occurred in the same area, the 1983 isolates clustered into genotype I67 and the 1995 isolate clustered with a Mexican isolate in genotype II.68 Genetic changes in HEV can occur over time in a given community. Shrestha et al.69 compared the HEV isolates in the Kathmandu valley of Nepal, recovered from 48 patients in 1997, 16 patients in 1999, 14 patients in 2000, and 38 patients in 2002. The annual frequency of cluster 1a-2 isolates declined from 63% in 1997, to 50% in 1999, to 7% in 2000 and none in 2002; cluster 1a-3 isolates were seen in all 4 years; and the annual frequency increased from 5% in 1997 to 95% in 2002. While cluster 1a-1 was only detectable in 1997, clusters 1a-4 and 1a-5 emerged in 2000 and 2002 respectively. HEV subtype 1c was identified in 1997, but not in 1999 and thereafter. To date, HEV 1c has been isolated from India and mainland China, suggesting that the 1c strain was imported from India or China to Nepal in 1997 or before, but was taken over by the cocirculating 1a strains in 1999. Also the observed changing prevalence of the various 1a clusters according to the year of disease onset suggests that the genetic variability of HEV in a community is due to continously occurring genetic changes and that takeover of existing strain(s) possibly occurs by the selected variant having an advantage in transmission in the community or the variant(s) that was imported from other communities. An alternate hypothesis for the variability observed in field isolates is ongoing evolution in alternative host species.69 Based on the phylogenetic relationships of the various isolates, many authors have proposed nomenclature for genotypes of HEV (Table 34-1).47,56,60,70
SEROTYPES AND ANTIGENICITY Despite the presence of genetically different isolates of HEV, there appears to be only one serotype. Antigenic variations have important implications for the serological detection of HEV infection. The type specificity of many epitopes was first recognized by Reyes et al.27 Antibody responses to individual viral antigens are highly variable, due to both strain-specific differences in some epitopes and differences in response to single antigens between individual patients. For example, pORF3 varies greatly between strains, and many experimentally infected animals and some patients fail to develop antibodies to ORF3 protein.71,72 This variable reactivity contributes to the poor sensitivity and concordance of HEV-diagnostic tests based on such antigens.73 Conversely, all isolates of HEV share some important cross-reactive antigens.. Immunization of prehuman primates with recombinant pORF2 proteins conferred immunity to both homologous and heterologous challenge, suggesting that major protection epitopes are common among HEV genotypes (see Active immunoprophylaxis, below).
EPIDEMIOLOGY Hepatitis E is often mentioned among the “novel” diseases,74 which is right only in a sense of “newly described” or “newly identified” and should not be interpreted as a “newly emerged” disease.75 Hepatitis occurring in previous centuries and the early 20th century and known variously as “campaign jaundice” and “infective hepati-
Table 34-1. Proposed Classification of Hepatitis E Virus Genotypes Isolates
Bur1 (Burmese) (2,3,4), Bur2 (Burmese) (2,3,4), I2 (Indian) (2,3,4), I1 (Indian) (3) C1 (Chinese) (2,3,4), C2 (Chinese) (2,3,4), C3 (Chinese) (2,3,4), C4 (Chinese) (2,3,4), C5 (Chinese) (2), C6 (Chinese) (2), P1 (Pakistani) (2,3,4) B1 (Chinese) (2), B2 (Chinese) (2), B6 (Chinese) (2,4), B7 (Chinese) (2,4), S1 (Chinese) (2), S10 (Chinese) (2), S11 (Chinese) (2), S13 (Chinese) (2,4), H8 (Chinese) (2) Tash (Uzbekistan) (2,4), Osh (Kirgizia) (2,4) Mor12 (Morocco) (2,4), Mor23 (Morocco) (2,4), Chad (2,4) Mex (Mexico) (2,3,4) Ni (Nigerian) (4) SwUS1 (swine USA) (3,4) US1 (USA) (2,3,4) US2 (USA) (2,3) S15 (Chinese) (2,3,4), H3 (Chinese) (2,4) B3 (Chinese) (2,4), B4 (Chinese) (2,4), S5 (Chinese) (2,4) Sw NZ1 (swine New Zealand) (4) It1 (Italian) (3,4) Gr1 (Greek) (3,4), Sp1 (Spain) (4), Sp2 (Spain) (4) Gr2 (Greek) (3,4) Ar1 (Argentina) (3,4), Ar2 (Argentina) (3,4) Au1 (Australia) (4) Ct1 (Chinese) (4) Cs9 (Chinese) (4) Ct705 (Chinese) (4), Ct825 (Chinese) (4), Ct845 (Chinese) (4)
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Arankalle70 (1999) (1)
Wang60 (1999) (2)
Schlauder56 (2000) (3)
Schlauder47 (2001) (4) scheme 1
Schlauder47 (2001) (4) scheme 2
IA
1a
1
I
1
IB
1b
1
I
1
IC
1c
I
1
ID
1d 1e 2
I I II II III III
1 1 2 12 3 3
IV IV III III III III III III IV IV IV
9 10 4 4 5 6 7 7 8 11 10–11
II III
3a 3b 4a 4b
2 3 3 3 4
5 6 7 8
Chapter 34 HEPATITIS E Figure 34-2. Countries with high endemicity for hepatitis E virus (HEV) or sites of reported outbreaks. (Data from Aggarwal R, Krawczynski K. Hepatitis E: an overview and recent advances in clinical and laboratory research. J Gastroenterol Hepatol 2000; 15:9–2000, ©2000, with permission of Blackwell Publishing.368)
Table 34-2. Characteristics of Endemic versus Non-endemic Areas of Hepatitis E Virus (HEV) Features
Endemic
Non-endemic
Incidence of hepatitis E Epidemiological manifestations General sanitation Spread through contaminated water Non-human HEV reservoirs
High Outbreaks
Low Only sporadic cases
Poor Frequent
Adequate Absent
Probable
If exist, do not play essential role Undocumented Moderate Good
Subclinical infection Climatic conditions Hepatitis E reporting system
Frequent Hot Poor
tis” was probably hepatitis E and not hepatitis A, because the epidemiological descriptions of such disease resemble those of hepatitis E, not hepatitis A.6 Therefore it is possible that HEV infection may once have been prevalent in various parts of the world and has only recently become restricted to certain geographic areas, mostly underdeveloped regions with poor sanitation.
INCIDENCE AND PREVALENCE Worldwide, two geographic patterns can be differentiated: (1) areas of high HEV prevalence (endemic regions), in which major outbreaks and a substantial number of sporadic cases occur; and (2) non-endemic regions, in which HEV accounts for a few cases of acute viral hepatitis, mainly among travelers to endemic regions. In connection with epidemiological studies, a concept of endemic versus non-endemic areas is appropriate. Several features have been suggested for characterization of these areas (Table 34-2).75 Endemic disease is geographically distributed around the equatorial belt, including Central America, Africa, and the Middle East, subcontinental India, Asia, and the south-east Pacific (Figure 34-2). In these areas, which are generally characterized by inadequate envi-
ronmental sanitation, major outbreaks of HEV infection involving thousands of cases have been reported. A large epidemic of waterborne viral hepatitis was reported from India in 1955 and 1956, when raw sewage from the flooding Yamuna river resulted in 30 000 cases of jaundice.76 Although initially presumed to be caused by HAV, subsequent analysis of stored serum samples indicated that this epidemic was caused by HEV. It is now recognized that HEV is the commonest cause of epidemic enterically transmitted hepatitis, and HEV is viewed as a significant health problem by the World Health Organization. The largest reported outbreak occurred in the Xinjiang region of China between 1986 and 1988 and involved 120 000 cases.77 Studies of this and other large outbreaks of HEV infection have provided important information on the clinical and epidemiologic features of HEV infection. During the outbreaks, overall attack rates range from 1% to 15%, being much higher among adults 15–40 years (3–30%) than children (0.2–10%).1,78–80 The reason for this pattern of age distribution, which is unusual for an enteric infection, is unknown. It has been suggested that HEV runs a predominantly anicteric course in young age groups followed by gradual loss of immunity.1 It has also been speculated that HEV somehow has a selective tropism for liver cells of adults.81 Nevertheless, young children are susceptible to infection with HEV, because clinical disease has occurred with a similar frequency in all age groups in some epidemics,82 and sporadic clinical hepatitis E in children has been reported.82–91 A male preponderance of cases has been observed in most reports (the male to female ratio varies from 1.5 to 3.5:1).76 It is unclear whether this reflects the greater involvement of men in professional and social activities and, accordingly, their greater exposure to risk factors, or a true difference in susceptibility. Outbreaks of hepatitis E are characterized by high attack rates and mortality in pregnant women.1,76,78 An outbreak may be singlepeaked and short-lived, or multi-peaked and prolonged, lasting for more than a year. In endemic areas, hepatitis E accounts for a substantial proportion of cases of acute sporadic hepatitis in both children and adults.
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In India, HEV infection accounts for 50–70% of all cases of sporadic viral hepatitis.1,92,93 The demographic and clinical features of patients with acute sporadic hepatitis E closely resemble those during epidemics of hepatitis E.94 In endemic areas, outbreaks have a periodicity of 5–10 years, which in part reflects the patterns of heavy rainfall. The reservoir for HEV during interepidemic periods is unknown. Sporadic HEV infection in endemic areas may be sufficient to maintain the virus within the community during the interepidemic periods. Another possibility is that a non-human HEV reservoir exists. HEV has been isolated from swine, and antibodies to HEV have been detected in a number of animal species, including swine, sheep, cattle, chickens, rats, and captive monkeys. Moreover, viruses recovered from swine have been identified as variants related to human HEV strains found in the same geographic regions (see Is HEV a zoonosis? below). In non-endemic countries, cases of acute HEV infection are uncommon and most of the cases reported in industrialized countries occur in travelers recently returned from an endemic area.95–106 Secondary transmission has not been reported in these cases. Acute hepatitis E without a history of travel has also been described,107–110 and the source of HEV infection in such cases has not yet been determined. In most non-endemic areas, the disease accounts for fewer than 1% of reported cases of acute viral hepatitis. Although there are still questions about the relative sensitivity and specificity of serologic tests for anti-HEV, a more complete picture of the worldwide distribution and seroprevalence of HEV infection is emerging. Surprisingly, the prevalence of antibody to HEV in developing and documented endemic regions is much lower than expected (3–27%) (Table 34-3).111–143 The paucity of anti-HEV seropositivities in normal human populations of endemic countries has been difficult to explain. Various explanations could be: 1. Anti-HEV immunoglobulin G (IgG) does not persist for a long period of time and disappears early after the acute infection. 2. HEV causes, predominantly, a clinically overt disease: subclinical or silent forms are rare. 3. Currently available serological tests do not capture the whole spectrum of anti-HEV antibodies. Clinical HEV infection in developed countries is frequently associated with recent travel to areas of endemic infection. However, swine HEV strains have a worldwide distribution and infection in pigs from commercial farms is almost ubiquitous. In view of the demonstrated potential of swine HEV to cross species barriers and the close relationship between swine HEV and strains isolated from some humans, HEV infection should be considered a possibility in acute hepatitis patients who do not have a relevant travel history or markers of other hepatitis viruses (see Is HEV a zoonosis? below). Also, the prevalence of anti-HEV in nonendemic regions has been much higher than anticipated (1–28%) (Table 34-4)147–165 and thus, the true rate of HEV infection in developed countries remains controversial. Serological assays based on truncated pORF2 protein expressed using the baculovirus system have yielded seroprevalence rates of over 20% in blood donors from Baltimore, Maryland, USA,144 whereas rates lower than 2% were found in Australian blood donors when using the ORF2.1 protein expressed in Escherichia coli.145 Differences in assay specificity (favoring the ORF2.1 based
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assay) are a major problem in the detection of HEV prevalence, but differences in assay sensitivity (favoring baculovirus-based assay) may also be important.146 In many studies those individuals who showed the presence of anti-HEV did not differ from the seronegatives with regard to history of recent travel to endemic areas or exposure to local risk factors such as contact with imported hepatitis E cases. Possible explanations for these phenomenon include: 1. inadequacy of the methods employed for detection of antiHEV IgG (false-positivity) 2. circulation of an unrecognized agent capable of eliciting antibodies cross-reacting with HEV 3. a situation where the same virus causes a certain pathology in one climatic zone and a completely different disease in another (e.g., the Epstein–Barr virus causes infectious mononucleosis in moderate climates and Burkitt lymphoma in Africa). In endemic areas, the prevalence of anti-HEV in infants and children has been much lower than expected for a virus transmitted by the fecal–oral route.82,111,113,129,133,135,136,139,143,166 However, in a report from India, anti-HEV was detected in more than 60% of children below 5 years of age.90 Such differences may be related to varying epidemiological conditions or differences in the diagnostic tests used. An increase in the prevalence of anti-HEV has been found among young adults, but not older adults (constant between 10 and 40%).87,111,129,143,166 Although such a pattern of age-specific anti-HEV might suggest a cohort effect representing the disappearance of HEV from endemic regions, as was seen for HAV previously,6 similar age-specific anti-HEV patterns have been reported for sera collected 10 years apart from the same population residing in India, an area that is highly endemic for HEV.143,166 Thus, HEV appears to have epidemiologic characteristics that are quite different from those of most viruses that are transmitted by the fecal–oral route.
TRANSMISSION HEV is transmitted by the fecal–oral route. The most common vehicle of transmission during epidemics has been the ingestion of fecally contaminated water.1,76,78,80 Outbreaks in endemic areas occur most frequently during the rainy season, after floods and monsoons, or following recession of flood waters.76,80,167,168 These climatic conditions in conjunction with inadequate sanitation and poor personal hygiene lead to epidemics of HEV infection, when the sewage waters gain access to open-water reservoirs.76,78,169 In several regions of HEV-endemicity, a pattern of recurrent epidemics has been observed, which is probably related to the permanent existence of conditions in which drinking water is fecally contaminated. In South-East Asian regions, the disposal of human excreta into rivers and the use of river water for drinking, cooking, and personal hygiene have been shown to be significantly associated with a high prevalence of HEV infection: the use of river water over years for various activities can lead to recurrent epidemics.170,171 Both in epidemic and sporadic HEV there is a low rate of clinical illness among household contacts of infected patients, an unexpected finding because the virus is transmitted by the fecal–oral route. Reported secondary attack rates in households of HEVinfected persons range from 0.7% to 2.2%, in contrast to secondary
Chapter 34 HEPATITIS E
Table 34-3. Prevalence of Antibodies to Hepatitis E Virus Among Normal Humans in Endemic Areas Country
Number of samples tested
Taiwan, China
384 adults > 20 years 600 < 20 years 184 urban pregnant woman (Caracas) 204 rural populations (San Camilo, Edo Apure) 223 rural Amerindians (Padamo, Edo Amazonas) 1350 overall 105 second decade 464 third decade 308 fourth decade 157 adults attending sexually transmitted disease clinic 61 129 adults 593 blood donors 174 blood donors 252 blood donors 155 residents of a semiurban village 555 canoeists 227 medical students 1360 blood donors 72 health care workers 241 inmates in state jails 100 Araucanian indians 1850 villagers 503 Inuits 407 urban 360 rural 996 ethnic population 934 134 355 997 165 blood donors with ALT < 2 ¥ ULN 40 blood donors with ALT > 2 ¥ ULN 117 health care workers 53 cleaning service workers Women from a rural area 98 children 100 adults 10 026 rural population 1065 urban children 1005 rural children 487 children 699 1046 185 children 139 adults 96 blood donors 936 884 urban high-socioeconomic group 1497 urban lower-middle-socioeconomic group 1710 rural lower-middle-socioeconomic group 250
Venezuela
Turkey
Central African Republic Brazil Burundi Saudi Arabia Chile Uruguay Egypt South Africa Chile
Egyptian Nile delta West Greenland South Africa French Guiana Tadjikistan Kirghizstan Hong Kong Taiwan Brazil
Tanzania Pakistan Egypt India Amazonian basin, Brazil Rio de Janeiro, Brazil Ankara, Turkey. Tamil Nadu, India Thailand Korea Hong Kong India
India
% seropositive
Reference
10.7 0.3 1.6 3.9 5.4 5.9 0 3.7 9.1 24 4.9 14 16.9 4 1.2 57 1.8 2.6 8 12.5 7.5 17 17.2 3 6.6 15.3 6.4 8.5 4.6 16.1 6.4–8.8 3 7.5 2.6 13.2 7 19.4 16 67.7 28.7 23.8 4.5 2.4 3.8 5.3–16.7 6.5 18 18.8 6.9 10.6 14 4
111 112
113
114 115 116 117 118 119 120 121 122
123 124 125 126 127 128 129 130 131
132 133 134 135 136 137 138 139 140 141 142 143
92
ALT, alanine aminotransferase; ULN, upper limit of normal.
attack rates of 50–75% in households of HAV-infected individuals.85,86,172,173 The reasons for this difference may be related to instability of HEV in the environment, differences in infectious dose needed to produce infection, or a higher frequency of subclinical disease among persons secondarily infected with HEV. Even when multiple cases occur among members of a family, such occurrence
is related to exposure to a common source of contaminated water rather than to person-to-person spread.174 The mode of transmission responsible for sporadic HEV infections is unclear. Contaminated water is probably responsible for most of the cases in this setting. However, foodborne hepatitis E infection after eating uncooked liver of pig or wild boar and meat
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Section V. Liver Diseases Due to Infectious Agents
Table 34-4. Prevalence of Antibodies to Hepatitis E Virus Among Normal Humans in Non-endemic Areas Country
Number of samples tested
% seropositive
Reference
Northern California (USA) Baltimore, Maryland (USA)
5000 blood donors
1.2–1.4
147
15.9 23 21.3
144
2.5 2.6 0.4 2.81 1.81 2.6 5.4 9.3
148 149 150 151
1 2.1 0.9 2.2 1.1 1.5 1.2 0.5 1.7 6.7 4.6
153 153 153 153 106 154 155 127 156 157
3.8 2.1 0.89 14.8
158 159 160 161
1.7 3 0.4 18 29 25 20 14 9 2.4
162 163
Portugal Italy Australia Israel Italy
UK Germany France Spain Netherlands Belgium Russia Ukraine New Caledonia Japan
Norway Istanbul, Turkey Antalya, Turkey Italy Bolivia Japan USA
Canada
300 homosexual males 300 intravenous drug users 300 blood donors 1473 blood donors 653 279 1139 Jews 277 Arabs 1889 general population 279 intravenous drug users 193 chronic hepatitis disease 1500 972 1007 775 1275 394 168 1721 351 military recruits 478 in a hepatitis C virus-endemic area 775 in a non-hepatitis C virus-endemic area 208 flying personnel 909 children 338 1005 refugee Kurds from Iraq and Turkey 1393 children 200 adults 246 infants 400 blood donors >60 years (55) 50–59 years (65) 40–49 years (104) 30–39 years (95) 10%
Uncommon: 1–10%
Very rare: 45 000/mm3; international normalized ratio = 1.7, stop anticoagulation medications and aspirin 1 week and 2–3 days prior to procedure, respectively. ERCP, endoscopic retrograde cholangiopancreatography; IV, intravenous.
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Section V. Liver Diseases Due to Infectious Agents
no major complications or death over a 22-year observation period.28 Percutaneous drainage was carried out if platelets were >45 000 cells/mm3, international normalized ratio was 1.7 or less, and anticoagulation medications and aspirin were discontinued 1 week and 2–3 days before the procedure, respectively. Thus, percutaneous aspiration and drainage appear to be safe in patients with mild to modest coagulation disorders. Transient hyperthermia (up to 40°C) may occur in the first 24 hours after liver abscess drainage.28 Other complications, including bleeding, pneumothorax, septicemia caused by manipulation of abscess cavity, catheter blockage, and dislodgement, are infrequent.16,44 In most instances, the percutaneous catheter is left in place for 1–2 weeks or until drainage has reduced to small volumes and is stable. Percutaneous aspiration without catheter placement may require repeated aspirations; however, approximately 40% of aspirations do not require repeat aspiration, and only two attempts are needed in another 40%.6,16,68 Some studies suggest that continuous catheter drainage is preferred for patients with multiple abscesses,9 although this has been disputed by others.8 For example, there was no difference in treatment outcome among 64 patients randomly assigned to continuous catheter drainage or intermittent needle aspiration,16 including time to defervescence and normalization of liver-associated enzyme levels. Of three outcome measures studied, the length of hospital stay (11 versus 15 days), treatment success rate (93.8 versus 84.4%), and mortality (3.1 versus 12.5%) favored the intermittent needle aspiration group, although the results were not statistically significant.16 Abscess characteristics (number, size, or loculation) did not appear to influence the effectiveness of either intermittent or continuous percutaneous drainage.8,16,68 Intermittent aspiration utilizes a smaller needle that is potentially more acceptable to the patient, and it eliminates problems related to an indwelling catheter. Thus, intermittent needle aspiration appears to be safe and effective, and many consider this the first line of therapy for percutaneous liver abscess drainage.3,16 However, it is important to appreciate that multiple aspirations may be required using this method.
ENDOSCOPIC DRAINAGE Endoscopic placement of a nasobiliary stent has been shown to be effective for liver abscesses of biliary tract origin.22 However, this approach requires concomitant percutaneous abscess drainage. The main disadvantage of primary nasobiliary drainage is that no definitive invasive cultures can be obtained. Furthermore, most abscesses with biliary tract communication respond to percutaneous drainage.22 If there is an obstruction within the biliary tree or the abscess is not responding appropriately to percutaneous drainage alone, endoscopic therapeutic procedures and drainage may eliminate the need for surgical procedures.3,17,22 Although ERCP may have a limited therapeutic role, it is helpful in determining the extent of biliary tract disease and may assist in determining if surgical resection is required.6
SURGICAL DRAINAGE Although surgical drainage of liver abscesses was considered standard therapy in the past, this approach has steadily declined in frequency. In the past 10 years only 2% of people with liver abscess
754
had primary surgical drainage.8 Surgery is now reserved for patients with a complicated abscess, including rupture on presentation, association with hepatolithiasis, or in those who fail to respond to percutaneous therapy.7,15,16,40,44,67 Surgical drainage may be used in patients with concurrent intra-abdominal pathology requiring surgical management or when percutaneous drainage is felt to be contraindicated because of abscess location or factors such as severe ascites or bleeding tendency.7,15,16,44 In rare instances, abscess may lead to necrosis of a segment or a lobe of the liver requiring surgical resection.14 Conventionally, open surgery is used; however, laparoscopic surgery is increasingly being used in patients with liver abscess.15 Intraoperative ultrasonograpy is usually used to localize the abscess, guide the collection of samples for culture and pathologic examination, and to facilitate debridement and irrigation of the abscess cavity as well as to confirm that no residual abscesses are present.15 In general laparoscopic surgery is a safe and effective alternative to open surgery in patients who have failed conventional therapy with percutaneous drainage.15
PROGNOSIS AND NATURAL HISTORY As noted, untreated liver abscesses are invariably fatal. Currently, in spite of effective antimicrobials, enhanced diagnostic imaging capability, and improved drainage methods, mortality remains substantial, with an overall mortality ranging from 3% to 15%.2–8 Increased mortality was associated with multiple lesions; however, this association does not appear to apply with current drainage and antibiotic management.2,3,7–9,14 Nonetheless, complications of liver abscesses may still affect the outcome, with septicemia, underlying illnesses, and general condition of the patient strongly predicting poor outcome or death.2,7,9,40 Of these factors, cirrhosis and underlying malignancy, in particular hepatobiliary or pancreatic carcinomas, are significantly associated with increased mortality.3,4,7–9,11,32,40 Hypoalbuminemia, anemia, and high Acute Physiology and Chronic Health Evaluation (APACHE II) score have also been shown to predict poor outcome.2,9 Less favorable outcomes are observed when biliary tract disorders are the underlying process when compared to cryptogenic abscesses.2,4,7–9
CONCLUSIONS The clinical diagnosis of pyogenic liver abscesses is challenging and requires a high degree of suspicion. Hepatic imaging is essential for the diagnosis of liver abscesses, and ultrasound is usually the preferred method, although contrast-enhanced CT is required in patients for whom the initial evaluation is negative or inconclusive. Treatment options are varied and continue to evolve. Percutaneous intermittent needle aspiration is emerging as the preferred drainage procedure for most patients, and surgical management is not usually required. For patients who require surgical intervention, laparoscopic surgery is becoming a more widely accepted alternative to open surgery. In suspected biliary tract disease, endoscopic evaluation and procedures are complementary to percutaneous drainage.
Chapter 37 BACTERIAL AND MISCELLANEOUS INFECTIONS OF THE LIVER
ENDOTOXIN AND INFLAMMATORYRELATED HEPATITIS Six to 65% of neonates, children, and adults with bacterial infections have abnormal levels of liver-associated enzymes and bilirubin.69,70 This is referred to as “parainfectious hepatitis.” These hepatic laboratory abnormalities may accompany the syndrome of generalized systemic inflammation response (SIRS: defined as two or more changes in four factors: (1) body temperature; (2) heart rate; (3) respiratory function; and (4) peripheral leukocyte count). SIRS is frequently, but not always, associated with bacterial infection. Clinically, elevated serum bilirubin levels are frequently out of proportion with the serum alkaline phosphatase and aminotransferase levels, and hyperbilirubinemia is usually more severe among patients with underlying hepatobiliary disease, and may precede positive blood cultures by up to 9 days.69 Mortality is highest in patients with underlying liver disease, and mortality approaches 100% in patients with persistent or increasing hyperbilirubinemia.71 In patients without pre-existing liver disease, mortality is usually lower,70 presumably related to differences in patient populations. The etiology of the underlying infection is most commonly E. coli, although hyperbilirubinemia may occur in association with virtually any bacterial organism. Prognosis appears to be significantly worse when SIRS accompanies S. aureus sepsis.72 Although the primary site of infection is usually from an intra-abdominal source, this syndrome may accompany pneumonia, endocarditis, pyelonephritis, and soft-tissue and pelvic abscesses.73 Sepsis-associated cholestasis should always be considered in the differential diagnosis of jaundice in hospitalized and critically ill patients. If a patient has disproportionate elevations in bilirubin compared to serum alkaline phosphatase and serum aminotransferases, sepsis-associated cholestasis should be considered, even in the absence of fever, leukocytosis, or other signs or symptoms. Early recognition and treatment of the underlying process are critical for reducing morbidity and mortality. Unless prolonged hypotension occurs, liver failure or hepatic necrosis is uncommon. Parainfectious hepatitis appears to be caused by the systemic effects of the release of inflammatory mediators, and active infection is not required for the syndrome to occur. The outer membrane of Gram-negative bacteria contains lipopolysaccharides (LPS or endotoxin), which may be released into the bloodstream even without bacteremia. LPS stimulates macrophages (or, in the case of the liver, Kupffer cells), hepatocytes, and bile duct epithelial cells to release a variety of cytokines, including tumor necrosis factor-a (TNF-a), interleukin-1 (IL-1), 6, and 8, directly affecting hepatocyte function.74 Therapeutic use of TNF-a and IL-2 and administration of LPS to human volunteers results in intrahepatic cholestasis.74 Bacterial products other than LPS can also trigger release of proinflammatory cytokines, resulting in parainfectious hepatitis. Patients with alcoholic liver disease frequently have detectable endotoxin in their portal and systemic venous systems,75 and this may be associated with increased plasma cytokine levels.76 Interventions that block TNF-a (pentoxifylline or anti-TNF-a antibodies) appear to block alcoholic liver injury in experimental animal models, suggesting that these agents may be useful in alcoholic hepatitis. Endotoxin may also play a role in the jaundice associated with
total parenteral nutrition (TPN),77 which may explain why TPNrelated hepatitis can sometimes be prevented or improved with antibiotic therapy.78 In addition to endotoxin-induced parainfectious hepatitis, hepatitis is commonly a complication of bacterial toxin-mediated syndromes, including staphylococcal and streptococcal toxic shock syndrome (TSS). Reduced hepatic function and gastrointestinal symptoms of vomiting and diarrhea are included as criteria for the diagnosis of TSS (reviewed by Hughes and Stapleton79).
PYELOPHLEBITIS Suppurative thrombophlebitis of the portal vein, or pyelophlebitis, is a life-threatening complication of intra-abdominal infections. While pyelophlebitis may not directly involve the liver, bacteremia via the portal system can lead to liver abscesses. Although currently rare, pyelophlebitis was common in the pre-antibiotic era, and was a common cause of liver abscess. Diverticulosis is the most common predisposing cause of portal vein suppurative thrombophlebitis, although it may also develop following appendicitis, necrotizing pancreatitis, inflammatory bowel disease, or any process resulting in bacteremia of the portal vein.80 Signs and symptoms (fever, chills, abdominal pain, diarrhea, jaundice, tender hepatomegaly) are not specific, but suggest an intra-abdominal process or a fever of unknown origin.81 Leukocytosis and blood cultures are positive in 50–80% of cases. A radiographic clue to the diagnosis is gas in the portal area; however, this is relatively insensitive. Contrast CT, ultrasonography, MRI, magnetic resonance angiography and positron emission tomography scanning have all been successfully used to diagnose portal vein thrombosis.82–84 The bacteria isolated reflect bowel flora, with E. coli, Proteus mirabilis, Bacteroides fragilis, and other aerobic Gram-negative bacilli commonly isolated. Therapy consists of prompt initiation of broad-spectrum antibiotics to cover Gram-negative aerobic bacilli, anaerobic bacteria, and streptococcal species. The duration of antibiotics is not clear, although most authors recommend a minimum of 4 weeks, and longer courses in conjunction with surgical or percutaneous drainage may be needed if abscesses complicate the pyelophlebitis.85 The role for anticoagulation therapy is controversial, and no formal studies have been conducted to provide evidence from which to determine their efficacy and safety. In spite of appropriate therapy, pyelophlebitis has a mortality rate of >30%.83
BACTERIAL AND FUNGAL HEPATITIS A variety of bacteria and fungi are capable of directly infecting the liver. Clinical presentation of bacterial and fungal hepatitides may be indistinguishable from viral hepatitis and thus, individuals presenting with elevated liver-associated enzymes or jaundice with fever should be evaluated with blood cultures and appropriate serology. In contrast to viral hepatitis, it is unusual for liver-associated enzymes to be elevated to more than 10 times the upper limits of normal in bacterial or fungal-related hepatitis. Table 37-8 lists specific infections associated with hepatitis. Bacteria are historically classified by size, shape, staining properties, and biochemical properties, and bacterial infections are classified as Gram-positive
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Section V. Liver Diseases Due to Infectious Agents
Table 37-8. Etiologic Agents of Bacteria and Fungal Hepatitis Bacteria, Chlamydia, and Rickettsia Gram-positive bacilli Clostridium, Listeria, actinomycosis Gram-positive cocci Staphylococcus, Streptococcus Gram-negative bacilli Salmonella, Shigella, Yersinia, brucellosis, legionellosis, Burkhoderia pseudomallei (meliodosis), bartonellosis Gram-negative cocci Meningococcus, Gonococcus Mycobacteria Tuberculosis, non-tuberculous mycobacteria Spirochetes Treponema pallidum, borelliosis, leptospirosis Rickettsia Rocky Mountain spotted fever, rickettsial pox, typus ehrlichiosis, Q fever Fungi Systemic mycoses
Invasive mycoses
Cryptococcosis, coccidioidomycoses, histoplasmosis, paracoccidioidomycosis, Pneumocystis spp. Candida, Fusarium, Aspergillus, Rhizopus, etc.
or Gram-negative bacilli or cocci. However, this approach does not account for many bacterial agents, including mycobacteria, treponemes, other spirochetes, Mycoplasma, Rickettsia, Chlamydia, and Actinomyces. Fungal infections may also be associated with hepatitis, including the systemic mycoses or the invasive fungal infections that frequently result from iatrogenic complications of medical care.
GRAM-POSITIVE BACILLI Clostridium Clostridium species are generally not related to hepatitic disease, and although jaundice frequently occurs in immunocompromised patients with C. perfringens and C. septicum bacteremia, this is due to the release of a toxin, hemolysin, which results in significant hemolysis.86 Clostridium is a contributor to polymicrobial infections of the liver (abscesses) and biliary tract.
Listeria Listeria monocytogenes infection is uncommon except in neonates, pregnant women, elderly individuals, immunosuppressed transplant recipients, and others with impaired cell-mediated immunity. Listeria primarily causes bacteremia and meningoencephalitis, although it may also cause endocarditis, gastrointestinal disease, and pneumonitis. The bacteria is an important zoonosis, and it is increasingly associated with foodborne outbreaks. It is recovered in up to 5% of stool specimens obtained from healthy adults.87 A major source of L. monocytogenes is contaminated foods, including raw vegetables, raw milk, cheese, and meats, including chicken and beef.88 Listeria infection results in disseminated microabscesses and granulomas in neonates, particularly in the liver and spleen. This complication is rare in adults, and when it does occur, it has a predilection for people with underlying liver disease.89
Actinomycosis Actinomycosis is an indolent infection caused by higher bacterium Actinomyces. These organisms are anaerobic or microaerophilic, Gram-positive, filamentous bacilli that most commonly cause
756
oral–cervical disease, although up to 20% of reported cases involve the abdomen.90,91 Hepatic infection is present in approximately 5% of all cases of actinomycosis91 and 16% of cases of abdominal actinomycosis.92 Actinomycosis spreads to the liver either from a contiguous focus or via hematogenous spread, and in one case was associated with a pancreatic stent.93 Single or multiple abscesses are the usual findings, and the indolent nature often leads to mistaken diagnosis of malignancy.94 Actinomycosis typically presents with fever, weight loss, abdominal pain, and anorexia. Rarely, actinomycosis presents with cholangitis, portal vein occlusion, cholestasis, or extension into the thorax.95–97
GRAM-POSITIVE COCCI Staphylococci and streptococci Staphylococci and streptococcal infections involve the liver either as liver abscesses or alternatively, they may induce parainfectious hepatitis via inflammation and/or toxin release, as noted above.
GRAM-NEGATIVE BACILLI Salmonella Hepatitis is common among individuals with acute salmonella infection, particularly Salmonella typhi.98,99 Presenting symptoms, signs, and laboratory studies may mimic acute viral hepatitis, although a history of high fever (>40°C or 104°F) and travel, relative bradycardia, and a left shift on the white blood cell differential all increase the likelihood of typhoid hepatitis. The liver histology is consistent with a parainfectious etiology.100 Undiagnosed and untreated typhoid hepatitis has a mortality rate of 20%, although mortality is much lower with appropriate antibiotic therapy.
Brucellosis Brucellosis is a worldwide zoonosis that is most prevalent in the Mediterranean basin, the Indian subcontinent, and in parts of Mexico and Central and South America.101 Brucellosis presents as a non-specific illness with fever, night sweats, malaise, anorexia, headache, and back pain. Characteristically, the fever has an undulating pattern that begins 2–4 weeks after inoculation. Depression and somatic complaints predominate, and physical abnormalities are less common, leading to consideration of malingering. Symptoms referable to the gastrointestinal tract are common, although liverassociated enzyme levels are normal or mildly elevated. Brucellus abortus is associated with granulomatous hepatitis that can be indistinguishable from sarcoidosis,102 and B. melitensis may have lymphocytic infiltrates resembling mild viral hepatitis.103 Alternatively, epithelioid granuloma may accompany brucellosis.104 Liver abnormalities usually resolve with appropriate antibacterial therapy.
Legionellosis Legionella species are a common cause of pneumonia. Legionella are classified as Gram-negative bacteria, although their staining characteristics are poor and they may be missed on sputum Gram-stain examination. Although no evidence for direct hepatic involvement has been reported, up to 50% of patients with documented L. pneumophila infection will have elevations in liver-associated enzymes, and up to 5% will have jaundice.105,106
Chapter 37 BACTERIAL AND MISCELLANEOUS INFECTIONS OF THE LIVER
Burkhoderia pseudomallei Burkhoderia pseudomallei is the causative agent of meloidosis. B. pseudomallei is a water and soil bacterium predominantly found in South-East Asia, Madagascar, Australia, and regions of Central America. Infection may present as an asymptomatic infection or be confined to localized skin ulcers without systemic illness.107,108 At the other end of the spectrum, abscesses may develop throughout the lungs and organs, including the liver. Hepatic involvement may include microabscesses, focal necrosis, and occasionally granulomatous hepatitis.108 B. pseudomallei, like tuberculosis, has the potential for reactivation from a latent focus, and latency periods have been documented to be up to 29 years.109 The infection is diagnosed primarily by culture of involved organs or secretions.
Bartonellosis and peliosis hepatis Bartonella species are closely related to Brucella based on ribosomal RNA sequence.110 Bartonella species have been associated with a number of clinical entities, including Oroya fever, trench fever, catscratch fever, and peliosis hepatis. There are several species within the genus, including B. bacilliformis, the causative agent of Oroya fever, an acute illness with bacteremia occurring 3–12 weeks after inoculation, and a spectrum of disease ranging from mild illness to a profound, systemic disease.111,112 Jaundice due to hemolysis is frequent in severe cases; however, hepatic dysfunction is frequent and appears to result from microvascular thrombosis and ischemia.112 B. quintana is associated with trench fever in human immunodeficiency virus (HIV)-negative, frequently homeless people.113 Trench fever may have many associated symptoms, including fever, hepatosplenomegaly, and elevated liver-associated enzymes. B. henselae causes cat-scratch disease, and may be localized, or lead to persistent bacteremia, more frequently in HIV-positive and other immunosuppressed individuals. Liver lesions may be identified on imaging studies (Figure 37-4) and histological examination may reveal microabscess formation in both liver and lymphoid tissue. Bacillary angiomatosis is pathologically characterized by neovascular proliferation, typically in the skin and regional lymph nodes of HIVinfected or immunosuppressed individuals.114 Bacillary peliosis (BP) has also been identified in the liver and spleen of HIV-infected and other immunosuppressed individuals.115 Numerous blood-filled cystic structures as large as several millimeters may virtually replace the hepatic parenchyma.116 Non-specific symptoms of fever,
A
B
malaise, and associated skin lesions and/or adenopathy are the usual presenting signs and symptoms. It appears that both B. quintana and B. henselae can cause bacillary angiomatosis, although only B. henselae appears to cause bacillary peliosis. Bartonella organisms can be seen on histologic specimens when stained with the Warthin–Starry silver stain.
GRAM-NEGATIVE COCCI Neisseria species Although unusual, Neisseria meningitides sepsis may resemble severe viral hepatitis.117 More commonly, liver enzyme elevations associated with meningococcemia are related to inflammatory mediators released in response to lipo-oligosaccharide. In contrast, N. gonorrhoeae bacteremia is associated with abnormalities in liverassociated enzymes in virtually all patients.118 Jaundice is uncommon, and perihepatitis (Fitz-Hugh–Curtis syndrome or FHC) is the most common complication of disseminated gonococcal infection. FHC syndrome occurs in up to 25% of women with pelvic inflammatory disease (PID). Patients typically present with right upper quadrant abdominal pain and fever, usually in the setting of PID, although some women will lack pelvic signs and symptoms. Fever, hepatic tenderness, right upper quadrant peritoneal inflammatory signs, and occasionally a friction rub over the liver are observed on physical examination.119 FHC syndrome should be considered in the differential diagnosis of right upper quadrant pain in young, sexually active women, and is commonly mistaken for acute cholecystitis or viral hepatitis. Perihepatitis occurs by direct extension of N. gonorrhoeae or Chlamydia trachomatis from the fallopian tubes to the liver capsule and overlying peritoneum, although some lymphatic or hematogenous spread may occur. The latter routes of dissemination explain the rare cases of FHC syndrome that occur in men. Laparoscopic examination may identify violin-string-like adhesions between the liver capsule and peritoneal wall.119
Mycobacteria Mycobacterium tuberculosis. The most common cause of liverassociated enzyme elevation in patients with tuberculosis results from antituberculous therapy-related hepatotoxicity.120 Direct liver involvement is present in up to 50% of patients with miliary tuberculosis.121 Tuberculomas and tubercular abscesses are uncommon, occur in people with disseminated tuberculosis, and may be diffi-
C
D
Figure 37-4. (A–D) Multiple microabscesses in splenectomized patient with disseminated bartonellosis.
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cult to diagnose. In the absence of disseminated disease, liver involvement is unusual. When it occurs, hepatic lesions represent localized granulomas, often with central necrosis. Signs and symptoms commonly include right upper quadrant or generalized abdominal pain, hepatomegaly, fever, and weight loss.121–123 Laboratory studies reveal a cholestatic pattern of liver-associated enzyme elevations, and fewer than 25% of patients have jaundice. The diagnosis of hepatic tuberculosis is often not suspected unless active pulmonary disease is present, as it is in approximately 65% of patients. Hepatic lesions may be hypoechoic or hyperechoic on ultrasound.124 Diagnosis relies upon liver biopsy with histologic demonstration or culture of acid-fast bacilli. Up to 33% of patients will not have bacteria identified on histologic specimens, and culture and polymerase chain reaction (PCR) are helpful in these cases. Non-tuberculous mycobacterium. The most common nontuberculous mycobacteria that affects the liver is the Mycobacterium avium-intracellulare complex (MAC).125,126 MAC dissemination with liver involvement is common in HIV-positive individuals, although this is decreasing in countries where effective antiretroviral therapy is common. Disproportionate elevation of serum alkaline phosphatase (up to 40 times the upper limits of normal) may be seen in up to 5% of patients. The other liver-associated enzymes may be remarkably normal, or minimally elevated.126 Patients have non-specific symptoms and liver histology is relatively normal, suggesting that MAC interferes with enzyme metabolism rather than causing cholestasis or hepatic necrosis. Dissemination of mycobacteria, including M. kansasii, M. genavense, and others has been observed,127,128 usually in people with HIV infection or less commonly in organ transplant recipients, patients receiving chronic steroids, or in children with congenital defects in the interferon-g and IL-12 receptor.129
Spirochete diseases Treponema pallidum. Syphilis may affect the liver in any of its various clinical phases.130 Diagnosis of syphilis relies upon serologic testing and maintaining a high index of suspicion. Intrahepatic epithelioid granulomas may complicate congenital syphilis and may on occasion lead to portal or interstitial fibrosis.131,132 In these cases T. pallidum may be visualized in histologic examination using a silver stain. If granuloma formation is extensive, hepatomegaly may occur with development of portal hypertension and ascites. Intrahepatic calcifications may develop, and these should raise the diagnosis of congenital syphilis. Primary syphilis is not associated with hepatic involvement, although treatment may lead to a Jarisch–Herxheimer reaction with systemic inflammation and perihepatitis.133 Although Jarisch–Herxheimer reactions are more frequent with secondary syphilis, they may occur while treating any stage.134 Liver disease is uncommon in secondary syphilis, although mild elevations of serum alkaline phosphatase may occur. Rarely, jaundice and right upper quadrant pain are present, and liver biopsy demonstrates lymphocytic infiltration with organisms identified by silver stain. Late syphilis is a slowly progressive inflammatory disease affecting any organ, including the liver. Clinical illness often occurs years to decades after initial infection.135 Liver disease is related to syphilitic “gummas,” indolent, non-specific, granulomatous-like
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lesions that vary in size from microscopic defects to large tumor-like masses. The center of large gummas may become necrotic. Gummatous hepatitis may cause fever, epigastric pain, and tenderness, and lead to cirrhosis. Gummas respond rapidly to penicillin therapy, and are quite rare in the antibiotic era. T. pallidum may also cause liver disease through vasculitis or endarteritis. Borreliosis. Borrelia burgdorferi causes Lyme disease, a tick-borne illness consisting of several clinical phases. The first stage usually begins in the summer (stage 1), and presents as an expanding skin lesion called erythema migrans, occurring at the site of a tick bite. Within days to weeks, the spirochete may spread to other sites (stage 2), including skin, heart, joint, and central nervous system.136,137 During stage 2, recurrent erythema migrans may develop, and up to 40% of patients have elevated liver-associated enzymes.138 Stage 2 borrelliosis may include jaundice and tender hepatosplenomegaly.139 Following long periods of latent infection, B. burgdorferi may cause disease in the joints, heart, or central nervous system (stage 3), although liver involvement is unusual. All three stages are usually curable with appropriate antibiotic therapy (summarized by Steere136). Leptospirosis. Leptospirosis is globally distributed and is probably the most common zoonosis.140 Leptospirosis is caused by pathogenic spirochetes of the Leptospira genus, and the disease is thought to be greatly under-reported.140 Human infection results from direct contact with infected animal urine or tissues, or more commonly by indirect exposure to the organisms present in soil and water. The wide spectrum of illness ranges from asymptomatic infection to fatal, multisystem disease. In approximately 90% of people with leptospirosis, the illness is a self-limited febrile illness. In the remaining 10%, any combination of renal failure, liver failure, and pneumonitis may occur.141,142 Weil reported this biphasic illness (consistent with leptospirosis) in 1886, and it is now termed Weil’s disease. This biphasic illness has the initial stage of viral-like illness (associated with bacteremia) after an incubation period of 5–14 days. Five to 7 days later, the fever temporarily declines but is followed in 4–30 days by an immune phase with severe symptoms,141 including the abrupt onset of high fever, headache, chills, rigors, myalgias, conjunctival suffusion without discharge, abdominal pain, anorexia, nausea, and vomiting. Diarrhea, cough, pharyngitis, and a pretibial maculopapular rash may also occur. Conjunctival suffusion and myalgias are the most common findings.143 Forty-six to 85% of patients develop jaundice, frequently with renal failure, headache (with aseptic meningitis), photophobia, and hepatosplenomegaly.143,144 The most significant complication of the immune phase is hepatic and renal failure, and patients developing renal disease have mortality rates as high as 40%.143,144 The rise in liver-associated enzymes is disproportional to the mild pathological findings, with serum bilirubin levels relatively higher than transaminase elevations.143 Liver biopsy reveals cholestasis, and hepatocyte degenerative changes are seen in the absence of hepatocellular necrosis. Erythrophagocytosis and mononuclear cell infiltrates may also be present.145 Leptospira can be recovered from blood and cerebrospinal fluid during the acute phase, and urine sediment abnormalities and organisms can be recovered 5–7 days after the onset of symptoms.143
Chapter 37 BACTERIAL AND MISCELLANEOUS INFECTIONS OF THE LIVER
There are insufficient data on which to develop treatment guidelines;146 however, severe disease is usually treated with intravenous penicillin or ceftriaxone, and less severe disease with oral doxycycline. Similar to syphilis and Lyme disease, Jarisch–Herxheimer reactions may occur with therapy. Prophylactic treatment for individuals with unavoidable exposure to leptospirosis in endemic environments consists of using doxycycline.147
RICKETTSIA SPOTTED FEVERS Spotted fevers and other rickettsial infections are caused by a group of 12 Rickettsia species transmitted by ticks, fleas, and mites.148 Rickettsia rickettsii is the etiologic agent of Rocky Mountain spotted fever (RMSF) and is transmitted by ticks. RMSF is classically recognized as the triad of fever, headache, and rash; however, only about half of patients with RMSF have the rash develop in the first 3 days of illness. Gastrointestinal symptoms (nausea, vomiting, abdominal pain, diarrhea, jaundice, hepatosplenomegaly) may occur before the rash, and up to 60% of patients have elevated serum aspartic aminotransferase levels.149,150 Diagnosis is usually based on clinical suspicion, as serologic approaches to diagnosis are negative early in infection and may not be reliable. Detection of antigen in skin biopsies by immunofluorescence is a sensitive approach to diagnosis, although this is not widely available.151 PCR for R. rickettsii is insensitive early in disease.152
TYPHUS R. prowazekii (epidemic typhus) is transmitted by lice, and causes devastating epidemics associated with conditions of poor personal hygiene such as war, poverty, natural disasters including floods and earthquakes, and homelessness.153 In epidemic areas the disease is readily diagnosed, and consists of fever, headache, and myalgia, usually with a rash. In non-endemic areas, the diagnosis may be confused with typhoid fever, hemorrhagic viruses, syphilis, measles, and meningococcemia.154 R. typhi is found worldwide, and is most prevalent in tropical and subtropical regions where the vectors (fleas) and reservoirs (rats) are most common.155,156 Scrub typhus (caused by Orientia tsutsugamuchi) is transmitted by chigger bites and is found in a triangular region of the world bordered by Japan, Australia, and India.157,158 Epidemic, murine, and scrub typhus may present with severe multisystem illness, commonly with neurologic signs, pneumonia, fever, and rash. Jaundice and elevated liver-associated enzymes are present in up to 24% of patients with murine typhus and may suggest viral hepatitis.157 Multiple organ dysfunction, hemorrhage, and elevated liver-associated enzymes occur in severe cases.159,160 Diagnosis of typhus relies on a fourfold rise in convalescent antibody titers, and PCR may be useful for epidemic and murine typhus, although the test is not usually available in regions where it is needed.161,162 The diagnostic sensitivity and specificity of PCR have not been determined for murine typhus.162 Treatment with doxycycline is usually effective, although relapse may occur.163 Due to the retrospective nature of diagnostic approaches, therapy should be initiated based on clinical suspicion.
ERLICHIOSIS Members of the genera Erhlichia, Anaplasma, and Neorickettsia within the family Anaplasmataceae cause human illness referred to as ehrlichiosis, although the clinical manifestations and etiologic agents are distinct.148 Ehrlichiosis are zoonoses transmitted by ticks, and are generally classified as “human monocytic erhlichiosis” (HME) caused by Ehrlichia chaffeensis164 or “human granulocytic anaplasmosis” (HGA; previously human granulocytic ehrlichiosis), caused by Anaplasma phagocytophilum.165 Disease in immunocompetent people ranges from mild systemic illness to severe multisystem illness;165,166 however, in immunocompromised patients, E. chaffeensis commonly causes fatal, multisystem disease.166,167 Both illnesses typically start with fever, headache, myalgia, and malaise, and subsequently progress to include gastrointestinal symptoms including nausea, anorexia, vomiting, and diarrhea. HME is more frequently complicated by abdominal pain, and elevations in liverassociated enzymes occur in up to 86%.168,169 Hepatosplenomegaly is common, and a macular rash occurs in fewer than 50% of HME and rarely in HGA. Neutropenia is common in HGA and HME, while leukopenia and lymphopenia are also common in HME. Diagnosis is based on epidemiology and clinical and laboratory findings. In patients with fever, leukopenia, thrombocytopenia, elevated liver-associated enzymes, and a history of a tick bite in endemic regions during the late spring to mid-summer, HME and HGA should be suspected. HME cannot be distinguished from RMSF clinically, although a rash is less common and leukopenia more common in HME. E. chaffeensis morulae in circulating mononuclear cells visualized by Wright-stained blood smears are detected in fewer than 7% of patients with HME,170 and diagnosis is made by acute and convalescent serologic testing.171 PCR testing for E. chaffeensis (HME) appears promising.172 Morulae in polymorphonuclear cells occur in up to 80% of patients with HGA, and this is diagnostic.173 While culture may be successful, PCR detection of A. phagocytophilum may also be useful (55% and 86% sensitive, >95% specific).174,175 Serology provides a retrospective diagnosis. Doxycycline or tetracycline therapy should be initiated based on clinical suspicion, and delay may result in progression to multiorgan failure and significant mortality. Rifampin is active in vitro, and has been used in pregnant women, although there are insufficient data on therapies other than tetracyclines. Up to 21% of patients with HGA have serologic evidence of Borrelia burgdorferi or Babesia microti infection, as these pathogens and A. phagocytophilum are transmitted by Ixodes spp. ticks.176 It is unclear if concurrent infection alters the severity, incubation period, duration of illness, or likelihood of sequelae.177
COXIELLA BURNETII (Q FEVER) Q fever is a worldwide zoonosis causing a systemic febrile illness. The etiologic agent is Coxiella burnetii,178,179 which is found in cattle, sheep, and goats. Infected animals shed organisms into urine, feces, milk, and placenta178,179 and humans become infected by inhaling the rickettsia. The most common illness is a self-limited febrile illness lasting 2–14 days, although pneumonia, endocarditis, hepatitis, osteomyelitis, and neurological syndromes may occur. Hepatitis is the most common manifestation of Q fever in France and approximately 60% of US cases manifest as hepatitis.179–182
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Jaundice occurs in up to 30% of infected patients, and alkaline phosphatase is disproportionately elevated compared to other liverassociated enzymes. Histologically, a characteristic “ring granuloma” consisting of fat vacuoles surrounded by a ring of fibrinoid necrosis, histiocytes, and lymphocytes may be seen. This lesion may also be seen in several other diseases, including cytomegalovirus and lymphoma.183,184
FUNGAL HEPATITIS Fungal hepatitis may occur with any of the disseminated mycoses in immunocompetent hosts, although the frequency of liver involvement and diversity of organisms increase in immunocompromised patients. Cell-mediated immune defects, particularly diseases and/or drugs affecting CD4+ T-lymphocyte function, are the major immune factor predisposing to disseminated mycoses. In contrast, invasive mycoses rarely involve the liver in the absence of severe immunocompromise, and typically require defects in neutrophil function or number, or defects in the natural barriers to infection (skin and gastrointestinal mucosa).
HISTOPLASMOSIS Histoplasmosis is acquired by inhalation of Histoplasma capsulatum, one of the commonest causes of infection in the midwest and southeast USA. Although histoplasmosis is distributed in various regions of the world, the most endemic regions are the Ohio and Mississippi river valleys. H. capsulatum is a soil-based fungus that prefers growth in moist soil containing guano. Bats carry and shed the fungus, whereas birds do not.185 The predilection for H. capsulatum growth in soils containing high nitrogen content explains the high concentration of organisms found in environmental sources associated with chickens, and other birds. H. capsulatum resides in macrophages and dendritic cells186 and is distributed throughout the reticuloendothelial system. Most individuals who acquire histoplasmosis do not develop clinical disease, and retrospective diagnosis is made serologically.187 Hepatic calcifications are common in endemic areas, and these occasionally contain H. capsulatum. Immunecompetent people with acute histoplasmosis may develop a mild, influenza-like illness, although occasionally severe pulmonary disease with systemic illness occurs with transient elevations in liverassociated enzymes, particularly alkaline phosphatase. Severe disease may result when the inhaled inoculum is large. Progressive disseminated histoplasmosis (PDH) occurs in immune-competent individuals; however, it is more common in people with cellmediated immune defects, especially HIV infection. PDH may cause hepatosplenomegaly, elevated liver-associated enzymes, and rarely isolated hepatic lesions.188 Liver histology may reveal portal lymphocytic and histiocytic infiltrates or granulomas. Diagnosis may be made by the detection of yeast forms in liver tissue; however, non-invasive diagnosis is possible using blood cultures and serum and urine histoplasma antigen detection enzyme-linked immunosorbent assay methods.187 Antibodies are common in people from endemic regions, limiting the diagnostic utility of serology. Treatment usually consists of a combination of amphotericin B and oral azoles, and practice guidelines for histoplasmosis therapy have been published.189
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CRYPTOCOCCOSIS Cryptococcus neoformans is acquired by inhalation, and frequently causes subclinical infection in healthy people. The extent of disease is primarily determined by the immune status of the host, although virulence factors have been identified190 and the inoculum size may influence the outcome. Disease usually presents as pneumonia or meningitis,190,191 although C. neoformans infects any organ. Hepatitis, though rarely severe, is described in both immune-competent and compromised hosts and histology reveals a lymphocytic infiltrate or granulomatous hepatitis.191 Diagnosis can be made by identification of yeast in liver biopsy materials and cultures; however, non-invasive diagnosis can be made by culture or detection of serum or cerebrospinal fluid cryptococcal antigen using latex agglutination.190,191 Treatment varies based on immune status and must include attempts to restore host immunity. Treatment strategies may utilize a combination of amphotericin (or liposomal amphotericin) frequently combined with flucytosine, frequently followed by oral azole therapy.192,193 Specific recommendations for patients with HIV infection have been published.194 Relapse of cryptococcal disease is common in HIV-positive people in whom HIV is not well controlled with antiretroviral therapy. Drug resistance testing should be considered in individuals not responding to therapy or with severe disease.195
COCCIDIOIDO- AND PARACOCCIDIOIDOMYCOSES Coccidiodes immitis infection occurs in the south-western USA and the San Joaquin valley of California. Paracoccidiodes brasiliensis is found in South and Central America. Like H. capsulatum, these two organisms are dimorphic fungi that commonly cause subclinical infection. Most disease occurs in immunocompromised hosts, and consists of pneumonia, fever, and dissemination via the reticuloendothelial system. Elevated liver-associated enzymes are commonly found in disseminated infection, and hepatic granuloma formation may occur.196 While hepatomegaly is common, jaundice is infrequent. Diagnosis of coccidioidomycosis requires a variety of serologic tests to detect antibodies197 or C. immitis antigen in blood,198 and PCR-based methods appear promising.199 Paracoccidioidomycosis diagnosis is made by culture and direct examination of histologic specimens, although PCR is under development.200
PNEUMOCYSTIS SPECIES Pneumocystis species are unicellular fungi acquired by most humans during early childhood.201 Infection is controlled by host immunity, and disease is unusual except in cases of impaired cellular immunity. HIV infection is the commonest cause of reactivation of Pneumocystis, although malnutrition and drug-related immunosuppression also result in disease. Although interstitial pneumonia is the commonest disease manifestation, extrapulmonary disease frequently occurs in HIV-infected people. Cholestatic jaundice is common in patients with HIV202–204 and less frequently in transplant recipients.107,205 Diagnosis is difficult, and relies upon identification of organisms in frothy, eosinophilic honeycombed materials found in affected tissues.206 Treatment involves reversing immune defects and trimethoprim-sulfamethoxazole, although alternative drugs are available when intolerance to trimethoprim-sulfamethoxazole occurs.204
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INVASIVE FUNGAL INFECTIONS Fungemia with Candida, Aspergillus, Fusarium, Zygomycetales and others is increasingly common. These organisms are normal flora of the skin, respiratory, gastrointestinal mucosa, or environment. Candida species gain access to the bloodstream by contamination of vascular catheters and devices, or by the loss of integrity of gastrointestinal or upper respiratory mucosa (frequently the result of cytotoxic therapy), or via abdominal perforation and surgery. Candidemia frequently results in liver infection; however, clinical disease is rare among patients with normal numbers of functional neutrophils. In contrast, patients with impaired neutrophil function or severe neutropenia develop microabscesses in the liver that may progress to frank hepatic candidiasis207,208 requiring prolonged therapy. Aspergillus, Fusarium, and Zygomycetales and other opportunistic fungi and molds may also reach the bloodstream by contaminated vascular devices or disrupted mucosal surfaces. These infections have a propensity to invade terminal vessels and tissues, causing either localized or widespread tissue and organ dysfunction.209–211
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GRANULOMATOUS DISEASES OF THE LIVER Shobha Sharma Abbreviations BCG bacille Calmette-Guérin FUO fever of unknown origin HCV hepatitis C virus HIV human immunodeficiency virus
IFN-g R1 IL MAI MTb
interferon-g receptor interleukin Mycobacterium avium intracellulare Mycobacterium tuberculosis
INTRODUCTION The interpretation of hepatic granulomata is more difficult than is generally appreciated, first because the etiology of such lesions can seldom be established on histologic grounds alone, and second because the presence of granulomata in the liver does not necessarily imply an underlying systemic granulomatous process. Indeed, unless these two points are borne in mind, the discovery of granulomata in the liver may prove to be misleading rather than diagnostic.1
DEFINITION Granulomas (Figure 38-1) are composed of discrete aggregates of epithelioid cells. Epithelioid cells are transformed macrophages that have abundant cytoplasm, rich in endoplasmic reticulum and with fewer phagolysosomes than macrophages. These characteristics are consistent with a secretory function for epithelioid cells. In contrast, macrophages are primarily phagocytic. Based on these differences, it is possible to distinguish an epithelioid cell granuloma from an aggregate of macrophages using the PAS (periodic acid–Schiff) stain. Epithelioid cell granulomas are PAS negative, in contrast to an aggregate of macrophages which may contain PAS-positive debris. Macrophages within an aggregate are always separate from one another, whereas epithelioid cells fuse to form a syncytium as well as multinucleated giant cells. In addition to epithelioid cells, granulomas also contain a variable number of multinucleated giant cells and other inflammatory cells, such as lymphocytes and macrophages.2
IMMUNOLOGY The purpose of a granuloma is to destroy or contain an injurious agent that cannot be disposed of either directly or indirectly by the humoral limb of the immune system. The injurious agent may be intracellular, as in Mycobacterium, extracellular as in schistosomiasis, or unknown as in sarcoidosis. It is well recognized that intracellular pathogens elicit a cytokine response that is distinct and different from that induced by extracellular pathogens. In a type 1
PAS PBC TNF
periodic acid-Schiff primary biliary cirrhosis tumor necrosis factor
or Th1 response, the cytokines secreted are interferon-g, interleukin-2 and interleukin-12 (IL-2, IL-12). The type 1 response develops against intracellular pathogens and is typically seen in mycobacterial and sarcoidal granulomas.3–5 Granulomas that develop under the influence of a Th1 response are larger, poorly formed, and more destructive than those that develop in response to Th2 cytokines. In contrast, the type 2 or Th2 response is characterized by secretion of interleukins-4, -5, -6 and -10 (IL-4, IL-5, IL-6, IL-10)3,6,7 and is primarily directed against extracellular antigens. Schistosomal ova induce a Th2 response against soluble egg antigens, contain prominent numbers of eosinophils, and may be associated with fibrosis, features that are attributed to IL-5 and IL4, respectively. Antigen-presenting cells such as macrophages and dendritic cells process antigens and present them to MHC class II restricted helper T cells. These macrophages also secrete interleukin-12, which stimulates the differentiation of CD4 lymphocytes, which in turn secrete INF-g and IL-2 (Th1 response).8 In a positive feedback loop, INF-g and IL-2 amplify the immune response by stimulating proliferation of T cells and the further production of INF-g and IL-2. In turn, these cytokines recruit monocytes and stimulate them to differentiate into macrophages. The activated macrophages secrete tumor necrosis factor-a (TNF-a), which up-regulates the expression of intercellular adhesion molecules (ICAM), allowing inflammatory cells to adhere to endothelial cells and localize to the antigenic stimulus. TNF-a also stimulates T-cell proliferation and the secretion of INF-g.9,10 In schistosomiasis, an initial type 1 response usually evolves into a type 2 response.9,11 This shift in cytokine profiles appears to be mediated by IL-10. Both IL-12 (Th1) and IL-10 (Th2) are cross-regulatory cytokines. For example, interferon-g and IL-12 secreted in a Th1 response can suppress the production of IL-4 and IL-10 associated with the Th2 response, and similarly IL-10 can suppress a Th1 response.8,12 Which response will predominate in a reaction depends on the antigen and the timing of the cytokines secreted in relation to the chronologic evolution of the granuloma. Thus, though the type 1 or type 2 responses may be present in an individual they are not mutually exclusive,3,4 and one response can evolve into the other. This flexibility may provide protection to the host. This is illustrated in the case of schistosomiasis, where an unabated Th1 response results in hepatotoxicity and death in a nude
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Figure 38-1. High-power photomicrograph of margin of epithelioid granuloma. Epithelioid cells (E) have abundant cytoplasm and indistinct cytoplasmic borders. Other inflammatory cells, such as lymphocytes (L) and macrophages (M), are present at the junction of the granuloma and the hepatocytes (H). (Hematoxylin and eosin, ¥480)
mouse model, whereas in other animals with schistosomiasis the evolution of a Th1 response into a Th2 response reduces the risk of hepatotoxicity.13 Type 1 and 2 cytokine responses are not restricted to T-helper cells, and similar but not identical responses have been demonstrated in cytotoxic T cells, B cells, natural killer cells and dendritic cells.3,4 In addition to immune induction, a non-immune granulomatous reaction can be induced around indigestible foreign material such as talc. However, talc more frequently accumulates in macrophages within the portal tract and Kupffer cells, rather than forming distinct granulomas.
morphologic grounds. An attempt should be made to examine granulomas for foreign material such as talc, which is birefringent under polarized light.14,15 The value of acid-fast and fungal stains is questionable in non-necrotizing granulomas and must be correlated with the indication for the biopsy, as well as the immune status and geographic background of the patient. The likelihood of finding acid-fast bacilli or fungal elements is low, particularly when the granulomas are found incidentally during the work-up for chronic hepatitis. Organisms are more likely to be found when the liver biopsy is performed in the investigation of pyrexia of unknown origin.16–18 Granulomas other than those associated with primary biliary cirrhosis, sarcoidosis and schistosomiasis are rarely destructive and not associated with consistent derangements of liver tests. Damage to bile ducts is seen in primary biliary cirrhosis and less frequently in sarcoidosis. Destruction of hepatic and portal veins with subsequent obliteration and scarring is implicated as a mechanism of portal hypertension that can develop in patients with granulomatous hepatitis.
LIPOGRANULOMAS Lipogranulomas are distinctive but inconsequential granulomas in the liver (Figure 38-2). These granulomas are composed of lipid-laden histiocytes, lipid vacuoles, and a variable number of chronic inflammatory cells. They are well circumscribed and are usually located around the central vein, though they may be present in the portal areas. The surrounding hepatic parenchyma may be normal or steatotic.2,22 In non-fatty livers, these granulomas appear to develop in response to exogenous mineral oils that are widely used in food processing. Using thin layer and gas–liquid chromatography studies, lipid extracted from liver tissue, candies and polished skins of apples and cucumbers show similar characteristics to mineral oil. Moreover, the incidence of lipogranulomas has increased over the years, which is consistent with the widespread use of mineral oils in the food industry.22
HISTOPATHOLOGY There is a discrepancy between the detection of granulomas in needle biopsies and that in autopsy samples,19 and this reflects the amount of tissue sampled as well as the ability to select grossly abnormal areas for sampling in autopsy material. Also, the likelihood of finding granulomas increases with the number of biopsies obtained. For example, the probability of finding granulomas increases from 50% when one biopsy core is obtained to 100% when three cores are obtained.20,21 The morphologic appearance of granulomas is very variable. They may be distinct and well formed, as in sarcoidosis, or they may be ill defined as in some drug reactions. They may be necrotizing in infections such as tuberculosis, or non-necrotizing as in sarcoidosis. The location of granulomas within the liver, i.e. portal or acinar, is unlikely to be of diagnostic use unless primary biliary cirrhosis (PBC) is the consideration. In general, granulomas in PBC are portal, though they can be found in the acini as well.1,2 In the absence of acid-fast organisms, fungi, parasites or foreign material, it is not possible to identify the etiology of a granuloma on
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INCIDENCE AND CAUSES The incidence of granulomas detected in liver biopsies varies from 0.8% to 15%.1,19,23–30 Hepatic granulomas may reflect a systemic granulomatous process, and the two major causes of granulomas in liver biopsies are tuberculosis and sarcoidosis (Table 38-1). Sarcoidosis is more frequent in developed countries, whereas tuberculosis is the more common cause in underdeveloped nations. Fungal and schistosomal granulomas are seen in areas endemic for these infectious agents.19,28 After excluding a pot pourri of diagnoses that includes drugs, malignancies, and a variety of infections listed in Table 38-2, there remains a category of granulomas of unknown cause and significance. These may be incidental findings in patients being staged for chronic viral hepatitis,1,24,31–33 though there is a small group of patients in whom idiopathic granulomatous hepatitis is responsible for the clinical symptoms and disordered liver tests. Thus, the determining the cause and the significance of hepatic granulomas is largely dependent on the clinical indication for the biopsy.
Chapter 38 GRANULOMATOUS DISEASES OF THE LIVER Figure 38-2. Lipogranuloma. Note small lipid droplets within the granuloma as well as within adjacent hepatocytes. (Hematoxylin and eosin, ¥480)
Table 38-1. Frequency of Granulomas in the US and Other Countries Ref
Year
City/Country
23 19 24 25 1 26 27 28 29
1953 1966 1970 1974 1977 1979 1988 1990 1994
Cincinatti, USA Texas, USA Scandinavia Cleveland, USA New York, USA Washington, DC Australia Saudi Arabia Ireland
No of bx
Bx with gran
Sarcoidosis (%)
Tb (%)
Unknown (%)
1100 1505 2813 2086 6075 N/A N/A 404 4124
54 35 21 50 565 73 59 59 163
11 23 29 22 38 55 12 0 18
24 20 48 10 12 12 4 32 18
18.50 37 36 21 3 17 0 11
Misc*(%)
54 schistosomiasis 55 PBC
*Miscellaneous causes of granulomas included syphilis,23 lymphogranuloma venereum,23 lymphoma,19,23,25–27 brucellosis,1,19,26,28 mycoses,19,26 drugs,26,27,29 Crohn’s disease,26,29 cytomegalovirus,26,27 berylliosis,1 temporal arteritis,1 Q fever,27 renal and hepatocellular carcinoma27 and typhoid.28
DISEASES ASSOCIATED WITH HEPATIC GRANULOMAS Granulomas may be found in liver biopsies performed for the following indications: ∑ ∑ ∑ ∑ ∑ ∑
Grading and staging in a patient with chronic viral hepatitis Investigation and staging of primary biliary cirrhosis Investigation of fever of unknown origin (FUO) Confirmation of diagnosis of sarcoidosis Investigation of portal hypertension Investigation of liver test abnormalities of undetermined etiology.
GRANULOMAS AND HEPATITIS C INFECTION Incidental hepatic granulomas have been documented in patients who undergo liver biopsy for the grading and staging of their hepa-
titis C infection (HCV), and the reported incidence varies from 0.73% to 10%,31–33 though it is probably closer to the latter. In one large study of 435 patients, non-necrotizing granulomas of unknown etiology were observed in 4.5% of biopsies from 155 patients with HCV infection.34 This was significantly higher than in the control groups of hepatitis B infection (0.66% of 151 patients) and alcoholic liver disease (none of 129 patients). The etiology of these granulomas is unclear, and patients do not appear to have symptoms attributable to this finding. There are anecdotal reports describing the development of non-necrotizing granulomas in the liver subsequent to treatment with interferon-a. In two patients the appearance of the granulomas appeared to correlate with a flare in transaminase activity after an initial 3–4-month biochemical response to interferon-a.35 In a third patient granulomas were no longer identified in sequential biopsies performed 6 months after withdrawal of treatment.36 The role of interferon-a in inducing hepatic granulomas is suggested by the absence of these granulomas prior to initiation of therapy, and their resolution following withdrawal of the drug.
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Table 38-2. Causes of Hepatic Granulomas Found incidentally in the evaluation of primary liver disease Hepatitis C – incidental Hepatitis B – incidental27 Associated with primary liver disease Primary biliary cirrhosis Sarcoidosis – destruction of bile ducts and portal veins Associated with portal hypertension Schistosomiasis Sarcoidosis Associated with pyrexia of unknown origin Usual Sarcoidosis Tuberculosis – miliary and pulmonary Atypical mycobacteria Q fever27 Brucellosis 1,23,28 Cat-scratch disease Mycoses, e.g. histoplasmosis, Coccidiodes immitis, South American blastomycosis, candidiasis Drugs Idiopathic granulomatous hepatitis Rare Cytomegalovirus26,27 Temporal arteritis1 Listeriosis Leprosy – reactional states Miscellaneous Lymphoma23,26,27 Hepatic adenomas Hepatocellular carcinoma27 Renal carcinoma27 Leprosy Syphilis23 Lymphogranuloma venereum23 Crohn’s disease26,29 Berylliosis1
Finally, there are reports of patients with hepatitis C who developed a sarcoidosis-like illness with dry cough, dyspnea, pulmonary interstitial infiltrates and nodules or hilar lymphadenopathy. In two patients the sarcoid-like illness became apparent approximately 6 years after the diagnosis of HCV infection was made, and responded to steroid therapy.37 Neither of the patients had been treated with interferon-a. In three other patients symptoms appeared 3–5 months after initiating treatment with interferon-a. Symptoms and radiologic abnormalities subsided 3–8 months after cessation of medication and without steroids.38
PRIMARY BILIARY CIRRHOSIS Granulomas are a common finding in the liver of patients with primary biliary cirrhosis. This disease predominantly affects middleaged Caucasian women who may be diagnosed during the investigation of non-specific symptoms such as fatigue, or in whom the diagnosis is suspected because of cholestatic symptoms and signs such as pruritus and jaundice. The laboratory investigations show a two- to fivefold increase in alkaline phosphatase and the presence of the diagnostic antimitochondrial antibodies. The granulomas
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are portal in location and may be intimately associated with interlobular bile duct damage.39,40 PBC is discussed more extensively in Chapter 42.
FEVER OF UNKNOWN ORIGIN Among the many causes of fever of unknown origin (FUO) in immunocompetent patients, intra-abdominal infections and neoplasms are diagnosed using increasingly sophisticated imaging modalities. Similarly, increasingly sensitive and specific serologic tests identify patients with a variety of collagen vascular disorders and infections. A thorough clinical history pertaining to medications and travel may provide clues to a drug-induced hepatitis and suggest the need to perform a serologic search for unusual infections. For these reasons, the diagnostic utility of a blind liver needle biopsy in the evaluation of FUO is arguable. In patients with FUO, hepatomegaly and abnormal liver tests, the diagnostic yield on liver biopsy for informative granulomas such as mycobacterial or fungal infection varies from 0 to 17%.16–18
HUMAN IMMUNODEFICIENCY VIRUS In contrast, hepatic granulomas are found in 16–75% of human immunodeficiency virus (HIV)-infected patients being investigated for the cause of an FUO and elevated liver tests or hepatomegaly.41–46 The most likely cause for these granulomas is infection with either Mycobacterium tuberculosis (MTb) or M. avium intracellulare (MAI). If the organisms are identified on AFB stains, a definitive diagnosis is quickly established and treatment instituted.42 Comparable to observations in non-HIV infected patients, acid-fast organisms are easier to find when the agent is MAI rather than MTb. The role of blood and tissue culture in HIVinfected patients is controversial but is useful to speciate the organism and to identify additional cases in which the liver or bone biopsy does not show diagnostic pathology.43 In both immunocompetent and immunocompromised patients the diagnostic yield for fungi and mycobacterial infections is greater in liver biopsy than in bone marrow biopsy.42,47
CAUSES OF HEPATITIC GRANULOMAS SARCOIDOSIS Sarcoidosis is a systemic granulomatous disease characterized by a Th1 response against an unknown antigen. Clustering of patients suggests a temporal and spatial relationship that would be expected if the cause was either a transmissible agent or a shared environmental factor. The fact that the clustering has been shown to be greater between relatives of patients than among their spouses suggests a genetic predisposition, as the latter would be considered most susceptible if contact or a shared environment were the only factors involved in the clustering of cases.48–51 This disease afflicts those in the 20–40-year age groups and affects both Caucasians and African-Americans, but Swedes, Danes and African-Americans have the highest prevalence rates. In most patients the disease is indolent, though in a small proportion patients may be quite symptomatic and the disease can result in death. The
Chapter 38 GRANULOMATOUS DISEASES OF THE LIVER
course tends to be more severe in African-Americans and Swedes, and poor prognostic features include African-American origin, age over 40 years, and involvement of three organ systems.52,53 Patients may present with fever of unknown origin, nonproductive cough and chest pain, malaise, and weight loss. The organs most commonly affected are the lungs (100%) and mediastinal lymph nodes (90%). The diagnosis is one of exclusion and the clinical history must exclude occupational or environmental factors that could cause granulomas. Other tests include chest Xray, pulmonary function testing, electrocardiogram, liver tests, ophthalmic examination and tuberculin skin test. These tests identify involvement of the lung, heart, liver and eye, and screen for tuberculosis. Confirmation of the diagnosis is by biopsy identification of the granulomas, and the preferred sites of biopsy are the tracheobronchial tree and the lung.52,53 Hepatic involvement is usually silent and liver dysfunction is an unusual manifestation of sarcoidosis. Presentation of the disease as either hepatitis or carditis is unusual and is seen in 4–7% of patients.53 Symptoms and signs suggestive of hepatic involvement include abdominal pain, hepatomegaly, jaundice and portal hypertension.54–56 Patients may present with FUO, and in a third of cases there is a disproportionate elevation in alkaline phosphatase in comparison to the amino transaminases.54,57–59 Non-necrotizing granulomas are identified in the liver in 24–75% of patients with sarcoidosis,57,60,61 and sarcoidosis is one of the commonest causes of hepatic granulomas (see Table 38-1). The incidence is slightly higher in biopsy series than in autopsies, and this is probably because a liver biopsy is more likely to be performed when there are symptoms and signs of liver involvement in a patient with established sarcoidosis.57,60,62 The granulomas are characteristically tight, well formed and nonnecrotizing. They are uniformly distributed throughout the liver, and although they tend to be periportal they are also seen within lobules. A component of lobular hepatitis and portal triaditis may be present.62 Most sarcoidal granulomas resolve spontaneously, though some heal by fibrosis and scarring. Cirrhosis may result because of scarring of granulomas or a coexistent disease such as viral hepatitis. Portal hypertension can develop in the absence of cirrhosis, and explanations for its development include presinusoidal portal hypertension secondary to scarring and obliteration of small portal and hepatic veins by portal granulomas, and arteriovenous shunts within the granulomas.54,58,63–65 Destruction of interlobular bile ducts produces a histologic picture that is indistinguishable from that of primary biliary cirrhosis.55,56,59,62 Bile duct damage resembling sclerosing cholangitis has also been described.59,66 Although patients with hepatic involvement may improve symptomatically and biochemically when placed on steroids, structural damage, particularly ductopenia, is irreversible.67,68 Ursodeoxycholic acid has also been used to alleviate cholestatic symptoms.69,70 Recurrence of the disease following liver transplantation has been documented, though this is unusual.71–73
Africa to the South Pacific and Southern China. Also, as a result of travel and migration 400 000 people with schistosomiasis live in the United States.74–77 Schistosomes are digenetic (sexual and asexual reproduction in alternating generations) trematodes, the most common species of which are S. hematobium (Africa and Middle East), S. mansoni (South America, Caribbean, Africa and Middle East) and S. japonicum (Far East). The adult worms reside in the mesenteric (S. mansoni and japonicum) and perivesical venous plexuses (S. hematobium). The adult worm is not immunogenic, but the schistosoma ova are highly antigenic.78–80 The ova elicit a Th2 cytokine response resulting in the development of eosinophil-rich granulomas in the portal tracts (Figure 38-3). In mice with severe combined immunodeficiency, the inability to elicit a granulomatous response leads to severe hepatotoxicity and death due to the egg antigens.13 Interestingly, though, this granulomatous response kills at least a third of eggs and protects the host from the toxicity of the egg antigens. It is also utilized by the parasite to protect the egg from further host damage and permit egg migration.13,78 Complications within the liver include portal fibrosis due to the release of fibrogenic cytokines such as IL-4. Granulomas and fibrosis cause obliteration and/or compression of the portal veins, resulting in presinusoidal portal hypertension. In a small proportion of patients extensive fibrosis develops along the portal venous system and is also known as the pipe-stem fibrosis of Symmers. The fibrosis follows the distribution of the portal veins but does not transect the hepatic parenchyma, and therefore is distinct from cirrhosis.81–84 Hepatic fibrosis does not develop in all patients. This complication is most often seen in young adults (5–15 years) who have had prolonged intense infection (15–20 years). Using segregation and linkage analysis, a genetic locus controlling for infection intensity was identified in a Brazilian cohort. This locus mapped to chromosome 5q31-q33. The genes for IL-4 and IL-5 are also located in this region.85 Similar studies identified a locus controlling for fibrosis on chromosome 6q22-q23. The gene on chromosome 6 is closely linked
SCHISTOSOMIASIS Schistosomiasis (bilharziasis) is another example of a granulomatous hepatitis that can be associated with portal hypertension. It affects 200 million persons worldwide and the infestation is seen in a wide geographic belt that extends from South America, across the Caribbean islands, sub-Saharan Africa and the Middle East, South
Figure 38-3. Portal granulomatous response and fibrosis around schistosome ova. (Hematoxylin and eosin, ¥55)
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Section V. Liver Diseases Due to Infectious Agents
to the IFN-g R1 (interferon-g receptor) gene. The exact genes and their protein products have yet to be identified.86 Certain polymorphisms within the interferon-g gene itself, such as IFN-g +2109, seem to be associated with severe fibrosis, whereas polymorphisms on IFN-g +3810 appear to protect against the development of fibrosis.87 Patients with hepatosplenic schistosomiasis have high levels of INF-g, TNF-a and soluble TNF receptors and low levels of IL-5 in their serum. This Th1 cytokine profile suggests that patients who do not develop a Th2 response are more likely to develop hepatic fibrosis.88 Understanding how cytokines modulate the development of granulomas may allow therapeutic immunomodulation to achieve the balance between isolation of the toxic egg antigens in a granulomatous response and limiting the development of fibrosis.80 A good clinical history that elicits origin or travel to endemic regions is perhaps the first clue to suspecting the clinical diagnosis. Though most patients are asymptomatic in the early phase of the infection, there may be a local skin rash at the site of entry of schistosomal cercaria (cercarial dermatitis). Persons living in endemic regions are usually asymptomatic. In contrast, visitors to endemic areas develop fever, chills, cough, diarrhea, malaise and arthralgias. These symptoms begin 4–10 weeks after infection. The physical examination of acute schistosomiasis is characterized by hepatosplenomegaly, and the blood work-up shows peripheral blood eosinophilia. Liver tests demonstrate a mild elevation of aminotransferases.76,77 In endemic areas, the diagnosis is made on examination of the stool and urine for ova. However, when the infestation is light, this may be an insensitive tool. In this situation detection of antibodies using sensitive and specific ELISA assays developed against microsomal antigens of the adult worm can be performed. Antibody testing can only be performed 6–8 weeks after exposure.89 The preferred treatment is with praziquantel, or oxamniquine is an alternative in patients infected with Schistosoma mansoni.76,77
TUBERCULOSIS Tuberculosis may manifest primarily as liver disease because of either hepatomegaly or abnormal liver tests. The majority of these patients have miliary tuberculosis, and caseating granulomas are seen in over 80% of cases.90 In one study of 36 patients with miliary tuberculosis, granulomas were present in 91% of the liver biopsies, 52% of which were caseating. In contrast, granulomas were only noted in 53% of the bone marrow biopsies performed on the same patients. Moreover, the hepatic granulomas were present in 78% of those whose bone marrow biopsies did not show granulomas. The conclusion of this study was that although bone marrow biopsies are safer to perform, the diagnostic yield of the liver biopsy in miliary tuberculosis is higher.47 Non-necrotizing hepatic granulomas are seen in 25% of patients with pulmonary tuberculosis, and it is unusual to find acid-fast organisms in these biopsies.91 Similarly, acid-fast bacilli are only identified in 9% of hepatic granulomas in miliary tuberculosis.90 Therefore, although the acid-fast stain should be performed in all cases of granulomatous inflammation associated with PUO, it should be recognized that the stain is insensitive in detecting acid-fast organisms and results vary from 0 to 35%. Similarly, culture of biopsied material yields organisms in less than 10% of cases. PCR for Mycobacterium tuberculosis has been performed on formalin-fixed
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paraffin embedded sections. Although it has a specificity of 96%, the sensitivity is only 53%. However, this is a relatively rapid method of detection, with a 90% positive predictive value and a 76% negative predictive value.92
ATYPICAL MYCOBACTERIA The characteristic appearance of Mycobacterium avium intracellulare (MAI) in immunocompromised patients and children is the presence of collections of foamy macrophages filled with acid-fast organisms. Infection with Mycobacterium gevanese has also been described in patients immunocompromised by HIV infection, and the morphology is similar.93 The morphology of MAI is different in immunocompetent persons in whom well-formed granulomas have been described in the liver and spleen. In these patients, it is unusual to demonstrate acid-fast organisms in the tissue sections and confirmation is by culture.94 A hepatitic presentation has been described in a patient infected with Mycobacterium scrofulaceum. Noncaseating granulomas were identified on liver biopsy and the diagnosis confirmed on tissue culture.95
BACILLE CALMETTE–GUÉRIN (BCG) BCG is an attenuated strain of Mycobacterium bovis that is used in the immunotherapy of superficial bladder carcinoma. Hepatic dysfunction and granulomatous hepatitis is a very rare complication of intravesical therapy.96 In a large series of 2602 patients tuberculous hepatitis/pneumonia developed in 18 (0.7%), although the frequency of hepatic granulomas in the absence of liver dysfunction is probably higher.96,97 It is rare to culture or to find acid-fast organism in the granulomas,98,99 and this has made the distinction between a hypersensitivity reaction and systemic mycobacteremia difficult. Therefore, the goal of treatment in patients who develop symptoms and signs of hepatic dysfunction is to cover both possibilities, and includes a 6-month course of rifampin and isoniazid, accompanied by steroids if indicated, and possibly cessation of immunotherapy.96
Q FEVER ‘Q’ or query fever was described in 1937 as an occupational disease among slaughterhouse workers and dairy farmers,100 and in 1999 it became a notifiable disease. It is a zoonotic infection caused by an intracellular Gram-negative rickettsial organism, Coxiella burnetii. The primary but not exclusive reservoirs are cattle, goats and sheep, and the infection is maintained in ticks and other arthropods. The organisms are excreted into the milk, urine and feces of the animals, and there is a high concentration in the placenta and amniotic fluid. For this reason, in sheep-farming communities the incidence rises during the lambing season. The organisms are resistant to heat and drying, and the most common method of infection is inhalation of aerosolized bacteria. Ingestion of contaminated milk and tick bites are other sources of infection. The incubation period is between 2 and 3 weeks.101 Most infected patients are asymptomatic. In those who do develop symptoms the illness is self-limited and characterized by a high spiking fever (38.5–40∞C), malaise, bifrontal headache, and pneumonia.102 In most patients there is resolution in 2–3 weeks, but in up to 16% chronic Q fever develops that is characterized by endocarditis.103
Chapter 38 GRANULOMATOUS DISEASES OF THE LIVER
Liver abnormalities are found in 11–65% of cases, and in a study of 72 patients 85% had abnormal liver tests and 65% had hepatomegaly.104 Liver biopsies may be performed in patients who present with fever of unknown origin. Although non-specific granulomas may be seen against a background of non-specific reactive hepatitis and steatosis, a characteristic fibrin ring granuloma (Figure 38-4) has been described. The granuloma surrounds a clear space felt to represent a lipid vacuole. A fibrin ring is present that either surrounds the lipid vacuole within the granuloma or is at the periphery of the entire granuloma.105 Variations on this histology include a granuloma around a lipid vacuole without a fibrin ring, or a granulomatous response around fragmented fibrin material.106,107 In fact, the recognition of this characteristic histology has led to serologic testing and confirmation of Coxiella burnetti infection in some patients with fever of unknown origin.106,108 In some patients who respond to treatment and are followed up with a repeat biopsy, there is resolution of the histologic findings in the liver. The granulomas do not show a preferential lobular or periportal distribution in the liver. Although there are individual reports of fibrosis developing in these patients, based on sequential biopsies, these reports are prior to the availability of hepatitis C testing and are so rare that an association between Q fever and the development of hepatic fibrosis cannot be made with any certainty.109 Although fibrin ring granulomas are characteristically described in acute Q fever and have led to serologic testing and confirmation of the diagnosis, they are not seen in all such patients108 and are not specific for this disease. Isolated case reports have described similar granulomas in liver biopsies from patients with viral hepatitis A, temporal arteritis, Epstein–Barr virus infection, cytomegalovirus infection, systemic lupus erythematosus, leishmaniasis and allopuri-
nol-induced hepatitis. Although Q fever was considered in all these cases, it was excluded on serologic testing.110–116 Confirmation of diagnosis is by identification of specific antibodies. The organism exists in two antigenic phases and antibodies to phase II antigens are seen early in the disease. Chronicity should be suspected if there are rising antibodies to phase I antigens with either constant or falling levels of phase II antibodies.117,118 The treatment of choice is doxycycline 100 mg twice daily for 15–21 days.100
BRUCELLOSIS Brucellosis is a zoonotic infection found in a variety of farm animals, including goats, pigs, cattle and dogs. Human infection is caused by Brucella abortus (cattle), B. suis (pigs) and B. melitensis (goats). Although B. abortus is most widely prevalent in the USA, worldwide the most clinically important of these species is B. melitensis.119 Human infection occurs through contact with animals or animal products, such as cheese made from unpasteurized milk. Similar to Q fever, the mode of entry is either through aerosols of the organisms, ingestion of foods or contamination of wounds. Hence those who consume these products and workers in abattoirs, animal inspectors and handlers, and veterinarians are at greatest risk. Based on information from the CDC, brucellosis is not a common infection in the US (50 cells after HAART. The antiretroviral drugs most often associated with drug toxicity include nevirapine and ritonavir.10,12 Indinavir can cause mild indirect hyperbilirubinemia and rarely acute hepatitis. Drug discontinuation is indicated when an allergic reaction occurs, whereas continuing these drugs may be reasonable in other settings if the elevations are only mild with close follow-up. The most feared liver injury associated with nucleoside reverse transcriptase inhibitors is mitochondrial damage and the lactic acidosis syndrome.12 The mechanism of toxicity is believed to be mitochondrial DNA polymerase depletion;13 stavudine has been implicated most frequently. The syndrome is characteristically a multisystem disease manifesting as liver injury (jaundice), lactic acidosis, and frequently death. Pancreatitis and myopathy are also common. Patients typically present with dyspnea, nausea, vomiting and abdominal pain, but may be asymptomatic; the liver tests are usually only mildly increased and hepatomegaly is often pronounced. Elevated serum lactate levels and a metabolic anion gap acidosis are characteristic. Prompt recognition with drug discontinuation is critical, as mortality rates up to 100% are common. No defined treatments are available except liver transplantation.
Trimethoprim–Sulfamethoxazole The high frequency of side effects, including hepatotoxicity, of trimethoprim–sulfamethoxazole in patients with AIDS is well recognized.7 Hepatitis occurs relatively soon after drug initiation, with features of an allergic reaction including rash, fever and eosinophilia. The most common liver test findings are a raised bilirubin associated with mild to marked increases in serum aminotransferases. Histologically, granulomas are commonly observed along with bile stasis and hepatocyte necrosis.
Antimycobacterial Therapy Antituberculous agents such as isoniazid and rifampin are common causes of liver injury. Drug-induced hepatotoxicity may be even more frequent in HIV-infected patients because of the frequent coexistence of alcoholism and the concomitant use of other drugs.13 Isoniazid and rifampin-induced liver injury usually occurs within the first several months of drug ingestion, but can occur at any time. Clinical features of hepatitis and jaundice are common. Prompt discontinuation of these medications usually results in complete resolution of symptoms, although failure to recognize isoniazid hepatotoxicity with continued drug administration may be fatal.
VIRAL DISEASES Among HIV-infected patients, viruses constitute the most important hepatic pathogens. The high prevalence of infection with hepatotropic viruses is not surprising, as the routes of transmission and the risk factors for acquiring these viruses and HIV are similar. Compared to immunocompetent hosts, a number of important differences in the natural history of viral hepatitis, as well as unique diagnostic and management challenges, are observed in HIVinfected patients.
HEPATITIS A VIRUS The epidemiology, natural history and outcome of hepatitis A virus (HAV) infection in HIV-infected patients have been understudied. Risk factors for the development of HAV infection in homosexual men include the number of sexual partners, as well as oral–anal and digital–rectal intercourse.14 Among an HIV-positive population of predominantly intravenous drug users (IVDU) and heterosexual patients, a higher prevalence of anti-HAV antibodies has not been observed compared to routine blood donors.15 Case reports and small case series have reported higher serum titers of HAV RNA, as well as more prolonged viremia and higher transaminase levels, among HIV-infected patients.16,17 Despite these observations, there are no data to suggest that HIV-infected patients develop more severe hepatic disease or worse outcomes than non-HIV-infected patients. Hepatitis A vaccine is safe in HIV-positive patients, although less immunogenic.18
HEPATITIS B VIRUS Epidemiology Prevalence Studies have shown that 65–96% of HIV-infected patients have had prior exposure to the hepatitis B virus (HBV), as defined by surface antibody (anti-HBs) or core antibody (anti-HBc) positivity.19 However, HBV seropositivity depends on the risk group studied, with the highest prevalence seen in homosexual men and IVDU.
Incidence Incidence studies of HBV infection in HIV-infected patients are limited. In one study of 57 patients, 6 (10%) acquired HBV over a median follow-up of 18 months.20 Decreasing rates of HBV infection have been observed among high-risk individuals (IVDU) with or without HIV, suggesting the effectiveness of public health efforts,
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including risk-behavior modification and vaccination, and perhaps HAART.21,22
Natural History Influence of HIV Infection on HBV Disease The outcome of HBV infection is strongly influenced by coexistent HIV infection. HIV-infected patients who develop acute HBV infection have a higher likelihood of chronic infection than do HIV-negative individuals, although no differences in the clinical presentation or severity of hepatitis have been observed.20 Studies have shown that HIV-infected patients are also more likely to lose antibodies and/or to have lower HBV antibody titers than are seronegative controls.20 Some patients who clear HBeAg and become anti-HBe positive and later become HIV-infected may have a return of HBV viremia and reactivation of disease. In addition, an increased frequency of positive isolated anti-HBc (with undetectable anti-HBs) has been observed in HIV-infected individuals, probably resulting from a failure to develop anti-HBs in the context of immunodeficiency.23 Studies examining the degree of HBV-related liver injury, as assessed by transaminase levels and hepatic histology, have yielded conflicting results. Most studies have found lower ALT levels and less severe histopathologic injury in HIV-infected patients than in controls. In addition, when stratified based on absolute CD4 lymphocyte count, there appears to be a positive correlation between hepatic injury and CD4 count. The impact of HIV co-infection on the outcome of chronic HBV infection is unclear, although some studies have suggested an accelerated progression towards decompensated cirrhosis in co-infected patients.24
Influence of HBV Infection on HIV Disease Studies have failed to demonstrate a major influence of HBV infection on progression of HIV infection to AIDS.25
Therapy a-Interferon therapy in HIV-infected patients has been associated with a poor response, which may be related to the overall immunosuppression and/or abnormalities in the cytokine profile related to HIV.26 Lamivudine promptly inhibits HBV replication, but the emergence of resistance to lamivudine is of concern.26 Adefovir has been shown to be effective in the treatment of lamivudine-resistant HBV in HIV/HBV-co-infected patients. Tenofovir has recently been shown to have significant activity against both HIV and HBV.26 Immune restoration with antiretroviral therapy has been associated with an acute elevation of serum transaminases (flare) followed by HBV seroconversion, but this phenomenon appears to be relatively infrequent.27 Nevertheless, when it occurs it may be severe, resulting in fulminant hepatic failure. As noted earlier, HBsAg seropositivity has been identified as an independent predictor of HAART-related hepatotoxicity.9
Prevention Given the increased prevalence of serological markers of prior HBV exposure and the high incidence of HBV infection, particularly among homosexual men and IVDU, vaccination has been recommended. However, as with other immunosuppressed hosts, seroconversion rates and, among responders, anti-HBs titers are lower,
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especially for patients with low CD4 counts. Therefore, it is important to implement vaccination in HIV-positive patients early in their disease to maximize the effectiveness of vaccination.21,28
HEPATITIS DELTA VIRUS Hepatitis delta virus infection is uncommon in the United States compared to other areas of the world. The primary risk factor for HDV infection is IVDU, although having an increased number of sexual partners has also been identified.29 HIV infection abrogates the suppressive effects of HDV on HBV replication, and higher ALT levels suggest more severe hepatic injury.30 The role of interferon in HIV-infected patients is not established, although promising results have been observed in HIV-negative patients.31 To date, there is no evidence that HDV infection alters the natural history of HIV infection.
HEPATITIS C VIRUS Epidemiology The prevalence of HCV infection among HIV-infected patients is highly dependent on the group studied: among homosexual or bisexual men prevalence rates vary from 1.6% to 11.7%, and among IVDU from 13% to 40%, and HCV positivity is very common in hemophiliacs, approaching 90–100%.32 Some patients with HCV/HIV co-infection may lose HCV antibody over time, thus potentially underestimating the true prevalence of HCV infection. HCV infection has important implications for both HCV and HIV transmission. HIV infection, probably related to higher levels of HCV viremia, increases the risk of sexual and parenteral transmission of HCV to HIV-seronegative female sex partners, and to newborns of HIV-infected mothers.32
Natural History Influence of HIV Infection on HCV Disease The outcome and clinical presentation of acute HCV infection in HIV-infected patients are not well defined. However, HIV infection is now recognized as an important cofactor in an accelerated progression of HCV disease.33–37 HCV RNA is more commonly detected and levels are higher in HIV-infected patients than in HIVnegative controls, which suggests an increased rate of viral replication and/or decreased clearance by the host, perhaps owing to underlying immunosuppression. No difference in ALT levels between HCV+/HIV+ compared to HCV+/HIV- controls can be shown,38 although liver biopsy specimens have demonstrated a higher degree of necroinflammatory activity and fibrosis in patients with HIV infection, especially in those with lower CD4 cell counts and HCV genotype 1b.34,37,39 Factors that predict fibrosis and progression to cirrhosis in co-infected patients include older age at time of infection, higher alanine aminotransferase levels, higher inflammatory activity, alcohol consumption of >50 g/day, and CD4+ T-cell count of 200/mm3. In
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most patients, disease results from reactivation rather than primary infection. Regardless of the stage of immunodeficiency, pulmonary disease is the most common presentation, and dissemination to almost any organ, including the liver, has been described. Hepatic involvement may be suspected by liver test abnormalities (raised alkaline phosphatase) in the setting of disseminated tuberculosis. Similar to MAC, clinical expression of liver disease is rare. Focal liver disease may present with right upper quadrant abdominal pain and fever. CT findings of disseminated TB include lymphadenopathy with evidence of central necrosis.56 In contrast to MAC, focal lesions often occur in other organs and characteristically are necrotic. Pulmonary disease may be absent. The diagnosis may be established by fine needle aspiration of liver lesions, lymph nodes or other focal lesions, with appropriate staining. Blood cultures are often positive. Unlike the therapy for MAC, multidrug regimens for tuberculosis yield a clinical and microbiologic cure, with relapse of infection being infrequent. Monitoring of the liver tests in selected patients is important to detect hepatotoxicity early. In the HIV-infected patient with a positive PPD, prophylactic therapy with isoniazid has been recommended,59 and although it decreases the incidence of both pulmonary and disseminated tuberculosis, liver toxicity has been reported in ª10% of patients.59
FUNGAL DISEASES Despite the prevalence of fungal diseases in other organ systems in AIDS patients, involvement of the liver is uncommon clinically. Fungal diseases have important differences in prevalence, based on the epidemiologic and geographic setting. For example, disseminated histoplasmosis is much more common in the central USA, whereas coccidiodomycosis is a common opportunistic pathogen in the southwest. Cryptococcus is a frequent pathogen in AIDS, and the most common fungus reported to involve the liver.6 Disseminated disease is rare in those with meningitis but it can involve lung, liver, or bone marrow. Amphotericin B is effective for acute disease, whereas fluconazole is administered chronically to maintain a remission. Disseminated histoplasmosis is an AIDS-defining illness and most often involves the lung and hematopoietic organs at the time of presentation. In immunocompetent hosts, pulmonary involvement is the most common manifestation; pulmonary involvement is observed in most AIDS patients with disseminated histoplasmosis.60 The most common clinical presentation is recurrent high fevers, and in some patients a clinical picture consistent with disseminated intravascular coagulation. High LDH concentrations >600–1000 IU/l suggest the diagnosis.61 The diagnosis can be established by staining of peripheral blood smears, where the organism may be observed in polymorphonuclear leukocytes; bone marrow biopsy with culture and occasionally liver biopsy may be diagnostic. Amphotericin B and itraconazole are effective for acute treatment and chronic prophylaxis, respectively. Immune reconstitution with HAART will negate the need for long-term maintenance antifungal therapy.57 Other fungi reported to involve the liver in AIDS include blastomycosis, coccidiodomycosis, and rarely candida. Penicillium marneffie is a fungus endemic to Southeast Asia and has been increasingly
Chapter 39 HIV-ASSOCIATED HEPATOBILIARY DISEASE
recognized as an opportunistic infection that can involve the liver in patients with AIDS.6
BACTERIAL INFECTIONS Bacterial infections of the liver are rare. Peliosis hepatica is now recognized as being caused by the bacteria Bartonella henselae. Patients with marked immunodeficiency are at greatest risk and present with disseminated disease involving the skin, bones and liver, similar to cat-scratch disease.62 Fever, abdominal pain and hepatosplenomegaly are common with hepatic involvement. The serum alkaline phosphatase is usually markedly elevated. Abdominal CT scan demonstrates hepatomegaly with low-density lesions representing vascular lakes. Biopsy of these lesions reveals vascular channels, and specific stains (Warthin–Starry) aid in identifying these bacteria.63 Culture of liver tissue may also be positive. Antibiotic therapy with doxycycline or erythromycin is usually effective; relapse may occur.
Figure 39-2. Hepatic non-Hodgkin’s lymphoma: multiple filling defects of variable size. Ultrasound-guided biopsy demonstrated high-grade lymphoma.
PROTOZOAL INFECTIONS Protozoa are distinctly uncommon hepatic pathogens. Although rare cases of microsporidial or cryptosporidial hepatitis have been noted, cryptosporidia and microsporidia typically involve the biliary tree rather than hepatic parenchyma (see below). Disseminated Pneumocystis carinii (PCP) may involve the liver, usually in patients using inhaled prophylactic pentamidine to prevent PCP. Liver involvement is diffuse and thus hepatic mass lesions seen on CT are uncommon. Other reported causes of liver infection in AIDS include Isospora belli, Leishmania64 and Toxoplasma. Amebic liver abscess has been observed in high-prevalence areas.
NEOPLASMS Neoplasms may be the initial manifestation of AIDS and can present with isolated liver involvement. In early reports of the AIDS epidemic non-Hodgkin’s lymphoma (NHL) was present in up to 2.3% of patients,65,66 but the rate of most lymphomas has decreased in the HAART era.67 In contrast to immunocompetent hosts, AIDSrelated lymphomas typically present in extranodal sites, are more advanced, and have a poor prognosis.66 HIV-associated lymphoma may have a primary presentation in the liver, and autopsy studies show hepatic lymphoma which was unsuspected antemortem.68 Hepatic lymphoma is usually manifested by abnormal liver tests, often with striking elevations of the alkaline phosphatase or LDH; jaundice is common. Hepatomegaly with or without abdominal pain is common, whereas fever is inconsistent. Peripheral lymphadenopathy is uncommon. Although abdominal ultrasound is frequently diagnostic, abdominal CT is the diagnostic modality of choice.68 Liver involvement appears as one or more focal lesions of variable size (see Figure 392). Abdominal lymphadenopathy and splenic involvement are common, whereas mediastinal adenopathy is rare. Percutaneous radiographically directed biopsy of identified mass lesions will safely and reliably establish the diagnosis. With HAART, the response rate and long-term disease-free survival following chemotherapy have dramatically improved.69
Kaposi’s sarcoma (KS) was recognized early on as a common initial manifestation of AIDS. This neoplasm, caused by human herpes virus-8 (HHV-8), becomes manifest under conditions of immunodeficiency.70 This virus can be detected in blood, and sexual contact is an important route of transmission. The incidence of KS has fallen dramatically, coincidental with the use of effective antiretroviral therapies.71 Cutaneous involvement is the most common presentation; visceral involvement is common and usually asymptomatic. Hepatic KS is generally clinically silent and was a frequent finding at autopsy before HAART. The lesions are generally multiple and hyperechoic. Chemotherapy is relatively effective in controlling disease but, remarkably, tumor regression occurs with HAART therapy alone, making it the treatment of choice.72 Several other tumors involving the liver have been reported in HIV-infected patients, including metastatic adenocarcinoma, cholangiocarcinoma, melanoma and hepatoma. Whether these neoplasms are related to HIV infection or are a complication of other diseases (excluding HCV) is unknown.
CAUSES OF ASCITES As in any patient, ascites may be caused by hepatic (portal hypertension), extrahepatic or peritoneal disease(s). The most common cause of ascites in these patients is cirrhosis and portal hypertension secondary to chronic viral hepatitis. However, in about 25% of patients opportunistic infections and neoplasms are causative, usually from disseminated disease with peritoneal involvement. A syndrome of non-specific peritonitis has been described.73 These reported patients presented with abdominal pain and overt ascites. Ascitic fluid analysis demonstrated high protein concentrations and leukocytosis, but no specific identifiable pathogens. Laparoscopy and laparotomy also failed to disclose a specific etiology; in some patients adhesions and peritonitis were found. The cause of peritoneal disease in these patients is unknown. The evaluation of ascites should generally parallel that in other patients. Patients with high-protein ascites should also have a sample
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submitted for cytological analysis to exclude NHL. CT may be useful to identify peritoneal mass lesions (e.g. NHL) or other intraabdominal processes; mass lesions may then be safely biopsied percutaneously under CT guidance. The presence of chylous ascites suggests underlying disruption of the lymphatic system caused by KS or tuberculosis. Laparoscopy with inspection of the liver and peritoneum may play a role when other tests are non-diagnostic.
BILIARY TRACT ABNORMALITIES ETIOLOGY Disorders of the biliary tree in AIDS patients, termed AIDS cholangiopathy, were recognized early in the AIDS epidemic. However, like other opportunistic processes in AIDS, the frequency of this entity has fallen coincidentally with the introduction of HAART. The cause of ductal disease in most patients is infection. Liver biopsies, which included biliary ductal epithelium, biopsies of the common bile duct and/or papilla after endoscopic sphincterotomy, biliary brushings and cytology, as well as periampullary small bowel biopsies, have all identified an infectious cause in selected cases. However, in a substantial number no cause(s) can be found. The most frequent identifiable pathogen is Cryptosporidium; other infectious causes include microsporidia (E. bienusi, Septata intestinalis),74 Cyclospora, CMV, MAC and Giardia. Based on autopsy studies and bile duct biopsies at ERCP, these infections cause severe inflammatory changes that result secondarily in the observed cholangiographic abnormalities. Non-infectious causes of biliary disease, both benign and malignant, include stones and strictures.
CHOLANGIOGRAPHIC PATTERNS
A
The most frequent cholangiographic finding is papillary stenosis in association with intrahepatic sclerosing cholangitis, occurring in approximately 50% of patients with the AIDS cholangiopathy syndrome (Figure 39-3). The next most frequent pattern is intra- and extrahepatic sclerosing cholangitis without papillary stenosis, followed by papillary stenosis alone, or intrahepatic sclerosing cholangitis alone. Isolated strictures of the common bile duct may result from primary common bile duct lymphoma, or pancreatic disease caused by chronic pancreatitis, infections, or neoplasms. Pancreatic duct lesions have also been described, perhaps related to ampullary obstruction.75
CLINICAL PRESENTATIONS The typical presentation of AIDS cholangiography is right upper quadrant pain; papillary stenosis is usually present in patients with severe pain and clinical cholangitis, whereas intrahepatic cholangitis results in milder pain or may be asymptomatic. Diarrhea is common and related to coexistent small bowel involvement with cryptosporidial or microsporidial infection. Asymptomatic elevation of liver tests may be the first clue to the diagnosis. Serum alkaline phosphatase is usually elevated, with mean values in most series of 700–800 IU/l. Mild increases in ALT are common, but jaundice is rare. Rarely liver tests may be normal.
DIAGNOSIS When using ERCP as the gold standard, ultrasound has a sensitivity of approximately 75–87%76 and may demonstrate striking ductal
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B
Figure 39-3. AIDS cholangiopathy. (A) The common bile duct and cystic duct are markedly dilated to the level of the ampulla. There is also nodularity of the duct. (B) The intrahepatic ducts have areas of stricture and dilation typical for sclerosing cholangitis. A biliary sphincterotomy was performed.
thickening in some patients. HIDA scanning may suggest biliary obstruction when there is delayed excretion of the tracer. In the symptomatic patient without jaundice ultrasound should be the initial study, reserving CT for the patient with jaundice where intrahepatic mass lesions, abdominal adenopathy, and biliary dilation can be identified. ERCP should be reserved for those in whom endoscopic therapy is anticipated.
Chapter 39 HIV-ASSOCIATED HEPATOBILIARY DISEASE
TREATMENT Treatment of AIDS cholangiopathy is primarily endoscopic. For those patients with abdominal pain or cholangitis associated with papillary stenosis, endoscopic sphincterotomy generally provides benefit, with long-term symptom relief in most patients following sphincterotomy for papillary stenosis. It is possible that coexistent pancreatic disease may be responsible for the incomplete pain relief in some patients.75 On long-term follow up the serum alkaline phosphatase may continue to rise owing to the progression of associated intrahepatic sclerosing cholangitis. Sphincterotomy is not indicated for sclerosing cholangitis in the absence of papillary stenosis and common bile duct dilation, and may be associated with a higher complication rate. For those with a dominant common bile duct stricture, endoscopic stenting and/or balloon dilatation should be performed after sampling the stricture. Several reports show some improvement or even resolution of cholangiographic abnormalities with antimicrobial therapy, usually in conjunction with HAART.77 This would suggest that, depending on the chronicity of disease, the cholangiographic findings are inflammatory and reversible. As with all AIDS-related opportunistic infections, the prognosis of disease is linked to the severity of immunodeficiency.78
GALLBLADDER DISEASE As with AIDS-associated sclerosing cholangitis, disorders of the gallbladder are caused primarily by infections: Isospora belli, CMV, cryptosporidia, microsporidia and Candida albicans are reported etiologies. The most common manifestation of gallbladder disease is acalculous cholecystitis, although symptomatic cholelithiasis may also be seen. The clinical presentation of acalculous cholecystitis includes right upper quadrant pain and fever; abnormal liver tests suggest concomitant sclerosing cholangitis. As in the normal host, ultrasound or isotopic imaging may be used for diagnosis. Laparoscopic cholecystectomy is curative and is the operative technique of choice.79
APPROACH TO THE DIAGNOSIS OF HEPATOBILIARY DISEASE The evaluation of hepatobiliary disease in HIV-infected patients must be tailored to the presenting symptoms and signs, pattern of liver test abnormalities, and severity of immunodeficiency. Patients with a CD4 lymphocyte count >200/mm3 are unlikely to have an opportunistic infection, although some infections (e.g. TB) and neoplasms, including NHL and KS, may occur at only modest levels of immunodeficiency. Striking ALT elevations suggest hepatitis, possibly from drugs or viral disease, depending on the clinical setting. Alcoholic liver disease can generally be suspected by the history, physical examination and pattern of liver test abnormalities. Disproportionate elevations of the alkaline phosphatase suggest infiltrative disorders or biliary tract disease, but may be present in the absence of any obvious identifiable biliary or hepatocellular disease. Jaundice most commonly results from drug-induced hepatitis, highgrade biliary obstruction or NHL.
Abdominal imaging studies provide valuable diagnostic information. CT is most useful to evaluate for mass lesions, adenopathy, peliosis and peritoneal diseases. Infections resulting in diffuse parenchymal liver disease, such as TB, MAC and parasitic diseases, rarely have focal abnormalities found on these studies. The utility of ultrasound compared to CT in various clinical settings has not been well studied. We employ ultrasound in the anicteric patient in whom AIDS cholangiopathy is suspected, reserving CT for those with marked hepatomegaly, jaundice, or suspected mass lesions or intra-abdominal processes. Invasive techniques should be used judiciously. ERCP is most appropriate in the patient with biliary ductal dilation where endoscopic therapy for papillary stenosis, choledocholithiasis or ductal strictures is likely. As mentioned previously, because the majority of opportunistic infections involve the liver secondarily, liver biopsy rarely uncovers disorders not identified in other tissues. Both liver biopsy and bone marrow biopsy may be helpful in the patient with fever of unknown origin when other modalities are non-diagnostic.55,80 As blood cultures may take at least 2 weeks to become positive, liver and/or bone marrow biopsy may expedite the diagnosis. Several studies document the high yield of liver biopsy in HIV-infected patients with fever and abnormal liver tests. Many of these studies are from developing countries where TB is a common histologic finding. When MAC is suspected and blood and bone marrow cultures are negative, liver biopsy may establish the diagnosis. At many centers, however, empiric therapy for MAC, rather than liver biopsy, is often administered pending culture results, given the efficacy and tolera-
Table 39-1. Overview of the Evaluation of Hepatobiliary Disease in Patients with HIV infection 1. The extent and rapidity of the evaluation should be tailored to the clinical presentation and pattern of liver tests. 2. The CD4 lymphocyte count is essential in formulating the differential diagnosis. Opportunistic infections and neoplasms are most prevalent when the CD4 count is 50 years) 41
LKM1 Cytochrome P450 (CYP)2D6 UDP-glucuronosyltransferase UGTIA 20% in Europe 4% in the USA pediatric (2–14 years) 34
58
B14, DR3, C4AQO 82
Unknown 75
Extrahepatic associated diseases (%) HLA association Progression to cirrhosis (%)
B8, DR3, DR4 45
3.0 –2 1.53 0 2.0 +3 1.5–2.0 +2 1.0–1.5 +1 1:80 +3 1:80 +2 1:40 +1 200 U/l) requires the exclusion of superimposed viral or drug-induced hepatic injury. Serum total bilirubin levels often rise during disease progression but are commonly within normal limits at diagnosis. Levels reaching 20 mg/dl are unusual, but can be associated with advanced hepatic disease. Elevations in serum total bilirubin, hypoalbuminemia, and prolongations in prothrombin time are associated with poor clinical outcome and often justify consideration for liver transplantation. Hypercholesterolemia is observed in up to 85% of cases at diagnosis. Serum IgM levels may also be elevated in patients with PBC.61
SEROLOGIC FEATURES Between 90% and 95% of patients with PBC will test positive for serum antimitochondrial antibody (AMA) in titers greater than or equal to 1:40. The finding of AMA seropositivity is not organ specific but remains highly sensitive (98%) as a diagnostic test.61 Reduced specificity and sensitivity for AMA detection by indirect immunofluorescence (IIF) is partially resolved with the use of enzyme-linked immunosorbent assays (ELISA).65 Conversely, three recent investigations describe a 13–17% prevalence rate for AMAnegative PBC, which is higher than previously accepted values.66,67
Chapter 41 PRIMARY BILIARY CIRRHOSIS
PBC patients may also exhibit serum antinuclear antibody (ANA) and/or smooth muscle antibody (SMA) in 35–66% of cases. The finding of serum ANA and clinical features suggestive of PBC in the absence of a positive AMA has been termed AMA-negative PBC. No evidence for cross-reactivity with ANA and the anti-M2 subunit of AMA has been observed.68,69 Serum anticentromere antibodies in PBC patients with the CREST syndrome in the absence of scleroderma have also been noted between 10% and 15% of the time. Other autoantibodies found in PBC include rheumatoid factor (70%) and antithyroid (antimicrosomal, antithyroglobulin) antibodies (40%).61,69 Antigp210 antibodies, which are formed against nuclear envelope proteins, have been declared to be highly specific and diagnostic of PBC. The prevalence rate is estimated at 25%. Anti-p62 antibodies were recently observed in association with advanced fibrosis in PBC.68,70
Stage I
Stage II
RADIOLOGIC FEATURES Ultrasonography or cross-sectional imaging with computed tomography (CT) or magnetic resonance imaging (MRI) is useful to exclude biliary tract obstruction in patients suspected to have PBC. Increased hepatic echogenicity and/or features of portal hypertension (splenomegaly, intra-abdominal varices, reversal of portal vein flow) may also be observed but are generally absent at diagnosis. Non-progressive periportal adenopathy detected by CT has been described in up to 88% of individuals with PBC.71 Large or bulky adenopathy warrants the exclusion of malignancy such as lymphoma or metastatic disease when present.
Stage III
HISTOLOGIC FEATURES Liver biopsy is needed to determine the stage of histologic disease at diagnosis and to confirm the existence of PBC in patients with persistently negative serum AMA testing. In the presence of serum alkaline phosphatase levels >1.5 times the upper limit of normal and AST values less than fivefold elevated, a liver biopsy is not essential for diagnosing PBC when serum AMA is positive.3 Histologic classification schemes developed by Ludwig and colleagues72 as well as Scheuer and associates73 are the most widely employed for staging PBC. Both systems describe the characteristic progression of liver injury in PBC that includes both focal and segmental destruction of intralobular bile ducts resulting in cholestasis and eventual biliary cirrhosis (Figure 41-1). Stage I PBC is associated with portal tract inflammation due to predominantly lymphoplasmacytic infiltrates, resulting in the destruction of septal and interlobular bile ducts up to 100 mm in diameter. Focal duct obliteration with granuloma formation has been termed the ‘florid duct lesion’ and is considered almost pathognomonic for PBC when present (Figure 41-2). Hepatic lobular involvement is uncommon at this stage of disease, but rare microgranulomas are seen in some cases. Most subjects with stage I PBC are clinically asymptomatic. Stage II PBC is consistent with the descriptions of periportal hepatitis by Ludwig et al.72 or ductular proliferation by Scheuer et al.73 An extension of the portal infiltrates observed in stage I disease to periportal areas is most commonly observed with associated interface hepatitis (piecemeal necrosis). Eosinophils may also be present within the inflammatory reaction. The histologic findings of chol-
Stage IV
Figure 41-1. Schematic representation of the staging system of primary biliary cirrhosis (Ludwig’s classification). Stage I is inflammation within the portal space, focused on the bile duct. Stage II is the inflammation extending into the hepatic parenchyma (interface hepatitis or piecemeal necrosis). Stage III is fibrosis, and stage IV is cirrhosis with regenerative nodules.
angitis, granulomas, and ductular proliferation are most commonly observed in stage II disease. Lobular involvement is similar to stage I disease. Individuals with stage II disease may be clinically asymptomatic, but at a lower frequency than in stage I involvement. Stage III PBC is dominated by the existence of septal or bridging fibrosis. The inflammatory features described with stage II disease are often seen in association with fibrosis spanning portal tracts. Ductopenia (defined as the loss of >50% of interlobular bile ducts)
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Section VI. Immune Diseases
Figure 41-2 Florid duct lesion in stage I primary biliary cirrhosis.
becomes more common, resulting in cholestasis within periportal and paraseptal hepatocytes. Increased hepatic copper deposition beginning in stage II becomes more apparent. The majority of patients with stage III disease are clinically symptomatic. Stage IV disease in both classification systems is consistent with biliary cirrhosis. Nodular regeneration in association with features of stage III disease is observed, with a ‘garland’ shaped appearance that is characteristic of advanced PBC. Portal tract involvement with cholangitis can also be seen in the remaining bile ducts. Cholestatic abnormalities involving the lobular parenchyma, as seen in stage III, remain present. Most subjects considered for liver transplantation have stage IV disease. When histologic disease in PBC is staged, the most advanced finding must be used to determine the extent of involvement. The presence of both non-advanced and advanced histologic features in patients undergoing liver transplantation for PBC provides further evidence of the great sampling variability that is observed from liver biopsy.74
Table 41-3. Differential Diagnosis of Primary Biliary Cirrhosis Extrahepatic biliary tract obstruction Choledocholithiasis Strictures Malignancy Primary sclerosing cholangitis Drug-induced cholestasis (e.g. estrogens, phenothiazines) Granulomatous hepatitis Autoimmune hepatitis Chronic hepatitis C Alcoholic hepatitis Sarcoidosis Celiac disease
syndrome compared to patients with typical PBC has not been reported.
DIFFERENTIAL DIAGNOSIS OVERLAP SYNDROME WITH AUTOIMMUNE HEPATITIS Selected patients with PBC may also have clinical and histologic features compatible with autoimmune hepatitis. This situation has been described as an ‘overlap’ syndrome between the two conditions. No consensus diagnostic criteria have been agreed upon for overlap syndrome. When patients have at least two of three serologic and histologic features of each condition, the estimated frequency can be as high as 20%.75 Refinement of diagnostic criteria using the International Autoimmune Hepatitis Group revised classification system, however, has reduced the prevalence rate of this condition to twofold ULN) or positive ASMA and a liver biopsy which showed moderate to severe periportal and periseptal lymphocytic piecemeal necrosis. Subsequently Talwalkar20 from the Mayo Clinic scrutinized a similar number of cases of PBC for features of AIH by applying the revised AIH scoring system.21 He was unable to find any patients with PBC who scored in the ‘definite’ range for AIH, though he did find that 19% of their PBC population scored in the ‘probable’ range.
IS IT PRIMARILY PBC OR AIH? The introduction of AMA testing greatly facilitated the diagnosis of PBC.22 However, AMA may be detected in other situations, and there are reports of patients with apparent autoimmune hepatitis who test positive for AMA.3 The report by Kenny et al.23 noted that AMA, when detected in the sera of patients thought to have AIH, tended to be of low titer and sometimes were really microsomal antibodies rather than mitochondrial antibodies. This confusion arises because anti-LKM1 antibodies (markers of type 2 AIH) stain similarly by immunofluorescence and may therefore be confused with AMA. Lohse et al.24 reported a series of 20 patients diagnosed with PBC, 20 patients diagnosed with autoimmune hepatitis, and 20 patients given a diagnosis of an overlap of PBC and AIH, and compared the biochemical, immunologic and histologic features of the three groups. The designation of ‘overlap’ was applied in 14 patients because they tested positive for AMA but had an ALT more than
Chapter 43 OVERLAP SYNDROMES
twice the upper limit of normal, and in six because they tested strongly positive for AMA and their liver histology showed features of both AIH and PBC. In their 20 cases of overt AIH, one tested positive for AMA but repeat testing by ELISA was negative, whereas 16 of the 20 ‘overlaps’ tested AMA positive by both immunofluorescence and ELISA, and all 20 patients given a primary diagnosis of PBC were AMA positive. From this heterogeneous population the authors concluded that those with ‘overlap’ in their series had more features in common with PBC, particularly in terms of elevation of serum alkaline phosphatase and serum IgM values. However, they did note that the HLA pattern was more typical of that seen in patients with AIH, namely more cases with HLA, DR3 and/or DR4, and so concluded that these patients indeed had a PBC/AIH overlap. In this retrospective study the therapy given to each patient was not standardized. Patients thought to have predominant AIH responded well to immunosuppressive therapy. Sixteen of the 20 patients diagnosed with an ‘overlap’ syndrome were also given immunosuppressive therapy for a minimum of 2 years. In followup, serum aminotransferase levels improved, as did the alkaline phosphatase levels in 12 of these 16, but all were also given additional UDCA. Of the 20 individuals given a primary diagnosis of PBC, four received immunosuppressive therapy, two with some biochemical benefit. The rest of this group was treated with UDCA, seven of whom had liver biochemical tests that returned to normal. No data beyond 2 years were presented and the numbers under study were small. Thus it cannot be said whether these treatments were appropriate or effective in the long term; neither do we know the natural history of these so-called overlaps. As both corticosteroid therapy and UDCA can be associated with an improvement in liver biochemical tests in almost all forms of liver disease, response to these therapies cannot be considered specific. It is interesting to note that none of the reports describing AIH/PBC overlap discuss any clinical features that distinguish overlapping PBC/AIH versus PBC or AIH alone – rather, the reports all focused on laboratory and histological features (Table 43-1).
AMA NEGATIVE PBC OR AIH/PBC OVERLAP? In 1987 Brunner and Klinge25 described three women who had clinical, histologic and biochemical criteria for PBC but who all tested AMA negative and instead had high-titer ANA. All were given treat-
ment with immunosuppressive therapy, and at least in the short term an improvement was seen. They were given a diagnosis of ‘immune cholangitis.’ Michielleti et al.26 later described 17 patients referred for a randomized controlled trial of UDCA for PBC who consistently tested negative for AMA by both immunofluorescence and immunoblotting, but who otherwise had all the clinical, biochemical and histologic features of PBC. These patients also all tested positive for ANA, generally in high titer. Subsequently several other series of AMA-negative individuals, all of whom appeared to have PBC but tested positive in high titer for ANA, were reported. These cases all had higher levels of IgG and lower levels of IgM, and somewhat higher serum aminotransferase levels than their AMA-positive counterparts, but in all, liver histology showed the typical histologic features of PBC, and such patients are now recognized as having AMA-negative PBC.27 Subsequently, a study by Kim28 reported that the response to UDCA in terms of changes in liver biochemistry was no different in AMA-negative PBC than in AMA-positive PBC. The natural history of AMA-negative PBC is similar to that of AMA-positive PBC, and to date the only difference appears to be their HLA associations. Whereas class 2 HLA DR8 is predominant in AMA-positive PBC, this is not the case in AMA-negative PBC.29 Thus although AMA-negative PBC is associated with certain features of AIH, namely high-titer ANA and higher IgG and aspartate aminotransferase (AST) levels, they probably do not represent a PBC/AIH overlap syndrome and should be treated as cases of PBC.
PSC WITH FEATURES OF AIH A large study, Boberg et al.30 reviewed cases of proven PSC and calculated their AIH score. The results suggested that features of AIH were indeed common in their population with PSC. However, although the AIH score may be helpful in confirming a case of AIH, it has not been validated in individuals with mixed forms of autoimmune liver disease. To rectify this confusion, this AIH score was revised. With the updated criteria21 the Mayo Clinic found that only 1.4% and 6% of patients with proven PSC had scores qualifying for ‘definite’ and ‘probable’ AIH, respectively.31 However, using the same revised scoring system, a group from the Netherlands in a somewhat younger cohort of patients with PSC found 8% with a ‘definite’ AIH score.32
PSC WITH FEATURES OF PBC Table 43-1. AIH, PBC, AIH/PBC Overlap—Laboratory and Histologic Features Laboratory
AIH
PBC
AIH/PBC overlap
ALP ALT AMA ANA SMA IgG IgM
+ +++ – +++ ++ +++ –
+++ + +++ + + + ++
++ ++ ++ ++ + ++ +/–
Liver histology Piecemeal necrosis Bile duct loss
+++ –
– +++
++ ++
There have been a few reported cases of radiologically documented PSC in whom the serum also tested positive for AMA and who had evidence of granulomatous bile duct destruction on liver biopsy.33 It is most likely that this represents two individual autoimmune liver diseases. Although there is a fairly wide differential diagnosis for the radiologic pattern of PSC, it does not include PBC. The ERCP findings in end-stage PBC may simulate PSC because external compression of the nodular liver may give an irregular appearance to the biliary tree; however, it should be easily distinguishable from true PSC. Such cases probably represent true PSC with superimposed early PBC. Liver histology in subjects who test positive for AMA but who have entirely normal liver biochemistry indicates that early lesions, including granulomatous bile duct destruction, are nearly always found on liver histology.34
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MANAGEMENT OF AIH WITH FEATURES OF PBC OR PBC WITH FEATURES OF AIH AND PSC WITH FEATURES OF PBC It has been known for 30 years that immunosuppressive therapy effectively improves survival in patients with severe AIH, but immunosuppressive therapy is not always recommended for those with mild AIH.35 The current standard of care for PBC is UDCA 15 mg/kg/day.36 This treatment leads to slowed progression of fibrosis37 and liver failure.38 Some would argue that treatment with UDCA increases survival free of liver transplantation,39 but none would acknowledge that this treatment cures PBC. UDCA has also been used effectively – at least in the short term – in patients with mild AIH, but it is not advised in the long term or for individuals with severe AIH. Thus there is much debate as to the appropriate management of AIH with overlapping features of PBC, and vice versa. Corpechot et al.40 have shown that in their patients with PBC outcome is worse in those with lymphocytic piecemeal necrosis on liver histology. They have advocated additional treatment with corticosteroid therapy in this patient population, but this has not been evaluated in a prospective manner. In a retrospective study of patients with PBC and features of overlapping AIH no difference in survival was observed in those patients with features of AIH, compared to those with PBC alone, when randomized to treatment with UDCA or placebo.41 Patients with PBC are at increased risk for osteoporosis, both because of their chronic cholestasis and because many are postmenopausal. Thus the major concern if steroid therapy is prescribed to those with PBC with or without overlapping features of AIH is that it may promote further osteoporosis. The same concern pertains to the treatment of AIH with features of PBC. For those patients in whom corticosteroid therapy is considered essential it may be advisable to introduce bisphosphonate therapy simultaneously with the corticosteroids. The management of patients with PSC who appear also to have PBC does not present any controversy as all forms of chronic cholestasis respond to treatment with UDCA, with an improvement in liver biochemistry.42 Recent evidence suggests that patients with PSC may benefit from a high dose >20 mg/kg/day UDCA. Similarly, the management of symptomatic cholestasis, namely pruritus, is the same for all, and appropriate management and prevention of osteoporosis does not vary according to the cause of cholestasis.
SEQUENTIAL PBC AND AIH There are a few individual case reports of patients who were first given a diagnosis of AMA-positive PBC with typical biochemical and histologic features that responded to UDCA treatment, and who subsequently had a complete change in their symptomatology, biochemistry and histology, changing to autoimmune hepatitis, even with loss of AMA.43,44 Only when treatment with immunosuppressive therapy was introduced were the features of the AIH adequately controlled. These cases are so rare that no definitive
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conclusions can be drawn, except to suggest that the immune response governed by the individuals’ genetic make-up may evolve in response to ongoing exposure to inciting antigen(s).
DIFFERENTIAL DIAGNOSIS IN OVERLAPPING SYNDROMES In any patients with a chronic liver disease it is important to consider numerous possible explanations for ‘atypical’ features before making a diagnosis of an overlapping syndrome. In the case of cholestasis in AIH, a common cause is the introduction of exogenous hormones such as the contraceptive pill, hormone replacement therapy or testosterone. Treatment with hormones may induce symptomatic or asymptomatic cholestasis, but importantly without duct injury. Duct injury but not duct loss is reported to be present in 30% of cases of otherwise typical AIH.45 True duct loss may occasionally be the result of a drug reaction, most commonly to antibiotics.46 Although patients with a background of liver disease are not known to be at increased risk of such untoward drug reactions, all patients with liver disease are more susceptible to the cholestatic effect of estrogen therapy simply because the liver disease itself impairs canalicular transport of bile, and in addition the effect of estrogen on bile flow promotes even more cholestasis. In individuals with PSC it is not unusual to see fluctuating levels of serum aminotransferases. This may be related to common duct stones or to the presence of sludge. Thus overlap with AIH should not be considered until the appropriate investigations have been performed and a longer-term perspective is available.
SUMMARY Despite ongoing rigorous investigation into the pathogenesis of the autoimmune liver diseases, their etiology remains obscure. The pathogenesis is clearly multifactorial, with both exogenous influences and genetics playing a role. There are so many potential targets within the liver, and thus it is not surprising that mixed pictures are sometimes observed. The rarity and the heterogeneous nature of overlap syndromes unfortunately means that there are no clear diagnostic criteria and little or no evidence on which to recommend appropriate management.
REFERENCES 1. Lenzi M, Bellentani S, Saccoccio G, et al. Prevalence of nonorgan-specific autoantibodies and chronic liver disease in the general population: a nested case–control study of the Dionysos cohort. Gut 1999;45:435–441. 2. Hooper B, Whittingham S, Mathews JD, et al. Autoimmunity in a rural community. Clin Exp Immunol 1972;12:79–87. 3. Shibata M, Momizne T, Tanaka A, et al. A case of autoimmune hepatitis with a high titre of antimitochondrial antibody and normal gamma-globulinemia. J Gastroenterol Hepatol 2001;16:830–831. 4. Wilschanski M, Chait P, Wade JA, et al. Primary sclerosing cholangitis in 32 children: clinical, laboratory, and radiographic features, with survival analysis. Hepatology 1995;22:1415–1422. 5. Gregorio GV, Portmann B, Karani J, et al. Autoimmune hepatitis/sclerosing cholangitis overlap syndrome in childhood: A 16 year prospective study. Hepatology 2001;33:544–553.
Chapter 43 OVERLAP SYNDROMES
6. Gohlke F, Lohse AW, Dienes HP, et al. Evidence for an overlap syndrome of autoimmune hepatitis and primary sclerosing cholangitis. J Hepatol 1996;24:699–705. 7. Luketic VAC, Gomez AG, Sanyal AJ, Shiffman ML. An atypical presentation for primary sclerosing cholangitis. Dig Dis Sci 1997;42:2009–2016. 8. Hatzis GS, Vassilious VA, Delladetsima JK. Overlap syndrome of primary sclerosing cholangitis and autoimmune hepatitis. Eur J Gastroenterol Hepatol 2001;13:203–206. 9. Abdo A, Bain VG, Kichian K, Lee SS. Evolution of autoimmune hepatitis to primary sclerosing cholangitis: a sequential syndrome. Hepatology 2002;36:1393–1399. 10. Goldstein NS, Soman A, Gordon SC. Portal tract eosinophils and hepatocyte cytokeratin 7 immunoreactivity helps distinguish early-stage, mildly active primary biliary cirrhosis and autoimmune hepatitis. Am J Clin Pathol 2001;116:846–853. 11. Talwalkar JA, Angulo P, Johnson CD, et al. Cost-minimization analysis of MRC versus ERCP for the diagnosis of primary sclerosing cholangitis. Hepatology 2004;40:39–45. 12. Lindor KD for the Mayo PSC–Ursodeoxycholic Acid Study Group. Ursodiol for primary sclerosing cholangitis. N Engl J Med 1997;336:691–695. 13. Mitchell SA, Bansi DS, Hunt N, et al. A preliminary trial of high-dose ursodeoxycholic acid in primary sclerosing cholangitis. Gastroenterology 2001;121:900–907. 14. Harnois DM, Angulo P, Jorgensen RA, et al. High-dose ursodeoxycholic acid as a therapy for patients with primary sclerosing cholangitis. Am J Gastroenterol 2001;96:1558–1562. 15. Guanabens N, Pares A, Ros I, et al. Alendronate is more effective than etidronate for increasing bone mass in osteopenic patients with primary biliary cirrhosis. Am J Gastroenterol 2003;98:2268–2274. 16. Adachi JD, Bensen WG, Brown J, et al. Intermittent etidronate therapy to prevent corticosteroid-induced osteoporosis. N Engl J Med 1997;337:382–387. 17. Geubel AP, Baggenstoss AH, Summerskill WHJ. Responses to treatment can differentiate chronic active liver disease with cholangitic features from the primary biliary cirrhosis syndrome. Gastroenterology 1976;71:444–449. 18. Zein CO, Angulo P, Lindor KD. When is liver biopsy needed in the diagnosis of primary biliary cirrhosis? Clin Gastroenterol Hepatol 2003;1:89–95. 19. Chazoullier O, Wendum D, Serfaty L, et al. Primary biliary cirrhosis – autoimmune hepatitis overlap syndrome: clinical features and response to therapy. Hepatology 1998;2002:296–301. 20. Talwalkar JA, Keach JC, Angulo P, Lindor KD. Overlap of autoimmune hepatitis and primary biliary cirrhosis: an evaluation of a modified scoring system. Am J Gastroenterol 2002;97:1191–1197. 21. Alverez F, Berg PA, Bianchi FB, et al. International Autoimmune Hepatitis Group Report: review of criteria for diagnosis of autoimmune hepatitis. J Hepatol 1999;31:929–938. 22. Walker JG, Doniach D, Roitt M, et al. Serological tests in diagnosis of primary biliary cirrhosis. Lancet 1965;1:827. 23. Kenny RP, Czaja AJ, Ludwig J, Dickson ER. Frequency and significance of automitochondrial antibodies in severe chronic active hepatitis. Dig Dis Sci 1986;31:705–711. 24. Lohse AW, Meyer zum Buschenfelde KH, Franz B, et al. Characterization of the overlap syndrome of primary biliary cirrhosis (PBC) and autoimmune hepatitis: evidence for it being a hepatitic form of PBC in genetically susceptible individuals. Hepatology 1999;29:1078–1084. 25. Brunner G, Klinge O. A chronic destructive non-suppurative cholangitis-like disease picture with antinuclear antibodies (immunocholangitis). Dtsch Med Wochenschr 1987;112:1454–1458. 26. Michieletti P, Wanless IR, Katz A, et al. Antimitochondrial antibody negative primary biliary cirrhosis: a distinct syndrome of autoimmune cholangitis. Gut 1994;35:260–265.
27. Invernizzi P, Crosignani A, Battezzati PM, et al. Comparison of the clinical features and clinical course of antimitochondrial antibody-positive and -negative primary biliary cirrhosis. Hepatology 1997;25:1090–1095. 28. Kim WR, Poterucha JJ, Jorgensen RA, et al. Does antimitochondrial antibody status affect response to treatment in patients with primary biliary cirrhosis? Outcomes of ursodeoxycholic acid therapy and liver transplantation. Hepatology 1997;26:22–26. 29. Stone J, Wade JA, Cauch-Dudek K, et al. Human leukocyte antigen class II associations in serum antimitochondrial antibodies (AMA)-positive and AMA negative primary biliary cirrhosis. J Hepatol 2002;36:8–13. 30. Boberg KM, Fausa O, Haaland T, et al. Features of autoimmune hepatitis in primary sclerosing cholangitis: an evaluation of 114 primary sclerosing cholangitis patients according to a scoring system for the diagnosis of autoimmune hepatitis. Hepatology 1996;23:1369–1376. 31. Kaya M, Angulo P, Lindor KD. Overlap of autoimmune hepatitis and primary sclerosing cholangitis: an evaluation of a modified scoring system. J Hepatol 2000;33:537–542. 32. van Buuren HR, van Hoogstraten HJF, Terkivatan T, et al. High prevalence of autoimmune hepatitis among patients with primary sclerosing cholangitis. J Hepatol 2000;33:543–548. 33. Burak KW, Urbanski SJ, Swain MG. A case of coexisting primary biliary cirrhosis and primary sclerosing cholangitis: a new overlap of autoimmune liver diseases. Dig Dis Sci 2001;46:2043–2047. 34. Mitchison HC, Bassendine MF, Hendrick A, et al. Positive antimitochondrial antibody but normal alkaline phosphase: is this primary biliary cirrhosis? Hepatology 1986;6:1279–1284. 35. Czaja AJ, Freese DK. Diagnosis and treatment of autoimmune hepatitis. Hepatology 2002;36:479–497. 36. Heathcote EJ. Management of primary biliary cirrhosis. Hepatology 2000;31:1005–1013. 37. Pares A, Caballeria L, Rodes J. Long-term ursodeoxycholic acid treatment delays progression of mild primary biliary cirrhosis. J Hepatol 2001;34(Suppl 1):187–188. 38. Gluud C, Christensen E. Ursodeoxycholic acid for primary biliary cirrhosis (Review). Cochrane Database Syst Rev 2004 Vol. 2. 39. Poupon RE, Lindor KD, Cauch-Dudek K, et al. Combined analysis of randomized controlled trials of ursodeoxycholic acid in primary biliary cirrhosis. Gastroenterology 1997;113:884–890. 40. Corpechot C, Carrat F, Poupon R, Poupon RE. Primary biliary cirrhosis: incidence and predictive factors of cirrhosis development in ursodiol-treated patients. Gastroenterology 2002;122:652–658. 41. Joshi S, Cauch-Dudek K, Wanless IR, et al. Primary biliary cirrhosis with additional features of autoimmune hepatitis: response to therapy with ursodeoxycholic acid. Hepatology 2002;35:409–413. 42. Paumgartner G, Beuers U. Ursodeoxycholic acid in cholestatic liver disease: mechanisms of action and therapeutic use revisited. Hepatology 2002;36:525–531. 43. Colombato LA, Alvarez F, Cote J, Huet PM. Autoimmune cholangiopathy: the result of consecutive primary biliary cirrhosis and autoimmune hepatitis? Gastroenterology 1994;107:1839–1843. 44. Weyman RL, Voigt M. Consecutive occurrence in primary biliary cirrhosis and autoimmune hepatitis: a case report and a review of the literature. Am J Gastroenterol 2001;96:585–587. 45. Czaja AJ, Carpenter HA. Autoimmune hepatitis with incidental histologic features of bile duct injury. Hepatology 2001;34: 659–665. 46. Al Traif I, Lilly L, Wanless IR, Heathcote J. Cholestatic liver disease with ductopenia (vanishing bile duct syndrome) following clindamycin and trimethoprim–sulfamethoxazole administration. Am J Gastroenterol 1994;89:1230–1234.
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GRAFT-VERSUS-HOST DISEASE AND THE LIVER
44
Daniel Shouval and Oren Shibolet Abbreviations aGvHD acute GvHD APC antigen presenting cells BMT bone marrow transplantation cGvHD chronic GvHD CMV cytomegalovirus GI gastrointestinal GvHD graft-versus-host disease GvL graft-versus-leukemia
HCV HLA HPCT IL-1 ICAM-1 LFA-1 MHC
hepatitis C virus human leukocyte antigens hematopoietic progenitor cell transplantation interleukin-1 intracellular adhesion molecule-1 alb2 integrin major histocompatibility complex
INTRODUCTION Bone marrow transplantation (BMT) and peripheral blood stem cell transplantation (PBSCT) may induce cholestatic as well as hepatocellular liver injury. Hepatic injury in these conditions may be the result of veno-occlusive disease (VOD), hepatotoxic chemotherapy, irradiation and infection, or it may be caused by graft-versus-host disease (GvHD). GvHD is an immunologically mediated clinical syndrome induced by immunocompetent cells from an organ donor against human leukocyte antigens (HLA) of the recipient. It may involve a single or multiple organs.1 It was first observed 50 years ago, when Barnes and colleagues recognized a wasting syndrome in mice following allogeneic BMT (“Runt disease”).2 Ten years later, Billingham defined the immunological requirements for the development of GvHD.3 These include: (1) the graft must contain immune competent cells; (2) the graft must recognize the host as non-self; (3) the recipient must be unable to mount an effective immune response against the graft; (4) the graft must survive in the host long enough to become sensitized and mount an immunological response against the host. If these requirements are met, donor lymphocytes may attack recipient organs, causing tissue injury, and resulting in significant morbidity and mortality. GvHD most commonly occurs following allogeneic BMT or hematopoietic progenitor cell transplantation (HPCT) for hematological malignancies4 and seldom following solid organ transplantation.5 It is rarely reported following blood transfusion in immune-competent patients.6 Sporadic cases were reported in patients receiving immune suppression for other causes.7 GvHD has two distinct forms, acute and chronic, which differ in their time of onset (with some overlap), organ involvement, and presentation. The disease involves the skin, liver, gastrointestinal (GI) tract, and the hematopoietic and immune systems, with epithelial injury being the most common histopathological manifestation. GvHD is graded as mild (grade I and II), moderate (grade III), and severe (grade IV) according to the severity of organ involve-
PBSCT TCR TOR TNF-a VCAM-1 VLA-4 VOD
peripheral blood stem cell transplantation T-cell receptor target of rapamycin molecule tumor necrosis factor-a vascular cell adhesion molecule-1 a4b1 integrin veno-occlusive disease
ment, with mortality rates ranging between 100 days Following BMT Elevated Alkaline phosphatase, gamma glutamyl transpeptidase and bilirubin levels. Mild elevation of ALT and AST
M>F Highest incidence, 4–5th decade Carriers may develope fluctuating serum ALT levels; One third of patients have persistently normal ALT.
Auto-antibodies
AMA; rheumatoid factor; ASMA (25%), anti-thyroid, ANA (22%).
Histological findings
Sparing of large bile ducts, absence of granulomas, progression to cirrhosis rare.
F>M 4–5th decade Elevated Alkaline phosphatase, gamma-glutamyl transpeptidase, mild elevated AST, ALT. Bilirubin gradual increases over years AMA (95%). Other autoantibodies less frequent: ASMA, anti-thyroid antibodies, ANA. Epithelial injury to small bile ducts; ductopenia; epitheloid granuloma; progression to cirrhosis
Treatment
Immunosuppressive agents (see text); T cell depletion and anti-T-cell antibodies
No effective treatment, UDCA may delay progression.
Mixed cryoglobulins (30–50%). ANA (20%), ASMA (20%), anti-LKM (5%). Common-Steatosis, lymphoid aggregates, bile duct damage. Range-active hepatitis, bridging fibrosis, hepatocyte necrosis, cirrhosis Pegylated interferon alpha and Ribavirin.
Chapter 44 GRAFT-VERSUS-HOST DISEASE AND THE LIVER
DIFFERENTIAL DIAGNOSIS Hepatic injury following BMT or HPCT is common.43,44,46,48,56 Some of the various characteristics of a GvHD and a cGvHD are shown in Table 44-1. Furthermore, cGvHD may occasionally present with clinical, laboratory or histological features resembling PBC or chronic hepatitis C infection as shown in Table 44-2. However, a variety of etiological factors other then GvHD may contribute to post-transplantation liver damage and mandates a careful and systematic approach to the differential diagnosis in these patients.70,71 One such practical approach is to consider etiologies based on the time interval following transplantation.
EARLY HEPATIC COMPLICATIONS FOLLOWING TRANSPLANTATION NON-INFECTIOUS CAUSES OF LIVER DYSFUNCTION FOLLOWING HPCT Veno-occlusive disease (VOD) (see Chapter 46) This is a life-threatening complication of BMT, manifesting as jaundice, tender hepatomegaly, and ascites. It is thought to be secondary to injury to the hepatic sinusoidal epithelial cells, leading to central vein occlusion. The incidence varies from 4% to 50%, depending on the chemotherapy regimen used and genetic susceptibility. The disease usually occurs prior to day 20 posttransplantation but approximately 25% of cases occur later.72 The diagnosis is usually based on clinical signs and symptoms. Imaging modalities may be employed to rule out infiltrative lesions and serve to visualize the hepatic veins as well as the biliary tree. Although Doppler ultrasound examination may occasionally demonstrate portal flow reversal, it is an insensitive modality for diagnosis of VOD.73 Recently it was suggested that measurement of the hepatic artery resistance index might be a more sensitive marker for VOD.74 Liver biopsy plays an important role in establishing the diagnosis. Treatment is based on either antithrombotic or thrombolytic agents (such as tissue plasminogen activator, heparin, defibrotide, and antithrombin III), with a marginal success rate,75 or mechanical relief of obstruction with transjugular portosystemic shunt.76 Liver transplantation was successfully performed in a few selected patients with VOD.77–78
EFFECTS OF HEPATOTOXIC AGENTS Several of the drugs employed in the setting of BMT may cause liver function disturbances. These include antibiotics (i.e., clavulinic acid, trimethoprim-sulfamethoxazole and antifungal agents), chemotherapeutic agents (azathioprine, mycophenolate mofetil, and ciclosporin) and miscellaneous other agents. Total parental nutrition may also lead to cholestatic dysfunction, either by inducing steatosis or by biliary obstruction secondary to sludge formation.
BILIARY DISEASE Cholestatic liver dysfunction may be the presenting manifestation of malignancy recurrence. Acalculous cholecystitis may occur following organ transplantation.79
SYSTEMIC INFECTIONS Infections are common following BMT and HPCT.80–82 Chronic infections with HBV and HCV are the most important infectious agents to consider in diagnosing disturbed liver functions in long-term survivors of BMT.
Hepatitis B Virus (HBV) Infection Mild to severe hepatitis may develop in patients chronically infected with HBV who undergo BMT, regardless of whether they have been asymptomatic carriers or already had active liver disease at the time of transplantation. Disease manifestations can range from mild abnormal cholestatic liver dysfunction through fibrosing cholestatic hepatitis to fulminant hepatitis and death.83 In hepatitis B surface antigen carriers, corticosteroid treatment may enhance viral replication leading to HBV reactivation and hepatitis, which usually occurs during the immune reconstitution phase within 60 days post-transplantations.84 Diagnosis is based on serum HBV-DNA determination as well as immunohistochemistry staining of HBcAg in liver biopsies. Treatment of reactivation may include nucleoside analogues such as lamivudine and adefovir dipavoxil. Evidence has recently been provided justifying pre-emptive nucleoside analogue treatment prior to organ transplantation in patients at risk in order to avoid HBV reactivation. However, the length of posttransplantation treatment is not established.85 Furthermore recipient of nucleoside (tide) analogues and in particular of lamivudine may develop viral escape mutants (i.e., YMDD). HPCT recipients are also at risk of contracting de novo HBV infection. Therefore, pretransplantation active immunization against hepatitis B is highly recommended. There is evidence to suggest that immunity to HBV in bone marrow donors, regardless of whether acquired through natural infection followed by recovery and immunity or acquired through active immunization, may lead to protection of organ transplant recipients against HBV infection.86–88 However, GvHD may abrogate immune memory leading to loss of immunity to HBV following transplantation.88
Hepatitis C Virus (HCV) Infection A mild increase in aminotransferases may be observed in HCVinfected patients following BMT. Acute hepatitis C infection may also rarely occur.89,90 Recent data suggest that fibrosis and cirrhosis are important survival risk factors in HCV-infected BMT patients.81,82 Diagnosis is based on serological and nucleic acid viral markers and occasionally on liver histology. Treatment is currently unsatisfactory as the only partially effective therapy is combined interferon and ribavirin treatment. Administration of interferon following BMT is associated with an increased risk of GvHD.91
Cytomegalovirus (CMV) Infection CMV infection is common in BMT recipients. It usually occurs between 40 and 100 days post-transplantation but can occur later. Hepatic manifestations include fever, jaundice, and disturbed liver functions, but the disease can involve other organ systems (lungs, GI). Diagnosis is based on serological markers and viral nucleic acids, or biopsy of involved organs. Ganciclovir is the most effective treatment. Prophylaxis with this drug is sometimes used.92
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Other Viral Infections Adenovirus, hepatitis G virus, herpes simplex virus, human herpes virus-6, human parvovirus B-19, Epstein–Barr virus, and transfusiontransmitted virus have all been reported in BMT recipients. Some can cause severe fulminant hepatitis (herpes simplex virus) or post-transplantation-associated lymphoproliferative disease (Epstein–Barr virus), while the role of others (transfusion-transmitted viruses such as, hepatitis G virus) in inducing disease is currently unknown.93
POST-TRANSPLANTATION LYMPHOPROLIFERATIVE DISEASE Lymphoproliferative disease may affect the liver post-transplantation and is occasionally associated with Epstein–Barr virus infection. It may occur at any time following transplantation, but usually does so within the first 2 years. Reduction in immune suppression and donor T-lymphocyte infusion may result in disease regression. Diagnosis is based on Epstein–Barr virus DNA detection by polymerase chain reaction or detection of viral particles by immunostaining of liver biopsies.97
Fungal Infections Fungal infections usually occur in the first weeks post-transplantation, when immune suppression is most profound. Candida albicans is the most common infecting fungus, but invasive Aspergillus fumigatus is also an important infection in BMT recipients. A raised alkaline phosphatase level may be the earliest sign of infection. Imaging studies are helpful in detecting liver lesions. Diagnosis is based on liver biopsy and culture. Amphotericin, fluconazole, and newer antifungal agents such as caspofungin are useful in eradicating infection.94,95
Mycobacterial Infection Although rare, mycobacterical infection is significantly more prevalent in BMT recipients following transplantation in countries where tuberculosis is prevalent. Diagnosis is based on culture, polymerase chain reaction, imaging, and liver biopsy. Mycobacterial treatment is useful in eradicating the disease.96
Miscellaneous Bacterial Infections The liver may become involved during systemic infection. Diagnosis is usually based on clinical evidence and primary infection is rarely limited to the liver. Ascending cholangitis may be the primary source for septicemia, leading to cholestatic dysfunction early posttransplantation. Treatment is with antibiotics.
LATE POST-TRANSPLANTATION LIVER COMPLICATIONS IRON OVERLOAD Iron overload is a late complication of organ transplantation resulting from intensive transfusion treatment. Manifestations include hepatomegaly, hepatic fibrosis, and cirrhosis. Diagnosis is based on measurements of body iron stores and assessment of iron content. Treatment includes an aggressive phlebotomy regimen and chelation.53
RECURRENT DISEASE IN BMT RECIPIENTS TRANSPLANTED FOR HEMATOPOIETIC MALIGNANCY The original hematologic disorder may recur and invade the liver, as may be proven by a liver biopsy.
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TREATMENT ACUTE GVHD Prevention and Treatment The development of moderate or severe GvHD is associated with significant morbidity and reduced survival. It was shown that, once GvHD develops, treatment modalities to prevent progression are limited.98 Therefore major efforts have been invested into preventing GvHD. The two basic strategies of GvHD prophylaxis are donor (graft) lymphocyte depletion and pharmacological therapy. T-lymphocyte depletion of the graft is an effective means of eliminating or reducing the risk of GvHD.99 However, most lymphocyte separation methods may cause loss of stem cells needed for successful engraftment, thereby reducing engraftment rates. Other problems encountered are increased leukemia recurrence rates and higher rates of infection.100 Initial trials of T-cell depletion used exvivo incubation of donor bone marrow with broadly reactive antilymphocyte antibodies such as anti-TCR antibodies (i.e., anti-CD3, anti-CD2, anti-CD5, anti-CD8 and anti-CD52 (Campath 1)). It was hoped that complement fixation combined with direct antibody-dependent cellular toxicity would cause donor T-cell destruction. However, most of these trials were unsuccessful.101 Newer methods include antibodies bound to toxins, antibodies conjugated to magnetic beads, lectins, agglutination, and E-rosette formation or addition of donor lymphocytes following recovery from the acute phase of induction regimen toxicity.21 Although these techniques are effective in considerably reducing the numbers of T cells in the graft, they are ineffective in improving disease-free survival because of increased leukemia recurrence rates and death secondary to infectious complications.102–104 Therefore, these treatments are usually reserved for high-risk patients (i.e., mismatched transplants).
Pharmacological Treatment Depending on clinical presentation and severity, treatment of GvHD may require a systemic as well as an organ directed approach. The most commonly used method for prevention and treatment of GvHD employs immunosuppressive agents. This arm of treatment acts either specifically or non-specifically to inhibit T cell activation. Non-specific immunosuppressive drugs. Corticosteroids have been the cornerstone of anti-GvHD treatment, often used in combination with other medications. Their mechanism of action in GvHD remains unknown, but may involve anti-inflammatory effects, direct lymphotoxic properties, and decreasing proinflam-
Chapter 44 GRAFT-VERSUS-HOST DISEASE AND THE LIVER
matory cytokines. Although widely used, a recent report showed no added105 or only limited benefit to corticosteroids when added to methotrexate for patients with moderate to severe GvHD.106 Trials assessing the use of newer steroids which undergo extensive first pass in the liver, such as budesonide,107 or directly administrating medications to the intestine and the liver via a catheter placed in the splanchnic vasculature are currently underway.108 Methotrexate. This drug is an antimetabolite analog of aminopterin. It interferes with critical metabolic pathways via its action as a folic acid antagonist. Methotrexate is used in GvHD patients in various combinations with steroids, ciclosporin, tacrolimus, sirolimus, sacrolimus, and mycophenolate mofetil. Combination therapy was shown to improve survival.109 Recently, the administration of lowdose methotrexate intra-arterially to the hepatic artery was shown to be beneficial for patients with severe hepatic GvHD.110 Side effects of methotrexate include bone marrow suppression and renal, hepatic, and GI toxicity. Long-term administration of the drug can cause dose-dependent hepatic fibrosis. This is an uncommon side effect in the setting of BMT because of the relatively short duration of administration and the low cumulative dose. Elevations of bilirubin and transaminases are commonly seen during methotrexate treatment. Thalidomide. Thalidomide is a glutamic acid derivative. The mechanism of action of the drug is unknown. It may act as an antiangiogenic, mild anti-TNF agent, or an inhibitor of lymphocyte–APC interactions. Various studies have shown decreased overall survival and increased incidence of cGvHD when the drug was initiated early after BMT. In contrast, its use in cGVHD in combination with steroids and other immunosuppressive drugs has been shown to relieve cGvHD effectively in up to 50% of patients. Major side effects of the drug include sedation and constipation, myelosuppression, skin rashes and ulceration, and toxic peripheral neuritis that may be irreversible.111 Azathioprine. This drug has not been specifically used as an antiGVHD medication, although it is mentioned in several immunosuppressive regimens. Topical azathioprine may be beneficial in the treatment of severe oral mucosal involvement of GvHD.112 Ursodeoxycholic acid (UDCA). Several reports have shown that the incidence and severity of aGvHD, especially grade III and IV liver and intestinal GvHD, was reduced with UDCA treatment. Administration of UDCA showed an improvement in hepatic enzyme serum levels in patients with hepatic GvHD.52 The most common side effect is GI irritation.113 UDCA may be beneficial in improving biochemical markers of cholestasis and pruritus, irrespective of the etiology.114,115 Hydroxychloroquine. This drug has been used in cGvHD with promising initial results.116
T-cell-specific Immunosuppressive Drugs Ciclosporin. Ciclosporin is currently the mainstay treatment for the prevention of GvHD, in combination with other immunosuppressive drugs.117,118 It is a fungal-derived hydrophobic peptide with potent immunosuppressive activity through inhibition of increased
expression of high affinity IL-2R by activated T-cells. It inhibits the intracellular activation molecule calcineurin, which is essential for T-cell activation. Major side effects include nephrotoxicity, elevated bilirubin levels, hypertension, and seizures. Rarely the development of a thrombocytopenic thrombotic purpura-like syndrome has been described. The drug is extensively metabolized and cleared mainly in the bile, with lesser amounts in the urine. It has multiple interactions with other drugs. Tacrolimus. Tacrolimus is a fungal macrolide antibiotic. Similarly to ciclosporin, it interferes with T-cell activation by inhibiting the calcineurin pathway. In clinical trials it reduced the occurrence of aGvHD, although overall survival was unchanged. There are no data to suggest that tacrolimus is more efficacious than ciclosporin; however, several reports suggest that tacrolimus can be used as a salvage drug for patients who continue to have progressive GvHD while being treated with ciclosporin. The side effect profile of this drug is very similar to the one described for ciclosporin.119 Rapamycin (sirolimus). Rapamycin is a fungal macrolide devoid of antibiotic activity. Its action is mediated via the target of rapamycin molecule (TOR), and its activation leads to arrested cell cycle maturation. It also inhibits several intracellular transduction pathways essential for T-cell activation. Preliminary studies with rapamycin have shown encouraging results in patients who were unresponsive to steroids. One side effect is GI irritation, with diarrhea and vomiting. Liver enzyme abnormalities, hyperlipidemia, and blood dyscrasias, including thrombocytopenia and neutropenia, have been reported.120 Mycophenolate mofetil. Mycophenolate mofetil is a fungal-derived morpholinethyl ester that possesses antibiotic, antifungal, and antiviral properties. It has a synergistic effect with ciclosporin and has been shown to enhance engraftment. Its main toxicity is myelosuppression and GI irritation.121 Antibodies. In view of the importance of T cells in inducing GvHD, several regimens using specific monoclonal or polyclonal antibodies have been assessed. Their use is currently under evaluation. Some molecules currently in use are described below: Muromonab-CD3 (OKT3). This is a mouse monoclonal antibody directed at the CD3 molecule present on T lymphocytes. Administration of the antibody causes activation and depletion of human T cells. Despite its anti-T-lymphocyte activity, results in clinical trials of this antibody in BMT patients and in preventing GvHD have been disappointing.122 Antithymocyte globulin. These are polyclonal antilymphocyte antibodies derived from various sources. Administration of antithymocyte globulin induced destruction of human lymphocytes. Several studies in BMT recipients have shown either no benefit or mild beneficial effect. The main side effects relate to lymphocyte destruction and include cytokine release, infections, and serum sickness.123 Intravenous immunoglobulin. These are polyclonal antibodies derived from human serum. Initial trials with intravenous immunoglobulin have shown promise in decreasing the incidence of GvHD, especially in younger patients (under 20 years of age), with
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a decrease in overall mortality. A recent trial has not found benefit for intravenous immunoglobulin over placebo in BMT recipients, with an increased incidence of VOD.124 Anti-TNF. TNF has been implicated in the pathogenesis of aGvHD. Several reports assessed the efficacy of a chimeric human–mouse anti-TNF agent (infliximab) in the treatment of aGvHD following failure of other conventional treatment modalities, with promising results.125 Anti-IL-2-receptor antibody (daclizumab). Daclizumab is a humanized monoclonal antibody against the IL-2 receptor expressed on activated T lymphocytes. Initial studies showed it to be well tolerated in patients with acute steroid-resistant GvHD. However, a recently published report suggested that the combination of daclizumab and steroids might decrease survival.126 Anti-co-stimulatory agents. CTLA4Ig is a fusion protein designed to block co-stimulatory signals important for T-cell activation, such as CD86 and CD80. Anti-CD40L antibodies are also important in blocking signal transduction through this pathway. These treatments have been successfully used in autoimmune diseases and have been shown to be effective in animal models of GvHD. Clinical trials assessing these agents are currently under way.127–129
New and Experimental Agents Monoclonal antibodies or receptor antagonists. These include anti-CD5, IL-1-receptor antagonist, murine anti-LFA-1 antibody, and anti-IL-2a receptor antibody (humanized and chimeric). Clofazimine, psoralen and photopheresis, radiation, peptides, and polymers. These drugs and treatment modalities were successfully tried in a limited number of patients with GvHD. Initial results are encouraging but further validation is needed.101,129
PREVENTION AND TREATMENT OF CHRONIC GVHD (NON-SPECIFIC AND T-CELL-SPECIFIC) A multidisciplinary approach with the involvement of dentists, ophthalmogists, gastroenterologists, and physiotherapists is important in managing the chronic manifestations of GvHD.13,102,103 Treatment of cGvHD is similar to that of aGvHD, with various modifications. Generally, patients who develop cGvHD require reinstitution of immunosuppressive medication (if already discontinued), increase in dosing, or addition of medication if still on such treatment. Common regimens include steroids, either systemically or topically, or in combination with other agents, such as ciclosporin. Other options include tacrolimus, mycophenolate mofetil, rapamycin, thalidomide, anti-T-cell antibodies, and photophoresis, all of which have been described for aGvHD (see above). Close attention should be given to infectious complications, as patients with cGvHD are severely immunodeficient. This should include routine assessment for CMV infection by monitoring CMV antigenemia. Patients suffering from recurrent respiratory infections secondary to immunoglobulin deficiency may benefit
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from intravenous immunoglobulin infusions. Routine prophylaxis against Pneumocystis carinii is recommended and prophylactic antibiotic treatment against encapsulated bacterial agents has also been suggested. The US Centers for Disease Control recommend a rigorous vaccination program, although patients with cGvHD may be poorly responsive to vaccination.130 Live attenuated vaccines are prohibited until at least 2 years following transplantation.
LIVER TRANSPLANTATION FOR SEVERE GvHD Liver failure is a rare complication of GvHD. A recent literature review identified 80 patients who underwent liver transplantation for GvHD. Data collected by the US United Network for Organ Sharing (UNOS) revealed a 1 and 5 year actuarial survival rate in 73 patients who underwent liver transplantation of 72.4% and 62.9% respectively. Thus, long-term disease free survival is an obtainable goal in BMT patients with hepatic failure as a result of GvHD.131
PROGNOSIS GvHD, whether acute or chronic, has a detrimental effect on survival. Approximately 30% of patients with moderate to severe aGvHD achieve cure, and about 70% progress to cGvHD.98 Approximately 50% of patients with cGvHD have limited disease and a good prognosis. Sixty percent of the patients having a more extensive disease respond to treatment and will eventually also have a good outcome. The remaining patients have a poor prognosis and will either die from infections or organ failure or will need prolonged immunosuppressive treatment.132
CONCLUSION GvHD is a systemic complication of BMT or PBSCT, which frequently involves the liver. Immunological mediated hepatic involvement is usually manifested clinically by cholestatic liver dysfunction and less frequently as hepatocellular injury. Although often presenting in the context of a life-threatening situation, liver failure is relatively rare, while immune suppression and systemic infection are the more frequent detrimental prognostic factors. Better technology for depletion of donor lymphocytes pretransplantation as well as pharmacological intervention through immunosuppressive agents have contributed to improved survival, while UDCA may be beneficial in ameliorating cholestasis. We would like to thank Dr O. Pappo for her comments on the histopathology section.
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108. Shapira MY, Bloom AI, Or R, et al. Intra-arterial catheter directed therapy for severe graft-versus-host disease. Br J Haematol 2002; 119:760–764. 109. Ringden O, Klaesson S, Sundberg B, et al. Decreased incidence of graft-versus-host disease and improved survival with methotrexate combined with ciclosporin compared with monotherapy in recipients of bone marrow from donors other than HLA identical siblings. Bone Marrow Transplant 1992; 9:19–25. 110. Bloom AI, Shapira MY, Or R, et al. Intrahepatic arterial administration of low-dose methotrexate in patients with severe hepatic graft-versus-host disease: an open-label, uncontrolled trial. Clin Ther 2004; 26:407–414. 111. Flowers ME, Martin PJ. Evaluation of thalidomide for treatment or prevention of chronic graft-versus-host disease. Leuk Lymphoma 2003; 44:1141–1146. 112. Epstein JB, Nantel S, Sheoltch SM. Topical azathioprine in the combined treatment of chronic oral graft-versus-host disease. Bone Marrow Transplant 2000; 25:683–687. 113. Ruutu T, Eriksson B, Remes K, et al. Ursodeoxycholic acid for the prevention of hepatic complications in allogeneic stem cell transplantation. Blood 2002; 100:1977–1983. 114. Mela M, Mancuso A, Burroughs AK. Review article: pruritus in cholestatic and other liver diseases. Aliment Pharmacol Ther 2003; 17:857–870. 115. Kowdley KV. Ursodeoxycholic acid therapy in hepatobiliary disease. Am J Med 2000; 108:481–486. 116. Khoury H, Trinkaus K, Zhang MJ, et al. Hydroxychloroquine for the prevention of acute graft-versus-host disease after unrelated donor transplantation. Biol Blood Marrow Transplant 2003; 9:714–721. 117. Ruutu T, Niederwieser D, Gratwohl A, Apperley JF. A survey of the prophylaxis and treatment of acute GVHD in Europe: a report of the European Group for Blood and Marrow, Transplantation (EBMT). Chronic Leukaemia Working Party of the EBMT. Bone Marrow Transplant 1997; 19:759–764. 118. Storb R, Martin P, Deeg HJ, et al. Long-term follow-up of three controlled trials comparing ciclosporine versus methotrexate for graft-versus-host disease prevention in patients given marrow grafts for leukemia. Blood 1992; 79:3091–3092. 119. Lee TJ, Kennedy LA. Tacrolimus: an alternative for graft-versushost disease prevention. Ann Pharmacother 2000; 34:377–381. 120. Cutler C, Antin JH. Sirolimus for GVHD prophylaxis in allogeneic stem cell transplantation. Bone Marrow Transplant 2004; 34:471–476. 121. Vogelsang GB, Arai S. Mycophenolate mofetil for the prevention and treatment of graft-versus-host disease following stem cell transplantation: preliminary findings. Bone Marrow Transplant 2001; 27:1255–1262. 122. Hebart H, Ehninger G, Schmidt H, et al. Treatment of steroidresistant graft-versus-host disease after allogeneic bone marrow transplantation with anti-CD3/TCR monoclonal antibodies. Bone Marrow Transplant 1995; 15:891–894. 123. Remberger M, Aschan J, Barkholt L, et al. Treatment of severe acute graft-versus-host disease with anti-thymocyte globulin. Clin Transplant 2001; 15:147–153. 124. Cordonnier C, Chevret S, Legrand M, et al. Should immunoglobulin therapy be used in allogeneic stem-cell transplantation? A randomized, double-blind, dose effect, placebo-controlled, multicenter trial. Ann Intern Med 2003; 139:8–18. 125. Couriel D, Saliba R, Hicks K, et al. Tumor necrosis factor-alpha blockade for the treatment of acute GVHD. Blood 2004; 104:649–654. 126. Lee SJ, Zahrieh D, Agura E, et al. Effect of up-front daclizumab when combined with steroids for the treatment of acute graftversus-host disease: results of a randomized trial. Blood 2004; 104:1559–1564.
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127. Wallace PM, Johnson JS, MacMaster JF, et al. CTLA4Ig treatment ameliorates the lethality of murine graft-versus-host disease across major histocompatibility complex barriers. Transplantation 1994; 58:602–610. 128. Jacobsohn DA. Emerging therapies for graft-versus-host disease. Expert Opin Emerg Drugs 2003; 8:323–338. 129. Jacobsohn DA, Vogelsang GB. Anti-cytokine therapy for the treatment of graft-versus-host disease. Curr Pharm Des 2004; 10:1195–1205. 130. Dykewicz CA, National Center for Infectious Diseases, Centers for Disease Control and Prevention, et al. Guidelines for
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preventing opporturistic infections among hematopoietic stem cell transplant recipients: focus on community respiratory virus infections. Biol Blood Marrow Transplant 2001;7:19S– 22S. 131. Barshes NR, Myers GD, Lee D, et al. Liver transplantation for severe hepatic graft versus host disease: an anlysis of aggregate survival data. Liver Transpl 2005;11:525–531. 132. Martin PJ, Schoch G, Fisher L, et al. A retrospective analysis of therapy for acute graft-versus-host disease: secondary treatment. Blood 1991; 77:1821–1828.
Section VII: Vascular Disease of the Liver
BUDD–CHIARI SYNDROME Dominique-Charles Valla
Abbreviations BCS Budd–Chiari syndrome IVC inferior vena cava PNH Paroxysmal nocturnal hemoglobinuria
TIPS
45
transjugular intrahepatic portosystemic stent shunt
INTRODUCTION Budd–Chiari syndrome (BCS) is a heterogeneous disorder characterized by an obstacle to the hepatic venous outflow, be it at the level of the small hepatic veins, large hepatic veins, or the suprahepatic portion of the inferior vena cava (IVC).1 Variation in the level of obstruction is one of the factors explaining the heterogeneity of the disease. Therefore, optimal denomination should include the level of predominant occlusion (i.e., BCS due to IVC occlusion, or BCS due to small hepatic vein obstruction).1,2 The definitions for these levels are presented in Table 45-1.2 BCS is further defined as primary when the obstructing process arises from the venous wall (phlebitis or fibrous thickening) or the venous lumen (thrombosis).1 BCS is considered secondary when the veins are compressed or invaded by a lesion arising outside these veins. Veno-occlusive disease is a misleading term that should be replaced either by “sinusoidal obstruction syndrome” for toxic injury to sinusoidal endothelial cells (particularly in the setting of myeloablative therapy or following ingestion of pyrrolizidine alkaloids),3 or by BCS where appropriate.
EPIDEMIOLOGY BCS is a rare disease: surveys performed in the early 1990s in Japan4 and France (unpublished data from the Observatoire National du Syndrome de Budd–Chiari) could identify only a few hundred cases over one to two decades. However, in some places such as Nepal a high prevalence has been recorded.5 Current estimates of the prevalence may be higher due to increased awareness and improved diagnostic methods.
CAUSES PRIMARY BUDD–CHIARI SYNDROME Following a systematic investigation, one or several thrombotic risk factors are found in about 90% of patients with primary BCS; two or more of these factors are found in at least 25% of patients.6–10 Estimates of the frequencies or odds ratios for these risk factors in BCS patients are presented in Table 45-2.
Primary myeloproliferative disorders account for about 50% of cases of primary BCS.6,10,11 Conversely, only 1–3% of patients with primary myeloproliferative disorders are likely to develop BCS.12,13 In most cases the myeloproliferative disorder can be classified as essential thrombocythemia or polycythemia rubra, but forms that are difficult to classify are encountered as well. Most patients are women (80%) and of a young age (30 years on average). It is poorly understood why some patients with myeloproliferative disorders develop hepatic vein or IVC thrombosis. Association with other risk factors for thrombosis (mainly factor V Leiden or antiphospholipid antibodies) can be identified in about 30% of patients. At the time of presentation with BCS, the myeloproliferative disorder has not previously been recognized in 90% of patients. Moreover, in half of the patients, peripheral blood cell counts at presentation are not suggestive for a myeloproliferative disease.6,10,14 These occult or latent forms are explained by hypersplenism, iron deficiency, and dilution of blood element due to a marked increase in plasma volume – all factors related to portal hypertension.15 Diagnosis is made by showing clusters of dystrophic megakaryocytes in a bone marrow biopsy, or by showing formation of so-called spontaneous or endogenous colonies in cultures of erythroid progenitors on erythropoietinpoor media (in the subject without primary myeloproliferative disorder, erythroid colonies only form upon adding erythropoietin to the culture medium).6,14–16 In the author’s experience, 12% of patients have discordant results by bone marrow biopsy compared to the assessment of erythroid colonies. BCS patients with myeloproliferative disorders have, on average, higher platelet counts and a larger spleen than those without myeloproliferative disorders, but a large overlap makes these features of little practical utility. However, in a patient with features of marked portal hypertension, platelet counts over 300 000/fl strongly suggest that a myeloproliferative disorder is present. Except in the setting of a systemic inflammatory syndrome, secondary thrombocytosis has not been reported to cause BCS. Isotopic determination of total red cell mass shows an increased total red cell mass in many patients with a normal hematocrit. Such a finding is strong evidence for a primary myeloproliferative disorder as BCS complicating secondary erythrocytosis appears exceptional. Late transformation into leukemia has been reported in some of these patients17,18 but the incidence remains unclear. No specific information is available on the treatment of primary myeloproliferative disorders complicated by BCS.
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decision on bone marrow transplantation. Liver transplantation has been successfully performed in anecdotal BCS patients with PNH but recurrence has been observed.22 All known inherited thrombophilias have been implicated in BCS.6–10 As a rule, the thrombophilias are cured by liver transplantation. Activated protein C resistance, generally related to heterozygous or homozygous factor V Leiden mutation, is found in about 25% of patients with BCS, whereas its prevalence is about 5% in the Caucasian population.7,10 When present, factor V Leiden mutation is usually associated with another thrombotic risk factor. Factor V Leiden mutation is particularly frequent in some subgroups of BCS patients: those with progressive IVC obstruction; those with acute BCS related to pregnancy; and among oral contraceptive users.10 Diagnosis is ruled out by a normal test for activated protein C resistance. Confirmation of the mutation is obtained by molecular biology techniques. The G20210A mutation in the prothrombin gene is responsible for increased plasma levels of prothrombin and a moderate increase in the risk of venous thrombosis. This mutation is found in about 5% of BCS patients, whereas the background prevalence in western countries is about 2%.7,10 Diagnosis is made by molecular biology techniques. Antithrombin, protein C, or protein S is produced by the liver and their plasma level is non-specifically decreased in many patients with BCS. Family studies are necessary to establish a low plasma level of these proteins as a primary deficiency. The high number of mutations in different families precludes direct molecular testing at present. Formulas taking into account the degree of liver insufficiency have yielded a prevalence estimate of about 25% for primary protein C deficiency in patients with BCS.7,10 However, these formulas require further validation. These three inherited thrombophilias are cured with liver transplantation from an unaffected donor. Afibrinogenemia may paradoxically predispose to thrombosis and has been implicated as a cause for BCS.23
Paroxysmal nocturnal hemoglobinuria (PNH) accounts for about 5% of cases of primary BCS, a surprisingly high proportion when the extreme rarity of this blood disease is considered. Indeed, retrospective surveys have indicated that up to 12% of patients with PNH will develop BCS.19 In many cases, thrombosis is limited to the small hepatic veins and the patient is asymptomatic.20 Diagnosis of PHN is made with flow cytometry, showing a decreased proportion of CD55- and CD59-positive blood cells. The mechanisms for hepatic vein or IVC thrombosis in PHN are not known. Treatment is not well established. The outcome is poor due to BCS and to the other complications of the blood disease. Resolution of BCS following bone marrow transplantation has been reported.21 The risk of severe deterioration from superimposed sinusoidal obstruction syndrome due to the conditioning regimen use before stem cell transplantation should be strongly considered before making a
Table 45-1. Level of Hepatic Venous Outflow Obstruction2 Designation
Definition
Small hepatic veins
Veins that cannot be shown clearly in hepatic venograms or by ultrasound studies; they include terminal hepatic veins (central veins), intercalated veins, and interlobular veins (collecting veins) Veins that are regularly demonstrable on hepatic venograms and ultrasound studies; segmental hepatic vein branches are generally included A segment of the IVC that involves the entire area that is in contact with the right lobe and the caudate lobe of the liver up to the entry level of the right middle and left hepatic veins A segment of the IVC that extends from the entry level of the right, middle, and left hepatic veins to the junction between the IVC and the right atrium
Large hepatic veins Hepatic IVC
Suprahepatic IVC
IVC, inferior vena cava.
Table 45-2. Thrombotic Risk Factors in Budd–Chiari Syndrome (BCS) Patients Country
Myeloproliferative disorders Occult Classical Antiphospholipid syndrome Paroxysmal nocturnal hemoglobinuria Factor V Leiden mutation Factor II mutation Protein C deficiency Protein S deficiency Antithrombin deficiency Plasminogen deficiency Recent pregnancy Recent oral contraceptive use
Israel6 n positive/ n tested (%)
India9
France10 n positive/ n tested (%)
n positive/ n tested (%)
OR for BCS (95% CI)
n positive/ n tested (%)
OR for BCS (95% CI)
10/22 (45.4%) 8/22 (36.4%) 2/22 (9.0%) 5/22 (22.7%) 1/22 (4.4%)
NA NA 12/43 (27.0%) 2/43 (4.6%) 0/43
NA NA NA NA NA
NA NA NA 6/53 (11.3%) NA
NA NA NA NA NA
31/61 (50.8%) 15/61 (24.6%) 16/61 (26.2%) 9/63 (14.3%) 1/63 (1.6%)
6/22 (27.2%) NA 7/22 (31.8%) 4/22 (18.2%) 5/22 (22.7%) NA NA NA
11/43 (25.6%) 2/43 (4.7%) 4/43 (9.3%) 0/43 0/43 NA NA 12/20 (60%)
11.3 (4.6–26.5) 2.1 (0.4–9.6) 6.8 ( 1.9–24.4) — — — — 2.4 (0.9–6.2)
14/53 (26.4%) 0/53 7/53 (13.2%) 3/53 (5.7%) 2/53 (3.8%) NA 2/17 (11.7%) 1/17 (5.9%)
14.4 (11.9–35.7) — 8.3 (2.1–10.9) 4.4 (2.2–20.6) 4.4 (2.1–10.9) NA NA NA
20/63 (31.7%) 3/47 (6.4%) 4/21 (19.0%) 2/30 (6.6%) 0/47 1/21 (4.7%) 3/47 (6.4%) 23/47 (48.9%)
OR, odds ratio; CI, confidence index; NA, not available.
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Netherlands7,9
Chapter 45 BUDD-CHIARI SYNDROME
Increased plasma levels of factor VII, factor VIII, or homocysteine are risk factors for thrombosis.24 Plasma levels of all three substances are altered in patients with liver disease. Therefore, the significance of their plasma level in patients with BCS is unclear. The antiphospholipid syndrome associated with BCS is the primary type, i.e., the American Rheumatism Association criteria for the diagnosis of systemic lupus were not fulfilled.25,26 Anticardiolipin antibodies have been reported in about 25% of primary BCS patients.6–11 Antibeta-2 glycoprotein I antibodies, lupus anticoagulant, and association with other autoantibodies are more convincing evidence for the diagnosis of antiphospholipid syndrome than the mere presence of low-titer anticardiolipin antibodies. Behçet’s disease, an established cause for venous thrombophlebitis, accounts for less than 5% of BCS patients in western countries.27 In countries where the disease is prevalent, such as Turkey, the corresponding figure is close to 40%.28,29 Conversely, the incidence of BCS in patients with Behçet’s disease is 5–10%. Over 80% of affected patients have IVC involvement with or without extension to the hepatic veins. The outcome of BCS patients with Behçet’s disease seems to be particularly poor. Liver transplantation has been performed successfully in a few patients. Idiopathic hypereosinophilic syndrome was associated with a few cases of idiopathic BCS.30 The endothelial toxicity of certain eosinophil constituents has been incriminated.31 Eosinophilia associated with 5q deletion has also been reported.32 Idiopathic granulomatous venulitis has been described in several case reports.33–35 The criteria for a diagnosis of sarcoidosis were fulfilled in some,33 but not in all, cases. A good response to corticosteroid therapy has been reported.34 Various gastrointestinal diseases have been anecdotally reported in association with BCS, including celiac disease36,37 and ulcerative colitis.38,39 The mechanisms involved in celiac disease are unknown. As to ulcerative colitis, thrombosis has been attributed to the hypercoagulable state related to the inflammatory syndrome, but reports of an association with an underlying myeloproliferative disorder cast doubts on this simple explanation.40 The link between pregnancy and BCS is based on the time relationship with the onset of acute, and generally severe, clinical manifestations of hepatic vein thrombosis.10,41–43 However many pregnant patients have other thrombotic risk factors.11 Similar to deep vein thrombosis at other sites, BCS related to pregnancy is strongly associated with factor V Leiden mutation.10 Drug-related BCS appears to be exceptional.44 It is of note that, in drug- and plant-related sinusoidal obstruction syndrome, the obstructive process begins in the sinusoids and may affect central veins but does not progress to involve the major hepatic veins.3 A case of hepatic vein thrombosis attributed to a slimming drug preparation was recently reported.45 A case–control study has established oral contraceptives as a risk factor for BCS.46 However, this study was performed in the era of oral contraceptives with a high estrogen content. In a more recent study, the increased risk in oral contraceptive users fell short of statistical significance.7 In both studies, oral contraceptive users generally had other risk factors for thrombosis. Therefore, the role of current oral contraceptives in BCS should be re-evaluated.
SECONDARY BUDD–CHIARI SYNDROME Compression by Space-Occupying Lesions Compression alone is unlikely to induce thrombosis, unless an underlying prothrombotic state is present.11 The local and systemic prothrombotic state associated with inflammation is probably involved in BCS cases reported in association with amebic45 and pyogenic48 liver abscess. Cystic hydatic disease due to Echinococcus granulosus45 rarely produces BCS. By contrast, alveolar hydatid disease due to E. multilocularis commonly obstructs of the hepatic veins or IVC via direct invasion.49 Following blunt abdominal trauma, hepatic veins or IVC can be compressed by an intrahepatic hematoma50 or by a ruptured right hemidiaphragm, allowing herniation of the liver into the thorax51 and compression of the hepatic veins. Hepatic vein ligation during hepatic resection can be followed by hepatic decompensation when asymptomatic obstruction of the remaining veins is overlooked.52,53 Following liver transplantation, misplacement of the graft resulting in a torsion of the hepatic veins or IVC, or stenosis of the hepatic venous anastomosis can result in hepatic venous outflow block.54 Secondary liver cancer, the most common cause of malignant liver tumor, is an uncommon cause of hepatic venous outflow block.55 Compression by a thoracoabdominal aortic aneurysm has been reported.56 In polycystic dominant kidney disease, compression of the hepatic veins by large-sized or infected liver cysts is the cause for portal hypertension, occasionally found in this disease.57 A solitary simple cyst of the liver or a nodule of focal nodular hyperplasia may compress one or several of the three major hepatic veins, albeit without giving rise to signs or symptoms. This occurs when the cyst or nodule is large-sized and located in the upper central part of the liver. A large collateral circulation usually develops, circumscribing the space-occupying lesion.
Endoluminal Invasion by a Tumor This complication is specific to those malignant tumors which tend to progress inside the lumen of their venous outflow tract, toward the IVC, up to the point where they obstruct the hepatic vein ostia, producing an acute form of BCS. These malignant tumors include Wilm’s tumor58 and renal cell carcinoma,59 hepatocellular carcinoma,60 adrenocortical carcinoma,61 and leiomyosarcoma of IVC,62 and hepatic angiosarcoma.63 Right atrial myxoma can obstruct the IVC by a similar but retrograde mechanism.64 The endoluminal tumor can usually be cleaved from the venous walls. This feature allows an urgent operation to be performed to relieve the obstruction. However, prognosis is dismal because lung metastasis is almost always present. Protracted remission or cure is likely only when an effective systemic therapy is available.
EVALUATION FOR ETIOLOGY Whether or not there is evidence for an underlying disorder from clinical data and routine laboratory tests, a comprehensive investigation is recommended in order to allow the recognition of the associated risk factors for thrombosis which then allows for better monitoring, earlier identification of subsequent complications of the underlying disease, and counseling relatives with inherited
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A
B
Figure 45-1. Pathology of venous lesions in a 31-year-old female patient with essential thrombocythemia. Cross-section of the ostium of the median hepatic vein in the inferior vena cava (IVC) along the axis of the hepatic vein (on the right) and across the axis of the IVC (on the left). (A) Gross aspect. There is ostial stenosis but, upstream, the vein is patent. (B) Mounted slide at the level of the ostium. There is a membrane-like fibrous material obstructing the ostium which is stenosed by fibrous subendothelial thickening. Such lesions are considered the late stage of a thrombus. Similar short-length stenosis in the vicinity of the ostium is encountered on at least one major hepatic vein in about 25% of patients with Budd–Chiari syndrome seen in western countries. The reason why thrombosis develops at this uncommon site is not known. (Courtesy of late Dr. Molas, Pathology Department, Hôpital Beaujon.)
Table 45-3. Initial Workup for Causes of Budd–Chiari Syndrome (BCS)
Figure 45-2. Obstruction of the suprahepatic inferior vena cava (IVC). Inferior vena cavography through the transfemoral route. The catheter in seen in the IVC at the bottom center of the figure. There is complete interruption of the IVC and obstruction of the ostium of the enlarged middle hepatic vein (which remains patent upstream). There is a complex intrahepatic collateral circulation connecting the hepatic veins with the lower portion of the IVC. There are also upward-winding collaterals connecting the veins with the superior vena cava territory. Although the IVC obstruction is membranous-like, it is currently considered the late transformation of a thrombus.
thrombophilia.65 A proposal for the initial workup is presented in Table 45-3.
PATHOLOGY AND PATHOGENESIS OF VENOUS LESIONS THROMBOSIS AS A CAUSE FOR PRIMARY BCS The role of thrombosis in causing primary BCS has long been debated. Primary endophlebitis and congenital malformation have been proposed as alternative hypotheses. Indeed, fresh thrombi are
880
1. For local factors or tumors Abdominal imaging (otherwise needed for BCS diagnosis) 2. For systemic diseases Clinical history and examination 3. For myeloproliferative disorders and paroxysmal nocturnal hemoglobinuria Complete blood cell counts, bone marrow biopsy. Where available, endogenous erythroid colony assays Flow cytometry for CD55- and CD59-deficient cells 4. For antiphospholipid syndrome Lupus anticoagulant, anticardiolipin antibodies, antibeta2glycoprotein 1 and antinuclear factors 5. For inherited coagulopathies Activated protein C resistance (or factor V Leiden mutation) and factor II gene mutation; protein C, protein S, and antithrombin plasma levels in patients without decreased clotting factor levels 6. For hyperhomocysteinemia Blood folate and vitamin B12 levels rather than serum homocysteine level or MTHFR polymorphism
not the most common finding in explanted livers. Rather, fibrous subendothelial thickening is found, involving various length of the hepatic veins or IVC, as illustrated by Figure 45-1, with or without superimposed thrombosis.55 Short-length stenoses can even take the aspect of a membrane, as illustrated by Figure 45-2, suggesting a congenital anomaly. However, short-length stenoses are now considered a sequela of thrombosis, based on the following observations: the transformation of recent thrombi into short-length stenoses;66,67 the presence of venous wall remnants within the stenosed segment;68 the similarly high prevalence of underlying thrombophilias in patients with and without short-length stenoses; and adulthood presentation of most patients with short-length stenoses, dismissing a congenital malformation.68–70 Short-length stenoses are found in about 25% of patients with hepatic vein obstruction and over 60% of patients with IVC obstruction.68–70
Chapter 45 BUDD-CHIARI SYNDROME
Table 45-4. Main Clinical and Laboratory Features of Budd–Chiari Syndrome Patients According to the Area Country Predominant site of venous obstruction Number of patients Ascites (%) Abdominal pain (%) Hepatomegaly (%) Dilated veins over body, trunk (%) Leg edema (%) Jaundice (%) Platelet count (103/mm3) ALT (¥ ULN) Serum bilirubin (mmol/l) Serum albumin (g/l) Serum creatinine (mmol/l) Prothrombin Child–Pugh score Cirrhosis (%)
India41 Mostly IVC
Japan139 Mostly IVC
France, Netherlands, USA85 Mostly hepatic veins
119 86 57 89 49 41 18 NA NA > 17 in 51% < 30 in 37% NA < l70% in 19% NA
157 31 23 55 27 32 6 121 ± 64a NA 27.4 ± 14.8a NA NA NA NA
NA
NA
237 84 NA 79 NA NA NA 265 (10–896)b 1.0 (0.1–86)b 28 (3–301)b 34 (17–57)b 80 (35–469)b INR > 2.3 in 26% A 24%, B 54%, C 22% 11
IVC, inferior vena cava; NA, not available; ALT, alanine aminotransferase; ULN, upper limit of normal; INR, international normalized ratio. a Mean ± SD. b Median, range.
DIFFERENTIATION OF HEPATIC VEIN THROMBOSIS AND IVC THROMBOSIS As compared with pure hepatic vein thrombosis, IVC thrombosis is more common in the Far East; it runs a more indolent course and is more commonly complicated by hepatocellular carcinoma (Table 45-4).70 Thus, the recently proposed distinction of primary hepatic vein thrombosis (“true BCS”) from primary IVC thrombosis (“obliterative hepatocavopathy”) is well grounded for clinical and therapeutic purposes.70 However, causes of these two conditions are similar. Indeed, IVC thrombosis is generally associated with obstruction of a variable length of the hepatic veins, as illustrated in Figure 45-2, or may follow hepatic vein thrombosis.70,71 Moreover, comprehensive investigations show that underlying risk factors are similar for both sites.8,9,72 What remains to be elucidated is why the IVC is so frequently involved in the Far East and relatively spared in the west.
LOCAL FACTORS It is not known why, in the setting of a generalized thrombotic diathesis, thrombosis develops in the hepatic veins or the suprahepatic IVC. A local factor is found in fewer than 5% of cases, whereas such a factor is found in at least 25% of patients with portal vein thrombosis unrelated to cirrhosis or cancer. It has been proposed that the motion of the diaphragm can induce sufficient trauma to the endothelium of the IVC and large hepatic veins and that it can induce local activation of the coagulation process in susceptible patients. Indeed, IVC thrombosis may follow blunt abdominal trauma in patients with an underlying thrombotic risk factor.73 Furthermore, postmortem studies using inflation of the lungs have shown that, during inspiration, the descent of the diaphragm leads to compression of the termination of the large hepatic veins near the edges of the foramen where the IVC penetrates the
diaphragm.69 Indeed, in many patients, the terminal part of the large hepatic veins is the predominant site of involvement.
SIMULTANEOUS OBSTRUCTION VERSUS SEQUENTIAL INVOLVEMENT OF THE MAJOR HEPATIC VEINS Combining the available data from clinical, imaging, and pathological studies, it is clear that the usual scenario for primary BCS due to hepatic vein obstruction is that of the successive, albeit in a random order, progressive involvement of the hepatic veins. This scenario is illustrated in Figure 45-3. By contrast, the simultaneous, abrupt, formation of a thrombus in all three major veins appears to be extremely rare.
PATHOPHYSIOLOGY AND HISTOPATHOLOGY OF LIVER DAMAGE Former autopsy studies47,55 and more recent analyses in explanted liver at transplantation74,75 have allowed a detailed description of the hepatic lesions at an advanced stage of the disease. Liver biopsy provides information on earlier stages of the disease41,76–78 but with considerable sampling variation due to inhomogeneous distribution of the lesions.74,75 The obstruction of a single main hepatic vein is clinically silent.55 As depicted in Figure 45-4, the obstruction of two or three main hepatic veins produces two hemodynamic changes: an increased sinusoidal blood pressure and a reduced sinusoidal blood flow. Raised sinusoidal pressure explains liver enlargement, pain, and ascites through several mechanisms.
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1
2
3
4
Figure 45-3. Scenario likely accounting for most cases of hepatic vein thrombosis. (1) The first step consists of the rapid or progressive formation of a thrombus, usually in one major hepatic vein, close to its ostium in the inferior vena cava. This event is followed by the formation of a short-length stenosis (as depicted) or by complete obliteration of the vein (not shown). Collaterals develop connecting the obstructed territories to patent hepatic or extrahepatic veins in the vicinity. (2) In the next step, there is a rapid or progressive development of a thrombus in another major hepatic vein. Short-length or diffuse stenosis again develops, together with collaterals. There may be atrophy of the parenchyma in the obstructed territory, with compensatory hypertrophy of the preserved territories (most commonly the caudate lobe). (3) A thrombus can then develop in a vein that had remained partially (as depicted) or completely (not shown) patent. (4) Alternatively a thrombus can develop in the portal vein. Probably depending on the velocity of the obstructive process, steps 1–2 can take place without notice. Symptoms would develop only when obstruction is rapid, or when most of the venous outflow tract is obstructed. Steps 3 and 4 are probably always associated with symptoms. The scenario where complete thrombosis develops simultaneously in all three major hepatic veins appears to be rare.
Hepatic venous outflow obstruction Increased sinusoidal pressure
Congestion Portal hypertension Ascites
Decreased liver perfusion
Centrilobular necrosis Fibrosis hepatic atrophy/failure
Figure 45-4. Pathophysiology of Budd–Chiari syndrome. Increased sinusoidal pressure upstream from the obstruction is almost constant but, due to compensatory mechanisms, not always very marked. Decreased hepatic perfusion occurs when there is abrupt obstruction but, due to compensatory mechanisms, it is usually transient. Congestion is likely to potentiate the effects of ischemia.
882
1. There is sinusoidal dilatation and congestion, predominantly in the central area of the hepatic lobules (Figure 45-4).79 2. The filtration of interstitial fluid increases, which leads to its passage through the liver capsule when the capacity for lymph drainage is exceeded. Usually, the filtrated fluid has a high protein content, due to the high permeability of sinusoidal wall to proteins. 3. Portal pressure increases, leading to transudation of fluid from the splanchnic bed into the abdominal cavity.80 Changes in total hepatic blood flow have not been well characterized. Indirect evidence suggests that, in the microcirculation of the areas with impaired outflow, perfusion with portal blood decreases while perfusion with arterial blood increases.81,82 These alterations are unevenly distributed. Ischemic-type damage to the liver cells (noninflammatory centrilobular cell necrosis as shown in Figure 45-5) is
Chapter 45 BUDD-CHIARI SYNDROME
Figure 45-5. Liver biopsy findings in a patient with recent Budd–Chiari syndrome (Masson’s trichrome). The lesions affect the centrilobular area whereas the immediate periportal area is well preserved. There is a loss of the hepatocytes. The sinusoidal spaces are dilated. There is no fibrosis. These features are only diagnostic of Budd–Chiari syndrome if cardiac or pericardial disease has been ruled out. (Courtesy of Prof. C. Degott, Pathology Department, Hôpital Beaujon.)
(200) (192)
ALT (x ULN)
120
50 20 10 5 2 6
12
20
30
Days Figure 45-6. Course of serum alanine aminotransferase (ALT) changes following presentation in Budd–Chiari syndrome patients with initial ALT levels > 5 ¥ upper limit of normal (ULN). There is a rapid decrease in ALT levels except in 3 patients (red lines) who, within weeks, died or were transplanted. Ischemia–reperfusion injury may explain a rapid decrease in most patients, whereas the absence of reperfusion may explain the progressive increase and poor outcome in some. ALT, alanine aminotransferase; ULN, upper limit of normal. (Denié C, Valla D, personal data.)
found in about 70% of cases.55 Reperfusion injury may participate in liver cell damage, which would explain the rapid return of alanine aminotransferase values to normal in the majority of patients (Figure 45-6).83 Centrilobular necrosis is likely worsened by congestion.84 When hepatic veins and portal veins are both obstructed in the same region, confluent cell loss occurs in regions supplied by preterminal and larger portal tracts.74 Liver failure resulting from ischemic liver cell necrosis is rarely fulminant. Within a few weeks of obstruction of the hepatic veins, fibrosis develops and can predominate either in the centrilobular area (when there is pure hepatic vein obstruction) or in the periportal area (when there is associated obstruction of the
Figure 45-7. Liver biopsy findings in a patient with long-standing Budd–Chiari syndrome (Masson’s trichrome). The parenchymal architecture is destroyed. Still, portal tracts (center bottom) and hepatic veins (upper left) are recognizable. There are regenerative nodules. The area extending from the hepatic veins to the portal tract is fibrotic and devoid of hepatocytes (“parenchymal extinction”). (Courtesy of Prof. C. Degott, Pathology Department, Hôpital Beaujon.)
portal veins).74,79 Within a few months, nodular regeneration may take place, predominantly in the periportal area,55,74,75,79 as illustrated in Figure 45-7. Cirrhosis can eventually develop but it is found in only 10–20% of patients coming to transplantation.55,74,75 Whereas there is evidence for increased sinusoidal pressure being a stable, permanent state, decreased hepatic perfusion appears inconstant. Indeed, centrilobular necrosis is lacking in 20–45% of patients.78,85 Moreover, at the time of presentation, alanine aminotransferase levels are normal in over half of the patients (as illustrated in Figure 45-8) and transiently elevated in most (Figure 45-6). Natural mechanisms able to compensate for decreased perfusion may therefore be involved. These mechanisms are illustrated in Figure 45-9. They include: (1) development of venous collateral channels bypassing the obstructed veins (obvious in Figures 45-2 and 45-10);86,87 (2) redistribution of portal flow from areas where outflow is impaired toward areas where outflow is preserved;81,82 (3) increased portal pressure; and (4) increased arterial flow. Superimposed thrombosis of the intrahepatic portal veins appears to be common at an advanced stage of BCS, which can be explained by the combination of a stagnant flow, and an underlying thrombophilic state. Obstruction of the extrahepatic portal vein is present in 10–20% of patients.85,88 Severe obliteration of intrahepatic portal veins (similar to those illustrated in Figure 45-11) can be found in over one-half of the cases of explanted livers.74,75 The areas where portal and hepatic veins are simultaneously obstructed undergo infarction (as illustrated in Figure 45-12) or parenchymal extinction (i.e., transformation into fibrotic areas devoid of parenchymal cells, as shown in Figure 45-7).74,75 Depending on whether only hepatic veins or both hepatic veins and portal vein are obstructed, bridging fibrosis can be found in a venovenous or in a portovenous or portoportal disposition, respectively.74,75 Because obstruction of the hepatic veins is usually asynchronous, atrophy of the areas of the liver affected early may coexist with the congested or hyperplastic areas that were affected more recently. In
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Section VII. Vascular Disease of the Liver
85 N = 49
Number of patients
N = 115
Figure 45-8. Distribution of serum alanine aminotransferase (ALT) values at presentation with Budd–Chiari syndrome. The distribution is bimodal with a boundary at a value of 5 times the upper limit of normal (ULN). Most patients present with ALT < 5 ¥ ULN. They are more likely to have no pain, lower serum bilirubin, and higher prothrombin levels. They are less likely to have abdominal pain, fever, and extensive necrosis at liver biopsy. However, the extent of fibrosis is similar. (Denié C, Valla D, personal data.)
0 0
1
2
3
4
5
6
7
8
9
10 20 30 50 100 200 300 400
ALT (xULN)
Hepatic venous outflow obstruction
Decreased low pressure (portal) inflow Redistributed portal flow Increased portal pressure Increased arterial inflow
Hepatic vein collaterals
Restored hepatic perfusion
Figure 45-9. Compensatory mechanisms tend to circumvent the effects of hepatic venous outflow obstruction. The development of the collateral venous circulation is crucial in relieving sinusoidal pressure. Patients with asymptomatic Budd–Chiari syndrome have numerous large-sized collaterals that may reach the size of the normal hepatic veins. The collateral circulation also allows the mechanisms that tend to restore hepatic perfusion to operate, including: (1) intrahepatic redistribution of the portal venous inflow from the obstructed territories to the territories where the outflow is preserved or restored; (2) increased portal pressure which improves portal perfusion; and (3) increased arterial inflow, the “buffer response” to decreased portal inflow.
80% of the cases, the caudate lobe is hypertrophied, causing IVC stenosis.89,90 Caudate lobe enlargement is explained by its veins draining directly into the IVC caudal to the ostia of the main hepatic veins; they are preserved from the thrombotic process affecting the main hepatic veins. Preservation of this drainage allows for compensatory hypertrophy and also for serving as an outflow path for intrahepatic venous collaterals draining the obstructed lobes of the liver. A frequent, and odd, feature of long-standing BCS is the development of multiple large regenerative nodules, some of them resembling focal nodular hyperplasia, as illustrated in Figure 45-13.74,75,91,92 These nodules can be viewed as a response to a focal loss of portal perfusion and hyperarterialization in areas with preserved hepatic venous outflow.74,75
884
Figure 45-10. Short-length stenosis demonstrated at retrograde hepatic venography in a young female patient with occult polycythemia rubra. The intrahepatic collateral circulation is well seen, connecting the median hepatic vein with the right part of the liver.
There are several reports of hepatocellular carcinoma developing in BCS patients.93–98 However there are few data to quantify the risk of malignancy. It is noteworthy that in most of the reported cases, there was long-standing, asymptomatic occlusion of the IVC; the patients originated from Asia, Africa, or Jamaica; and cirrhosis was documented in all of them. The growth of cancer seems to be much slower in patients with BCS than in patients with hepatitis B virus-related chronic liver disease.95 There is no evidence, at present, that hepatocellular carcinoma resulted from the transformation of benign regenerative macronodules.75,91
MANIFESTATIONS AND COURSE Affected patients are 35 years on average, and most are female.85 The cardinal features of BCS in various geographical areas are presented in Table 45-1. Presentation varies from a picture of acute hepatic failure to an asymptomatic condition recognized fortuitously.86 Several classifications into acute, subacute, and chronic
Chapter 45 BUDD-CHIARI SYNDROME
A
B
Figure 45-11. Obstruction of the intrahepatic portal veins in patients with Budd–Chiari syndrome (BCS). (A) Medium-sized portal vein. There is marked subendothelial thickening. (B) Small-sized portal vein. The arrows point to the original outline of the portal vein. The original lumen is completely obstructed by cellular and fibrillar material. There are several recanalization channels. Such lesions affect about half the intrahepatic portal veins in explanted livers from BCS patients.
A
C
B
Figure 45-12. Intrahepatic portal vein thrombosis occurring in a Budd–Chiari syndrome patient. (A) After contrast injection, lack of opacification of the segmental portal vein indicates recent thrombosis (arrow). There is an associated infarct in the corresponding territory, seen as an unenhanced area (asterix). Rapid deterioration in this patient led to emergency liver transplantation. (B) Gross examination of the sliced explant confirmed recent portal vein thrombosis and hepatic infarction (arrows). (C) There is a well-circumscribed area of infarction (arrows) in the vicinity of a thrombosed portal vein (arrowhead). (Reproduced from Valla DC. The diagnosis and management of the Budd– Chiari syndrome: consensus and controversies. Hepatology 2003; 38:793–803, with permission.)
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Section VII. Vascular Disease of the Liver
A
B
C
D
E
F
Figure 45-13. Focal nodular regenerative hyperplasia-like lesions in a Budd–Chiari syndrome liver. (A) T1-weighted magnetic resonance imaging (MRI) showing multiple hyperintense nodules (arrowhead). (B) T2-weighted MRI showing that the nodules are hypointense, some of them with a central scar (arrowhead); the inhomogeneous area of hyperintensity is congestive (asterix). (C) Hepatic arteriography showing arterialization: the hepatic artery (arrow) is larger than in the splenic artery; there are multiple hypervascular areas. (D) The sliced native liver at transplantation shows multiple nodules in an otherwise congestive parenchyma, some of them with a central scar (arrow). (E) The fixed liver slice shows multiple pale nodules, some of them harboring a central scar (arrow). (F) Low-power view of a nodule showing the central scar devoid of portal vein (arrow); the neighboring liver parenchyma is congestive with two thrombosed hepatic veins. (Reproduced from Valla DC. The diagnosis and management of the Budd–Chiari syndrome: consensus and controversies. Hepatology 2003; 38:793–803, with permission.)
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Chapter 45 BUDD-CHIARI SYNDROME
Table 45-5. Presenting Forms of Budd–Chiari Syndrome (BCS): Definitions and Distributions Reference: n
Fulminant
Acute
Subacute
Chronic
115
n = 22
Acute portal hypertension. Massive increase in AST or ALT. Death within days
Ascites +++. Moderate increase in AST/ALT. Death within months
Ascites ++. Splenomegaly +. AST and ALT near normal. Moderate progression but variable
117
0 Acute symptoms with fulminant hepatic failure
27% Acute symptoms without fulminant hepatic failure
72% Duration of illness < 6 months. No evidence of cirrhosis
11% Severe pain. Distension. Jaundice. Ascites. Hepatomegaly. Encephalopathy. Severe hepatocellular dysfunction. In general, < 4 weeks’ duration 7% —
11% Pain. Distention. Tender hepatomegaly. Ascites. No encephalopathy
39% —
28% ALT or AST > 5 ¥ ULN. No combination of lobar atrophy/hypertrophy, no gross irregularity of the liver surface 7%b
— —
Ascites +. Malnutrition. AST and ALT increased. Cirrhosis. Death inevitable 0 >6 months’ duration. Evidence of portal hypertension and cirrhosis 39% Symptoms and signs of portal hypertension. Large nodular liver. Distended veins. Relatively preserved hepatocellular function 65%a° ALT or AST £ 5 ¥ ULN. Combination of lobar atrophy/hypertrophy, or gross irregularity of the liver surface 46%b
n = 44
41
n = 177
99
n = 72
—
—
AST, aspartate aminotransferase; ALT, alanine aminotransferase. a 20% of the patients with chronic BCS could recall an episode of acute BCS in the past. b An additional form, so-called acute-on-chronic, was defined by the following features: AST or ALT > 5 ¥ ULN (upper limit of normal), and combination of atrophy/hypertrophy of liver lobes, or gross irregularity of the liver surface. This form accounted for 47% of the patients.
presentation have been proposed because prognosis and management differ accordingly. These classifications are presented in Table 45-5. Schematically, the acute presentation is characterized by a short illness, abdominal pain and fever, ascites, marked elevation in serum aminotransferases and markedly decreased coagulation factors; whereas the chronic presentation is characterized by the indolent development of ascites, or portal hypertensive bleeding, with normal or mildly increased serum aminotransferases, and moderately decreased plasma coagulation factors; jaundice is uncommon. The chronic presentation is more frequent than the acute presentation. Except for descriptive purposes, however, the utility of these classifications is uncertain for several reasons.1 First, definitions have differed among the proposed classifications. Second, these classifications combine duration and severity while these two features can be dissociated. Third, the relationship between clinical presentation and hepatic parenchymal or hepatic venous lesions is not straightforward. Indeed, extensive parenchymal fibrosis is commonly found in patients with an acute presentation.41 However, combining clinical and pathological features may be of prognostic value. It was recently shown that an acute presentation in patients with anatomic features of long-standing disease is associated with a poor outcome as compared with patients presenting acutely but without evidence of long-standing disease or patients with a chronic presentation.99 Presentation appears to depend both on the extent and on the speed of the obstructive process (Figure 45-3).55 Thus, obstruction of only one major hepatic vein usually develops without symptoms; slow obstruction of two or three major veins produces a chronic presentation or, when accompanied with extensive collaterals, no symptoms at all;86 both rapid obstruction of at least two major veins,
and a fresh thrombus superimposed on a long-standing but partial obstruction, gives rise to an acute presentation.
DIAGNOSIS BCS should be suspected when ascites, liver enlargement, and upper abdominal pain are simultaneously present, or when intractable ascites contrasts with moderate alteration of liver tests; when liver disease occurs in a patient with known risk factors for thrombosis; or when liver disease remains unexplained after other common or uncommon causes have been excluded.
DIRECT EVIDENCE The diagnosis of BCS is established when an obstructed hepatic venous outflow tract is demonstrated. X-ray venography remains a reference for evaluation of the hepatic veins.100 Three patterns of opacification are regarded as specific at retrograde catheterization: 1. spreading out from the catheter tip wedged into a blocked vein, a fine “spider-web” network pattern without filling of venous radicals; 2. when there is incomplete occlusion of the hepatic veins, a coarse network of collateral veins which arch outward from the catheter tip and then come together again near the site of entry of the hepatic vein into the IVC (Figure 45-10); 3. a patent vein upstream from a stricture (Figure 45-10). Diagnostic pitfalls include failure to cannulate the hepatic vein ostia, and a distorted appearance of hepatic veins. These two features are
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Section VII. Vascular Disease of the Liver
encountered in patients with cirrhosis of other origin. Direct percutaneous venography can show a localized obstruction in the vicinity of the ostia when the hepatic veins cannot be entered using retrograde cannulation. Inferior venacavography allows for demonstration of caval stenosis or occlusion (Figure 45-2). In many patients with pure hepatic vein thrombosis, the IVC appears narrowed at its intrahepatic portion, because of the enlargement of the caudate lobe. An increased pressure gradient across the IVC stenosis or the presence of a collateral cavacaval circulation are better indications of the impact of intrahepatic stenosis on caval hemodynamics than the apparent degree of stenosis. Sonography must be combined with color Doppler imaging and pulse Doppler analysis of hepatic vein wave form. The following features can be considered specific for hepatic vein obstruction: 1. a large hepatic vein appearing void of flow signal, or with a reversed, or turbulent flow; 2. large intrahepatic or subcapsular collaterals with continuous flow connecting to the hepatic veins or the diaphragmatic or intercostal veins (as illustrated in Figure 45-14); 3. a spider-web appearance usually located in the vicinity of hepatic vein ostia, together with the absence of a normal hepatic vein in the area; 4. an absent or flat hepatic vein wave form without fluttering; 5. a hyperechoic cord replacing a normal vein.82,101 Absence of visualization, or tortuosity of the hepatic veins by grayscale real-time sonography, albeit with flow signals at Doppler imaging, are common in BCS but not specific, being also observed in advanced cirrhosis of other causes. A distinctive feature, however, is the association with intrahepatic or subcapsular hepatic venous collaterals. This collateral circulation is the most sensitive feature for the diagnosis, being found in over 80% of cases of BCS.82,101 Magnetic resonance imaging (MRI) with spin-echo and gradientecho sequences, and intravenous gadolinium injection allows visualization of obstructed hepatic veins and IVC, intrahepatic, or subcapsular collaterals (as shown in Figure 45-15), as well as the spider-web network pattern.81,82 MRI, however, is not as effective
A
as sonography in demonstrating the intrahepatic collaterals. MRI does not allow for an easy determination of the flow direction as well. If computed X-ray tomography fails to visualize the hepatic veins, then this suggests hepatic vein obstruction. However, false-positive and indeterminate results are found in approximately 50% of cases.82
INDIRECT EVIDENCE Liver biopsy usually shows the characteristics of congestion of the sinusoids, liver cell loss, or fibrosis in the centrilobular area (Figures 45-5 and 45-7).2 The differential diagnoses for hepatic congestion are heart failure and constrictive pericarditis; and for centrilobular liver cell loss with mild to marked congestion, circulatory failure whatever its cause, and sinusoidal obstruction syndrome. Isolated perivenular fibrosis is encountered in alcoholic or diabetic patients.
Figure 45-14. Diagnostic features at color Doppler-ultrasonography in a patient with Budd–Chiari syndrome. A large collateral is seen in red (flow directed toward the probe), connecting the obstructed right hepatic vein (with bright walls) to the patent median hepatic vein, seen in blue (flow directed away from the probe).
B
Figure 45-15. Diagnostic features at magnetic resonance imaging in an asymptomatic patient with a fortuitous diagnosis of Budd–Chiari syndrome. T1-weighted sequences. There is an increased number of tubular (black) structures in an abnormal location. These structures are large-sized hepatic vein collaterals. (A) In the left lower corner, the right accessory hepatic vein is seen enlarged, connecting to the patent inferior vena cava (lower left of the panel). (B) In the center, a large subcapsular vein makes up for the left hepatic vein.
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Chapter 45 BUDD-CHIARI SYNDROME
The absence of congestion in the centrilobular area is a strong argument against the diagnosis of hepatic vein thrombosis. Surprisingly, small vein thrombosis is relatively uncommon in biopsy specimens from most patients with BCS. The rare form of BCS due to involvement of the small hepatic veins with patent large veins is only recognized at liver biopsy.2 Differentiation of this form from sinusoidal obstruction syndrome is not always feasible on the basis of pathological examination.2,3 At a late stage, differentiation of hepatic vein thrombosis complicated by cirrhosis from cardiac cirrhosis, and from cirrhosis complicated by hepatic vein thrombosis, may become difficult on pathological grounds.102 Liver specimens can be obtained through transcapsular needle puncture under sonographic guidance, or during laparoscopy. Serious consideration should be given to the risk of bleeding from the puncture site in these patients who are likely to receive early anticoagulation or, urgently, thrombolytic therapy. Liver imaging can also provide indirect evidence for hepatic vein thrombosis. Based on quantitative measurements, hypertrophy of the caudate lobe is found in about 80% of patients.90 This hypertrophy is explained by the preservation of the multiple hepatic veins draining this lobe directly into the IVC. However, caudate lobe enlargement is also common in many cases of cirrhosis of other causes. Liver scintiscanning is non-specific and insensitive. Altered parenchymal perfusion pattern is seen at computed tomography or magnetic resonance following bolus intravenous contrast injection. The most characteristic pattern is early homogeneous central enhancement (particularly at the level of the caudate lobe) together with delayed patchy enhancement and prolonged retention of the contrast agent in the periphery of the liver.82 This pattern is suggestive but neither sensitive nor specific for BCS, being observed in many other situations where portal venous perfusion is compromised.103 Among the latter situations, constrictive pericarditis deserves a special mention because, clinically, it closely mimics hepatic venous obstruction, and diagnosis can be missed at echocardiography.104
DIAGNOSTIC WORKUP Color Doppler imaging combined with pulsed Doppler should be the first test performed, and magnetic resonance should only be used in patients in whom the diagnosis is unclear. The major advantages of the ultrasound study, beyond sensitivity and specificity, are relatively low cost, wide availability, complete lack of harm, and minimal technical difficulty. Limitations lie in the patient’s body habitus which may preclude complete sonographic evaluation, and in a lack of experience on the part of the examiner in diagnosing BCS. MRI is a minimally invasive investigation with no harm from contrast injection to kidney function. Techniques can be standardized and the results are not examiner-dependent. In a minority of patients, mostly cirrhotics, where uncertainty persists, the third investigation can be liver biopsy because it is expected to give information not only for a diagnosis of hepatic vein thrombosis but also for important differential diagnosis: sinusoidal obstruction syndrome, cirrhosis of other origin, and diffuse spreading of malignant cells within the microcirculation. When coagulation disorders preclude liver biopsy through the transcapsular approach, an attempt at retrograde cannulation of the hepatic veins for
venography and liver biopsy using the transjugular route should be performed. X-ray venography is no longer considered necessary for establishing the diagnosis.1 In planning treatment, however, X-ray venography remains a gold standard, permitting a precise delineation of outflow obstruction, which is facilitated by prior non-invasive imaging. Therefore, venography can be reserved for patients where interventional therapy is deemed necessary, and will allow for the performance of a decompressive intervention in the same session if needed.1 The deleterious effect of iodinated contrast injection in patients with renal insufficiency must be kept in mind.
THERAPY TREATMENT FOR THE UNDERLYING CONDITION Treatment for the underlying conditions is logical. Preventing extension of thrombosis into other hepatic veins, collaterals, and into the intrahepatic or extrahepatic portal venous system is aimed at preventing deterioration, and at keeping feasible all the therapeutic options that require a patent portal vein.65 The evidence for the efficacy of anticoagulation therapy is circumstantial, including: 1. an improved outcome since the introduction of systematic anticoagulation, in non-transplant as well as in transplant patients;78,105–107 2. reports on recanalization of thrombosed hepatic veins, and of thrombosed portal vein associated with thrombosed hepatic veins;108 3. the efficacy in patients with portal vein or other deep vein thrombosis.109 The type and duration of optimal anticoagulation have not been established. In most centers, heparin followed by long-term administration of a vitamin K antagonist have been used, whatever the underlying thrombotic risk factor. Low-molecular-weight heparins are generally preferred to unfractionated heparins because of a lower risk of heparin-induced thrombocytopenia and easier administration. Newer agents and alternative protocols require further evaluation.110
MANAGEMENT OF COMPLICATIONS Treatments recommended for portal hypertension and ascites in cirrhotic patients likely apply to BCS patients, albeit with some caution. For example, anticoagulant therapy increases the risk of bleeding from paracentesis. During active bleeding from esophageal varices, the reduction in splanchnic blood flow induced by exogenous vasoconstrictors might precipitate portal venous thrombosis in the patient with BCS. Lastly, anticoagulation therapy may increase the risk of endoscopic therapy for esophageal varices.
LIVER DECOMPRESSION As illustrated in Table 45-6, decompression aims to decrease sinusoidal pressure by restoring the outflow of blood from the liver by means of recanalizing the obstructed venous outflow, or by side-toside portacaval shunting. Recanalization can be attempted using thrombolytic therapy for recent thrombosis, or percutaneous angioplasty, or surgery for more long-standing disease. The benefit/risk ratio of thrombolysis is still unclear. From the limited experience thus far reported, in situ thrombolysis combined with angioplasty
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Section VII. Vascular Disease of the Liver
Table 45-6. Therapy for Budd–Chiari Syndrome Recanalization of obstructed outflow tract
Decompression through side-to-side portosystemic shunting
Recent thrombosis Short-length stenosis Stenosis + recent thrombosis Patent IVC
Obliterated or compressed IVC
Compressed IVC Replacement of the liver and its vessels
Thrombolysis (local or systemic) Angioplasty ± stenting Angioplasty ± stenting + local thrombolysis Surgical • Portacaval • Mesocaval TIPS Surgical • Mesoatrial • Mesoinnominate • Portacaval + cavoatrial TIPS Liver transplantation
IVC, inferior vena cava; TIPS, transjugular intrahepatic portosystemic stent shunt. Rationale for the various forms of therapy for primary Budd–Chiari syndrome. Recanalization procedures (thrombolysis, angioplasty, stenting) would relieve the outflow block. Side-to-side portosystemic shunting (surgery or TIPS) would decompress the liver and improve perfusion through the hepatic artery. Transplantation would both correct the block and replace the failing liver.
for a pre-existing stenosis appears superior to thrombolysis alone, whether systemic or in situ.107,111 Percutaneous angioplasty, primarily or secondarily associated with stenting, has achieved long-term patency rates of the order of 80–90% both for IVC and for hepatic vein thrombosis.107,112,113 An immediate relief of symptoms has been reported. Rethrombosis and restenosis are amenable to in situ thrombolysis and reangioplasty, respectively. Data on long-term effects are encouraging for IVC obstruction but are still scarce for hepatic vein obstruction. The indications for primary stenting need to be clarified. Surgical angioplasty and the hepatoatrial anastomosis (Senning’s procedure)114 have been abandoned with the development of percutaneous maneuvers. Side-to-side portacaval shunting aims to transform the portal vein into an outflow tract, with the expectation that loss of portal inflow will be compensated for by an increased arterial flow. When the IVC is patent, porta- or mesocaval shunts have given better results than other types of surgical shunts.76,115–120 Interposition of a venous or prosthetic graft is usually necessary. Reported in-hospital mortality averages 20%, which may be explained by the poor general condition of some patients. In an experimental model, however, studies on sham operation and portacaval shunting suggest that surgery by itself is particularly risky in the setting of BCS.121 Most surviving patients have no ascites and improved liver function at 1 year of follow-up. Reversal of fibrosis has been reported.76,122 Shunt dysfunction, occurring in about 30% of patients, can result from thrombosis, stenosis, or caval compression by the caudate lobe.123 The outcome of the patient with shunt dysfunction is poor. Stenosis can be amenable to percutaneous stenting.124 Hemodynamically significant compression of intrahepatic IVC can be corrected with the insertion of a stent before or after porta- or mesocaval shunting.125 In patients with obliteration of the IVC at its terminal portion, or severe compression at its intrahepatic portion, when percutaneous maneuvers were not possible, long prosthetic interposition grafts have been used to construct porto- or mesoatrial, cavoatrial with portoatrial, or mesoinnominate anastomoses.120,122 Postshunt encephalopathy seems to be rare.76 The impact of surgical shunting on survival remains unclear. Two multivariate analyses failed to show
890
a survival advantage in surgically shunted patients as compared to patients receiving only medical treatment;78,99 however, a benefit was suggested in a third report.85 High operative mortality and late shunt dysfunction could cancel the long-term beneficial impact of surgical decompression. Except when terminal IVC is obliterated, transjugular intrahepatic portosystemic stent shunt (TIPS) appears to be technically successful in most cases of BCS.126–128 The shunt can be constructed through the suprahepatic IVC when no hepatic vein stump is available.129,130 Intrahepatic caval stenosis is bypassed by the TIPS. Insertion of a TIPS has been successful in patients with partial portal vein thrombosis. Secondary thrombosis or shunt dysfunction requiring revision occurs in about 70% of cases by 6 months.128,131 A recent report suggests improved 5-year survival as compared to the reported findings in similarly severe patients managed differently. Interestingly, TIPS dysfunction was not always associated with deterioration of the patient’s condition.128 However, another study found no survival difference between patients treated with surgical shunt or TIPS.85 TIPS insertion is more difficult and hazardous in BCS patients than in cirrhotic patients.127 Intrahepatic hematomas have been reported.132,133 Secondary stenosis of the terminal IVC has required stenting or liver transplantation.134 More experience is needed to evaluate this new technique, particularly in patients in a poor condition.
LIVER TRANSPLANTATION Liver transplantation has been used as an alternative to, or after failure of, surgical shunting.18,105,116,135,136 Ten-year survival rates are about 75%, on average, which is encouraging when compared with a survival rate below 55% in patients with severe BCS in recent reports on large cohorts.85,99 Further cohort studies, including transplant and non-transplant patients, are needed to evaluate the results according to baseline prognostic factors. The risk of recurrence of BCS is acceptably low when anticoagulant therapy is instituted early.18 The apparently low risk of exacerbating malignant transformation of an underlying myeloproliferative disorder because of the use of immunosuppression requires further assessment.18,136
Chapter 45 BUDD-CHIARI SYNDROME
Anticoagulation
Figure 45-16. Algorithm for the management of patients with Budd–Chiari syndrome according to the European group for the Study of Vascular Disorders of the Liver (EN-Vie consortium: www.envie-project.org).
Severe Manifestations
No Yes
Anticoagulation Medical therapy
Angioplasty Thrombolysis Stenting
Tips
Transplantation
MANAGEMENT In this area, where evidence is scarce, there appear to be some areas of consensus.1 First, treatment of the underlying condition and lifelong anticoagulation therapy should be initiated without delay. Second, patients in a stable condition without symptoms should not undergo any interventional therapy. Third, portosystemic shunting or transplantation should not be proposed to patients whose condition improves rapidly on medical therapy, as judged from recovery in liver function and easy control of ascites on low-salt diet and diuretics. In these patients, finding a short-length stenosis in the IVC or a large hepatic vein may prompt angioplasty with or without stenting. Currently, there is no established timeframe to define what a rapid improvement is. Reasonably, in patients with an acute presentation and severe liver disease it should be a matter of days, whereas in patients with a chronic presentation it could be a matter of weeks. Fourth, patients who do not steadily improve, or whose symptoms recur on medical therapy, should be considered for decompression. The order, or the combination, in which the various options for decompression should be proposed remains debated. Still, there appear to be some further areas of consensus.1 First, the possible need for prompt transplantation should be kept in mind because deterioration in the patient’s condition can be rapid. Stent placement should be in a position that will not hamper subsequent transplantation if needed. Preservation of renal function deserves attention so that optimal immune suppression regimens can be used early post-transplant. Patients are better managed at, or in close connection with, centers where all possible treatment options, including transplantation, are readily available. Second, TIPS insertion is currently preferred to surgical shunting because of a low operative mortality and because efficacy is not compromised by caudate lobe enlargement.127,128 Third, pharmacological thrombolysis and angioplasty or TIPS are usually considered together. Indeed, thrombolysis should always be available when angioplasty or TIPS insertion is undertaken because of the risk of early hepatic or portal venous thrombosis. Moreover, angioplasty or TIPS insertion immediately following thrombolysis is currently preferred to thrombolysis alone on the unproved basis that this will permit a high-velocity blood flow to be maintained, which in turn will prevent rethrombosis.111 Therefore, in order to reduce the risk of bleeding induced
by thrombolysis, all non-essential invasive procedures should be avoided when percutaneous intervention is considered. As to patients not responsive to medical therapy alone, there remain areas of dispute including: (1) whether TIPS or angioplasty should be used as a first-line procedure in patients still in a good condition; and (2) whether hepatic transplantation is an alternative to portosystemic shunting, a rescue operation after failed shunting, or a primary operation in patients with severe liver disease. Furthermore, the criteria for severe liver disease need to be clarified. The algorithm recently proposed by the European Group for the Study of Vascular Disorders of the Liver is presented in Figure 45-16.65 Finally, the problem patient is one with associated portal vein thrombosis who, on average, is in poor condition and in whom derivative procedures and transplantation will be impossible or hazardous. When portal vein thrombosis is recent, TIPS combined with thrombolysis may be the only option.111,137
SURVIVAL AND PROGNOSIS Data on the natural history of BCS are limited. In early studies where no therapy was administered, diagnosis was difficult and most cases were diagnosed postmortem. One-year mortality was estimated to be about 60%. Since these early days, however, noninvasive imaging now allows recognition of asymptomatic forms with an excellent spontaneous outcome in up to 20% of patients. Furthermore, various forms of therapy have been implemented. Large cohorts of patients have shown an overall 5 year-survival rate of 65–69%.78,85,99 Figure 45-17 shows the overall survival curve in the largest cohort of patients so far reported.85 There are two portions to the survival curve. The downward slope is steep during the first 2 years, which accounts for half of the mortality. Thereafter, the slope is less steep, but still, does not appear to plateau. The main causes of death in that cohort were liver failure (n = 17), postoperative multiorgan failure (n = 12), sepsis (n = 4), newly developed malignancy (n = 2), cardiovascular disease (n = 3), cerebrovascular accident (n = 2), variceal bleeding (n = 1), combinations of the above (n = 3), or unknown (n = 8). Recent multivariate analyses have assessed the prognostic significance of disease characteristics at the time of diagnosis as well as
891
Section VII. Vascular Disease of the Liver
Table 45-7. Prognostic Factors in Budd–Chiari Syndrome Patients. Results from Multivariate Analyses Reference
78
Encephalopathy Ascites
present versus absent present versus absent Score 1, 2, or 3a INR £ 2.3 versus INR mmol/l
Prothrombin < 2.3) Bilirubin Pugh score Creatinine Acute-on-chronic form ALT Prognostic indexb Portosystemic shunting
P
2.11
0.04
1.33 —
mmol/l present versus absent IU/l yes versus no
77
RR
—
0.005 0.29
0.344
RR
99 P
NA
0.009
NA
0.008
NA
0.008
RC
85 P
2.15
0.006
1.26
0.0001
RR
P
3.58 2.83
< 0.001 0.08
2.05 1.004
0.02 0.7
RR, risk ratio; RC, regression coefficient; INR, international normalized ratio; ALT, alanine aminotransferase. a Ascites score: 1, absent without diuretics; 2, absent on diuretics; 3, refractory. b Prognostic index (PI) is according to Zeitoun et al.78: PI = 0.75 ascites score + 0.28 Pugh score + 0.037 age + 0.0036 creatinine.
Transplant-free survival
1
Table 45-8. Prognostic Scores for Budd-Chiari Syndrome
82%
1984-2000 (n=237) Langlet et al.99
69% 0.75
62%
59% Murad et al.85
0.50
INR, international normalized ratio. a Ascites score: 1, absent without diuretics; 2, absent on diuretics; 3, refractory. b 0, absent; 1, present.
0.25
0 0 1
5
10
15
20
Years Figure 45-17. Transplant-free survival in Budd–Chiari syndrome (BCS) patients. This multicenter cohort of western patients with a diagnosis of BCS made between 1984 and 2000 was followed up until January 2001. (After Murad SD, Valla DC, de Groen PC, et al. Determinants of survival and the effect of portosystemic shunting in patients with Budd–Chiari syndrome. Hepatology 2004; 39:500–508.)
treatment-related variables. Their results, and the prognostic indices that were derived, are presented in Table 45-7. Several points should be kept in mind before extrapolating these data. First, most of the patients included in these studies were from western countries. Occlusion of the suprahepatic IVC is less common than hepatic vein thrombosis in the west than in Africa or in Asia. The clinical features of BCS related to IVC occlusion differ from those of hepatic vein obstruction. However, the level of obstruction was not found to affect survival in western studies.78,85 Moreover, causal factors may differ in western and eastern patients, although this view has not been substantiated. Second, the impact of causal factors on prognosis has not been adequately assessed due to a lack of systematic investigations. Some studies have suggested that patients with Behçet’s disease138 have a worse outcome than those with the
892
0.95 ascites score (1, 2, or 3)a + 0.35 Pugh score + 0.047 age (years) +0. 0045 creatinine (mmol/l) +2.2 acute-onchronic form (0 or 1)b -0.26 1.27 encephalopathy (0 or 1)b +1.04 ascites (0 or 1)b +0.72 prothrombin (INR) +0.004 bilirubin (mmol/l)
other, more common, causes for BCS. The excess mortality could be related to extrahepatic involvement. Third, the cohorts have been constituted over periods of 20 years or more, a lapse of time during which many aspects of the management have evolved. Fourth, the two most recent studies85,99 are partly redundant with two others,77,78 since a part of their patients had been included in the latter reports. Redundancy may partially explain the similarities of their results. These multivariate analyses brought significant information for therapy (discussed above) and prognosis (presented in Tables 45-7 and 45-8). All studies have identified Child–Pugh score or its components as the major prognostic variables. None of the four studies found liver biopsy data to be of independent prognostic value once adjustment for Child–Pugh score was performed. Other data suggest that portal vein thrombosis is a factor for a poor outcome in BCS patients.74,75,88 However, in multivariate analysis adjusting for the Child–Pugh score component, portal vein thrombosis fell far from having an independent prognostic value (P = 0.9).85 This does not mean that superimposed portal vein thrombosis is not a factor in deterioration. Rather, this finding can be interpreted as indicating that portal vein thrombosis, by inducing deterioration in the Child–Pugh score components, loses its independent prognostic value. Further studies addressing this issue are needed. In the study that evaluated a clinicopathological classification, the acute-onchronic form had an extremely poor outcome as compared to the purely acute or purely chronic forms. The independent value
Chapter 45
Transplant-free survival
BUDD-CHIARI SYNDROME
1
9.
0.75
10. 11.
0.50
12. 0.25
Class I (n = 55) Class II (n = 95) Class III (n = 55)
13.
0 0
5
10
15
20
Years Figure 45-18. Survival in Budd–Chiari syndrome patients according to the prognostic classification of Murad et al. See Table 45-7 for a description of the score. Class I includes patients with a score between 0 and 1.1; class II, patients with a score 1.1–1.5; and class III, 1.5 and higher. (After Murad SD, Valla DC, de Groen PC, et al. Determinants of survival and the effect of portosystemic shunting in patients with Budd–Chiari syndrome. Hepatology 2004; 39:500–508.)
14.
15.
16.
persisted after adjustment for other prognostic variables.99 Most importantly, these studies have allowed a better appreciation of the extreme heterogeneity of BCS with regard to outcome. This is illustrated in Figure 45-18. A subgroup of patients with a 15-year survival of about 90% can be identified at the time of diagnosis; and, at the other end of the spectrum, a subgroup with a 5-year survival only 49% can also be identified.85
17. 18. 19.
20.
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Chapter 45 BUDD-CHIARI SYNDROME
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95. Shin SH, Chung YH, Suh DD, et al. Characteristic clinical features of hepatocellular carcinoma associated with Budd–Chiari syndrome: evidence of different carcinogenic process from hepatitis B virus-associated hepatocellular carcinoma. Eur J Gastroenterol Hepatol 2004; 16:319–324. 96. Jang JW, Yoon SK, Bae SH, et al. Rapidly progressing Budd–Chiari syndrome complicated by hepatocellular carcinoma. Korean J Intern Med 2003; 18:191–195. 97. Havlioglu N, Brunt EM, Bacon BR. Budd–Chiari syndrome and hepatocellular carcinoma: a case report and review of the literature. Am J Gastroenterol 2003; 98:201–204. 98. Matsui S, Ichida T, Watanabe M, et al. Clinical features and etiology of hepatocellular carcinoma arising in patients with membranous obstruction of the inferior vena cava: in reference to hepatitis viral infection. J Gastroenterol Hepatol 2000; 15:1205–1211. 99. Langlet P, Escolano S, Valla D, et al. Clinicopathological forms and prognostic index in Budd–Chiari syndrome. J Hepatol 2003; 39:496–501. 100. Wilson MW, Ring EJ, LaBerge JM, et al. Percutaneous transhepatic hepatic venography in the delineation and treatment of Budd–Chiari syndrome. J Vasc Interv Radiol 1996; 7:133–138. 101. Ohta M, Hashizume M, Tomikawa M, et al. Analysis of hepatic vein waveform by Doppler ultrasonography in 100 patients with portal hypertension. Am J Gastroenterol 1994; 89:170–175. 102. Wanless I, Liu J, Butany J. Role of thrombosis in the pathogenesis of congestive hepatic fibrosis (cardiac cirrhosis). Hepatology 1995; 21:1232–1237. 103. Mathieu D, Vasile N, Grenier P. Portal thrombosis: dynamic CT features and course. Radiology 1985; 154:737–741. 104. Solano FX, Young E, Talamo TS, Dekker A. Constrictive pericarditis mimicking Budd–Chiari syndrome. Am J Med 1986; 80:113–115. 105. Halff G, Todo S, Tzakis AG, et al. Liver transplantation for the Budd–Chiari syndrome. Ann Surg 1990; 211:43–49. 106. Min AD, Atillasoy EO, Schwartz ME, et al. Reassessing the role of medical therapy in the management of hepatic vein thrombosis. Liver Transpl Surg 1997; 3:423–429. 107. Barrault C, Plessier A, Valla D, Condat B. (Non surgical treatment of Budd–Chiari syndrome: a review.) Gastroenterol Clin Biol 2004; 28:40–49. 108. Schmets L, Hagege H, Merlet C, et al. (Porto-hepatic thrombosis, revealing paroxysmal nocturnal hemoglobinuria, followed by regression induced by heparin therapy.) Gastroenterol Clin Biol 1993; 17:955–958. 109. Condat B, Pessione F, Hillaire S, et al. Current outcome of portal vein thrombosis in adults: risk and benefit of anticoagulant therapy. Gastroenterology 2001; 120:490–497. 110. D’Amico EA, Villaca PR, Gualandro SF, et al. Successful use of Arixtra in a patient with paroxysmal nocturnal hemoglobinuria, Budd–Chiari syndrome and heparin-induced thrombocytopenia. J Thromb Haemost 2003; 1:2452–2453. 111. Sharma S, Texeira A, Texeira P, et al. Pharmacological thrombolysis in Budd Chiari syndrome: a single centre experience and review of the literature. J Hepatol 2004; 40:172–180. 112. Fisher NC, McCafferty I, Dolapci M, et al. Managing Budd–Chiari syndrome: a retrospective review of percutaneous hepatic vein angioplasty and surgical shunting. Gut 1999; 44:568–574. 113. Xu PQ, Dang XW. Treatment of membranous Budd–Chiari syndrome: analysis of 480 cases. Hepatobiliary Pancreat Dis Int 2004; 3:73–76. 114. Vogt PR, Andersson LC, Jenni R, et al. Dorsocranial liver resection and direct hepatoatrial anastomosis for hepatic venous outflow obstruction: long-term outcome and functional results. Am J Gastroenterol 1996; 91:539–544.
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115. Bismuth H, Sherlock DJ. Portasystemic shunting versus liver transplantation for the Budd–Chiari syndrome. Ann Surg 1991; 214:581–589. 116. Shaked A, Goldstein RM, Klintmalm GB, et al. Portosystemic shunt versus orthotopic liver transplantation for the Budd–Chiari syndrome. Surg Gynecol Obstet 1992; 174:453–459. 117. Mahmoud AEA, Mendoza AEA, Meshokhes AN, et al. Clinical spectrum, investigations and treatment of Budd–Chiari syndrome. Q J Med 1996; 89:37–43. 118. Ahn SS, Yellin A, Sheng FC, et al. Selective surgical therapy of the Budd–Chiari syndrome provides superior survivor rates than conservative medical management. J Vasc Surg 1987; 5:28–37. 119. Pisani-Ceretti A, Intra M, Prestipino F, et al. Surgical and radiologic treatment of primary Budd–Chiari syndrome. World J Surg 1998; 22:48–53; discussion 53–54. 120. Kohli V, Pande GK, Dev V, et al. Management of hepatic venous outflow obstruction. Lancet 1993; 342:718–722. 121. Orloff MJ, Johansen KH. Treatment of Budd–Chiari syndrome by side-to-side portacaval shunt: experimental and clinical results. Ann Surg 1978; 188:494–512. 122. Orloff MJ, Daily PO, Orloff SL, et al. A 27-year experience with surgical treatment of Budd–Chiari syndrome. Ann Surg 2000; 232:340–352. 123. Panis Y, Belghiti J, Valla D, et al. Portosystemic shunt in Budd–Chiari syndrome: long-term survival and factors affecting shunt patency in 25 patients in western countries. Surgery 1994; 115:276–281. 124. Pelage JP, Denys A, Valla D, et al. Budd–Chiari syndrome due to prothrombotic disorder: mid-term patency and efficacy of endovascular stents. Eur Radiol 2003; 13:286–293. 125. Pisani-Ceretti A, Intra M, Prestipino F, et al. Surgical and radiologic treatment of primary Budd–Chiari syndrome. World J Surg 1998; 22:48–53; discussion 53–54. 126. Rossle M, Olschewski M, Siegerstetter V, et al. The Budd–Chiari syndrome: outcome after treatment with the transjugular intrahepatic portosystemic shunt. Surgery 2004; 135:394–403. 127. Mancuso A, Fung K, Mela M, et al. TIPS for acute and chronic Budd–Chiari syndrome: a single-centre experience. J Hepatol 2003; 38:751–754. 128. Perello A, Garcia-Pagan JC, Gilabert R, et al. TIPS is a useful long-term derivative therapy for patients with Budd–Chiari
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syndrome uncontrolled by medical therapy. Hepatology 2002; 35:132–139. Soares GM, Murphy TP. Transcaval TIPS: indications and anatomic considerations. J Vasc Interv Radiol 1999; 10:1233–1238. Gasparini D, Del Forno M, Sponza M, et al. Transjugular intrahepatic portosystemic shunt by direct transcaval approach in patients with acute and hyperacute Budd–Chiari syndrome. Eur J Gastroenterol Hepatol 2002; 14:567–571. Cejna M, Peck-Radosavljevic M, Schoder M, et al. Repeat interventions for maintenance of transjugular intrahepatic portosystemic shunt function in patients with Budd–Chiari syndrome. J Vasc Interv Radiol 2002; 13:193–199. Fickert P, Trauner M, Hausegger K, et al. Intra-hepatic haematoma complicating transjugular intra-hepatic portosystemic shunt for Budd–Chiari syndrome associated with antiphospholipid antibodies, aplastic anaemia and chronic hepatitis C. Eur J Gastroenterol Hepatol 2000; 12:813–816. Hasegawa S, Eisenberg LB, Semelka RC. Active intrahepatic gadolinium extravasation following TIPS. Magn Reson Imaging 1998; 16:851–853. Turnes J, Garcia-Pagan JC, Gonzalez-Abraldes J, et al. Stenosis of the suprahepatic inferior vena cava as a complication of transjugular intrahepatic portosystemic shunt in Budd–Chiari patients. Liver Transpl 2001; 7:649–651. Ringe B, Lang H, Oldhafer KJ, et al. Which is the best surgery for Budd–Chiari syndrome: venous decompression or liver transplantation? A single-center experience with 50 patients. Hepatology 1995; 21:1337–1344. Saigal S, Norris S, Srinivasan P, et al. Successful outcome of orthotopic liver transplantation in patients with preexisting malignant states. Liver Transpl 2001; 7:11–15. Opitz T, Buchwald AB, Lorf T, et al. The transjugular intrahepatic portosystemic stent-shunt (TIPS) as rescue therapy for complete Budd–Chiari syndrome and portal vein thrombosis. Z Gastroenterol 2003; 41:413–418. Orloff LA, Orloff MJ. Budd–Chiari syndrome caused by Behçet’s disease: treatment by side-to-side portacaval shunt. J Am Coll Surg 1999; 188:396–407. Okuda H, Yamagata H, Obata H, et al. Epidemiological and clinical features of Budd–Chiari syndrome in Japan. J Hepatol 1995; 22:1–9.
Section VII: Vascular Disease of the Liver
46
SINUSOIDAL OBSTRUCTION SYNDROME (Hepatic Veno-occlusive Disease) Laurie D. DeLeve Abbreviations GSH glutathione MMP-9 matrix metalloproteinase-9 NO nitric oxide
RILD SOS
radiation-induced liver disease sinusoidal obstruction syndrome
INTRODUCTION Sinusoidal obstruction syndrome (SOS) is a disease that has been rediscovered a few times and more recently renamed. It was first described in cattle1,2 as a complication of pyrrolizidine ingestion and the first publication about the human disease came from South Africa in 1920.3 The liver toxicity from pyrrolizidine alkaloids was rediscovered in Jamaica4 and named hepatic veno-occlusive disease based on the easily recognizable feature of fibrous obliteration of the small hepatic venules in some cases.5 The iatrogenic form of SOS was first noted with the advent of chemotherapy,6,7 but the disease remained a sporadic complication of chemotherapy until the introduction of stem cell transplantation (the current terminology for what was formerly referred to as bone marrow transplantation).8–10 Early on this liver disease was already postulated to be of primary vascular origin.6,11 In recent years it has become clear that the disease is initiated in the hepatic sinusoids12,13 and that it may be present in the absence of hepatic venular involvement,14 prompting the change in name to sinusoidal obstruction syndrome.15 In addition to SOS, hepatic veno-occlusive lesions may be seen in radiation-induced liver disease (RILD), alcoholic liver disease, and, rarely, after liver transplantation.16–19 Some case reports of liver disease with veno-occlusive lesions after liver transplantation may be due to azathioprine-induced SOS, but there are case reports of “hepatic veno-occlusive disease” occurring in patients who did not receive azathioprine. These other forms of “hepatic veno-occlusive disease” have different pathophysiology from SOS due to chemotherapy and pyrrolizidine alkaloids, but ascites is a prominent feature of each of these diseases. This chapter will focus on SOS due to chemotherapy, with a brief discussion of RILD and SOS due to ingestion of plant alkaloids.
EPIDEMIOLOGY
TIPS
transjugular intrahepatic portosystemic shunt
pyrrolizidine alkaloids. In some non-western nations there are still sporadic cases of SOS due to ingestion of so-called bush teas containing pyrrolizidine alkaloids and epidemics in regions where inadequately winnowed wheat is contaminated by plants containing pyrrolizidine alkaloids. In western countries infrequent cases occur due to ingestion of pyrrolizidine alkaloids present in teas from Crotalaria, Sennecio, and Heliotropium.
CHEMOTHERAPY AND IMMUNOSUPPRESSIVE AGENTS Table 46-1 lists the chemotherapeutic or immunosuppressive agents most closely associated with SOS unrelated to myeloablative preparative regimens for stem cell transplantation. Gemtuzumab ozogamicin, used to treat acute myeloid leukemia, comprises the toxin calicheamicin coupled to a humanized monoclonal antibody to CD33, an antigen present on myeloblasts. Gemtuzumab ozogamicin has been linked to SOS, particularly in patients who have been exposed to the drug either before or after stem cell transplantation.20,21 The incidence has been reported to be particularly high when the interval between gemtuzumab ozogamicin and stem cell transplantation is less than 3.5 months.21 The most common setting for SOS due to actinomycin D has been in patients with nephroblastoma (Wilms tumor), in particular in patients with right-sided tumors who received both actinomycin D and abdominal irradiation.22,23 The thiopurines, azathioprine and 6-thioguanine, have been associated with three forms of liver injury caused by damage to hepatic endothelial cells: (1) SOS; (2) peliosis hepatitis; and (3) nodular regenerative hyperplasia.24–31 Azathioprine-associated SOS has been most commonly described after long-term immunosuppression for kidney or liver transplantation. 6-Thioguanine, a metabolite of azathioprine, has been associated with SOS in case reports of exposure to the drug in chemotherapeutic or immunosuppressive regimens.
PYRROLIZIDINE ALKALOIDS
RADIATION-INDUCED LIVER DISEASE
Until the advent of chemotherapy the only recognized cause of SOS was the ingestion of teas or foodstuffs contaminated with
RILD is a form of hepatic veno-occlusive disease that differs from SOS in the clinical presentation, the time course, and some histo-
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Table 46-1. Standard Chemotherapy or Immunosuppressive Linked to Sinusoidal Obstruction Syndrome—Not in the Setting of Stem Cell Transplantation Actinomycin D ± abdominal irradiation Azathioprine Cytosine arabinoside Dacarbazine Gemtuzumab ozogamicin Mithramycin 6-Thioguanine Urethane
logical characteristics, but that has in common the characteristic veno-occlusive lesion. It is a complication that may occur when the mean liver dose of irradiation is greater than 31 Gy and when a larger portion of the liver is irradiated.32,33
SOS ASSOCIATED WITH STEM CELL TRANSPLANTATION Myeloablative stem cell transplantation for malignancy is the most common setting for SOS in North America and western Europe. The incidence of SOS varies greatly between transplantation centers and depends mainly on the choice of the preparative chemotherapeutic regimen, but also on diagnostic criteria for SOS and patient selection. The reported incidence ranges from 5 to 60%, with an overall case fatality rate of around 30%.34–37 The non-myeloablative stem cell transplantation regimens, which use reduced-intensity preparative chemotherapy, are an alternative approach to the traditional myeloablative regimens used in preparation for stem cell transplantation. SOS has been reported in some small series of non-myeloablative stem cell transplantations,38,39 but one large case series did not find any cases of SOS.40
PATHOGENESIS CLINICAL STUDIES The clinical presentation and histological features of SOS associated with stem cell transplantation have been very well characterized and therefore provide some insights into the mechanisms involved. Several findings demonstrate that the preparative regimen for stem cell transplantation initiates the injury in SOS. In stem cell transplantation a chemotherapeutic preparative regimen is given over several days followed by infusion of stem cells. The onset of symptoms of SOS can be as early as the day of infusion of stem cells, which indicates that it is the chemotherapy rather than the infusion of stem cells that initiates SOS. In cyclophosphamide-based regimens, cyclophosphamide metabolism is one of the risk factors for developing SOS,41 again demonstrating that the chemotherapy itself initiates the injury. The incidence of SOS varies dramatically between transplantation centers and the intensity of the chemotherapeutic regimen seems to be the greatest determinant of this variability. Unlike other intrinsic liver diseases, the manifestations of portal hypertension precede evidence of parenchymal disease. This indicates that SOS is a primary circulatory disorder. Careful analysis of
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histology has shown that involvement of the hepatic veins is not essential to the development of clinical signs of SOS, although occlusion of the central veins is associated with more severe disease and the presence of ascites.14 Taken together, these findings suggest that the primary circulatory impairment occurs at the level of the sinusoid with exacerbation of the syndrome as the obstructive process extends into the central veins. The role of clotting in the pathogenesis of SOS has been one of the most controversial areas in the elucidation of the pathophysiology (see review by Korte42). Most of the currently available clinical evidence suggests that clotting is not involved. If intrahepatic coagulation contributes to the disease, the expected sequence of events would be damage to the sinusoidal and venular endothelium with initiation of coagulation by tissue factor. Damage to endothelial lining would certainly be expected to predispose to clotting. Studies have demonstrated increases in procoagulants and decreases in natural anticoagulants. The most consistent findings are decreases in protein C and factor VII levels. There is an association with low levels of protein C and factor VII prior to transplantation and the development of SOS and decreases have been seen in a number of studies following stem cell transplantation. However the low levels prior to transplantation may indicate underlying liver disease and the post-transplantation fall likely reflects hepatic dysfunction posttransplant. Increased fibrinogen is observed post-transplant, but this most likely occurs as an acute-phase response. Von Willebrand factor, factor VIII and tissue plasminogen activator increase after transplantation, which likely reflects endothelial damage. Immunohistochemical staining has demonstrated fibrinogen and factor VIII in the wall of the central veins.43 Given the disruption of the venular endothelial barrier and the accumulation of plasma proteins in the vessel wall, the presence of fibrinogen and factor VIII in the vessel wall likely reflects their presence in the blood as an acute-phase reactant and a marker of endothelial damage, respectively. There are reasons to believe coagulation does not play a role in the development of the disease. Fibrin was not observed in monocrotalineinduced SOS in humans44 and platelets could not be demonstrated by immunohistochemistry in SOS in stem cell transplantation patients.43 Prophylactic infusion of heparin or antithrombin III does not prevent SOS and thrombolytic therapy is beneficial in a minority of patients. Thus the preponderance of data in humans does not suggest that clotting is involved.
ANIMAL STUDIES In vitro studies have shown that hepatic sinusoidal endothelial cells are more susceptible than hepatocytes to various drugs that have been implicated in SOS clinically.26,45,46 In vivo studies have used a rat model of SOS induced by monocrotaline.12 Monocrotaline is a pyrrolizidine alkaloid, a class of plant compounds that cause SOS in humans who ingest these compounds in foods or herbal teas. The monocrotaline rat model shares the same characteristic signs, symptoms, and histology as the human disease.12 The earliest anatomic changes of SOS have been characterized by electron microscopy and in-vivo microscopy in this model.12,13 Kupffer cells disappear from the sinusoid, sinusoidal endothelial cells round up, and gaps form within and between sinusoidal endothelial cells. Red blood cells penetrate through the gaps in the endothelial barrier into the space of
Chapter 46 SINUSOIDAL OBSTRUCTION SYNDROME
Disse. With partial obstruction of the sinusoid by the rounded-up sinusoidal endothelial cells, the space of Disse becomes the pathway of least resistance and blood begins to flow within the space of Disse. Sinusoidal endothelial cells, stellate cells, and any remaining Kupffer cells are dissected off the space of Disse by the flow of blood and the sinusoid is largely denuded of lining cells. The sinusoidal cells embolize downstream, further obstructing sinusoidal flow. As the number of perfused sinusoids reaches a nadir, there is a parallel increase in necrosis of centrilobular hepatocytes. Mononuclear cells accumulate in the sinusoid and within the central vein and the mononuclear cell aggregates contribute to the sinusoidal obstruction. Fibrin was not identified by electron microscopy in a rat model of monocrotaline-induced SOS.12 In a rat model of monocrotalineinduced liver injury that is not SOS, fibrin has been demonstrated by immunohistochemistry47 and anticoagulants reduced the severity of hepatocyte necrosis.48 The biochemical underpinning of the changes described in the previous paragraph include changes in levels of nitric oxide (NO), glutathione (GSH), and matrix metalloproteinase-9 (MMP-9). Monocrotaline is P450-activated only within the liver.49–52 Hepatocytes, sinusoidal endothelial cells,26 and Kupffer cells all metabolize monocrotaline to monocrotaline pyrrole, the electrophilic metabolite. GSH is the major detoxification pathway for monocrotaline pyrrole26 and sinusoidal endothelial cells are selectively more susceptible than hepatocytes to toxicity because of weaker GSH defenses. One of the major adducts for monocrotaline pyrrole is the cytoskeletal protein, F-actin.53 Once sinusoidal endothelial glutathione is depleted, monocrotaline pyrrole binds to F-actin, leading to F-actin depolymerization.54 F-actin depolymerization leads to increased synthesis and activity of MMP-9.54 MMP-9 (gelatinase B) is exocytosed and digests extracellular matrix components. The depolymerization of the F-actin cytoskeleton allows sinusoidal endothelial cells to round up and the increased MMP-9 activity on the abluminal side of the sinusoidal endothelial cell digests extracellular matrix and allows the sinusoidal endothelial cell to let loose from the space of Disse. Normally there is tonic release of NO by sinusoidal endothelial cells and, to a lesser degree, by Kupffer cells.55 Basal release of NO reduces MMP-9 expression, whereas inhibition of NO synthesis increases cytokine-stimulated MMP-9 expression.56,57 As the sinusoidal endothelial cells and Kupffer cells disappear from the sinusoid, there is a parallel drop in hepatic vein NO. The decline in NO permits increased synthesis of MMP-9 by sinusoidal endothelial cells.55 MMP expression and activity are also regulated by redox status,58,59 so that the decline in sinusoidal endothelial cell GSH also permits up-regulation of MMP-9 activity. The events described here form a positive-feedback loop: F-actin depolymerization leads to up-regulation of sinusoidal endothelial cell MMP-9, MMP-9 up-regulation loosens the tethering of sinusoidal endothelial cells from the space of Disse, sinusoidal cells disappear from the sinusoid, and there is less NO from the sinusoidal endothelial cells and Kupffer cells to suppress MMP-9 synthesis, leading to the loss of more sinusoidal endothelial cells with further loss of NO production. The central role of the biochemical changes described in the pathogenesis of SOS are confirmed by the ability to prevent experimental SOS by infusion of GSH, of a liver-specific NO donor (V-PYRRO/NO), or of inhibitors of MMP-9.54,55,60
Table 46-2. Clinical Features Used to Diagnose Sinusoidal Obstruction Syndrome after Stem Cell Transplantation Seattle criteria34
Baltimore criteria61
Diagnosis requires two of three findings within 20 days of transplantation: Bilirubin >34.2 mmol/l (2 mg/dl) Hepatomegaly or right upper quadrant pain of liver origin >2% weight gain due to fluid accumulation
Hyperbilirubinemia plus two or more other criteria: Bilirubin >34.2 mmol/l (2 mg/dl) Hepatomegaly, usually painful ≥5% weight gain Ascites
CLINICAL FEATURES OF SOS AFTER MYELOABLATIVE THERAPY CLINICAL PRESENTATION The clinical presentation of SOS after myeloablative therapy for stem cell transplantation has been well characterized. The clinical features are tender hepatomegaly, hyperbilirubinemia, and fluid retention with weight gain. The diagnosis of SOS for patients who have undergone stem cell transplantation is based on these characteristic clinical features (Table 46-2) and diagnostic criteria have been published by investigators in Seattle and Baltimore.34,61 The Seattle criteria require two of three findings occurring within 20 days of transplantation: bilirubin >2 mg/dl, tender hepatomegaly, and >2% weight gain due to fluid accumulation. The Baltimore criteria require hyperbilirubinemia plus two of three other findings: bilirubin >2 mg/dl, (usually painful) hepatomegaly, >5% weight gain, and ascites. In myeloablative regimens that contain cyclophosphamide, features of SOS may present as early as day 0 (i.e., the day after the preparative regimen is completed and when the stem cells are administered). Most commonly, the onset of SOS is 10–20 days after completion of a cyclophosphamide-containing preparative regimen,34 whereas with other myeloablative regimens the symptoms may occur later.37,62,63 Thus the Seattle criteria, developed largely for patients receiving myeloablative regimens containing cyclophosphamide, have a temporal criterion that may not apply to non-cyclophosphamidecontaining regimens. In stem cell transplantation patients the major considerations in the differential diagnosis include sepsis-related cholestasis, hyperacute graft-versus-host disease, tumor infiltration, and cardiac failure. Cholestasis may also be caused by antibiotics, antifungals, ciclosporin, and parenteral nutrition. Although graft-versus-host disease usually occurs later than the typical time frame for SOS due to cyclophosphamide-containing regimens, hyperacute graft-versushost disease may be difficult to differentiate from SOS. Finally, congestive hepatopathy, e.g., due to cardiotoxic anticancer drugs, will also cause hepatomegaly, jaundice, and portal hypertension.
DIAGNOSTIC STUDIES Laboratory Studies Hyperbilirubinemia is a sensitive indicator of SOS, but jaundice post-transplantation may be due to other causes, such as sepsis,
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acute graft-versus-host disease, ciclosporin, or hemolysis. The level of total serum bilirubin correlates with non-relapse mortality from various causes, including SOS in this population.64 Serum aminotransferases are elevated in SOS and peak weeks after the treatment with the chemotherapeutic regimen. Serum aspartate aminotransferase >750 U/l carries a poor prognosis.65,66 Therapeutic monitoring and dose adjustment of busulfan may decrease the incidence of SOS, although studies have not reported a significant improvement in survival or relapse rate.67–69 Adjustment of cyclophosphamide dosing based on therapeutic monitoring of the metabolite, carboxyethylphosphoramide mustard, may reduce mortality rates.41,70
Imaging Studies Ultrasonography can confirm hepatomegaly and ascites, can exclude tumor infiltration in the liver parenchyma and vasculature, and can detect biliary tract disease, but probably does not improve the diagnostic yield over and above the use of clinical criteria in the early phase of SOS.71–73 Early studies suggested that increased resistive index of the hepatic artery and decrease or reversal of flow in the portal vein by duplex ultrasonography might be indicators of SOS. Two prospective studies that examined patients before and after stem cell transplantation could not confirm that any of these indicators were diagnostic,71,72 although the presence of these ultrasound findings may be supportive of the diagnosis. Liver biopsy may be particularly helpful in distinguishing SOS from graft-versus-host-disease. Percutaneous or laparoscopic liver biopsy carries a high risk in patients with thrombocytopenia posttransplantation. The risk from transvenous liver biopsy is lower than that of percutaneous liver biopsy, but mortality due to delayed bleeding from capsular perforation, subcapsular hematomas, and hemorrhage at the venepuncture site has been reported to be as high as 5% in post-transplantation patients.74 Tissue specimens obtained by the transvenous route are smaller than those obtained percutaneously, but the transvenous approach also allows measurement of the hepatic venous pressure gradient. A hepatic venous pressure gradient >10 mmHg has a specificity of 90%. Histological Features. The characteristic histologic features of SOS are found in the centrilobular region of the liver. These features are sinusoidal congestion, hepatocyte necrosis, subendothelial or periadventitial fibrosis of the central vein, and sinusoidal fibrosis. Not all features need to be present to make the diagnosis, but the presence of more of the histologic abnormalities correlates with more severe clinical SOS.14 As mentioned earlier, involvement of the central vein was the hallmark feature first identified in this disease. However a significant number of patients who develop SOS after myeloablative therapy do not have involvement of the central vein.14
DIFFERENTIAL DIAGNOSIS Patients with SOS present with jaundice, hepatomegaly, and weight gain. As described above, there are various causes of hyperbilirubinemia in the transplant setting that lead to jaundice, but in isolation these will not cause weight gain and will not usually be associated with hepatomegaly. The symptoms of SOS may be mim-
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icked by jaundice due to sepsis or cholestatic liver disease in association with intravenous hydration for hypotension causing weight gain or in association with congestive heart failure causing weight gain and hepatomegaly.
CLINICAL COURSE AND PROGNOSIS SOS is frequently classified as mild, moderate, or severe. Clinically, mild SOS is defined as SOS that requires no treatment and that resolves completely; moderate SOS requires medical therapy but resolves completely; and severe SOS requires treatment but does not resolve by day 100 or lead to the death of the patients. This classification is of course after the fact. Mortality from all causes by day 100 after infusion of stem cells in patients with mild, moderate, and severe SOS is 3, 20, and 98%, respectively.34 Using mathematical modeling, the severity of SOS due to cyclophosphamide-containing regimens can be predicted based on bilirubin levels and weight gain,75 but these models may not apply to regimens without cyclophosphamide. Poor prognostic features include higher serum transaminases, portal vein thrombosis, higher wedged hepatic venous pressure gradient, renal insufficiency, and hypoxia. The commonest cause of death from severe SOS is multiorgan failure, usually from renal and cardiopulmonary failure.
CLINICAL FEATURES OF SOS UNRELATED TO MYELOABLATIVE THERAPY SOS may also occur due to ingestion of pyrrolizidine alkaloids present in food sources, teas, or herbal supplements. Pyrrolizidine alkaloids are present in various botanically unrelated plant species such as Senecio, Crotalaria, and Heliotropium. In non-western nations, these pyrrolizidine alkaloids may contaminate inadequately winnowed wheat or may be present in bush teas. SOS after ingestion of pyrrolizidine alkaloids has a different time course than the chemoirradiation-induced syndrome: onset may be after 1–2 months of ongoing exposure and evidence of liver injury can persist for several months to years after the exposure. The features of SOS in this setting are hepatomegaly, ascites and, sometimes, abdominal pain, but jaundice is absent or mild. The histological features of SOS in this setting are sinusoidal congestion, hepatocyte necrosis, fibrotic occlusion of the central vein, and sinusoidal fibrosis. Another form of hepatic veno-occlusive disease is RILD. RILD occurs in adults after a mean liver dose of 31 Gy of conventional fractionation of irradiation.32,33 After partial hepatectomy the threshold dose of irradiation is lower. Three-dimensional radiation therapy treatment planning allows much higher doses of radiation to be delivered to the liver with a low incidence of RILD.76 Onset of RILD is usually 1–2 months after irradiation and the signs and symptoms of liver injury can persist for months. The clinical manifestations of RILD are hepatomegaly, ascites, and weight gain, but bilirubin elevations are minimal and the right upper quadrant pain is much less pronounced than in SOS after chemoirradiation. The histological features of RILD in the first 2 months after irradiation are centrilobular dropout of hepatocytes, congestion, and hemor-
Chapter 46 SINUSOIDAL OBSTRUCTION SYNDROME
rhage. In later months the atrophy of the hepatic cords persists, but congestion is minimal and there may be fibrotic occlusion of the central veins.77 Fibrin has been demonstrated by electron microscopy in the central veins after RILD,78 but not after SOS due to chemoirradiation or pyrrolizidine alkaloids.
PREVENTIVE STRATEGIES AFTER MYELOABLATIVE THERAPY The single most important strategy for preventing severe SOS is to avoid the use of high-risk myeloablative regimens in patients with major risk factors. The major risk factors for fatal SOS are: (1) SOS during a previous exposure to chemoirradiation; (2) a second stem cell transplant with myeloablative conditioning; (3) a short interval between gemtuzumab ozogamicin and stem cell transplantation;21 (4) viral hepatitis with elevated serum transaminases;34,79 and (5) extensive hepatic fibrosis or cirrhosis. The risk of SOS is substantially lower after non-myeloablative regimens.38,40,80,81 The commonest causes of hyperbilirubinemia after non-myeloablative regimens are graft-versus-host disease, sepsis, or a combination of both.40 In patients with hepatic fibrosis the risk for fatal liver decompensation remains high after non-myeloablative regimens.40 Although non-myeloablative regimens reduce overall hepatic toxicity, long-term studies will need to determine the effect on tumor relapse, graft-versus-host-disease, and infection. Use of prophylactic heparin to prevent SOS is standard practice in some stem cell transplantation centers. Prophylactic lowdose heparin can be used safely with careful monitoring of partial thromboplastin time;82,83 low-molecular-weight heparin is also safe in this setting.84,85 Two randomized studies showed a decrease in overall SOS with prophylactic low-dose heparin, but the studies were not powered to determine whether the incidence of fatal SOS was reduced.83,86 Four other studies did not find a reduction in overall SOS in patients treated with prophylactic heparin.82,87–89 Defibrotide is a large single-stranded polydeoxyribonucleotide that has numerous poorly understood effects. Defibrotide has an anticoagulant effect by reducing plasminogen activator inhibitor, increasing endothelial tissue plasminogen activator, and reducing endothelial expression of tissue factor. Defibrotide reduces leukocyte recruitment by reducing leukocyte rolling and adherence to endothelial cells. In two uncontrolled trials, prophylactic use of defibrotide reduced the incidence of SOS compared to historical controls,90,91 but this will need to be confirmed in randomized, controlled studies. Pentoxifylline, ursodeoxycholic acid, and prostaglandin E1 each initially showed promise as prophylactic agents. Several randomized controlled studies have found no effect of pentoxifylline.92,93 A large randomized study showed no benefit of ursodeoxycholic acid.94 Two studies found no effect of prostaglandin E1 in preventing fatal SOS.95,96 In summary, the best preventive strategy at present is to avoid high-risk myeloablative regimens in individuals at the greatest risk for SOS. There are currently no other prophylactic strategies that have been shown by randomized controlled studies to prevent fatal SOS.
TREATMENT FOR SOS AFTER MYELOABLATIVE THERAPY Some 70–80% of patients with SOS will survive without intervention or with conservative management. Fluid retention and ascites are treated with sodium restriction, diuretics, and therapeutic paracentesis as needed. In patients with multiorgan failure, hemodialysis and mechanical ventilation may be used, but these are unlikely to alter the outcome. Several interventions have been tried for severe SOS. Tissue plasminogen activator heparin infusion may improve the outcome in fewer than 30% of patients with severe SOS, but should not be used in patients at increased risk for pulmonary or intracerebral hemorrhage or in patients with renal or pulmonary failure. Defibrotide (see previous section) has been used therapeutically in uncontrolled trials of adults and children with severe SOS with multiorgan failure. The survival to day 100 post-transplantation in patients with severe SOS treated with defibrotide therapy was 35% (note: survival of severe SOS is historically around 5%).97,98 There have been case reports of improvement with treatment with Nacetylcysteine, prostaglandin E1, prednisone, topical nitrate, and vitamin E/glutamine, but these drugs have not been evaluated in formal studies. Transjugular intrahepatic portosystemic shunt (TIPS) has been used in SOS after stem cell transplantation to reduce portal pressure and to treat ascites.99–105 However, TIPS does not appear to alter the prognosis,101–103,106 and has been complicated by fatal acute respiratory distress syndrome.107 Two children who developed SOS underwent surgical portosystemic shunts that were effective in the treatment of ascites, but in both of these cases the surgical shunts were placed after resolution of liver dysfunction.108,109 It is uncommon for SOS to occur in the setting of stem cell transplantation for a benign disorder. When severe SOS occurs in a patient transplanted for a benign condition or in a patient with a malignancy with a good prognosis, liver transplantation may be an option.
CONCLUSIONS In North American and western Europe SOS most commonly occurs due to myeloablative chemotherapy prior to stem cell transplantation for malignancy. SOS is therefore an iatrogenic disease with a high case fatality rate. The incidence of the disease has declined in recent years as chemotherapy regimens, such as the nonmyeloablative regimens, have been chosen to avoid the risk of SOS. However it remains to be seen whether overall survival will improve or whether an increase in graft-versus-host disease, infection, and relapse mortality will offset the gains made by avoiding regimens with a high risk for SOS. Prophylactic medical interventions have not shown benefit and the main strategy to prevent SOS remains avoidance of high-risk regimens in individuals with risk factors for SOS. Management remains largely conservative. The most successful medical interventions that have been studied have shown efficacy in about one-third of patients in uncontrolled trials. Thus the challenge for the future
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will be to use our improved understanding of the pathophysiology to devise novel prophylactic and/or therapeutic strategies.
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Chapter 46 SINUSOIDAL OBSTRUCTION SYNDROME
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78. Fajardo LF, Colby TV. Pathogenesis of veno-occlusive liver disease after radiation. Arch Pathol Lab Med 1980; 104:584–588. 79. Locasciulli A, Testa M, Valsecchi MG, et al. The role of hepatitis C and B virus infections as risk factors for severe liver complications following allogeneic BMT: a prospective study by the Infectious Disease Working Party of the European Blood and Marrow Transplantation Group. Transplantation 1999; 68:1486–1491. 80. Nagler A, Or R, Naparstek E, et al. Second allogeneic stem cell transplantation using nonmyeloablative conditioning for patients who relapsed or developed secondary malignancies following autologous transplantation. Exp Hematol 2000; 28:1096– 1104. 81. Feinstein L, Sandmaier B, Maloney D, et al. Nonmyeloablative hematopoietic cell transplantation. Replacing high-dose cytotoxic therapy by the graft-versus-tumor effect. Ann NY Acad Sci 2001; 938:328–337. 82. Bearman SI, Hinds MS, Wolford JL, et al. A pilot study of continuous infusion heparin for the prevention of hepatic venoocclusive disease after bone marrow transplantation. Bone Marrow Transplant 1990; 5:407–411. 83. Attal M, Huguet F, Rubie H, et al. Prevention of hepatic venoocclusive disease after bone marrow transplantation by continuous infusion of low-dose heparin: a prospective, randomized trial. Blood 1992; 79:2834–2840. 84. Or R, Nagler A, Shpilberg O, et al. Low molecular weight heparin for the prevention of veno-occlusive disease of the liver in bone marrow transplantation patients. Transplantation 1996; 61:1067–1071. 85. Forrest DL, Thompson K, Dorcas VG, et al. Low molecular weight heparin for the prevention of hepatic veno-occlusive disease (VOD) after hematopoietic stem cell transplantation: a prospective phase II study. Bone Marrow Transplant 2003; 31:1143–1149. 86. Rosenthal J, Sender L, Secola R, et al. Phase II trial of heparin prophylaxis for veno-occlusive disease of the liver in children undergoing bone marrow transplantation. Bone Marrow Transplant 1996; 18:185–191. 87. Marsa-Vila L, Gorin NC, Laporte JP, et al. Prophylactic heparin does not prevent liver veno-occlusive disease following autologous bone marrow transplantation. Eur J Haematol 1991; 47:346–354. 88. Hagglund H, Remberger M, Klaesson S, et al. Norethisterone treatment, a major risk-factor for veno-occlusive disease in the liver after allogeneic bone marrow transplantation. Blood 1998; 92:4568–4572. 89. Carreras E, Bertz H, Arcese W, et al. Incidence and outcome of hepatic veno-occlusive disease after blood or marrow transplantation: a prospective cohort study of the European Group for Blood and Marrow Transplantation. European Group for Blood and Marrow Transplantation Chronic Leukemia Working Party. Blood 1998; 92:3599–3604. 90. Chalandon Y, Roosnek E, Mermillod B, et al. Prevention of veno-occlusive disease with defibrotide after allogeneic stem cell transplantation. Biol Blood Marrow Transplant 2004; 10:347–354. 91. Versluys B, Bhattacharaya R, Steward C, et al. Prophylaxis with defibrotide prevents veno-occlusive disease in stem cell transplantation after gemtuzumab ozogamicin exposure. Blood 2004; 103:1968. 92. Clift RA, Bianco JA, Appelbaum FR, et al. A randomized controlled trial of pentoxifylline for the prevention of regimenrelated toxicities in patients undergoing allogenic marrow transplantation. Blood 1993; 82:2025–2030. 93. Attal M, Huguet F, Rubie H, et al. Prevention of regimen-related toxicities after bone marrow transplantation by pentoxifylline – a prospective, randomized trial. Blood 1993; 82:732–736.
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Section VII: Vascular Disease of the Liver
47
PORTAL AND SPLENIC VEIN THROMBOSIS Hector Rodriguez-Luna and Hugo E. Vargas Abbreviations AT III Antithrombin III DUS Doppler ultrasound FVL Factor V Leiden HVPG Hepatic venous-portal gradient IPVT Isolated portal vein thrombosis
LT MRA MTHFR C677?T PTHRA20210
Liver transplantation Magnetic resonance angiogram Methylenetetrahydrofolate reductase Factor II prothrombin
PORTAL VEIN THROMBOSIS Portal vein thrombosis (PVT) was first reported by GW Balford and TG Stewart in 1869 in a patient who presented with ascites, splenomegaly, and varices.1 PVT is a rare condition affecting both children and adults with equal gender distribution, and is typically associated with myriad precipitating factors and subtle acute clinical manifestations.2 PVT represents the classic form of presinusoidal (infrahepatic) portal hypertension. In western countries this entity is the leading cause of extrahepatic portal hypertension in noncirrhotic patients.3 The incidence of PVT is not clearly defined and varies depending on the group of patients studied and the diagnostic methods used. In the United States, the overall incidence ranges from 0.05% to 0.5% in autopsy studies. Reported prevalence in candidates for liver transplantation (LT) with cirrhosis is between 0.6% and 26%.7,10 In patients with cirrhosis, the incidence of PVT at the time of LT has been reported to range from 10% to 21%.4–6 In Japan, Okuda et al. reported an incidence of 0.6% by angiography of 708 cirrhotic patients.7 After LT, the incidence of PVT varies from 1% to 2%.8,9
PATHOPHYSIOLOGY Under normal circumstances the portal vein (PV) contributes twothirds of the hepatic blood supply. However, PV occlusion with thrombosis often produces few acute clinical consequences or laboratory manifestations.2,12,13 Two mechanisms account for this ability of the liver to survive the loss of portal perfusion. The first consists of an arterial ‘buffer’ response manifested by immediate vasodilatation of the hepatic arterial bed in response to decreased portal vein flow.12,14,15 The second is the relatively rapid development of collateral veins that bypass the thrombosed portion of the PV (cavernous transformation).16 The latter process may take up to 12 months, although it has been reported as early as 5 weeks after the thrombotic event.16,17 Other collateral veins may also develop within the walls or at the periphery of the structures adjacent to the obstructed portion of the portal vein, such as the bile ducts, gallbladder, pancreas, gastric antrum, and duodenum.18 These collateral veins may
PV PVT SMV
Portal vein Portal vein thrombosis Superior mesenteric vein
alter the appearance of these surrounding structures during imaging, and occasionally lead to erroneous diagnoses of bile duct or pancreatic tumor, pancreatitis, or cholecystitis. In some instances these cavernous vessels can have clinical consequences: bile duct varices have been reported to cause obstructive jaundice.18 As a result of these hemodynamic compensations, the total hepatic blood flow is only minimally reduced, hepatic venous pressure gradient (HVPG) is initially preserved at normal levels, and the portal pressure is elevated.15 The increase in portal pressure allows portal perfusion to be maintained through the collateral veins. This initial state is not static, and portal pressure will increase further over time. The risk for bleeding esophageal varices develops when the HVPG rises to a threshold value of 10–12 mmHg.19 PVT patients can also experience the development of a compensatory hyperdynamic circulatory state akin to that seen in cirrhosis.15 The overall consequences of PVT are related to thrombus extension. Below the thrombus, there is little effect on the intestines so long as the mesenteric venous arches remain patent. Mesenteric ischemia results from extension of the thrombus into the mesenteric venous arches.12 When the ischemia is prolonged, intestinal infarction will ensue. In 20–50% of cases intestinal infarction is responsible for death due to peritonitis and multiple organ failure, even when the infarcted gut is resected.20,21 Above the thrombus, the consequences of PVT to the liver are hardly discernible and there are minimal laboratory abnormalities.22 Clinically, acute signs of liver disease are absent or transient unless the PVT occurs in a patient with cirrhosis.12 Concomitant PVT may be seen in 20% of patients with Budd–Chiari syndrome, and this may worsen their liver disease.23 PVT can be classified anatomically into four grades according to where the thrombus extends.24 These grades are reflective of the clinical consequences of the thrombus and have an impact on the selection of medical and surgical management options. 1. Grade 1: Minimally or partially thrombosed PV, in which the thrombus is minimal or, at most, confined to 2%) FVL mutation PTHR20210 mutation (Factor II prothrombin) MTHFR C677ÆT mutation Antiphospholipid syndrome Acquired hypercoagulable states Inflammatory bowel disease Pregnancy Oral estrogens Miscellaneous Non-cirrhotic portal hypertension Abdominal surgery, shunt surgery Splenectomy Liver transplant
acts in conjunction with factors VII/VIIa to activate factor X. The enzymatic function of cancer procoagulant is the activation of factor X. Another postulated procoagulant mechanism in cancer patients is impaired fibrinolysis, with a subsequent increase in plasminogen activator inhibitor.25,34
Myeloproliferative Disorders The hypercoagulable state due to myeloproliferative disorders accounts for 3–12% of adult patients with PVT.2,30,35 Some patients with idiopathic PVT have a latent myeloproliferative disorder that becomes evident only years after the diagnosis of PVT. Valla and coworkers found that 48% of adult patients with non-malignant PVT originally classified as idiopathic had either overt or latent myeloproliferative disorders.30
Infection In the adult population, infection accounts for 10–25% of PVT cases in non-cirrhotic, non-cancer patients.2 Septic PVT (pyelophlebitis) is usually related to appendicitis, cholecystitis, or diverticulitis.1,29,31
Chapter 47 PORTAL AND SPLENIC VEIN THROMBOSIS
However, PVT as a result of infection is infrequent in the adult population, with a decreasing incidence because of earlier diagnosis and earlier initiation of effective antibiotic therapy.36 Interestingly, Bacteroides species bacteremia of unknown origin is so strongly associated with PVT that culture of this organism from the blood should prompt a search for portal or mesenteric vein thrombosis.12,37 In children, infection is the most common etiologic factor for PVT, accounting for 43–52% of all cases.31 Neonatal umbilical sepsis, the single most frequent infectious cause, is present in 10–26% of children with PVT.1,2,12,31 Neonatal thrombosis is well documented after omphalitis or umbilical vein cannulation complicated by septic pyelophlebitis. However, infants with infection of the umbilical vein in the absence of prothrombotic disorder infrequently go on to develop PVT.38 The first clinical manifestations of neonatal PVT are frequently delayed until adulthood.
intrahepatic portosystemic shunt (TIPS) carries a risk for PVT of approximately 10%.55 PVT following endoscopic variceal ligation or sclerotherapy was a matter of controversy in the past, as most published studies did not document the patency of portal vessels prior to the initiation of therapy.56,57 In an effort to further clarify this issue, Politoske et al. studied the incidence of PVT in patients treated with sclerotherapy and band ligation for variceal bleeding in cases without pre-existing PVT; the study found no significant difference between the groups after therapy.58 Finally, several authors have suggested an association between PVT and congenital cardiovascular abnormalities, such as atrial septal defect, ventricular septal defect, and deformed inferior vena cava.59
CLINICAL MANIFESTATIONS Thrombophilias Inherited or acquired prothrombotic states may predispose to the development of PVT. The presence of more than one deficiency seems to be the rule rather than the exception.26,27,33 Inherited prothrombotic disorders are subclassified into two groups according to the prevalence in the population. The first group includes deficiencies in protein C, protein S, and antithrombin III (AT III). The prevalence of these deficiencies is very low in the general Caucasian population (2%) in the general population. FVL gene mutation and deficiencies of anticoagulant proteins have been associated with PVT.33,45–47 Janssen et al. reported that the relative risk of PVT for individuals with FVL mutation was 2.7, 1.4 for those with PTHR A20210, and 4.6 for those with protein C deficiency. Protein C and S deficiency has been reported in up to 30% of cirrhotic patients with PVT.45 Antithrombin III deficiency has been less frequently associated with PVT.27,45,47 Antiphospholipid syndrome has been reported in up to 11% of patients with PVT.27
Other Inflammatory disorders such as pancreatitis and inflammatory bowel disease (IBD) have also been implicated in PVT.30,39 Pancreatitis accounts for 3–5% of cases of PVT, via either a contiguous inflammatory process, direct compression of the PV by a pseudocyst, or a combination of both. Chronic pancreatitis can also lead to splenic vein thrombosis and a unique form of ‘left-sided’ segmental portal hypertension with the development of isolated gastric varices.30,40 Other associated factors include pregnancy and oral intake of estrogens.48,49 PVT can also be seen in the setting of blunt abdominal trauma, surgery in the absence of septic complications,2 or non-surgical treatment for hepatocellular carcinoma, such as radiofrequency ablation or microwave coagulation therapy.50 Splenectomy carries a PVT risk ranging from 0.7 to 8%.51–54 In patients with underlying myeloproliferative disorder or cirrhosis, splenectomy carries a particularly increased risk of PVT, ranging from 13–18%.51,54 Transjugular
Patients with PVT typically present with abdominal pain, increased abdominal girth, or, more dramatically, hematemesis.2,12,13 Neonatal PVT can present many years later with complications of the ensuing portal hypertension, such as ruptured gastroesophageal varices or splenomegaly.1,2,59 Most patients with PVT diagnosed before the index gastrointestinal bleed eventually go on to bleed within a mean of 4 years from diagnosis.1 However, 10% of patients with PVT never bleed.1 The presence of acute-onset abdominal pain can be an ominous sign and should prompt aggressive work-up for bowel ischemia.2 Bowel ischemia patients may also present with gastrointestinal bleeding. Other common complaints of patients with PVT include nausea, vomiting, diarrhea, anorexia, weight loss, and abdominal distention.28,31,59 Some patients may also experience lowgrade fever.51 Portosystemic encephalopathy is rare unless the patient has underlying liver disease. A rare presentation of PVT is the occurrence of bile duct compression due to collateral veins which develop in the hepatoduodenal ligament.18,60 On physical examination, splenomegaly is seen in 75–100% of cases. Hepatomegaly may also be present. Ascites is an uncommon finding in PVT, and when present is usually mild and transient. Ascites typically develops immediately after the thrombotic event, before the patient has had time to develop a collateral circulation.22,28,59 A rare presentation of PVT is bile duct compression due to collateral veins which develop in the hepatoduodenal ligament.18,60 Some patients may also experience low-grade fever.51 Hepatic enzymes and liver injury tests are usually within normal limits in patients without underlying liver disease.2,12 However, mild elevations in transaminases, alkaline phosphatase, and bilirubin have been reported.12 A mild decrease in red cell count, white cell count, and platelets due to hypersplenism may also be seen.2,13,28,59 Histologically, there is little alteration in the hepatic architecture when the obstruction is limited to the extrahepatic portal vein and its largest intrahepatic branches. Non-cirrhotic patients typically show normal histology, with increased reticulin around the portal tracts. Experimentally, apoptosis of the liver cells can be demonstrated in rats with graded portal vein ligation.61 The degree of apoptosis is related to the grade of portal vein obstruction. There is a simultaneous increase in mitotic activity in the remaining well-perfused liver.61,62 Wanless and colleagues have postulated a mechanism by which the vascular changes seen with chronic PVT may lead to the development of venoportal bridging fibrosis and eventual cirrhosis.63
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NATURAL HISTORY The natural history of PVT remains shrouded in mystery, as the patient population is heterogeneous and the management is predicated by the time of diagnosis and the underlying etiology. The clinical course is characterized by repeated bouts of variceal hemorrhage, with an average of 2.5–5 episodes per patient.64,65 In an analysis of non-cirrhotic non-neoplastic PVT, the incidence of gastrointestinal bleeding was found to be approximately 12.5 per 100 patient-years. In this study, the only independent predictive factor for bleeding was the size of the esophageal varices.66 In cases of neonatal PVT, the bleeding episodes tend to increase in severity and frequency at puberty, followed by abatement after the development of spontaneous splenorenal or splenogastric shunts in 10–20% of patients.60 The overall prognosis for patients with chronic PVT and recurrent gastrointestinal bleeding in the absence of cirrhosis or malignancy is good, with a mortality rate of approximately 10%.2,67 In a Dutch study, non-cirrhotic patients with PVT had an overall survival rate of 70% at 1 year and 63% after 5 years.68 In the case of acute PVT due to intra-abdominal sepsis, pre-existing liver disease, or abdominal surgery, the mortality rate approaches 50%.31 In children the prognosis is much better, with a 10-year survival rate greater than 70%. Extensive PVT with mesenteric venous thrombosis with bowel infarction is invariably fatal without prompt surgical intervention. Mortality in this scenario can approach 20% even with expedient bowel resection.69
High index of suspicion for portal vein thrombosis
Portal vein Doppler ultrasound
PVT confirmed
PVT ruled out
Evaluate patient for risk factors Refer to Table 1 Screen for portal hypertension Establish age of thrombus
Non-diagnostic ultrasound
MRI or CT scan of the hepatic vessels to confirm or rule out PVT
Determine therapy: thrombolytics vs. chronic anticoagulation vs. no treatment Treat portal hypertension according to manifestations Figure 47-1. Suggested diagnostic work-up for patients with suspected PVT. Refer to text for further details on diagnostic work-up.
DIAGNOSIS The key to the diagnosis of PVT is a high index of suspicion (Figure 47-1). For confirmation, a variety of radiologic techniques can be used to investigate the suspected thrombosis. Invasive angiographic techniques, such as ‘indirect’ portography (venous phase of superior mesenteric artery angiogram) and ‘direct’ portal venography (transhepatic or transjugular) are the time-honored diagnostic techniques for PVT.70 However, a variety of non-invasive techniques, such as color Doppler ultrasound (DUS), computed tomographic angiography (CT angiography), and magnetic resonance angiography (MRA), have become available for the screening of patients suspected of having PVT.70–75 Nowadays, ultrasonography is the first-line diagnostic modality because of its accuracy, affordability, and non-invasiveness. An echogenic thrombus within the portal lumen is the key finding for the ultrasonographic diagnosis of PVT.76 Other signs include dilatation of the proximal vessel, the presence of collateral vessels (best seen near the porta hepatis), or an unidentifiable portal vein.74–76 The lack of variation in portal venous diameter with respiration, coupled with a portal vein diameter greater than 13–15 mm, is also highly indicative of portal vein occlusion. These hallmarks may be less reliable when the thrombus is long-standing. The sensitivity of ultrasonography ranges from 70 to 90%, with a specificity of 99%.24,75 The presence of arterial flow signal in the thrombus typically correlates with a malignant thrombus.75 Major limitations to ultrasonography include obesity, fatty liver, and non-visualization secondary to bowel gas. In addition, ultrasonography is operator dependent, and PVT might be missed if the examiner is not specifically asked to look for it.74–76 CT scans can be used to confirm and follow the course of PVT. On CT scan, the thrombus within the portal vein shows decreased
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intraluminal density (filling defect in the contrast-enhanced lumen) or as total portal occlusion with or without the development of periportal collaterals creating a ‘train-track’ appearance on enhancement. This ‘train-track’ appearance is associated with proliferation of the vasa vasorum and is associated with old thrombus. A nonenhanced CT scan of the liver will show a high luminal density within the portal vein when the thrombus is less than 10 days old.77 The presence of periportal collaterals suggests that the occlusion is chronic and the thrombus is organized, also known as ‘cavernous transformation’ of the portal vein77 (Figure 47-2). This process may take up to 12 months to occur, although it has been demonstrated as early as 5 weeks after the thrombotic event.12 Contrast-enhanced CT has the advantage over ultrasound of displaying varices (sensitivity 65–85%) and parenchymal hepatic abnormalities.71 The falsepositive rate in one small series was 16%, possibly owing to poor bolus injection. CT is not operator dependent; however, the radiation dose, cost, and need for intravenous contrast make it a less than ideal test.71,78,79 Contrast-enhanced MR angiography, spin-echo MR, and gradientecho MR have been introduced for the diagnosis of PVT. Spin-echo MR images usually shows PVT as an area of abnormal signal within the lumen of the portal vein. PVT appears hyperintense on T1 and T2 images when the thrombus has been formed recently. Old thrombus appears isointense on T1 images. Gradient-echo MR gives a sharper delineation of vascular structures and helps clarify any confusion on spin-echo images. MR angiography shows flow patterns and patency of the portal vein. Tumor thrombi can be differentiated from bland thrombi because they appear more hyperintense on T2weighted images and enhance with gadolinium. The sensitivity of MRI is 85% and the specificity is 90–95%.72,80
Chapter 47 PORTAL AND SPLENIC VEIN THROMBOSIS Figure 47-2. High-resolution CT scan coronal reconstruction of portal anatomy in a patient with ductal adenocarcinoma of the body of the pancreas. Note the portal vein occlusion with cavernous collateral transformation (narrow arrow). In addition, the patient has liver metastases with bile duct obstruction and malignant ascites. (Photo courtesy of Dr Joseph Collins.)
More invasive radiographic techniques are currently reserved for cases when non-invasive testing is inconclusive, immediately before anticipated percutaneous interventional treatment, or when a meticulous preoperative assessment is necessary.70
ISOLATED SPLENIC VEIN THROMBOSIS Isolated splenic vein thrombosis (ISVT) deserves special attention as the etiology and clinical manifestations differ from those of PVT. ISVT usually results in left-sided portal hypertension and isolated gastric fundal varices.81 The most common cause of ISVT is chronic pancreatitis, with a reported incidence up to 45%.82 Pancreatitis-associated ISVT is believed to result from perivenous inflammation.82,83 The prevalence of splenic vein complications in relation to the CT scan severity index of pancreatitis has shown an inversely proportional significant increase in the prevalence of thrombosis.83 Other known factors include pancreatic masses and cancer, splenectomy,84 portal hypertension, renal disorders, and inflammatory disorders.81 The diagnostic test of choice to assess the presence of ISVT is late-phase celiac angiography.82 However, endoscopic ultrasonography (EUS) has emerged as a fairly sensitive and non-invasive diagnostic tool.85,86 Splenoportography was previously used to make this diagnosis (Figure 47-3).
The natural history of ISVT is uncertain and literature reports are few. Most cases of ISVT are asymptomatic and require no treatment.82 In an effort to further define the natural history of pancreatitis-induced ISVT, Heider and co-workers87 studied 53 patients with a history of pancreatitis and ISVT and found that 77% of isolated gastric varices were evident on CT scanning, 31% by esophagogastroduodenoscopy (EGD), and 28% by the combined modalities. The risk of variceal bleeding was 5% for patients with CT-identified varices and 18% for EGD-identified varices.87 Of those patients, only 4% had a gastric variceal bleeding episode and required splenectomy.87 Other investigators have reported a gastric variceal bleeding risk of approximately 10%.88 The treatment of ISVT is conservative, given the low risk of associated gastric variceal bleeding.87 Once there is an index episode of gastrointestinal bleeding, splenectomy is the treatment of choice.82 The surgical and medical teams need to ensure that hepatic fibrosis has been carefully evaluated. Splenectomy in the setting of cirrhosis should be avoided if possible, as the development of PVT post splenectomy may complicate or eliminate the opportunity for liver transplantation in the future.
LIVER TRANSPLANTATION AND PVT In the past, PVT has been considered a relative contraindication to liver transplantation because of the technical difficulties it added to
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Figure 47-3. Splenoportogram of a patient with splenic vein thrombosis. The needle is within the spleen (closed arrow). Contrast material can be seen to flow from the spleen to the portal vein (PV) via large collaterals (C). The pool of contrast material (open arrow) is within numerous gastric varices.
the procedure. However, in recent years innovative surgical techniques have been introduced and many technical obstacles have been overcome.89–94 As a result, patients with non-neoplastic PVT routinely undergo LT. The first successful LT in a patient with PVT using venous conduits to bypass the thrombotic segment was reported in 1985.93 Since then, many centers have reported several techniques to tackle the problem of PVT in liver transplant candidates. The surgical technique to re-establish portal blood flow in liver transplant recipients with PVT depends on the extent of the thrombosis and the experience of the transplant team. Partial PVT with less than 25% luminal obstruction has no clinical repercussions because it can be treated with resection, whereas more than 25% luminal obstruction requires extensive thrombectomy.95 Thromboendovenectomy and/or direct venous anastomosis has been performed in patients when the thrombosis involves the portal vein with or without extension into the SMV. If the portal vein is completely thrombosed and the proximal SMV is occluded but the distal part is patent, then the preferred method is a graft to the proximal SMV using donor iliac vein.6,24,94 In cases where the portal vein is not amenable to thrombectomy and the SMV is thrombosed, the coronary vein or any large accessible collateral vein can be used to join the donor portal vein to the SMV.94 For patients with extensive and complete occlusion of the portal vein and SMV, cavoportal hemitransposition (a procedure that diverts caval blood to the liver as replacement of portal inflow and without compromise of hepatic function) has been described as an innovative and successful procedure.90 De novo PVT after liver transplantation is rare and usually occurs at the anastomotic site in the early postoperative period. In patients with pretransplant PVT the early outcome seems to be satisfactory, although there remains a risk of the portal vein rethrombosing which ranges from 4.2 to 38.5%.24,89 The greater risk for rethrombosis has been reported in grafts that were not preserved with University of Wisconsin solution, or when venovenous bypass was used.95 In an effort to prevent recurrence of PVT in this group of patients, therapeutic or prophylactic anticoagulation for 3 months has been advocated.6,95,96
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Liver transplant candidates with PVT, especially those who have more than 50% of the portal vein occluded with or without superior mesenteric vein (SMV) occlusion, are more prone to develop severe perioperative complications, and have a high mortality rate and decreased long-term survival.89 The mortality rate is influenced by the extent of thrombosis before liver transplantation. Mortality has been reported to be greater in patients with PVT and splanchnic vein involvement than in those with only PVT (45.5% vs 36.1%) or associated periphlebitis (83.3% vs 9.4%). Nevertheless, the vast majority of patients with PVT can be technically transplanted, with a survival comparable to that of patients without PVT.89 Acute graft failure due to early occurrence of PVT after liver transplantation, bleeding from esophageal varices, and massive ascites can be serious complications from PVT after liver transplantation.89
MANAGEMENT A thorough etiological investigation and assessment of thrombus chronicity is paramount in the management of PVT in order to identify those conditions amenable to treatment and to tailor treatment. Investigation of the local factors is carried out with Doppler ultrasonography, MRI scan, abdominal CT, or endoscopic ultrasound. When portal cavernous transformation is recognized, portal hypertension can be assumed. Investigation of general thrombophilic factors must be comprehensive because in most patients there are usually several factors that contribute to the hypercoagulable state. Factors such as myeloproliferative disorders should be systematically investigated as they can have subtle presentations before becoming fully apparent on hematological grounds. One feature of a myeloproliferative disorder that may be present before the disease is clinically manifest is the spontaneous formation of erythroid colonies on culture of the circulating or bone marrow precursors in the absence of erythropoietin added to the culture medium. A similar test has also been developed for spontaneous colonies of megakaryocytes.30 Where these tests are not easily available, diagnostic information can also be obtained using isotopic determination of the total red cell volume
Chapter 47 PORTAL AND SPLENIC VEIN THROMBOSIS
coupled with determination of serum erythropoietin levels, provided that iron deficiency has been corrected. Bone marrow biopsy is another means to demonstrate primary myeloproliferative disorder when the peripheral blood picture is not suggestive, but this procedure is too invasive to serve as a screening procedure.12 Other considerations include coagulation factor gene mutations, coagulation inhibitor deficiencies, and the antiphospholipid syndrome. Interpretation of the results of antithrombin, protein C and protein S is particularly difficult in the context of PVT because their plasma levels may be non-specifically decreased whenever there is underlying liver disease or coagulation activation. Therefore, comparisons with the results of prothrombin determination and familial studies are necessary before the conclusion of a primary (inherited) deficiency can be reached.12 Factor V Leiden mutations can be assessed directly using molecular techniques, or indirectly by evaluation of the resistance to activated protein C. Identification of factor II G20210A mutation requires molecular techniques. The antiphospholipid syndrome is diagnosed when high titers of antiphospholipid antibodies are found on two separate occasions, or when a lupus anticoagulant is demonstrated. However, determination of anti-/32 glycoprotein-1 antibodies may be both more sensitive and more specific than the first two tests.98 Hyperhomocysteinemia is difficult to ascertain once PVT has developed because the plasma level is dependent on normal hepatic function. The C677T mutation of the methylene tetrahydrofolate reductase gene is associated with an increased plasma homocysteine; however, it is not clear whether this genetic marker alone is as good a marker for the increased risk of thrombosis as is the plasma homocysteine level.26,44 Gastrointestinal lesions that may be a source of bleeding need to be identified for adequate prophylactic measures to be taken. To date, there has been no controlled study specifically addressing bleeding in the setting of PVT. However, the available uncontrolled data indicate that the measures of established efficacy in patients with cirrhosis in good condition, such as propranolol and endoscopic therapy, can be applied to patients with PVT.26,44,99–101 Therapy for active gastrointestinal bleeding should, likewise, follow the guidelines for patients with intrahepatic portal hypertension regarding sclerotherapy and band ligation.101 There is, however, a matter of concern about the use of vasoconstrictive agents. Theoretically, the profound decrease in splanchnic blood flow induced by bleeding and by the therapeutic vasoconstrictive agents may trigger recurrence, or favor the extension of thrombosis in the portal venous system and precipitate intestinal ischemia. Indeed, peripheral vasopressin infusion has been reported to cause portal and mesenteric vein thrombosis, leading to intestinal ischemia in bleeding cirrhotic patients.102
SURGICAL MEASURES The place of surgery and the optimal type of operation is still being debated. A shunting procedure that would efficiently and permanently decompress the portal venous system with a low risk of encephalopathy would appear ideal. Some authors report a success rate in excess of 80% in shunt procedures, with a rebleeding rate as low as 4%.11,64 Unfortunately, the risk of shunt thrombosis or stenosis is predictably high, between 8 and 24%.11,103 Indeed, several precipitating factors are often present: underlying thrombophilia, surgery for portal hypertension, and splenectomy. Only the largest
veins (superior or inferior mesenteric veins or splenic veins) should be used because of the high risk of shunt thrombosis when using smaller veins.104 However, veins as small as 4 mm can be used.103 Because it leaves the spleen in place and preserves portal perfusion with a lower risk of encephalopathy, distal splenorenal shunt appears most suited for cirrhotic patients.11 Unfortunately, the splenic vein is frequently involved in the thrombotic process. TIPS has been used for the control of intractable bleeding as a bridge to liver transplantation or in patients with non-cavernous PVT as an adjunct to thrombolysis.70,105,106 The Sugiura procedure (transthoracoabdominal esophageal transection) has also been used to manage PVT, but it carries a surgical mortality as high as 20%.107 Splenectomy, only indicated for the management of gastrointestinal bleeding from gastric varices associated with splenic vein thrombosis, is contraindicated in patients with PVT because it may preclude the option of splenorenal shunt surgery at a later stage if needed.109
MEDICAL MEASURES The role of anticoagulant therapy for PVT is not well understood, although a large body of data continues to accumulate. Ideally, recent and old PVT must be differentiated before instituting therapy, as the recent PVT might benefit from thrombolytic therapy.70 In a retrospective study, Condat et al. showed a beneficial role of anticoagulation in recanalization and prevention of thrombus extension in patients with cavernous transformation of the portal vein. A major observation was that anticoagulant therapy reduced the risk of thrombotic events by two-thirds without an increase in the risk or severity of bleeding. Therefore, they suggest anticoagulation in those patients with a demonstrable prothrombotic state, absent or small varices that have never bled, and no predictable bleeding sites outside the gastrointestinal tract.66,110 To what extent spontaneous recanalization can be expected is not known. Current experience suggests that it is possible but uncommon, whereas complete or extensive recanalization can be achieved with anticoagulant therapy in more than 80% of patients.12,111 Recanalization prevents ischemic intestinal injury in the short term and extrahepatic portal hypertension in the long term. Malkowski et al. reported on the efficacy of thrombolytic agents in 28 patients and concluded that if it is administered early after the diagnosis of PVT, 82% of patients will have restitution of portal vein flow. Of those, 36% will have complete recanalization and 46% partial recanalization, with normal hepatopedal flow. The greatest benefit was seen in those patients with PVT of less than 4 weeks’ duration.112 Septic pyelophlebitis represents a special case in which recanalization can follow effective antibiotic therapy even in the absence of anticoagulant therapy. Drainage of associated hepatic, pancreatic, or splenic abscesses to achieve faster control of infection is recommended to allow recanalization by removing the inflammatory process.70 In the case of established PVT with portal hypertension due to cavernous transformation, anticoagulant therapy increased neither the risk of gastrointestinal bleeding nor the severity of bleeding. Valla et al. found that there were no deaths due to bleeding with anticoagulant therapy and no recurrent thrombosis.30 Therefore there is mounting evidence of a positive benefit–risk ratio with anticoagulant therapy. Some investigators only recommend anticoagula-
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tion in specific clinical scenarios, such as a demonstrable prothrombotic state, concomitant mesenteric vein thrombosis, or portosystemic shunt (to prevent thrombosis).68 Successful thrombolytic therapies, with or without mechanical thrombectomy, have been reported by several investigators in case reports and small series. Thrombolytic agents can be infused via selective SMA or via the transhepatic route. The reported complication rate is low, ranging from none to rectal bleeding.70,113,114 There have been many reports of successful treatment of postliver transplantation PVT with percutaneous portal vein thrombolysis, angioplasty, and endovascular stent placement. Immediate retransplantation is required when serious deterioration of liver function occurs after early PVT.89 TIPS is not recommended because it reduces the effective portal flow and may deteriorate liver function further over the long term.97 In summary, PVT should be considered a clue to the presence of one or several prothrombotic disorders, whether or not a local precipitating factor is identified. Acute PVT can and probably should be treated with anticoagulation or thrombolytic agents in an effort to prevent extension of the thrombus, mesenteric vessel occlusion, and portal hypertension. On the other hand, chronic PVT should be treated conservatively with measures to control major consequences related to portal hypertension. The duration of anticoagulation therapy should be tailored to the identified predisposing factors.
REFERENCES 1. Webb LJ, Sherlock S. The aetiology, presentation and natural history of extrahepatic portal venous obstruction. Q J Med 1979;48:627–639. 2. Cohen J, Edelman RR, Chopra S. Portal vein thrombosis: a review. Am J Med 1992;92:173–182. 3. Belli L, Romani F, Riolo F, et al. Thrombosis of portal vein in absence of hepatic disease. Surg Gynecol Obstet 1989;169:46–49. 4. Nonami T, Yokoyama I, Iwatsuki S, Starzl TE. The incidence of portal vein thrombosis at liver transplantation. Hepatology 1992;16:1195–1198. 5. Lerut JP, Mazza D, van Leeuw V, et al. Adult liver transplantation and abnormalities of splanchnic veins: experience in 53 patients. Transpl Int 1997;10:125–132. 6. Stieber AC, Zetti G, Todo S, et al. The spectrum of portal vein thrombosis in liver transplantation. Ann Surg 1991;213:199–206. 7. Okuda K, Ohnishi K, Kimura K, et al. Incidence of portal vein thrombosis in liver cirrhosis. An angiographic study in 708 patients. Gastroenterology 1985;89:279–286. 8. Langnas AN, Marujo W, Stratta RJ, et al. Vascular complications after orthotopic liver transplantation. Am J Surg 1991;161:76–82;discussion 82–83. 9. Lerut J, Tzakis AG, Bron K, et al. Complications of venous reconstruction in human orthotopic liver transplantation. Ann Surg 1987;205:404–414. 10. Orozco H, Takahashi T, Mercado MA, et al. Postoperative portal vein obstruction in patients with idiopathic portal hypertension. J Clin Gastroenterol 1990;12:607. 11. Warren WD, Henderson JM, Millikan WJ, et al. Management of variceal bleeding in patients with noncirrhotic portal vein thrombosis. Ann Surg 1988;207:623–634. 12. Valla DC, Condat B. Portal vein thrombosis in adults: pathophysiology, pathogenesis and management. J Hepatol 2000;32:865–871.
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13. Valla DC, Condat B, Lebrec D. Spectrum of portal vein thrombosis in the West. J Gastroenterol Hepatol 2002;17 (Suppl 3):S224–S227. 14. Henderson JM, Gilmore GT, Mackay GJ, et al. Hemodynamics during liver transplantation: the interactions between cardiac output and portal venous and hepatic arterial flows. Hepatology 1992;16:715–718. 15. Lebrec D, Bataille C, Bercoff E, Valla D. Hemodynamic changes in patients with portal venous obstruction. Hepatology 1983;3:550–553. 16. Ohnishi K, Okuda K, Ohtsuki T, et al. Formation of hilar collaterals or cavernous transformation after portal vein obstruction by hepatocellular carcinoma. Observations in ten patients. Gastroenterology 1984;87:1150–1153. 17. Zalcman M, Van Gansbeke D, Matos C, et al. Sonographic demonstration of portal venous system thromboses secondary to inflammatory diseases of the pancreas. Gastrointest Radiol 1987;12:114–116. 18. Perlemuter G, Bejanin H, Fritsch J, et al. Biliary obstruction caused by portal cavernoma: a study of 8 cases. J Hepatol 1996;25:58–63. 19. Garcia-Tsao G, Groszmann RJ, Fisher RL, et al. Portal pressure, presence of gastroesophageal varices and variceal bleeding. Hepatology 1985;5:419–424. 20. Clavien PA, Huber O, Rohner A. Venous mesenteric ischaemia: conservative or surgical treatment? Lancet 1989;2:48. 21. Clavien PA, Durig M, Harder F. Venous mesenteric infarction: a particular entity. Br J Surg 1988;75:252–255. 22. Macpherson AI. Portal hypertension due to extrahepatic portal venous obstruction. A review of 40 cases. J Roy Coll Surg Edin 1984;29:4–10. 23. Vogelzang RL, Anschuetz SL, Gore RM. Budd–Chiari syndrome: CT observations. Radiology 1987;163:329–333. 24. Yerdel MA, Gunson B, Mirza D, et al. Portal vein thrombosis in adults undergoing liver transplantation: risk factors, screening, management, and outcome. Transplantation 2000;69:1873–1881. 25. Falanga A, Rickles FR. Pathophysiology of the thrombophilic state in the cancer patient. Semin Thromb Hemost 1999;25:173–182. 26. Rosendaal FR. Venous thrombosis: a multicausal disease. Lancet 1999;353:1167–1173. 27. Denninger MH, Chait Y, Casadevall N, et al. Cause of portal or hepatic venous thrombosis in adults: the role of multiple concurrent factors. Hepatology 2000;31:587–591. 28. Brown KM, Kaplan MM, Donowitz M. Extrahepatic portal venous thrombosis: frequent recognition of associated diseases. J Clin Gastroenterol 1985;7:153–159. 29. Thompson EN, Sherlock S. The aetiology of portal vein thrombosis with particular reference to the role of infection and exchange transfusion. Q J Med 1964;33:465–480. 30. Valla D, Casadevall N, Huisse MG, et al. Etiology of portal vein thrombosis in adults. A prospective evaluation of primary myeloproliferative disorders. Gastroenterology 1988;94:1063–1069. 31. Witte CL, Brewer ML, Witte MH, Pond GB. Protean manifestations of pylethrombosis. A review of thirty-four patients. Ann Surg 1985;202:191–202. 32. Belli L, Romani F, Sansalone CV, et al. Portal thrombosis in cirrhotics. A retrospective analysis. Ann Surg 1986;203:286–291. 33. Amitrano L, Brancaccio V, Guardascione MA, et al. Inherited coagulation disorders in cirrhotic patients with portal vein thrombosis. Hepatology 2000;31:345–348. 34. Arkel YS. Thrombosis and cancer. Semin Oncol 2000; 27:362–374. 35. Lang R, Inbal A, Jutrin I, Ravid M. Recurrent venous thrombosis: the sole manifestation of an occult myeloproliferative disease. Isr J Med Sci 1982;18:705–797.
Chapter 47 PORTAL AND SPLENIC VEIN THROMBOSIS
36. Sobhonslidsuk A, Reddy KR. Portal vein thrombosis: a concise review. Am J Gastroenterol 2002;97:535–541. 37. Ni YH, Wang NC, Peng MY, et al. Bacteroides fragilis bacteremia associated with portal vein and superior mesentery vein thrombosis secondary to antithrombin III and protein C deficiency: a case report. J Microbiol Immunol Infect 2002;35:255–258. 38. Schwartz DS, Gettner PA, Konstantino MM, et al. Umbilical venous catheterization and the risk of portal vein thrombosis. J Pediatr 1997;131:760–762. 39. Reh TE, Srivisal S, Schmidt EH 3rd. Portal venous thrombosis in ulcerative colitis: CT diagnosis with angiographic correlation. J Comput Assist Tomogr 1980;4:545–547. 40. Bernades P, Baetz A, Levy P, et al. Splenic and portal venous obstruction in chronic pancreatitis. A prospective longitudinal study of a medical–surgical series of 266 patients. Dig Dis Sci 1992;37:340–346. 41. Bertina RM. Factor V Leiden and other coagulation factor mutations affecting thrombotic risk. Clin Chem 1997;43:1678–1683. 42. Margaglione M, Brancaccio V, Giuliani N, et al. Increased risk for venous thrombosis in carriers of the prothrombin GÆA20210 gene variant. Ann Intern Med 1998; 129:89–93. 43. Brown K, Luddington R, Williamson D, et al. Risk of venous thromboembolism associated with a G to A transition at position 20210 in the 3¢-untranslated region of the prothrombin gene. Br J Haematol 1997;98:907–909. 44. Margaglione M, D’Andrea G, d’Addedda M, et al. The methylenetetrahydrofolate reductase TT677 genotype is associated with venous thrombosis independently of the coexistence of the FV Leiden and the prothrombin A20210 mutation. Thromb Haemost 1998;79:907–911. 45. Janssen HL, Meinardi JR, Vleggaar FP, et al. Factor V Leiden mutation, prothrombin gene mutation, and deficiencies in coagulation inhibitors associated with Budd–Chiari syndrome and portal vein thrombosis: results of a case–control study. Blood 2000;96:2364–2368. 46. Mahmoud AE, Elias E, Beauchamp N, Wilde JT. Prevalence of the factor V Leiden mutation in hepatic and portal vein thrombosis. Gut 1997;40:798–800. 47. Egesel T, Buyukasik Y, Dundar SV, et al. The role of natural anticoagulant deficiencies and factor V Leiden in the development of idiopathic portal vein thrombosis. J Clin Gastroenterol 2000;30:66–71. 48. Capron JP, Lemay JL, Muir JF, et al. Portal vein thrombosis and fatal pulmonary thromboembolism associated with oral contraceptive treatment. J Clin Gastroenterol 1981; 3:295–298. 49. Donaldson LB, Plant RK. Pregnancy complicated by extrahepatic portal hypertension: review of literature and report of two cases. Am J Obstet Gynecol 1971;110:255–264. 50. Kojima Y, Suzuki S, Sakaguchi T, et al. Portal vein thrombosis caused by microwave coagulation therapy for hepatocellular carcinoma: report of a case. Surg Today 2000;30:844–848. 51. Broe PJ, Conley CL, Cameron JL. Thrombosis of the portal vein following splenectomy for myeloid metaplasia. Surg Gynecol Obstet 1981;152:488–492. 52. Eguchi A, Hashizume M, Kitano S, et al. High rate of portal thrombosis after splenectomy in patients with esophageal varices and idiopathic portal hypertension. Arch Surg 1991;126: 752–755. 53. Fujita F, Lyass S, Otsuka K, et al. Portal vein thrombosis following splenectomy: identification of risk factors. Am Surg 2003;69:951–956. 54. Rossi P, Passariello R, Simonetti G. Portal thrombosis: high incidence following splenectomy for portal hypertension. Gastrointest Radiol 1976;1:225–227.
55. Freedman AM, Sanyal AJ, Tisnado J, et al. Complications of transjugular intrahepatic portosystemic shunt: a comprehensive review. Radiographics 1993;13:1185–1210. 56. Leach SD, Meier GH, Gusberg RJ. Endoscopic sclerotherapy: a risk factor for splanchnic venous thrombosis. J Vasc Surg 1989;10:9–12;discussion 12–13. 57. Deboever G, Elegeert I, Defloor E. Portal and mesenteric venous thrombosis after endoscopic injection sclerotherapy. Am J Gastroenterol 1989;84:1336–1337. 58. Politoske D, Ralls P, Korula J. Portal vein thrombosis following endoscopic variceal sclerotherapy. Prospective controlled comparison in patients with cirrhosis. Dig Dis Sci 1996;41:185–190. 59. Alvarez F, Bernard O, Brunelle F, et al. Portal obstruction in children. I. Clinical investigation and hemorrhage risk. J Pediatr 1983;103:696–702. 60. Dilawari JB, Chawla YK. Spontaneous (natural) splenoadrenorenal shunts in extrahepatic portal venous obstruction: a series of 20 cases. Gut 1987;28:1198–1200. 61. Harada H, Imamura H, Miyagawa S, Kawasaki S. Fate of the human liver after hemihepatic portal vein embolization: cell kinetic and morphometric study. Hepatology 1997;26:1162–1170. 62. Bilodeau M, Aubry MC, Houle R, et al. Evaluation of hepatocyte injury following partial ligation of the left portal vein. J Hepatol 1999;30:29–37. 63. Tanaka M, Wanless IR. Pathology of the liver in Budd–Chiari syndrome: portal vein thrombosis and the histogenesis of venocentric cirrhosis, veno-portal cirrhosis, and large regenerative nodules. Hepatology 1998;27:488–496. 64. Grauer SE, Schwartz SI. Extrahepatic portal hypertension: a retrospective analysis. Ann Surg 1979;189:566–574. 65. Maddrey WC, Sen Gupta KP, Mallik KC, et al. Extrahepatic obstruction of the portal venous system. Surg Gynecol Obstet 1968;127:989–998. 66. Condat B, Pessione F, Hillaire S, et al. Current outcome of portal vein thrombosis in adults: risk and benefit of anticoagulant therapy. Gastroenterology 2001;120:490–497. 67. Voorhees AB Jr, Price JB Jr. Extrahepatic portal hypertension. A retrospective analysis of 127 cases and associated clinical implications. Arch Surg 1974;108:338–341. 68. Janssen HL. Changing perspectives in portal vein thrombosis. Scand J Gastroenterol Suppl 2000:69–73. 69. Zimmerman D, Bell WR. Venous thrombosis and splenic rupture in paroxysmal nocturnal hemoglobinuria. Am J Med 1980;68:275–279. 70. Uflacker R. Applications of percutaneous mechanical thrombectomy in transjugular intrahepatic portosystemic shunt and portal vein thrombosis. Tech Vasc Intervent Radiol 2003;6:59–69. 71. Taylor CR. Computed tomography in the evaluation of the portal venous system. J Clin Gastroenterol 1992;14:167–172. 72. Taylor CR, McCauley TR. Magnetic resonance imaging in the evaluation of the portal venous system. J Clin Gastroenterol 1992;14:268–273. 73. Parvey HR, Raval B, Sandler CM. Portal vein thrombosis: imaging findings. AJR Am J Roentgenol 1994;162:77–81. 74. Tanaka K, Numata K, Okazaki H, et al. Diagnosis of portal vein thrombosis in patients with hepatocellular carcinoma: efficacy of color Doppler sonography compared with angiography. AJR Am J Roentgenol 1993;160:1279–1283. 75. Ueno N, Tomiyama T, Tano S, et al. Color Doppler ultrasonography in the diagnosis of portal vein invasion in patients with pancreatic cancer. J Ultrasound Med 1997;16:825–830. 76. Van Gansbeke D, Avni EF, Delcour C, et al. Sonographic features of portal vein thrombosis. AJR Am J Roentgenol 1985;144:749–752.
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77. Haddad MC, Clark DC, Sharif HS, et al. MR, CT, and ultrasonography of splanchnic venous thrombosis. Gastrointest Radiol 1992;17:34–40. 78. Mathieu D, Vasile N, Grenier P. Portal thrombosis: dynamic CT features and course. Radiology 1985;154:737–741. 79. Mathieu D, Vasile N, Dibie C, Grenier P. Portal cavernoma: dynamic CT features and transient differences in hepatic attenuation. Radiology 1985;154:743–748. 80. Zirinsky K, Markisz JA, Rubenstein WA, et al. MR imaging of portal venous thrombosis: correlation with CT and sonography. AJR Am J Roentgenol 1988;150:283–288. 81. Koklu S, Koksal A, Yolcu OF, et al. Isolated splenic vein thrombosis: an unusual cause and review of the literature. Can J Gastroenterol 2004;18:173–174. 82. Weber SM, Rikkers LF. Splenic vein thrombosis and gastrointestinal bleeding in chronic pancreatitis. World J Surg 2003;27:1271–1274. 83. Mortele KJ, Mergo PJ, Taylor HM, et al. Splenic and perisplenic involvement in acute pancreatitis: determination of prevalence and morphologic helical CT features. J Comput Assist Tomogr 2001;25:50–54. 84. Petit P, Bret PM, Atri M, et al. Splenic vein thrombosis after splenectomy: frequency and role of imaging. Radiology 1994;190:65–68. 85. Lewis JD, Faigel DO, Morris JB, et al. Splenic vein thrombosis secondary to focal pancreatitis diagnosed by endoscopic ultrasonography. J Clin Gastroenterol 1998;26:54–56. 86. Wiersema MJ, Chak A, Kopecky KK, Wiersema LM. Duplex Doppler endosonography in the diagnosis of splenic vein, portal vein, and portosystemic shunt thrombosis. Gastrointest Endosc 1995;42:19–26. 87. Heider TR, Azeem S, Galanko JA, Behrns KE. The natural history of pancreatitis-induced splenic vein thrombosis. Ann Surg 2004;239:876–880;discussion 880–882. 88. Makowiec F, Riediger H, Emmrich J, et al. [Prophylactic splenectomy for splenic vein thrombosis in patients undergoing resection for chronic pancreatitis]. Zentralbl Chir 2004;129:191–195. 89. Manzanet G, Sanjuan F, Orbis P, et al. Liver transplantation in patients with portal vein thrombosis. Liver Transpl 2001;7: 125–131. 90. Tzakis AG, Kirkegaard P, Pinna AD, et al. Liver transplantation with cavoportal hemitransposition in the presence of diffuse portal vein thrombosis. Transplantation 1998;65:619–624. 91. Orlando G, De Luca L, Toti L, et al. Liver transplantation in the presence of portal vein thrombosis: report from a single center. Transplant Proc 2004;36:199–202. 92. Molmenti EP, Roodhouse TW, Molmenti H, et al. Thrombendvenectomy for organized portal vein thrombosis at the time of liver transplantation. Ann Surg 2002;235:292–296. 93. Shaw BW Jr, Iwatsuki S, Bron K, Starzl TE. Portal vein grafts in hepatic transplantation. Surg Gynecol Obstet 1985;161:66–68. 94. Seu P, Shackleton CR, Shaked A, et al. Improved results of liver transplantation in patients with portal vein thrombosis. Arch Surg 1996;131:840–844;discussion 844–845. 95. Moreno Gonzalez E, Garcia Garcia I, Gomez Sanz R, et al. Liver transplantation in patients with thrombosis of the portal, splenic or superior mesenteric vein. Br J Surg 1993;80:81–85.
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96. Figueras J, Torras J, Rafecas A, et al. Extra-anatomic venous graft for portal vein thrombosis in liver transplantation. Transpl Int 1997;10:407–408. 97. Fujimoto M, Moriyasu F, Someda H, et al. Recovery of graft circulation following percutaneous transluminal angioplasty for stenotic venous complications in pediatric liver transplantation: assessment with Doppler ultrasound. Transpl Int 1995;8:119–125. 98. Greaves M. Antiphospholipid antibodies and thrombosis. Lancet 1999;354:1031. 99. Kahn D, Terblanche J, Kitano S, Bornman P. Injection sclerotherapy in adult patients with extrahepatic portal venous obstruction. Br J Surg 1987;74:600–602. 100. Kahn D, Krige JE, Terblanche J, et al. A 15-year experience of injection sclerotherapy in adult patients with extrahepatic portal venous obstruction. Ann Surg 1994;219:34–39. 101. Abraldes JG, Bosch J. Novel approaches to treat portal hypertension. J Gastroenterol Hepatol 2002;17(Suppl 3):S232–S241. 102. Brearley S, Hawker PC, Dykes PW, Keighley MR. A lethal complication of peripheral vein vasopressin infusion. Hepatogastroenterology 1985;32:224–225. 103. Bismuth H, Franco D. Portal diversion for portal hypertension in early childhood. Ann Surg 1976;183:439–446. 104. Galloway JR, Henderson JM. Management of variceal bleeding in patients with extrahepatic portal vein thrombosis. Am J Surg 1990;160:122–127. 105. Opitz T, Buchwald AB, Lorf T, et al. The transjugular intrahepatic portosystemic stent–shunt (TIPS) as rescue therapy for complete Budd–Chiari syndrome and portal vein thrombosis. Z Gastroenterol 2003;41:413–418. 106. Blum U, Haag K, Rossle M, et al. Noncavernomatous portal vein thrombosis in hepatic cirrhosis: treatment with transjugular intrahepatic portosystemic shunt and local thrombolysis. Radiology 1995;195:153–157. 107. Orozco H, Takahashi T, Mercado MA, et al. The Sugiura procedure for patients with hemorrhagic portal hypertension secondary to extrahepatic portal vein thrombosis. Surg Gynecol Obstet 1991;173:45–48. 108. Harnar T, Johansen K, Haskey R, Barker E. Left-sided portal hypertension from pancreatic pseudotumor. Am J Gastroenterol 1982;77:639–641. 109. Condat B, Valla D. [Portal vein thrombosis: is anticoagulation indicated?]. Gastroenterol Clin Biol 2001;25:507–508. 110. Baril N, Wren S, Radin R, et al. The role of anticoagulation in pylephlebitis. Am J Surg 1996;172:449–452;discussion 452–453. 111. Malkowski P, Pawlak J, Michalowicz B, et al. Thrombolytic treatment of portal thrombosis. Hepatogastroenterology 2003;50:2098–2100. 112. Walser EM, NcNees SW, DeLa Pena O, et al. Portal venous thrombosis: percutaneous therapy and outcome. J Vasc Interv Radiol 1998;9:119–127. 113. Rosen MP, Sheiman R. Transhepatic mechanical thrombectomy followed by infusion of TPA into the superior mesenteric artery to treat acute mesenteric vein thrombosis. J Vasc Interv Radiol 2000;11:195–198.
Section VII. Vascular Disease of the Liver
48
LIVER INVOLVEMENT IN OSLER–WEBER–RENDU DISEASE (HEREDITARY HEMORRHAGIC TELANGIECTASIA, HHT) Martin Caselitz, Siegfried Wagner, and Michael P. Manns Abbreviations ALK-1 activin receptor-like kinase-1 AVMs arteriovenous malformations BMPs bone morphogenetic proteins
HCC HHT MRI
hepatocellular carcinoma hereditary hemorrhagic telangiectasia magnetic resonance imaging
GENERAL ASPECTS AND DIAGNOSTIC CRITERIA Hereditary hemorrhagic telangiectasia (HHT) or Osler–Weber– Rendu disease is a rare hereditary autosomal dominant disorder of blood vessels. Mucocutaneous and visceral fibrovascular dysplasia leads to various arteriovenous malformations (AVMs) and telangiectasia in different organs. These manifestations predominantly involve the skin, mucosa, liver, GI tract, lung, and brain (Figure 481). In particular angiodysplasia of the mucosa often leads to recurrent bleeding. These features are summarized in the classical triad of Osler’s disease: 1. multiple mucocutaneous telangiectasia (Figure 48-2) 2. epistaxis 3. positive family history The association of these features was described by Rendu in 1896 and independently by Osler in 1901 and Weber in 1907.1–3 The names of these authors appear in various orders in the common eponymous labels for this condition. In 1909 Hanes coined the term “hereditary hemorrhagic telangiectasia” in acknowledgment of the three features that by then defined the disorder.4 Initially it was suspected that visceral involvement in HHT was a rare condition, but interpretation was based on the frequency of symptomatic presentation to an astute physician. With the onset of modern imaging methods and asymptomatic screening programs a much higher visceral involvement was seen.4 Moreover, visceral involvement of HHT is responsible for the high mortality rate of affected patients.5,6 With regard to the importance of visceral involvement, an actual diagnostic score was developed based on the criteria described in Table 48-1.5 These criteria permit a high level of clinical suspicion with limited diagnostic procedures. However, symptoms of HHT are progressive over time. Family members of a
PAVMs TGF-b TGF-bR
pulmonary AVMs transforming growth factor-b TGF-b receptors
patient with HHT can only be informed that they do not have HHT if a molecular diagnosis is applied (see below).
EPIDEMIOLOGY HHT occurs in many ethnic groups with a wide geographic distribution, including Asia, Africa, and the Middle East.7,8 The prevalence of HHT is in the range between 1 in 2000 and 1 in 40 000. There are considerable differences in the geographic distribution of HHT. HHT has been found to occur in 1 in 2351 persons in the French department of Ain, 1 in 3500 on the Danish island Fünen,9 1 in 5155 in the Leeward Islands (West Indies), 1 in 16 500 in the state of Vermont, USA,10 and 1 in 39 000 in northern England.11
GENETIC BACKGROUND HHT is inherited in an autosomal dominant manner, with variable expression of clinical symptoms even among family members.12 However, penetrance of HHT is high (>95%), with an agedependent phenotype that is nearly complete by the age of 40–45 years.11,13 Up to 20% of patients lack a family history of HHT. The homozygous state appears to be lethal.14 Studies of families with HHT have identified two genes whose defects are believed to be responsible for the majority of cases of HHT. The phenotype has thus been classified as HHT-1 or HHT-2, depending on the mutated gene underlying the disorder. Both genes code for two receptors of the transforming growth factor-b (TGF-b) superfamily: endoglin and activin receptor-like kinase-1. TGF-b is a member of a supergene family of polypeptide growth factors, which include activins, inhibins, and bone morphogenetic proteins (BMPs). These growth factors share homology at a group of cysteine residues that are held together by intramolecular disul-
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Table 48-1. Diagnostic criteria (Curaçao criteria), as presented by Shovlin and Tarte5 The hereditary hemorrhagic telangiectasia (HHT) diagnosis is: • “Definite” if three or four criteria are present • “Possible” or “suspected” if two criteria are present • “Unlikely” if fewer than two criteria are present Criteria Multiple telangiectasia at characteristic sites – Lips (Figure 48-2) – Oral cavitiy – Fingers – Nose • Epistaxis: spontaneous, recurrent nose bleeds • Family history: a first-degree relative with HHT according to these criteria • Visceral manifestation – Gastrointestinal telangiectasia (with or without bleeding) – Pulmonary arteriovenous malformations (AVMs) – Hepatic AVMs – Cerebral AVMs – Spinal AVMs
•
Figure 48-1. Distribution of affected organs in hereditary hemorrhagic telangiectasia.
Figure 48-2. Telangiectasia on lips and tongue.
fide bonds. TGF-b is produced by nearly every cell and each cell expresses the corresponding receptors. This factor plays a significant role in the regulation of cell proliferation and differentiation, wound-healing, angiogenesis, and embryonic development. In endothelium, TGF-b modulates endothelial cell functions (e.g., migration, proliferation, adhesion) interactions between endothelium and smooth-muscle cell layers, and vascular tone. Three highly conserved isoforms of TGF-b exist, each encoded by a separate gene: TGF-b1, TGF-b2, and TGF-b3. The cellular action of TGF-b is mediated through cell surface receptors that have intrinsic serine/threonine kinase activity. Several TGF-b receptors
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(TGF-bR) have been identified so far: TGF-bRI, TGF-bRII, and TGF-bRIII (also known as g-glycan) and endoglin (see below). Endoglin is homologous to TGF-bRIII in its transmembrane and cytoplasmic tail regions.14 HHT-1 is caused by mutations of the endoglin gene on chromosome 9q3. Confirmation that endoglin (CD 105, ENG) mutations are causative is available from experiments in transgenic mice. Some mice carrying one normal and one mutated copy of the endoglin gene display features of HHT.15 Endoglin is the most abundant TGFb-binding protein found on endothelial cells. It consists of 658 amino acids and is an integral transmembrane protein that associates with TGF-b ligand-binding receptors and modulates cellular responses. Endoglin interacts as a binding protein with affinity to several proteins, including activin-A and several BMPs (BMP-2 and BMP-7).16 Endoglin interacts with, and becomes a component of, the TGF-b1 and TGF-b3 receptor complexes involved in endothelial cell signaling. More than 70 mutations have been identified to date and disease severity has not been correlated with type of mutation. The mutant endoglin products seen in HHT-1 are transient intracellular molecules that show no cell surface expression. Measurable levels of normal endoglin in HHT patients are thus reduced by 50%, even in “normal” vessels, suggesting that a single copy of the gene confers susceptibility to the disease but that a “second hit” or modifier genes likely contribute to the development of vascular abnormalities.17 HHT-2 – the second genetic defect – is mapped on chromosome 12q13. This results in a mutation in the activin receptor-like kinase1 (ALK-1) gene, which has a similar affinity to the TGF-b complex as endoglin. Significant expression of this gene product only occurs in endothelium, but it may also be found in peripheral blood leukocytes. ALK-1 is a type 1 cell surface receptor in the TGF-b superfamily. It modulates TGF-b signaling during the regulation of angiogenesis and plays an important role in controlling blood vessel development and repair. ALK-1 has the properties of a type I serine/threonine kinase receptor that binds to TGF-b1 only in asso-
Chapter 48 LIVER INVOLVEMENT IN OSLER-WEBER-RENDU DISEASE
ciation with TGF-b receptor type II in vitro; however the ALK-1 ligand in vivo remains unknown. About 50 mutations are identified so far. Similar to endoglin, reduced levels of functional ALK-1 are seen, suggesting a haploinsufficiency mechanism of disease.18 The presence of two disease loci provided the basis for genotype/phenotype studies of the disease.19 Recently, a large questionnaire-based study led Berg et al. to the conclusion that the HHT-1 phenotype is distinct from, and more severe than, the HHT-2 phenotype.20 In this study, an earlier onset of epistaxis and telangiectasis was present in patients with HHT-1. Additionally, pulmonary AVMs (PAVMs) were only seen in HHT-1,20 a fact that confirmed earlier observations.21 However, PAVMs can also be encountered in some patients with HHT-2.22,23 Additionally, ALK-1 mutations were also identified in kindreds with pulmonary hypertension and HHT.24
GENETIC BACKGROUND OF LIVER INVOLVEMENT Nikolopoulos et al.25 and Piantanida et al.26 described HHT families with an accumulation of liver involvement, suggesting a genotype/ phenotype correlation of hepatic manifestation in HHT. With respect to liver involvement in HHT, Olivieri et al. hypothesized from their observations on the presence of intrahepatic arteriovenous shunts in six of 10 patients with HHT-2 that mutations in the ALK-1 gene may be associated with a higher risk of liver AVMs.27 A significantly higher liver involvement had also previously been reported for two large HHT-2 families.2,28 These observations prompted another group to screen systematically for mutations in the ENG and ALK-1 genes in a group of HHT patients with and without liver involvement from Germany. The researchers found that hepatic manifestation in HHT patients is associated with mutations in the ALK-1 gene, but rarely also caused by ENG mutations.29
PATHOPHYSIOLOGY In HHT the malformations consist of aberrant vascular development with multiple dilated vessels that are lined by a single layer of endothelium that is attached to a continuous basement membrane. The smallest of the hallmark telangiectases are focal dilatations of postcapillary venules. In fully developed telangiectases the venules are markedly dilated and convoluted with excessive layers of smooth muscle without elastic fibers. These venules often connect directly to dilated arterioles. This aberrant development is also associated with a perivascular mononuclear cell infiltrate. However, no single pathognomonic histological characteristic for the telangiectasia in HHT exists. Various explanations for the characteristic bleeding of these vessels include insufficient smooth-muscle contractile elements, endothelial cell junction defects, perivascular connective tissue weakness, and endothelial cell degeneration.14 Changes of the liver in HHT are described below.
ORGAN MANIFESTATIONS The diverse manifestations of HHT involve mostly vascular abnormalities of the nose, skin, lung, brain, and gastrointestinal tract.
Table 48-2. Clinical manifestations of hereditary hemorrhagic telangiectasia Affected organ or system
Type of lesion
Frequency
Nose, skin, and oral cavity Lung Gastrointestinal tract
Telangiectases Arteriovenous malformations Telangiectases, angiodysplasia, arteriovenous malformations Telangiectases, arteriovenous malformations, arterial aneurysm, cavernous angioma Arteriovenous malformations telangiectases
80–100% 15–30% 11–44%
Central nervous system (brain and spinal cord)
Liver
8–31%
8–30%
Table 48-2 summarizes the most important organ manifestations of persons with HHT. However, angiodysplasia may occur in every organ. Cases of urogenital,11,30,31 ophthalmological,32,33 and splenic34 involvement of HHT have been reported in the literature. Furthermore, aneurysms of the coronary artery35 and the aorta36 are described.
NOSE Epistaxis caused by spontaneous bleeding from telangiectases of the nasal mucosa is the most common (95%) and earliest manifestation of HHT occurring in the majority of affected persons, but not in all. It may be so severe as to require multiple transfusions and oral iron supplementation,37 and on the other hand so mild that HHT is never suspected. Recurrent epistaxis begins by the age of 10 years in many patients and by the age of 21 years in most (> 90%), becoming more severe in the later decades in about two-thirds of affected persons.
SKIN Telangiectases of the skin typically present later in life than epistaxis. By the age of 40, most affected persons (70%) have multiple telangiectasis of the lips, tongue, palate, fingers, face, nail beds, or combination of these.4,38 There may be bleeding from cutaneous telangiectasis, but it is rarely clinically important. In these cases or for cosmetic concern laser ablation can be effective.
LUNG PAVMs are thin-walled abnormal vessels that replace normal capillaries between the pulmonary arterial and venous circulations, often resulting in bulbous sac-like structures.5,39 They are often multiple and appear in both lungs, with a predilection for the lower lobes.40 These “capillary-free” shunts provide three main clinical consequences:41 1. Pulmonary arterial blood cannot be oxygenated, leading to hypoxemia. 2. The absence of a filtering capillary bed allows embolic particles to reach the systemic circulation where it impacts on other capillary beds (e.g., central nervous system). 3. The fragile vessels may lead to hemorrhage into a bronchus or the pleural cavity. Embolic cerebral events (cerebral abscess and embolic stroke) occur in patients regardless of the degree of respiratory symptoms and still carry significant morbidity and mortality.5
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Complications of PAVMs can be limited if this condition is diagnosed and treated with transcatheter embolization, which offers the safest method of treatment. Whether asymptomatic patients should be treated is a matter of debate but excellent safety profiles in experienced centers supported a trend towards earlier treatment using transcatheter embolization. Surgical management of PAVMs may be an alternative option in selected patients.5 Long-term follow-up of treated patients is important, because the growth of malformations may require further treatment. In addition, prophylactic antibiotics are recommended at the time of dental and surgical procedures to reduce the risk of brain abscess. Screening methods are based on non-invasive procedures to image PAVMs (thoracal radiography, computed tomography) or detection of right-to-left shunt (e.g., contrast echocardiography, radionuclide perfusion, arterial blood-gas measurements).
CENTRAL NERVOUS SYSTEM About 15% of HHT patients may have cerebral involvement with telangiectases, cerebral AVMs, aneurysms, or cavernous angiomas.5 However, asymptomatic HHT patients are not routinely investigated with regard to cerebral involvement, so a higher incidence (up to 23%) is presumed.42 Cerebral involvement can lead to migraine headache, seizures, ischemia of the surrounding tissue due to a steal effect, hemorrhage, and, less commonly, paraparesis.43 An important cause for neurological complications is pulmonary embolism due to PAVMs. It is estimated that in up to two-thirds of those in whom neurological symptoms develop, PAVMs are the source of the symptoms. In the remaining third complications arise from cerebral AVMs. HHT patients with neurological symptoms suggestive of cerebral involvement or pulmonary embolism deserve further assess-
ment, as in the non-HHT population, by experienced neurointerventional centers. Cerebral magnetic resonance imaging (MRI) is currently the most sensitive non-invasive test. The question of whether asymptomatic HHT patients should be screened for cerebral AVM remains debated.
GASTROINTESTINAL TRACT The gastrointestinal tract is the second most common organ system involved in HHT following the respiratory system. Gastrointestinal manifestation of HHT can be found in every section of the gut.38,44 Characteristically, gastrointestinal symptoms do not appear until the fifth or sixth decade of life.4 Telangiectasia or AVM may be seen in about 60% of patients with HHT (Figure 48-3). Hemorrhage, the most common gastrointestinal manifestation, is seen in 10–45% of patients.5,45 In a retrospective study up to 40% of patients had an upper gastrointestinal source of bleeding, up to 10% had a lower gastrointestinal source, and 50% had an indeterminate bleeding side.45 So far no studies of capsular endoscopy are available in HHT patients. Requirements for the transfusion of more than 100 units of blood due to gastrointestinal bleeding have been documented.46 Hemobilia from hepatic telangiectasia has been proposed as the cause of gastrointestinal bleeding as well.47 The basic principles for the management of acute gastrointestinal bleeding in HHT patients are the same as in any other lesion in the first line. Early recognition of HHT is important for proper management. Endoscopy remains the main important tool in diagnosing and treating this condition. Endoscopic findings include AVMs or telangiectases similar to those seen in the oral or nasal mucosa.48 Tagged red-cell scans may diagnose the origin of a subacute bleeding, but are only of limited use because of the often intermittent
Figure 48-3. Angiodysplasia in the stomach.
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nature of bleeding. In the setting of severe acute hemorrhage, angiography may demonstrate the origin of bleeding as well.48 Recurrent hemorrhage is most difficult to manage. Endoscopic treatment techniques include the use of laser and argon plasma beamer; all methods have comparable results in the control of acute bleeding telangiectasia, with typical success rates exceeding 90%.49,50 However, the long-term results of endoscopy have been disappointing because of the multifocal nature of the disease, with recurrent episodes of bleeding from other sites in the gastrointestinal tract. Asymptomatic, non-bleeding lesions should not be treated because of the risk of inducing acute and/or delayed bleeding. Patients refractory to endoscopic treatment, or those with lesions not amenable to endoscopic therapy, may require angiography with arterial embolization or surgery after localization of the bleeding source. However, with further developments of endoscopic techniques, the need for surgical intervention has steadily decreased and is now rarely required. Long-term treatment of hemorrhage in HHT, regardless of the source, has been disappointing. In some small trials hormonal treatment with estrogen–progestogen combinations (ethinylestradiol 0.05 mg and norethisterone 1 mg daily, given orally) has been shown to decrease transfusion requirements in patients with gastrointestinal bleeding.46 The exact mechanism of hormonal therapy is not known so far. Epsilon-aminocaproic acid, a potent inhibitor of the fibrinolytic system, has been reported to decrease the frequency of bleeding episodes and the number of transfusions required.48 Patients should be advised to avoid nonsteroidal anti-inflammatory drugs and coumadin, which might increase the risk of bleeding.
HEPATIC MANIFESTATION OF HHT HISTORICAL ASPECTS AND EPIDEMIOLOGY OF LIVER INVOLVEMENT Liver involvement in HHT was originally suspected by Osler in 1901. The first case report was published by van Bogaert in 1935.51 Martini reviewed the literature in 197852 and grouped liver involvement in three histological subtypes: 1. telangiectasia with fibrosis or cirrhosis 2. cirrhosis without telangiectasia 3. telangiectasia without fibrosis or cirrhosis Group 2 often had a superimposed hepatopathy such as posttransfusion hepatitis or iron overload present, which leads to cirrhosis. Vascular dilatation and arteriovenous shunts with highoutput cardiac failure was found in group 1 and 3.14 Currently, it is estimated that liver involvement occurs in 8–31% of patients suffering from HHT.13,22,26,53 However, Reilly and Nostrant,45 who described hepatic manifestations in 31% of affected patients, defined this fact as elevated activity of liver enzymes and hepatomegaly. This definition of liver involvement is not appropriate in the context of Osler’s disease, because diagnosis of vascular malformations is based on imaging methods. Furthermore the prevalence of liver involvement depends on the age of the investigated patients, and patients with hepatic involvement, who remain asymp-
tomatic, may be unconsidered. Thus, the true prevalence of hepatic involvement in HHT is unknown.
HISTOPATHOLOGY AND PATHOPHYSIOLOGY OF HEPATIC INVOLVEMENT The liver has a unique vascular supply. Blood enters the liver from two sources, the portal vein and the hepatic artery, merging at the level of hepatic sinusoids and exiting through the hepatic veins. Therefore hepatic changes in HHT can be complex and multiple, including sinusoidal ectasia, arteriovenous shunts (direct communication between arterioles and sinusoids), arterioportal shunts (direct shunt between the branches of the hepatic artery and the portal vein causing portal hypertension), and portovenous shunts (connections between portal veins and sinusoids). Three forms of angiodysplasia have been identified: 1. Telangiectases are focal dilatations that originate from capillaries and postcapillary venules (or sinusoids in the liver) (Figure 48-4). These dilated vessels are lined by a single layer of endothelium attached to continuous basement membrane. The vessels are often surrounded by a mononuclear cell infiltrate. 2. AVMs are larger dilated tortuous vessels of both arterial and venous elements with interrupted elastica lamina and variable thickness in the smooth-muscle layers. AVMs are devoid of interlinking capillaries; therefore significant shunting occurs. In the liver arteriovenous shunting, arterioportal shunting and portovenous shunting may occur. The different types of shunt may explain the wide variety of clinical symptoms in HHT patients with hepatic involvement. The lesions are embedded in dense fibrous tissue. These fibrous bands may link, entrapping the hepatocytes, leading to a fine or course nodular appearance similar to cirrhosis (pseudocirrhosis) (Figure 485). The hepatocellular architecture is preserved within these nodules, including central veins and portal areas. There may be little or no hepatocellular necrosis or inflammation. Reilly and Nostrant45 performed liver biopsy in 10 HHT patients with suspected liver involvement. They found hepatic telangiectasia in 30%, iron overload in 50%, and periportal fibrosis in 80% of the affected patients. However, no case with fully developed cirrhosis, bridging necrosis, or chronic active hepatitis was found. These findings were confirmed by other authors.54 3. Aneurysms form large vessels secondary to the fragmentation of the elastic lamina and loss of the vessel muscularis. Focal nodular hyperplasia – independent of hormonal therapy – in HHT is presumed to be parenchymal hyperplasia secondary to hyperperfusion by large anomalous hepatic arteries. Hepatomegaly (>15 cm medioclavicular line), sometimes accompanied by splenomegaly, is found as a consequence of portal hypertension or intrahepatic AVMs in 44% of affected patients.55 Primary involvement of the liver has to be distinguished from secondary hepatic complications occurring in patients affected by HHT. Viral hepatitis following transfusion due to iron-deficiency anemia may cause liver cirrhosis in older HHT patients. Patients with unexplained elevated liver enzymes for more than 6 months should be
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Figure 48-4. Microscopic aspect of hepatic involvement in hereditary hemorrhagic telangiectasia with dilated vessels. (Hematoxylin & eosin 100¥).
Figure 48-5. Nodular pattern of hepatic involvement in hereditary hemorrhagic telangiectasia (pseudocirrhosis).
evaluated with hepatitis B and C serologies and sonographic imaging (see below). HHT patients requiring blood transfusions should be vaccinated against hepatitis B. Hepatocellular carcinoma (HCC) is described in a few patients with hepatic manifestation of HHT.56 However, HHT with liver involvement itself cannot be considered
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as a risk factor for HCC, but viral hepatitis in the affected patients may lead to HCC. Furthermore, hepatic iron overload is described as a complication of repetitive blood transfusions or iron supplementation in HHT. Secondary involvement of the liver in the form of peliosis hepatis,
Chapter 48 LIVER INVOLVEMENT IN OSLER-WEBER-RENDU DISEASE
hepatic adenoma, and focal nodular hyperplasia may develop as a result of long-term hormonal therapy (see section on gastrointestinal tract, above) as well. Systemic AVMs may lead to a high-output cardiac failure and congestion of the liver. The increased pressure in the sinusoids may induce fibrosis of the liver. Therefore cirrhosis in some HHTaffected patients may be caused by right-sided congestive heart failure (cardiac cirrhosis). Up to 70% of HHT patients suffering from symptomatic liver involvement are female and in their fourth or fifth decade.5,13,57,58 These findings were confirmed by other authors, referring pulmonary40,59 and cerebral5,60 vascular malformations. The increasing magnitude of hepatic vascular malformations36,61 and pulmonary arteriovenous fistulas during pregnancy indicates the potential role of hormones in the pathogenesis of vascular malformations.62,63 This is supported by the observation that gastrointestinal bleedings are treated successfully with estrogen–progestogen combinations46 and recurrent episodes of epistaxis depend on menstruation.5 However, so far the exact mechanism of hormonal influence remains unclear.
CLINICAL PRESENTATION Manifestations of HHT are generally not present at birth, but telangiectases and malformations develop with increasing age. Therefore the clinical course can be divided into three periods: 1. an asymptomatic period during the childhood 2. a hemorrhagic period with episodes of severe and recurrent epistaxis from puberty up to the third decade 3. a period of manifest organ involvement (e.g., pulmonary, hepatic) with clinical symptoms and secondary complications in some patients53 Vascular involvement of the liver may be asymptomatic in up to 50% of patients with HHT.51 However, clinical features of hepatic manifestations in HHT patient show a wide variety, including hepatomegaly, pulsatile hepatic nodules, palpable thrill, abdominal bruit, ascites, and variceal bleeding due to portal hypertension. Right upper quadrant pain is encountered in some patients, presumably because of thrombosis in telangiectasia. Recurrent hepatic encephalopathy is described in patients with liver failure, after gastrointestinal bleeding, and in patients with portovenous shunts.64 The development of encephalopathy depends on the magnitude of the shunt; if it is small, encephalopathy is unusual. Other clinical findings include dyspnea due to pulmonary hypertension, high-output congestive cardiac failure, or a hyperdynamic circulation without heart failure secondary to left-to-right shunting of blood within the liver. Abdominal AVMs may further cause a “steal syndrome” which may lead to symptoms of abdominal angina with consequent loss of weight.57 Laboratory results reveal anicteric cholestasis (elevated gglutamyltranspeptidase and alkaline phosphatase) in up to 73% of patients.57 In cases with advanced cirrhosis an impaired liver function can be observed.
DIAGNOSIS OF HEPATIC INVOLVEMENT IN HHT Diagnosis of liver involvement is based both on clinical diagnostic criteria and on imaging methods. Though anicteric cholestasis is well
described by several authors, laboratory tests are not appropriate to diagnose hepatic manifestation of HHT. Percutaneous liver biopsy may reveal typical features such as telangiectasia, hepatic congestion, and periportal fibrosis.45 However, due to a considerable risk of bleeding following biopsy and improvement of imaging methods, liver biopsies are no longer required in most patients. A transjugular wedge biopsy may be an alternative to a percutaneous biopsy in selected patients. As liver involvement in patients with HHT is a complication with a potentially life-threatening outcome, e.g., due to the massive increase of cardiac output, an early diagnosis is desired. Although a dilated hepatic artery and vascular lesions are present in most HHT patients with liver involvement, the picture of hepatic involvement in HHT is highly variable. It may be confused with other comorbidities (Table 48-3) like liver cirrhosis, focal nodular hyperplasia, and hepatic congestion. The situation is further complicated by the fact that a considerable number of older HHT patients are infected with hepatitis C virus due to a history of blood transfusions. Thus, liver involvement in HHT must be differentiated from fibrotic liver disease due to HHT. Angiography is the “gold standard” for the diagnosis of hepatic AVMs (Figure 48-6). Characteristically, dilatation and tortuosity of the hepatic artery and its branches are seen with numerous telangiectatic lesions throughout the liver and early visualization of the hepatic veins and/or right heart chambers. The angiographic appearance depends on the stage of development of the AVM. The differential diagnosis includes conditions of reduced portal venous blood flow, cavernous hemangiomas, highly vascularized liver tumors or metastatic neoplasms, cirrhosis, and hemangioendothelioma of infancy.51,65,66 Dynamic computed tomography (Figure 48-7) and MRI are very sensitive and can confirm the diagnosis of liver involvement. Typical findings include hepatic artery dilatation, disseminated telangiectasia, early filling of the hepatic vein, and a pseudonodular pattern in the liver. The aspects of differential diagnosis are similar to those mentioned above.51,67–69 One should, however, consider that selective angiography of the hepatic artery – the current gold standard – is expensive, invasive, and not readily available for repeat measurements. Therefore, ultrasound has been proposed as a non-invasive approach to screen HHT patients and to diagnose and monitor hepatic HHT lesions.7,51,65 The current literature, mostly case reports, comprises about 35 papers describing the sonographic findings in about 100 patients with hepatic HHT lesions. The characteristic sonographic picture of liver
Table 48-3. Differential diagnosis of sonographic findings in hepatic involvement of hereditary hemorrhagic telangiectasia (HHT) Differential diagnosis of sonographic findings in hepatic involvement of HHT Cirrhosis of the liver: irregular surface of the liver Tumors of the liver (especially focal nodular hyperplasia): dilated and hypertrophic branches of the hepatic artery Caroli’s syndrome, sclerosing cholangitis: dilated intrahepatic bile ducts (B-mode sonography) Arteriovenous fistulas of other origin
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Figure 48-6. Angiography of hepatic involvement in hereditary hemorrhagic telangiectasia.
Figure 48-7. Computed tomography scan showing intrahepatic vascular malformations.
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Chapter 48 LIVER INVOLVEMENT IN OSLER-WEBER-RENDU DISEASE
lesions in HHT consists of an intrahepatic hypervascularization (Figure 48-8) caused by a lack of normal tapering of arterial branches combined with a tortuous course of the hepatic artery (Figure 489). To integrate different ultrasound findings in hepatic involvement of HHT, Caselitz and co-workers described a diagnostic scoring system (Table 48-4).55 The two major criteria (dilated common hepatic artery (Figure 48-10) and intrahepatic hypervascularization (Figure 48-8) detect hepatic HHT lesions with high sensitivity and specificity. Both should be positive to establish the diagnosis of hepatic HHT lesions. If one of the major criteria is negative or not measurable, the use of minor criteria may be suggested.
The minor criteria are highly specific, with a lower sensitivity to hepatic involvement in HHT. Furthermore, flow measurements are susceptible to investigator-dependent variability. This especially applies to the arterial parameters regarding the tortuous course of the hepatic artery in HHT. Therefore one of the major criteria with at least two of the minor criteria for the diagnosis of hepatic manifestation of HHT should be combined. In addition to the major and minor criteria, a group of facultative parameters was defined. These features are characteristic of hepatic involvement in HHT but may also be present in many other conditions. They therefore support the diagnosis of hepatic HHT lesions if major or minor criteria are present.55
Figure 48-8. Intrahepatic hypervascularization due to dilated branches of the hepatic artery. (Color Doppler, 3.75-MHz convex transducer.)
Figure 48-9. Tortuous course of the hepatic artery. (B-mode; 3.75-MHz convex transducer.)
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According to Caselitz and co-workers, one sonographic feature that appears to be linked to the more advanced stages of liver involvement in HHT is an irregular, nodular surface of the liver, usually addressed as pseudocirrhosis (Figure 48-11). This finding needs to be distinguished from regeneration nodules found in liver cirrhosis.55 In addition to the criteria mentioned above, sonographic findings may reveal right heart failure, portal hypertension, and different types of focal liver lesion, such as hemangiomas and focal nodular hyperplasia.51,65 Further sonographic methods include duplex sonography, which allows analysis of blood flow patterns. Characteristically, this shows a high-velocity signal in the main hepatic artery and a decreased resistance index if relevant arterial shunts are present. A pulsatile flow pattern in the portal vein together with a high velocity may indicate the presence of arterioportal shunts. Color Doppler can
Table 48-4. Sonographic criteria for hepatic involvement in hereditary hemorrhagic telangictasia according to Caselitz et al.55 Major criteria Dilated common hepatic artery >7 mm Intrahepatic arterial hypervascularization Minor criteria Vmax of the proper hepatic artery >110 cm/s Resistance index (RI) of the proper hepatic artery 25 cm/s Tortuous course of the extrahepatic hepatic artery Facultative findings Dilated portal vein >13 mm Dilated liver veins >11 mm Hepatomegaly >15 cm in mid clavicular line Nodular liver margin
prevent the confusion arising from B-mode sonography that may mimic biliary dilatation. Furthermore, extrahepatic vascular malformations can be visualized by Doppler sonography as well. Abnormalities of the bile duct similar to those in Caroli’s disease or those in sclerosing cholangitis are described in the literature.47,54,70,71 The intimate anatomic relationship of the vascular abnormalities to the dilated bile ducts suggests that external vascular compression could have caused their dilatation.70 However, these findings must be distinguished from anicteric cholestasis, found regularly in hepatic manifestation of HHT. Imaging methods such as computed tomography and sonography can show focal biliary dilatation in the liver. In case of inconsistent findings, biliary disease can be confirmed by endoscopic retrograde cholangiography or magnetic resonance cholangiography. It was shown that cardiac output in HHT correlated with both arterial and portal venous diameter. This correlation was even closer when both diameters were summed up, while the diameters did not correlate with each other. This suggests that the hepatic HHT lesions may induce both arterial and portal venous dilation. The proportion to which the supplying hepatic vessels contribute to the hepatic blood flow (and subsequently to cardiac output) differs in individual cases. However, the close correlation of the combined diameters with cardiac output points to the importance of hepatic HHT lesions for the circulatory changes in these patients. This is in contrast to the situation in liver cirrhosis where arterial flow compensates for missing portal venous flow, but the increase in cardiac output is mainly caused by extrahepatic shunts. However, measurement of cardiac output is required to evaluate symptomatic patients with hepatic AVM, especially before and after therapeutic procedures. Right heart catheterization with the use of thermodilution or non-invasive echocardiography can be used to calculate cardiac output.57 In conclusion, ultrasound is a method of high sensitivity and specificity to detect hepatic involvement in HHT. Sonography is a
Figure 48-10. Dilated common hepatic artery. (B-mode; 3.75-MHz convex transducer.)
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Chapter 48 LIVER INVOLVEMENT IN OSLER-WEBER-RENDU DISEASE Figure 48-11. Sonographic aspect of nodular patern of hepatic involvement in hereditary hemorrhagic telangiectasia (pseudocirrhosis). (B-mode; 3.75-MHz convex transducer.)
low-cost, non-invasive bedside method and does not require the application of contrast media or radiation. Thus, the risk of side effects and complications is minimized. Therefore, ultrasound is proposed as the first-line method to detect and monitor hepatic involvement in HHT patients.
THERAPY Clinical complications of hepatic AVMs may be indications for therapeutic interventions. These complications are grouped as follows:54 ∑ cardiac insufficiency induced by arteriovenous shunts ∑ complications of portal hypertension ∑ hepatic encephalopathy caused by portovenous shunts In addition, other complications of patients with HHT in combination with hepatic manifestation have been documented in the literature. A case of a female patient at the age of 33 years, who presented with symptoms of cardiac insufficiency followed by cholangiosepsis, has been documented by Bauer et al.61 Abdominal pain may be related to different causes. These pains may be caused by a tension of the hepatic capsule. On the other hand the pain may be induced by a special form of angina abdominalis followed by a steal syndrome due to hepatic vascular malformations. Since severe symptomatic hepatic involvement in HHT is quite rare and presented by multiple clinical features, a standard therapy cannot be recommended. The therapy of cardiac insufficiency and portal hypertension caused by increased cardiac output or arterioportal shunts should be a conservative one based on pharmaceutical intervention. Beta-blockers are the primary choice for these patients.14 Analgesics could be used in the case of abdominal complaints. In case of insufficient medical treatment, other therapeutic options must be considered. In general there are three options:
1. surgical ligation of the hepatic artery 2. transcatheter embolization of the hepatic artery or appropriate branches 3. liver transplantation A comparison of the different therapeutic approaches is so far based on case reports and small groups of patients. The empirical data of complications and successful outcome are rather inconsistent, so that general guidelines for therapy cannot be given at the moment. The different therapeutic options are based on different concepts and strategies. Liver transplantation is, by definition, an exchange of a pathologically altered organ by a normal substitute: as such, it may be called gene therapy. Surgical ligation and embolization of the hepatic artery are used to reduce the pathological hepatic blood flow. However, embolization of hepatic vessels is applied in various clinical indications. The method is, among others, indicated for liver tumors, bleeding, fistula, aneurysms of the hepatic artery, and intrahepatic vascular malformations. Given the various indications, the technique of embolization is different. The aim of embolization may be necrosis in cases of malignant liver tumors. In contrast, necrosis should be avoided in vascular malformations. Additional special features of arteriovenous shunts of the liver should be considered: The substrate of embolization could be transmitted into the pulmonary vessels, if shunts present with a large diameter. This may lead to right heart failure, especially in patients with latent right heart insufficiency. Therefore, the embolic particles should not pass the hepatic shunts. On the other hand, large particles may cause severe necrosis of the liver parenchyma. As a consequence, special attention should be paid to the size of the embolic particles. Whiting and coworkers used radioactive particles to avoid transmitting embolic particles to the pulmonary vessels.72 Using this method, Whiting et al. could exclude the presence of embolic material in pulmonary vessels. Ligation of the hepatic artery has been reported in only very select cases in the past 20 years, in comparison to alternative methods of
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therapy.73 The surgical ligation of the hepatic artery is so far regarded as a second- or third-line option in hepatic manifestations of HHT, if liver transplantation or embolization is not possible. Data published in the literature of embolization in patients with hepatic manifestation of HHT are based on 29 patients so far. Given all data, embolization was technically successful in more than 90%. However, the clinical results after embolization differ considerably. While some authors reported severe side effects or a fatal outcome,54,73,74 other authors described a successful outcome in up to 80% of the treated patients.57 Therefore, the efficiency of this therapy is controversial.14,74 These facts may be due to different techniques of embolization. Furthermore, it cannot be ruled out that different subtypes (e.g., shunt characteristics) of hepatic vascular malformations influence the result and rate of side effects. In a recent study intrahepatic portovenous shunts were characterized by small and multiple shunts in an apparent network on the periphery with or without a large shunt.75 In the presence of marked shunting from the portal into the hepatic vein, only the hepatic artery supplies blood to the liver. Hepatic necrosis after hepatic artery embolization supports the presence of shunting from the portal vein to the hepatic veins. Therefore, especially in patients with significant portovenous shunting in some cases causing encephalopathy, arterial embolization may be harmful to the liver. Since embolization may lead to necrosis of liver parenchyma, embolization should not be used in patients with reduced liver function. If patients with insufficiency of the liver undergo embolization, only (sub-)segmental embolization should be performed in order to support regeneration of the liver parenchyma. However, liver transplantation may be the method of choice for this subgroup.73,76 Trembath and co-workers described a coincidence of HHT type 2 and pulmonary hypertension.24 In our own experience an additional female patient with hepatic vascular malformations presented with pulmonary hypertension and severe dyspnea. Embolization was technically sufficient, but no improvement of clinical symptoms could be observed. The patient died due to respiratory failure. Retrospectively no alternative therapeutic option was available for this patient, since her health status did not permit a combination of lung and liver transplantation. Vasodilators, such as sildefanil, used to treat pulmonary hypertension may aggravate a steal syndrome in cases of severe vascular malformation. The small number of treated patients does not allow enough data for a general conclusion. An analysis of subgroups may be of interest in future embolization studies. If vascular hepatic malformations cannot be effectively treated by embolization, liver transplantation may be an alternative option. So far liver transplantations have been reported in 23 HHT patients with liver involvement. Six of these patients have been primarily treated by embolization, ligation, or banding, followed by severe complications requiring liver transplantation. In another case report new vascular malformations occurred after embolization.73 If liver transplantation is discussed as an option in hepatic vascular malformations one should exclude extrahepatic vascular malformations in other organs, especially in the abdomen. In cases of advanced extrahepatic vascular malformations a liver transplantation may not be sufficient to reduce a hyperdynamic circulatory state.
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Clinical observations of liver involvement in patients suffering from HHT are reported in most cases in the fifth to sixth decade. Therefore it is necessary to keep in mind that – in contrast to embolization – liver transplantation may be restricted by the age of the patient. Another problem of liver transplantation is a persisting lack of donor organs. Therefore, embolization therapy may be a bridge to liver transplantation in selected patients until a suitable organ becomes available. Different complications may be observed after liver transplantation and embolization. As described above, embolization may induce necrosis of the liver parenchyma, ischemic cholangitis, and cholecystitis, with potentially lethal outcome in some patients. After liver transplantation complications such as infections and insufficiency of biliary or vascular anastomosis may occur, requiring surgical intervention or retransplantation.73,76–78 A fatal outcome after embolization, surgical intervention,79 and liver transplantation was observed in singular cases.54 After successful liver transplantation a recurrence of hepatic vascular malformations should not be expected and this fact could be interpreted as a cure of HHT as regards the liver. Embolization of hepatic malformations may also have a long-lasting effect.79,80 After the acute and intermediate postintervention period (> 6 months), further complications after embolization occur rarely during longterm follow-up. In contrast, liver transplantation may be accompanied by several continuing problems such as infections or renal toxicity as a consequence of immunosuppression. Considering the heterogeneous data, it is difficult to present a proposal for general therapeutic guidelines. However, patients with symptomatic liver involvement in HHT should be presented to specialized centers, where interdisciplinary conferences are provided by hepatologists, transplant surgeons, and interventional radiologists. Multicenter trials are required to determine the efficacy of various treatment modalities for this disease. With the identification of specific gene defects, family investigations can identify members at risk.
SUMMARY HHT, also known as Osler–Weber–Rendu disease, is an autosomal dominant disorder that results in fibrovascular dysplasia with the development of telangiectases and AVMs. Telangiectases cause easy bleeding on skin and mucosal membranes, whereas AVMs may lead to serious complications when they are located in lungs, liver, and brain. Clinical manifestations vary over time, and are generally progressive. Phenotypes of HHT have been classified based on the recently identified mutated genes endoglin (HHT-1) and activin-like kinase receptor (HHT-2). Both genes encode for two receptors of the TGF-b families. Other families with phenotype HHT do not bear these mutations, therefore other genes are probably also involved. Liver involvement due to AVMs and pseudocirrhosis is reported in up to 30% of individuals affected by HHT. Generally, hepatic involvement becomes clinically symptomatic predominantly in women during the fifth to sixth decade. Large hepatic AVMs can lead to significant complications, including high-output congestive
Chapter 48 LIVER INVOLVEMENT IN OSLER-WEBER-RENDU DISEASE
heart failure, portal hypertension, hepatic encephalopathy, and abdominal ischemia. Hepatic malformations can be diagnosed by imaging methods such as Doppler sonography, computed tomography, MRI, and angiography. Ultrasound is a method of high sensitivity and specificity to detect hepatic involvement in HHT and is a low-cost, noninvasive bedside method that does not require the application of contrast media or radiation. Therefore, ultrasound is proposed as the first-line method to detect and monitor hepatic involvement in HHT patients. The therapy of hepatic involvement in symptomatic patients remains controversial. Treatment that includes segmental embolization of branch hepatic arteries may be helpful, but can lead to severe complications. An alternative option is liver transplantation, which eradicates malformations. It is crucial to select the appropriate treatment for the right patient.
REFERENCES 1. Rendu HJ. Epistaxis repetees chez un sujet porteur de petits angiomes cutanes et muqueux. Gaz Hop 1896; 13:731–733. 2. Osler W. On a family from recurrent epitaxis, associated with multiple telangiectases of the skin and mucous membranes. Bull Johns Hopkins Hosp 1901; 12:333–337. 3. Weber FP. Multiple hereditary developmental angiomata (telangiectases) of the skin and mucous membranes associated with recurrent haemorrhages. Lancet 1907; 2:160–162. 4. Guttmacher AE, Marchuk DA, White RIJ. Hereditary hemorrhagic telangiectasia. N Engl J Med 1995; 333:918–924. 5. Shovlin CL, Letarte M. Hereditary haemorrhagic telangiectasia and pulmonary arteriovenous malformations: issues in clinical management and review of pathogenic mechanisms. Thorax 1999; 54:714–729. 6. Kjeldsen AD, Vase P, Green A. Hereditary haemorrhagic telangiectasia: a population-based study of prevalence and mortality in Danish patients. J Intern Med 1999; 245:31–39. 7. Yamaguchi H, Azuma H, Shigekiyo T, et al. A novel missense mutation in the endoglin gene in hereditary hemorrhagic telangiectasia. Thromb Haemostat 1997; 77:243–247. 8. El-Harith EA, Al-Kharasani M, Ahmed MA, Stuhrmann M. Hereditary hemorrhagic telangiectasia: a case report. Saudi Med J 1999; 20:797–799. 9. Gallione CJ, Scheessele EA, Reinhardt D, et al. Two common endoglin mutations in families with hereditary hemorrhagic telangiectasia in the Netherland Antilles: evidence for a founder effect. Hum Genet 2000; 107:40–44. 10. Guttmacher AE, McKinnon WC, Upton MD. Hereditary hemorrhagic telangiectasia: a disorder in search of the genetics community. Am J Med Genet 1994; 52:252–253. 11. Porteous ME, Burn J, Proctor SJ. Hereditary haemorrhagic telangiectasia: a clinical analysis. J Med Genet 1992; 29:527–530. 12. Shovlin CL. Molecular defects in rare bleeding disorders: hereditary haemorrhagic telangiectasia. Thromb Haemostat 1997; 78:145–150. 13. Plauchu H, de Chadarevian JP, Bideau A, Robert JM. Age-related clinical profile of hereditary hemorrhagic telangiectasia in an epidemiologically recruited population. Am J Med Genet 1989; 32:291–297. 14. Larson AM. Liver disease in hereditary hemorrhagic telangiectasia. J Clin Gastroenterol 2003; 36:149–158. 15. Bourdeau A, Dumont DJ, Letarte M. A murine model of hereditary hemorrhagic telangiectasia. J Clin Invest 1999; 104:1343–1351.
16. Barbara NP, Wrana JL, Letarte M. Endoglin is an accessory protein that interacts with the signaling receptor complex of multiple members of the transforming growth factor-b superfamily. J Biol Chem 1999; 274:584–594. 17. Bourdeau A, Faughnan ME, McDonald ML, et al. Potential role of modifier genes influencing transforming growth factor-beta1 levels in the development of vascular defects in endoglin heterozygous mice with hereditary hemorrhagic telangiectasia. Am J Pathol 2001; 158:2011–2020. 18. Abdalla SA, Pece-Barbara N, Vera S, et al. Analysis of ALK-1 and endoglin in newborns from families with hereditary hemorrhagic telangiectasia type 2. Hum Mol Genet 2000; 9:1227–1237. 19. Johnson DW, Berg JN, Gallione CJ, et al. A second locus for hereditary hemorrhagic telangiectasia maps to chromosome 12. Genome Res 1995; 5:21–28. 20. Berg J, Porteous M, Reinhardt D, et al. Hereditary haemorrhagic telangiectasia: a questionnaire based study to delineate the different phenotypes caused by endoglin and ALK1 mutations. J Med Genet 2003; 40:585–590. 21. Berg JN, Guttmacher AE, Marchuk DA, Porteous ME. Clinical heterogeneity in hereditary haemorrhagic telangiectasia: are pulmonary arteriovenous malformations more common in families linked to endoglin? J Med Genet 1996; 33:256–257. 22. McDonald JE, Miller JF, Hallam SE, et al. Clinical manifestations in a large hereditary hemorrhagic telangiectasia (HHT) type 2 kindred. Am J Genet 2000; 93:320 –327. 23. Kjeldsen AD, Brusgaard K, Poulsen L, et al. Mutations in the ALK-1 gene and the phenotype of hereditary hemorrhagic telangiectasia in two large Danish families. Am J Med Genet 2001; 98:298–302. 24. Trembath RC, Thomson JR, Machado RD, et al. Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia. N Engl J Med 2001; 345:325–334. 25. Nikolopoulos N, Xynos E, Vassilakis JS. Familial occurrence of hyperdynamic circulation status due to intrahepatic fistulae in hereditary hemorrhagic telangiectasia. Hepatogastroenterology 1988; 35:167–168. 26. Piantanida M, Buscarini E, Dellavecchia C, et al. Hereditary haemorrhagic telangiectasia with extensive liver involvement is not caused by either HHT1 or HHT2. J Med Genet 1996; 33:441–443. 27. Olivieri C, Mira E, Delù G, et al. Identification of 13 new mutations in the ACVRL 1 gene in a group of 52 unselected Italian patients affected by hereditary haemorrhagic telangiectasia. J Med Genet 2002; 39:e39. 28. Lin WD, Wu JY, Hsu HB, et al. Mutation analysis of a family with hereditary hemorrhagic telangiectasia associated with hepatic arteriovenous malformation. J Formos Med Assoc 2001; 100:817–819. 29. Kuehl HK, Caselitz M, Hasenkamp S, et al. Hepatic manifestation is associated with ALK1 in hereditary hemorrhagic telangiectasia: identification of five novel ALK1 and one novel ENG mutations. Hum Mutat 2005; 25:320. 30. Hagspiel KD, Christ ER, Schopke W. Hereditary hemorrhagic telangiectasis (Osler–Rendu–Weber disease) with pulmonary, hepatic and renal disease pattern. Rofo Fortschr Geb Rontgenstr Neuen Bildgeb Verfahr 1995; 163:190 –192. 31. Cooke DA. Renal arteriovenous malformation demonstrated angiographically in hereditary haemorrhagic telangiectasia (Rendu–Osler–Weber disease). J R Soc Med 1986; 79:744–746. 32. Brant AM, Schachat AP, White RI Jr. Ocular manifestations in hereditary hemorrhagic telangiectasia (Rendu–Osler–Weber disease). Am J Ophthalmol 1989; 107:642–646. 33. Lepori JC, Thisse JY, Danchin N, Saudax E. Rendu–Osler familial hemorrhagic telangiectasia with retinal localization. Bull Soc Ophtalmol Fr 1981; 81:641–644.
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34. Secil M, Goktay A, Dicle O, Pirnar T. Splenic vascular malformations and portal hypertension in hereditary hemorrhagic telangiectasia: sonographic findings. J Clin Ultrasound 2001; 29:56–59. 35. Kurnik PB, Heymann WR. Coronary artery ectasia associated with hereditary hemorrhagic telangiectasia. Arch Intern Med 1989; 149:2357–2359. 36. Romer W, Burk M, Schneider W. Hereditary hemorrhagic telangiectasia (Osler’s disease). Dtsch Med Wochenschr 1992; 117:669–675. 37. Aassar OS, Friedmann CM, White RI Jr. The natural history of epistaxis in hereditary hemorrhagic telangiectasia. Laryngoscope 1991; 101:977–980. 38. Haitjema T, Westermann CJ, Overtoom TT, et al. Hereditary hemorrhagic telangiectasia (Osler–Weber–Rendu disease): new insights in pathogenesis, complications, and treatment. Arch Intern Med 1996; 156:714–719. 39. Kjeldsen AD, Oxhoj H, Andersen PE, et al. Prevalence of pulmonary arteriovenous malformations (PAVMs) and occurrence of neurological symptoms in patients with hereditary haemorrhagic telangiectasia. J Intern Med 2000; 248:255–262. 40. White RI Jr, Lynch-Nyhan A, Terry P, et al. Pulmonary arteriovenous malformations: techniques and long-term outcome of embolotherapy. Radiology 1988; 169:663–669. 41. Ference BA, Shannon TM, White RI Jr, et al. Life-threatening pulmonary hemorrhage with pulmonary arteriovenous malformations and hereditary hemorrhagic telangiectasia. Chest 1994; 106:1387–1390. 42. Fulbright RK, Chaloupka JC, Putman CM, et al. MR of hereditary hemorrhagic telangiectasia: prevalence and spectrum of cerebrovascular malformations. AJNR Am J Neuroradiol 1998; 19:477–484. 43. Roman G, Fisher M, Perl DP, Poser CM. Neurological manifestations of hereditary hemorrhagic telangiectasia (Rendu–Osler–Weber disease): report of 2 cases and review of the literature. Ann Neurol 1978; 4:130–144. 44. Vase P, Grove O. Gastrointestinal lesions in hereditary hemorrhagic telangiectasia. Gastroenterology 1986; 91:1079–1083. 45. Reilly PJ, Nostrant TT. Clinical manifestations of hereditary hemorrhagic telangiectasia. Am J Gastroenterol 1984; 79:363–367. 46. Van Cutsem E, Rutgeerts P, Vantrappen G. Treatment of bleeding gastrointestinal vascular malformations with oestrogen–progesterone. Lancet 1999; 335:953–955. 47. Costa MT, Maldonado R, Valente A, et al. Hemobilia in hereditary hemorrhagic telangiectasia: an unusual complication of endoscopic retrograde cholangiopancreatography. Endoscopy 2003; 35:531–533. 48. Sharma VK, Howden CW. Gastrointestinal and hepatic manifestations of hereditary hemorrhagic telangiectasia. Dig Dis 1998; 16:169–174. 49. Longacre AV, Gross CP, Gallitelli M, et al. Diagnosis and management of gastrointestinal bleeding in patients with hereditary hemorrhagic telangiectasia. Am J Gastroenterol 2003; 98:59–65. 50. Kitamura T, Tanabe S, Koizumi W, et al. Rendu–Osler–Weber disease successfully treated by argon plasma coagulation. Gastrointest Endosc 2001; 54:525–527. 51. Bernard G, Mion F, Henry L, et al. Hepatic involvement in hereditary hemorrhagic telangiectasia: clinical, radiological, and hemodynamic studies of 11 cases. Gastroenterology 1993; 105:482–487. 52. Martini GA. The liver in hereditary haemorrhagic teleangiectasia: an inborn error of vascular structure with multiple manifestations: a reappraisal. Gut 1978; 19:531–537. 53. Kirchner J, Zipf A, Dietrich CF, et al. Universal organ involvement in Rendu–Osler–Weber disease: interdisciplinary
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55.
56.
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58.
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61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
diagnosis and interventional therapy. Z Gastroenterol 1996; 34:747–752. Garcia-Tsao G, Korzenik JR, Young L, et al. Liver disease in patients with hereditary hemorrhagic telangiectasia. N Engl J Med 2000; 343:931–936. Caselitz M, Bahr MJ, Bleck JS, et al. Sonographic criteria for the diagnosis of hepatic involvement in hereditary hemorrhagic telangiectasia (HHT). Hepatology 2003; 37:1139–1146. Jameson CF. Primary hepatocellular carcinoma in hereditary haemorrhagic telangiectasia: a case report and literature review. Histopathology 1989; 15:550–552. Caselitz M, Wagner S, Chavan A, et al. Clinical outcome of transfemoral embolisation in patients with arteriovenous malformations of the liver in hereditary haemorrhagic telangiectasia (Weber–Rendu–Osler disease). Gut 1998; 42:123–126. Selmaier M, Cidlinsky K, Ell C, Hahn EG. Liver hemangiomatosis in Osler’s disease. Dtsch Med Wochenschr 1993; 118:1015–1019. Shovlin CL, Winstock AR, Peters AM, et al. Medical complications of pregnancy in hereditary haemorrhagic telangiectasia. Q J Med 1995; 88:879–887. Graf C, Perrett G, Torner J. Bleeding from cerebral arteriovenous malformations as part of their natural history. J Neurosurg 1983; 58:331–337. Bauer T, Britton P, Lomas D, et al. Liver transplantation for hepatic arteriovenous malformation in hereditary haemorrhagic telangiectasia. J Hepatol 1995; 22:586–590. Gammon RB, Miksa AK, Keller FS. Osler–Weber–Rendu disease and pulmonary arteriovenous fistulas. Deterioration and embolotherapy during pregnancy. Chest 1990; 98:1522–1524. Livneh A, Langevitz P, Morag B, et al. Functionally reversible hepatic arteriovenous fistulas during pregnancy in patients with hereditary hemorrhagic telangiectasia. South Med J 1988; 81:1047–1049. Fagel WJ, Perlberger R, Kauffmann RH. Portosystemic encephalopathy in hereditary hemorrhagic telangiectasia. Am J Med 1988; 85:858–860. Buscarini E, Buscarini L, Danesino C, et al. Hepatic vascular malformations in hereditary hemorrhagic telangiectasia: Doppler sonographic screening in a large family. J Hepatol 1997; 26:111–118. Hashimoto M, Tate E, Nishii T, et al. Angiography of hepatic vascular malformations associated with hereditary hemorrhagic telangiectasia. Cardiovasc Intervent Radiol 2003; 26:177–180. Buscarini E, Buscarini L, Civardi G, et al. Hepatic vascular malformations in hereditary hemorrhagic telangiectasia: imaging findings. AJR Am J Roentgenol 1994; 163:1105–1110. Kakitsubata Y, Kakitsubata S, Kiyomizu H, et al. Intrahepatic portal-hepatic venous shunts demonstrated by US, CT, and MR imaging. Acta Radiol 1996; 37:680–684. Saxena R, Hytiroglou P, Atillasoy EO, et al. Coexistence of hereditary hemorrhagic telangiectasia and fibropolycystic liver disease. Am J Surg Pathol 1998; 22:368–372. Hatzidakis AA, Gogas C, Papanikolaou N, et al. Hepatic involvement in hereditary hemorrhagic telangiectasia (Rendu–Osler–Weber disease). Eur Radiol 2002; 12 (suppl 4):S51–S55. Hillert C, Broering DC, Gundlach M, et al. Hepatic involvement in hereditary hemorrhagic telangiectasia: an unusual indication for liver transplantation. Liver Transpl 2001; 7:266–268. Whiting JHJ, Morton KA, Datz FL, et al. Embolization of hepatic arteriovenous malformations using radiolabeled and nonradiolabeled polyvinyl alcohol sponge in a patient with hereditary hemorrhagic telangiectasia: case report. J Nucl Med 1992; 33:260–262.
Chapter 48 LIVER INVOLVEMENT IN OSLER-WEBER-RENDU DISEASE
73. Pfitzmann R, Heise M, Langrehr JM, et al. Liver transplantation for treatment of intrahepatic Osler’s disease: first experiences. Transplantation 2001; 72:237–241. 74. Whiting JH Jr, Korzenik JR, Miller FJ Jr. Fatal outcome after embolotherapy for hepatic arteriovenous malformations of the liver in two patients with hereditary hemorrhagic telangiectasia. J Vasc Interv Radiol 2000; 11:855–858. 75. Matsumoto S, Mori H, Yamada Y, et al. Intrahepatic portohepatic venous shunts in Rendu–Osler–Weber disease: imaging demonstration. Eur Radiol 2004; 14:592–596. 76. Odorico JS, Hakim MN, Becker YT, et al. Liver transplantation as definitive therapy for complications after arterial embolization for hepatic manifestations of hereditary hemorrhagic telangiectasia. Liver Transpl Surg 1998; 4:483–490. 77. Boillot O, Bianco F, Viale JP, et al. Liver transplantation resolves the hyperdynamic circulation in hereditary hemorrhagic
telangiectasia with hepatic involvement. Gastroenterology 1999; 116:187–192. 78. Azoulay D, Precetti S, Emile JF, et al. Liver transplantation for intrahepatic Rendu–Osler–Weber ‘s disease: the Paul Brousse hospital experience. Gastroenterol Clin Biol 2002; 26:828–834. 79. Chavan A, Galanski M, Wagner S, et al. Hereditary hemorrhagic telangiectasia: effective protocol for embolization of hepatic vascular malformations – experience in five patients. Radiology 1998; 209:735–739. 80. Stockx L, Raat H, Caerts B, et al. Transcatheter embolization of hepatic arteriovenous fistulas in Rendu–Osler–Weber disease: a case report and review of the literature. Eur Radiol 1999; 9:1434–1437.
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PRETRANSPLANT EVALUATION AND CARE
49
Aijaz Ahmed, Emmet B. Keeffe Abbreviations AIDS acquired immunodeficiency syndrome AMA antimitochondrial antibody ANA antinuclear antibody AST aspartate aminotransferase BMI body mass index CAD coronary artery disease CMV cytomegalovirus CT computed tomography CTP Child–Turcotte–Pugh EBV Epstein–Barr virus ERCP endoscopic retrograde cholangiopancreatography FOBT fecal occult blood test
HAV HBV HCC HCV HDV HIV HSV IBD INR LDLT MELD MRI
hepatitis A virus hepatitis B virus hepatocellular carcinoma hepatitis C virus hepatitis D virus human immunodeficiency virus herpes simplex virus inflammatory bowel disease international normalized ratio living donor liver transplantation Model for End-Stage Liver Disease magnetic resonance imaging
INTRODUCTION Over the past 25 years there has been significant improvement in the 1-year patient survival rate following liver transplantation, from 30% in the early 1980s1 to the current rate of 85–90%.2 This is related in large part to judicious pretransplant evaluation and patient selection, improvements in surgical technique, the introduction of safer and more efficacious immunosuppressive regimens, and the prevention of infections historically associated with solid organ transplantation and potent immunosuppression.3
PRETRANSPLANT EVALUATION Prompt pretransplant evaluation is a critically important initial step that may influence the outcome of liver transplantation. In the current environment of an organ shortage, every effort should be made to avoid any delay in referral for liver transplant evaluation and listing. Patients with compensated cirrhosis should be monitored closely for the onset of hepatic decompensation or the development of hepatocellular carcinoma (HCC), which are triggers for referral for consideration for liver transplantation.
DEMOGRAPHICS During the last 5 years, the number of liver transplantations performed annually has remained relatively unchanged in the US, at approximately 5000 per year.4 By contrast, there has been an increase in the number of patients presenting with hepatic decompensation. Over the past few years, the number of patients listed for liver transplantation with the United Network for Organ Sharing (UNOS) has remained stationary, at 17 000–18 000. This imbalance
NIDDK OPOs PAP PBC PPD PSC PT TIPS UNOS VZV
National Institute of Diabetes and Digestive and Kidney Diseases organ procurement organizations pulmonary artery pressure primary biliary cirrhosis purified protein derivative primary sclerosing cholangitis prothrombin time transjugular intrahepatic portosystemic shunt United Network for Organ Sharing varicella-zoster virus
between demand and supply has resulted in a rise in the mortality rate among patients on the waiting list. Statistics from 2002 showed that 1777 listed patients died while awaiting liver transplantation, and 688 were removed from the list based on the development of contraindications to transplant surgery.5 These data demonstrate the limited options available to patients with hepatic decompensation and the importance of prompt pretransplant evaluation. The role of judicious patient selection for liver transplantation is even more important as physicians manage an increasing number of patients with end-stage liver disease, related in large part to the increased number of patients with chronic hepatitis C who have progressed through the natural history of this infection to end-stage liver disease or HCC.3,4
MEDICAL JUSTICE VERSUS UTILITY UNOS is committed to providing a prudent and optimal policy for organ allocation and distribution without compromising the ethical principles of medical justice and utility. In the field of liver transplantation, the concept of medical justice prioritizes the sickest patient with the longest waiting time, whereas medical utility prioritizes the patient with the highest likelihood of a successful post-transplant outcome. The Model for End-Stage Liver Disease (MELD), which is the criterion employed by UNOS for organ allocation since 2002, prioritizes medical justice and may thus predispose recipients to a higher risk of post-transplant complications. The recent surge in the number of patients presenting with hepatic decompensation over the past several years, together with the continued donor shortage, has prompted transplant centers to adhere to even more strict selection criteria in an attempt to identify patients who can withstand a prolonged waiting time and expect a reasonable outcome following transplantation. The donor short-
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age, major cuts in healthcare budgets, and reduced reimbursement rates for liver transplantation may result in a move by individual transplant selection committees to prioritize medical utility and cost-effectiveness by not listing or by delisting the sickest patients.6,7 It is difficult to predictably identify patients at higher risk for posttransplant complications, but more data are becoming available that may help exclude certain subgroups who are poor candidates for transplant surgery.8–11 For example, patients with recurrent hepatitis C virus (HCV) infection and severe allograft dysfunction who are being considered for retransplantation are at risk for a poor outcome after a second transplant and increased resource utilization.3,4 Thus, it remains controversial whether or not to offer a cadaveric organ for retransplantation to a patient with allograft failure, particularly due to recurrent hepatitis C, while an increasing number of patients wait for a donor organ to undergo their first transplant surgery.
AIMS OF LIVER TRANSPLANTATION The two major aims of transplantation for end-stage liver disease are to improve survival and to increase functional status. It is important that potential candidates for pretransplant evaluation do not have conditions that would prevent fulfillment of these aims. Statistics from UNOS on 24 000 adults who received liver transplantation between 1987 and 1998 showed 1-year, 4-year, and 10-year overall survival rates of 85%, 76%, and 61%, respectively.12 The highest 7year survival rates of 78–79% were noted in patients with the cholestatic liver disease, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC). On the other hand, patients who underwent liver transplantation for hepatic malignancies demonstrated a low 7-year survival rate of 34%. A more recent similar analysis of 17 044 recipients in the UNOS database transplanted from 1990 to 1996 showed nearly identical survival rates to the earlier analysis cited above: 83.0%, 70.2% and 61.9% at 1 year, 5 years and 8 years, respectively.13 In addition, this analysis showed that survival improved significantly over time: 74.8% in 1990 to 86.2% in 1996. This analysis also demonstrated the impact of chronic hepatitis C on liver transplantation: HCV infection was the reason for 20.0% of transplants in 1990–1992 and 30.8% in 1995–1996.13 The second goal of liver transplantation, to improve quality of life, was confirmed by a meta-analysis of health-related quality of life after liver transplantation that demonstrated a significant improvement in post-transplant cognitive, physical, and psychological functioning.14
INDICATIONS FOR LIVER TRANSPLANTATION The indications for liver transplantation can be divided into four major categories: hepatic decompensation secondary to chronic liver disease; acute liver failure; hepatic malignancy confined to the liver; and inborn errors of metabolism. Patients with metabolic disorders may present with hepatic decompensation (hereditary hemochromatosis, Wilson’s disease, and a1-antitrypsin deficiency) or a histologically intact liver with hepatic metabolic defect that results in extrahepatic organ damage (type I hyperoxaluria or familial homozygous hypercholesterolemia).15 Chronic hepatitis C is now the leading indication for liver transplantation in the US (Table 49-1). Pretransplant evaluation and care can be optimized with timely recog-
934
Table 49-1. Liver Disease of Adult Transplant Recipients in the United States (UNOS Database 1987–1998; n = 24 900) Primary Liver Disease Chronic hepatitis C Alcoholic liver disease Alcoholic liver disease and hepatitis C Chronic hepatitis B Cryptogenic cirrhosis Primary biliary cirrhosis Primary sclerosing cholangitis Autoimmune hepatitis Acute liver failure Hepatic malignancy Metabolic diseases Other Unknown
Number
%
5155 4258 1106 1368 2719 2317 2178 1194 1555 951 923 1050 126
20.7 17.1 4.4 5.5 10.9 9.3 8.7 4.8 6.2 3.8 3.7 4.2 0.5
Adapted and reprinted with permission from: Seaberg EC, Belle SH, Beringer KC, et al. Liver transplantation in the United States from 1987–1998: Updated results from the Pitt-UNOS liver transplant registry. In: Cecka JM, Terasaki PI, eds. Clin Transpl 1998. Los Angeles: UCLA Tissue Typing Laboratory, 1999:17–37.
nition and prompt referral of cirrhotic patients with clinical evidence of hepatic decompensation and/or abnormal biochemical parameters indicative of major impairment of hepatic synthetic function.3,16 Incapacitating fatigue and poor nutritional status are typically noted in association with other clinical or biochemical indications for liver transplantation. Acute liver failure is a relatively uncommon indication for transplantation, accounting for 6.2% of adult liver transplants.12 Acute liver failure must be recognized promptly and patients referred for an expedited evaluation for transplantation. Currently, there are two generally accepted listing criteria for acute liver failure, based on the reports of O’Grady et al.17 at King’s College Hospital and by Bernuau et al.18 at Villejuif. These criteria are useful for the early identification of patients with a poor prognosis who would thus benefit from timely liver transplantation (Table 49-2). The King’s College listing criteria provide a high specificity for poor outcome of acute liver failure, but a low negative predictive value.19 Therefore, patients with acute liver failure who do not meet the King’s College criteria should still undergo evaluation for transplantation as spontaneous recovery cannot be reliably predicted.19,20 Over the last decade, several factors have been identified as reliable predictors of improved allograft and patient survival following transplantation for acute liver failure, including early recognition, prompt referral, and multidisciplinary intensive care management. Hepatic malignancy, an uncommon indication in the past (3.8%),12 is now emerging as a growing indication for liver transplantation owing to the increased number of patients with chronic hepatitis C who develop end-stage liver disease, and the implementation of the MELD scoring system which grants priority to selected patients with HCC in the setting of cirrhosis.21 The selection of patients with HCC for either liver transplantation or hepatic resection has been controversial. However, several studies have demonstrated that liver transplantation provides improved survival and better costeffectiveness than hepatic resection in patients with cirrhosis.22 The long-term tumor-free survival following liver transplantation is over 80% for patients with solitary tumors less than 5 cm, or up to three tumors each less than 3 cm (so-called Milan criteria).23 Preliminary
Chapter 49 PRETRANSPLANT EVALUATION AND CARE
Table 49-2. Criteria for Liver Transplantation in Fulminant Hepatic Failure Criteria of King’s College, London1 Acetaminophen patients pH < 7.3, or Prothrombin time > 6.5 (INR) and serum creatinine > 3.4 mg/dl Non-acetaminophen patients Prothrombin time > 6.5 (INR), or Any three of the following variables: Age < 10 or > 40 years Etiology: non-A, non-B hepatitis; halothane hepatitis; idiosyncratic drug reaction Duration of jaundice before encephalopathy > 7 days Prothrombin time > 3.5 (INR) Serum bilirubin > 17.5 mg/dl Criteria of Hospital Paul-Brousse, Villejuif2 Hepatic encephalopathy, and Factor V level: < 20% in patient younger than 30 years of age, or < 30% in patient 30 years of age or older INR, international normalized ratio. 1 From: O’Grady JG, Alexander GJ, Hayllar KM, Williams R. Early indicators of prognosis in fulminant hepatic failure. Gastroenterology 1989; 97:439–445. 2 From: Bernuau J, Samuel D, Durand F, et al. Criteria for emergency liver transplantation in patients with acute viral hepatitis and factor V below 50% of normal: a prospective study [Abstract]. Hepatology 1991;14:49A. Adapted and reprinted with permission from: Yu AS, Ahmed A, Keeffe EB. Liver transplantation: evolving patient selection criteria. Can J Gastroenterol 2001;15:729–738.
data are promising regarding the role of preoperative chemoembolization to retard the progression of HCC and to reduce the risk of intraoperative tumor dissemination as patients await transplantation after listing. In one study, 1-year and 2-year survival rates of 91% and 84%, respectively, without tumor recurrence were noted in 27 recipients undergoing this protocol.24 If HCC is diagnosed incidentally on analysis of the explanted liver, survival is comparable to that in patients without tumor and the recurrence rate is lower than in patients with a pretransplant diagnosis of HCC.25 Fibrolamellar HCC is an uncommon and less aggressive variant, with a tendency to affect younger patients and with equal gender distribution.26 It is usually noted in the absence of pre-existing liver disease and is not associated with elevated a-fetoprotein levels. An aggressive approach toward surgical resection of these tumors and contiguous structures or transplantation for non-resectable tumors can yield acceptable results. The outcome following liver transplantation for other uncommon primary hepatic malignancies, such as epithelioid hemangioendothelioma and hepatoblastoma, is encouraging. However, survival following transplantation is dismal in patients with hemangiosarcoma and metastatic lesions to the liver, with the exception of neuroendocrine tumors. Budd–Chiari syndrome results in thrombotic or non-thrombotic blockage of the major hepatic veins, inferior vena cava, or both.27 The conditions that may cause Budd–Chiari syndrome include myeloproliferative syndrome (50%), tumors (10%), estrogen use and pregnancy (10%), hypercoagulable states (5%), and paroxysmal nocturnal hemoglobinura (5%), with the remaining cases (20%) being idiopathic. Progressive liver disease typically ensues and is characterized by congestion with centrilobular necrosis, fibrosis, and cirrhosis following occlusion of hepatic veins. The decision to proceed with transplantation may be facilitated by the performance of a liver biopsy. Liver transplantation is indicated for bridging fibro-
Table 49-3. Contraindications to Liver Transplantation Compensated cirrhosis without complications (CTP score 5–6) Advanced cardiopulmonary disease Human immunodeficiency (HIV) infection* Uncontrolled sepsis Cholangiocarcinoma* Extrahepatic malignancies Active alcoholism or substance abuse Anatomic abnormality precluding liver transplantation Inability to comply, e.g. psychosocial issues No potential for meaningful lifestyle post transplant, such as with irreversible and debilitating neuropsychiatric diseases CTP, Child–Turcotte–Pugh. *In most, but not all, centers cholangiocarcinoma and HIV infection are considered contraindications to liver transplantation. Transplantation for HIV is currently undergoing study at selected transplant centers. Adapted and reprinted with permission from: Yu AS, Keeffe EB. Orthotopic liver transplantation. In: Boyer T, Zakim D, eds. Hepatology, 4th edn. Philadelphia: Harcourt Health Sciences, 2002: 1617–1656.
sis and cirrhosis, whereas decompression by transjugular intrahepatic portosystemic shunt (TIPS) or shunt surgery, which is less often employed, is sufficient for mild liver disease.27,28 The prognosis following transplantation is excellent, but patients may need long-term anticoagulation. Polycystic liver disease is a rare indication for transplantation.29,30 Multiple liver cysts can be complicated by hemorrhage, infection, abdominal pain, massive cystic hepatomegaly, portal hypertension complicated by ascites, biliary obstruction, and rarely malignant transformation to cholangiocarcinoma despite intact hepatic synthetic function. Liver transplantation is curative, but remains controversial when performed in the absence of hepatic decompensation.30 The post-transplant survival rate is comparable to that for other indications of liver transplantation, with complete resolution of debilitating symptoms and complications.
CONTRAINDICATIONS TO LIVER TRANSPLANTATION: ABSOLUTE AND RELATIVE Contraindications to liver transplantation are listed in Table 49-3. A number of clinical situations, for example irreversible neurologic diseases and metastatic cancer, are obvious absolute contraindications. Other relative and absolute contraindications are discussed in the following section. In patients with one or more comorbid conditions, appropriate consultations and a thorough review by the transplant selection committee are helpful in determining whether or not to proceed with liver transplantation.
Active Alcohol or Substance Abuse Liver transplantation is contraindicated in patients with ongoing alcoholism and/or substance abuse, but can be performed once recovery has been convincingly demonstrated. The 1-year and 5-year actuarial survival rates following transplantation for patients with alcoholic liver disease in the US (82% and 68%, respectively) and in Europe (85% and 70%, respectively) are similar to the outcomes of patients transplanted for other types of chronic liver disease.31 In addition, post-transplant improvements in health-related quality of
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life are similar in patients transplanted for alcoholic liver disease versus other causes of end-stage liver disease.14 Specific improvements were also seen in employment, marital status, psychological health, and social activity following transplantation for alcoholic liver disease.32 Approximately 20% of patients transplanted for alcoholic liver disease use alcohol post transplant, with one-third of these individuals exhibiting repetitive or heavy drinking.31 However, a detrimental effect on graft or patient survival associated with resumption of drinking has only seldom been demonstrated. There are few reliable predictors of relapse in alcoholic patients after liver transplantation.33 Although not supported by all studies, abstinence of less than 6 months prior to transplantation appears to be a reasonable predictor of recidivism and is widely employed along with other criteria for listing for liver transplantation. Thus, patients are considered for liver transplantation upon completion of 6 months of sobriety, rehabilitation, and proof of compliance based on recommendations made by the transplant selection committee.31 There are no good data to determine whether some patients with sobriety less than 6 months might benefit from liver transplantation.
Older Age Liver transplantation is not contraindicated per se in patients with advanced age.34 However, pretransplant evaluation needs to be comprehensive, with emphasis on screening for comorbid conditions, such as cardiopulmonary or vascular disease and cancer. Review of the UNOS database from 1987 to 1998 showed a 1.5-fold relative risk of death following liver transplantation in recipients who were 50–59 years of age, with a further increase in mortality in those who were 60 or older.12,13 On the other hand, publications from individual liver transplant centers demonstrate comparable survival outcome between older and younger adult recipients. As many as two-thirds of older liver transplant recipients are fully functional in the post-transplant period and have improved quality of life. Paradoxically, older recipients have a senescent immune system, which results in decreased requirements for immunosuppressive drugs, and possibly a lower rate of acute allograft rejection. There is emerging evidence that despite favorable short-term survival in the elderly, long-term survival may be worse because of an increased rate of malignancy and heart disease, based on an age-related risk for these conditions. Thus, although advanced age is a negative risk factor for survival after liver transplantation, per se it should not exclude a patient from transplant surgery. However, a thorough pretransplant evaluation and careful long-term follow-up with attention to usual health maintenance issues in the elderly are mandatory.
Obesity The long-term survival rate following liver transplantation in patients with severe obesity has been controversial in reports from transplant centers. For example, in two studies survival in obese patients and non-obese controls was comparable.35,36 However, a study utilizing the UNOS database demonstrated adverse outcomes in patients with severe obesity.37 In an analysis of 18 172 recipients, 54% were classified as ranging from overweight to morbidly obese. Immediate, 1-year, and 2-year mortality was significantly higher in the morbidly obese group (body mass index (BMI) >40). In addition, 5-year mortality was significantly higher in both the severely obese (BMI 35.1–40) and the morbidly obese recipients, mostly as a result of
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adverse cardiovascular events. The authors concluded that weight loss should be recommended for obese patients awaiting a liver transplantation, especially if their BMI is more than 35.37 Observations in the post-transplant period are mixed regarding the tendency for patients to gain weight, with some studies demonstrating weight gain and others showing steady weight after transplantation.36 The risk of wound infections, respiratory failure and systemic vascular complications in the post-transplant period can be relatively high in obese patients.36
Cardiovascular Disease The decision regarding the candidacy of a patient with cardiac risk factors is dependent on several factors, and these patients should undergo a comprehensive pretransplant evaluation.16,38 Patients with end-stage liver disease maintain a lower baseline systemic vascular resistance. Following liver transplantation, patients with unremarkable pretransplant cardiac evaluation have been noted to develop congestive heart failure and cardiac arrhythmias, most likely resulting from resolution of low systemic vascular resistance with volume overload produced by blood products and fluids, and possibly the vasoactive actions of immunosuppressive agents resulting in increased afterload.39 Cardiac contraindications for liver transplantation include symptomatic coronary artery disease, severe ventricular dysfunction, advanced cardiomyopathy, severe pulmonary hypertension, severe valvular heart disease, and aortic stenosis with significant pressure gradient and poor ventricular function.16,40 The stress of liver transplant surgery can trigger myocardial ischemia or infarction in patients with significant underlying coronary artery disease.38 In this population, the overall morbidity can be as high as 81% and mortality can approach 50%.41 Patients with coronary artery disease can be listed for liver transplantation following correction by angioplasty or bypass surgery.41,42 It is recommended that patients with compensated cirrhosis should undergo angioplasty, as bypass surgery is associated with significantly higher risk of postoperative hepatic decompensation. The experience with combined coronary bypass grafting and liver transplantation is limited and associated with a high risk of complications; this approach has been employed in only a few circumstances.43
Renal Failure Candidates for liver transplantation with coexisting renal insufficiency should undergo an extensive pretransplant evaluation in consultation with a transplant nephrologist. Kidney biopsy may or may not be needed, and is associated with a significant risk of bleeding due to coagulopathy associated with hepatic decompensation.44 The etiology of renal dysfunction in the setting of end-stage liver disease is often multifactorial. Intrinsic renal disease is not a contraindication for liver transplantation, but may be an indication for combined liver–kidney transplantation, which has a slightly reduced but acceptable outcome. Statistics from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Liver Transplantation Database demonstrates that renal insufficiency associated with acute liver failure, occurring in patients undergoing dialysis, or leading to combined liver and kidney transplantation, results in reduced patient and graft survival rates, prolonged stay in the intensive care unit, and higher costs.45
Chapter 49 PRETRANSPLANT EVALUATION AND CARE
Pulmonary Disease Patients with severe pulmonary hypertension and advanced chronic obstructive pulmonary diseases or pulmonary fibrosis in the setting of end-stage liver disease are poor candidates for liver transplantation. Relative contraindications for liver transplant surgery include reversible pulmonary processes, such as reactive airway disease or asthma, hepatic hydrothorax, muscle wasting, and infectious processes.46 Patients with a1-antitrypsin deficiency have been shown to demonstrate an improvement in pulmonary function following liver transplantation. Patients with active tuberculosis should be treated for at least 2 weeks, and optimally for several months, in the pretransplant period, with continuation of treatment for 1 year after liver transplantation.39 Patients who are found to be PPD positive during pretransplant evaluation should undergo prophylaxis with isoniazid plus pyridoxine or ofloxacin for 6 months.47 Patients with cirrhosis are at risk for developing hepatopulmonary syndrome and portopulmonary hypertension. Patients with hepatopulmonary syndrome develop intrapulmonary vascular dilatation (with right-to-left shunting) and arterial hypoxemia in the setting of portal hypertension.48 Post-transplant mortality can be predicted by the severity of pretransplant hypoxemia, with a rise in the mortality rate from less than 5–30% if pretransplant PaO2 drops below 50 mmHg.49 The rapid progression of hepatopulmonary syndrome with worsening oxygenation may warrant consideration for priority listing and expedited liver transplantation. Following transplantation, hepatopulmonary syndrome can improve and completely resolve over several months.50 Patients may need continued oxygen supplementation for as long as 12–15 months while vascular remodeling takes place. The poor prognostic indicators for post-transplant outcome include refractory hypoxemia with multiorgan failure, intracerebral hemorrhage, sepsis, and portal vein thrombosis. Patients with portopulmonary hypertension in the setting of cirrhosis must undergo risk stratification during pretransplant evaluation.51 Several predictors for pulmonary hypertension have been identified, including systemic arterial hypertension, loud pulmonary component of the second heart sound, right ventricular heave on physical examination, right ventricular dilatation, right ventricular hypertrophy, and estimated systolic pulmonary artery pressure (PAP) higher than 40 mmHg on echocardiography.52 These predictors have greater than 90% specificity but a low sensitivity, and identify less than two-thirds of patients with pulmonary hypertension during pretransplant evaluation. On the other hand, it has been demonstrated that a systolic right ventricular pressure of 50 mmHg or higher is a reliable predictor of moderate to severe pulmonary hypertension, with a sensitivity of 97% and specificity of 77%.53 Suspected patients must undergo right-sided cardiac catheterization during the pretransplant evaluation.54 Patients with a mean PAP of 50 mmHg or higher have a 100% risk of cardiopulmonary mortality following liver transplantation. Therefore, severe pulmonary hypertension (PAP 50 mmHg or higher) is an absolute contraindication for liver transplantation. On the contrary, patients with a mean PAP of less than 35 mmHg are suitable candidates for liver transplant surgery with no added increase in risk. Patients with a mean PAP between 35 and 50 mmHg, or a pulmonary vascular resistance of 250 dynes/s/cm-5 or greater, have a 50% risk of cardiopulmonary mortality post transplant. Long-term epoprostenol infu-
sion may improve PAP in this subgroup of patients and lower the risk of mortality associated with liver transplant surgery.55
Human Immunodeficiency Virus Infection Human immunodeficiency virus (HIV) infection is considered an absolute contraindication for transplantation in most liver transplant programs in the US. However, antiretroviral drug therapy has changed the natural history of HIV infection, and the role of these drugs in improving post-transplant outcomes is under study.56 The risk of developing acquired immunodeficiency syndrome (AIDS) in HIV-positive patients was shown to be higher following liver transplantation than in non-transplanted patients in the early experience with transplantation in patients with HIV infection. In addition, post-transplant immunosuppression resulted in increased risk of progression to AIDS in HIV-positive patients. Similarly, HIVnegative patients who acquired HIV infection from donor organs or blood products at the time of liver transplantation demonstrated a poor outcome. However, recent data are more promising.56 The concerns of HIV progression have not been borne out by a growing anecdotal experience in the era of antiretroviral therapy. In addition, the severity of recurrent hepatitis C, which is almost universal in patients with HIV infection coming to transplantation, does not appear to be different from that in HIV-negative patients transplanted for chronic hepatitis C. Finally, immunosuppression does not appear to increase the risk of infectious complications or the development of AIDS.
Infections Both ongoing and untreated infections are contraindications to liver transplantation and must be treated adequately during the pretransplant period. Sepsis and pneumonia are considered absolute contraindications to liver transplantation. Other serious infections, including osteomyelitis, fungal diseases and deep abscesses, must be treated prior to transplantation.39 It is recommended that spontaneous bacterial peritonitis must be treated for at least 48 hours, with documented eradication of infection on repeat paracentesis before transplant surgery.38
Retransplantation Retransplantation is a consideration in patients with recurrent HCV infection following transplantation when progressive liver disease has developed.57 Recurrent hepatitis C is an independent predictor of poor survival following liver transplantation, based on multivariate logistic regression analysis.58 The outcome after retransplantation in the setting of recurrent hepatitis C is less than optimal, with less than 50% survival rate at 12 months. The poor survival rate was unrelated to the presence or absence of complications, such as graft failure due to hepatic artery thrombosis, biliary complications, or chronic ductopenic rejection.59 Hyperbilirubinemia and renal failure that are progressive in nature were independent predictors of poor outcome following retransplantation in the setting of recurrent hepatitis C.58
Anatomic Abnormalities The presence of isolated portal vein thrombosis was previously an absolute contraindication to liver transplantation. However, with the
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advent of techniques such as thrombectomy or jump grafts, portal vein thrombosis is categorized as a relative contraindication.60 Recent data have clearly identified portal vein thrombosis as a poor prognostic indicator with a significantly higher risk for acute graft failure and portal hypertensive complications following liver transplantation.61 In addition, post-transplant survival is sharply reduced if portal vein thrombosis extends into the superior mesenteric vein. Therefore, it is important to document the patency of the portal vein during the pretransplant evaluation.
Prior Hepatic Surgery Patients with a previous history of abdominal surgery are at risk for developing adhesions with portal hypertensive collaterals. This is associated with longer operative time and higher risk of blood loss, but comparable results are obtainable.
Cholangiocarcinoma Cholangiocarcinoma can be considered a relative or absolute contraindication, depending on the tumor stage. The post-transplant prognosis is highly variable, with 5-year survival rates of 30% for advanced disease and 70% for patients with stage I and II tumors.62 Preliminary data are promising, with the use of neoadjuvant external-beam radiotherapy, brachytherapy, and chemotherapy with fluorouracil if extrahepatic disease is excluded.63 A comprehensive pretransplant evaluation and staging is recommended, including body CT scan, bone scan and exploratory laparotomy to rule out metastases.
Extrahepatic Malignancy Liver transplantation is contraindicated in patients with extrahepatic malignancy except for those with liver metastases from innocuous neuroendocrine tumors, including gastrinoma, insulinomas, glucagonomas, somatostatinomas, and carcinoid tumors.64 Living donors can be considered to provide timely transplant surgery in this patient population.
Previous Non-Hepatic Malignancies Liver transplantation is not an absolute contraindication in patients with a myeloproliferative disorder or a previous non-hepatic malignancy.65 Data from the renal transplant experience have provided insights into the risk of tumor recurrence in patients with preexisting malignancies at the time of transplantation.66 A low recurrence rate of 10% was noted in the post-transplant period with incidental renal tumors, lymphomas, thyroid cancer, and testicular, uterine and cervical carcinomas. The recurrence rate was intermediate, i.e. 11–25%, in patients with uterine body carcinoma, Wilms’ tumor, and carcinomas of the colon, prostate, and breast. A high recurrence rate of more than 25% was noted with bladder carcinoma, sarcomas, malignant melanomas, symptomatic renal carcinomas, non-melanomatous skin cancers, and myelomas. It has been recommended that patients be monitored for a mandatory tumorfree period of 2 years following curative cancer treatment before they can be considered for liver transplantation. A longer time is advocated for malignancies such as malignant melanomas, and breast and colon carcinomas.
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TIMING OF LIVER TRANSPLANTATION Liver transplantation should be offered to a patient with cirrhosis at a point when the post-transplant survival rate exceeds the pretransplant life expectancy related to the presence of advanced liver disease. The 1-year mortality following liver transplantation is 10–15% in most transplant centers. Patients with Child’s class B cirrhosis have a 1-year survival rate of 85–90% based on the natural history of their liver disease.16,67 Therefore, it is appropriate to refer a cirrhotic patient with Child’s class B or C (or Child–Turcotte–Pugh (CTP) score 7 or higher) for consideration for liver transplantation. It is important to initiate the pretransplant evaluation as soon as the patient meets the minimal listing criteria. Delayed referral of a cirrhotic patient with severe hepatic decompensation (Child’s class C) characterized by progressive, irreversible complications or multiorgan failure is associated with poor post-transplant outcome. These patients should be precluded from costly, time-consuming evaluation based on their poor candidacy for transplantation. The Mayo model for PBC has been validated as a prognostic survival model and is a reliable tool to estimate the timing and outcome of liver transplantation. The Mayo model consists of five independent variables, including age, serum total bilirubin, serum albumin, prothrombin time, and the presence or absence of peripheral edema.68,69 Serum total bilirubin is a variable closely associated with survival in patients with PBC. Serum total bilirubin levels are within the normal range during the early stable phase of PBC and demonstrate a steady increase in the preterminal phase.70 In patients with serum total bilirubin 10 mg/dl or higher the estimated survival is 17 months. The Mayo model has demonstrated a favorable outcome with liver transplantation in patients with PBC during the progressive phase, and a poor prognosis with no change in natural history with medical management.69 The optimal time for liver transplantation with the best post-transplant survival is reflected by a Mayo risk score of 7.8 or lower. In addition to optimizing patient selection and the timing of liver transplantation, the Mayo model of PBC can predict the cost of resource utilization. The model has been validated and is clearly superior than CTP score in predicting outcome for patients with PBC.71 A modified version of the Mayo model incorporates the original five prognostic variables into a table format similar to that of the CTP score. Using the modified Mayo model, a score of 6 predicts a 1-year survival of 90.6%, which is the minimal listing criterion for liver transplantation.72 Patients with PSC may experience several complications, including recurrent episodes of bacterial cholangitis, jaundice due to a dominant stricture, or cholangiocarcinoma.73 A modified Mayo natural history model for PSC consists of five independent prognostic variables, including age, serum total bilirubin, serum aspartate aminotransferase (AST), serum albumin, and history of variceal bleeding.70,73 In contrast to the Mayo PBC model, the Mayo model for PSC is less reliable than the CTP score in predicting posttransplant survival.74
SELECTION CRITERIA FOR LIVER TRANSPLANT LISTING In 1964, Child and Turcotte proposed a system to categorize cirrhotic patients into three classes based on important clinical parameters, including serum total bilirubin, serum albumin, severity of
Chapter 49 PRETRANSPLANT EVALUATION AND CARE
ascites, degree of hepatic encephalopathy, and nutritional status, to estimate the risk of portosystemic shunt surgery in the setting of end-stage liver disease and variceal bleeding.75 Several years later, in 1973, Pugh and colleagues reevaluated and modified the Child’s classification by adding prothrombin time and eliminating nutritional status (CTP scoring system).76 The five clinical variables in the CTP classification can be scored based on their severity (score 1–3) with an aggregate score varying from 5 to 15. The total score allows classification into Child’s class A (CTP score 5–6), B (CTP score 7–10), or C (CTP score 11–15). The degree of hyperbilirubinemia in PBC and PSC uses a different point system when the CTP score is calculated. In addition, other indications for liver transplantation that may apply to patients with PBC and PSC include intractable pruritus, progressive bone disease with recurrent fractures and, in the case of PSC, recurrent bacterial cholangitis. The usefulness of CTP score in predicting the prognosis of patients with end-stage liver disease has been validated.77 Based on post-transplant 1-year survival of 85–90%, it has been recommended that initial listing of patients for liver transplantation should be pursued when the estimated 1-year survival rate of the underlying chronic liver disease is lower than 90%.16,67 A Child’s score of 7 or greater (Child’s class of B or C) is associated with a 1-year survival rate of 90% or lower. Patients with Child’s class A in the setting of cirrhosis with history of gastrointestinal bleeding caused by portal hypertension, or a single episode of spontaneous bacterial peritonitis, as well as patients with acute liver failure or non-metastatic primary hepatocellular cancer, can also be listed for liver transplantation. Despite the implementation of the MELD score for organ allocation, the previously employed minimal listing criteria for liver transplantation remains in effect.67
DIAGNOSTIC STUDIES AND CONSULTATIONS Patients being evaluated for liver transplantation should have no medical contraindications and meet the minimal listing criteria for transplantation. Patients should be considered for transplantation if there are no other alternative forms of treatment available to manage hepatic decompensation that might delay or prevent the need for transplantation.78 Following the initial screening, the transplant hepatologist and nurse coordinators proceed with a comprehensive evaluation to exclude any medical or psychosocial contraindications to liver transplantation (Table 49-4). Evaluation by a social worker is conducted to assess psychosocial status and the availability of social support, which plays an important role in the pretransplant care and management of the patient. Psychiatric consultation may be requested in patients with a history of psychiatric disorder, substance abuse, or alcoholic liver disease. Routine diagnostic tests include ABO blood typing, complete blood count, hepatic and renal chemistries, a-fetoprotein, viral serologies (hepatitis A, hepatitis B, hepatitis C, HIV, cytomegalovirus), chest X-ray, electrocardiogram, tuberculin skin test, and creatinine clearance (Table 49-4). The imaging tests include Doppler ultrasound, and CT or MRI to document the patency of hepatic vasculature and to screen for HCC. Patients over 50 undergo dobutamine echocardiography or the thallium stress test to rule out underlying silent coronary artery disease. Patients with abnormal stress tests or major risk factors for coronary artery disease are referred for cardiology consultation, and cardiac catheterization may be needed.41,42 Doppler imaging of the carotid
Table 49-4. Pretransplant Evaluation for Liver Transplantation Standard blood tests Complete blood count, liver chemistry, kidney profile, coagulation profile (PT, PTT) ANA, smooth muscle antibody, AMA Iron studies, ceruloplasmin, a1-antitrypsin phenotype CMV, EBV, HSV, VZV, HIV; syphilis; toxoplasmosis HAV-HDV serologies a-Fetoprotein Other standard tests Abdominal ultrasound with Doppler, electrocardiogram, chest X-ray, pulmonary function tests, endoscopic evaluations PPD skin tests Standard consultations Dietary Psychosocial Women’s health (Pap smear, mammogram in women over 35) Financial (insurance clearance must be obtained) Overall assessment of patient (clinical judgment in addition to biochemical parameters) Other optional tests CT or MRI (to exclude HCC); angiography if needed to exclude vascular abnormalities Carotid duplex scanning (for older or cardiovascular patients) Contrast echocardiography (for suspected hepatopulmonary syndrome) Cardiac catheterization (for suspected CAD) Colonoscopy (history of IBD, PSC, polyps, family history of colon cancer, (+) FOBT ERCP (in PSC) Liver biopsy Fungal serologies (in areas endemic for dimorphal fungi) Other optional measures Pretransplant vaccines, if needed (hepatitis A and B, pneumococcal vaccine, influenza vaccine, tetanus booster) PT, prothrombin time; ANA, antinuclear antibody; AMA, antimitochondrial antibody; EBV, Epstein–Barr virus; CMV, cytomegalovirus; HSV, herpes simplex virus; VZV, varicella-zoster virus; HIV, human immunodeficiency virus; HAV, hepatitis A virus; HDV, hepatitis D virus; PPD, purified protein derivative; CTP, Child–Turcotte–Pugh; MRI, magnetic resonance imaging; CT, computed tomography; CAD, coronary artery disease; IBD, inflammatory bowel disease; PSC, primary sclerosing cholangitis; ERCP, endoscopic retrograde cholangiopancreatography; FOBT, fecal occult blood test. Adapted and reprinted with permission from: Yu AS, Keeffe EB. Orthotopic liver transplantation. In: Boyer T, Zakim D, eds. Hepatology, 4th edn. Philadelphia: Harcourt Health Sciences, 2002: 1617–1656.
arteries and peripheral vessels is obtained based on clinical suspicion following an initial comprehensive consultation by the transplant hepatologist. Pulmonary function test is indicated for patients with a history of significant tobacco use or chronic lung disease. Rightsided cardiac catheterization is indicated in patients suspected of pulmonary hypertension. Patients with pulmonary hypertension can be categorized into mild, moderate and severe, based on the results of cardiac catheterization, and an estimation of post-transplant outcome can be established. Cancer screening is conducted based on individual risk factors (colonoscopy for occult fecal blood and in patients over age 50; endoscopic retrograde cholangiography in patients with PSC; and Pap smear and mammogram in female subjects). Upper endoscopy is performed to screen for gastroesophageal varices and other stigmata of portal hypertension. Additional testing and consultations are obtained based on the patient’s medical history.
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Patients must undergo financial clearance as part of the pretransplant evaluation to document adequate insurance coverage. A dedicated financial coordinator discusses with the patient and family members the available options to obtain adequate insurance coverage in an era of rising costs.78 Following successful completion of the evaluation, the patient is presented to the liver transplant selection committee by the primary hepatologist. The committee consists of transplant hepatologists, liver transplant surgeons, transplant nurse coordinators, social worker, psychiatrist, financial coordinator and a nutritionist/dietician. The selection committee determines the candidacy of a patient and may recommend approval for listing, deferral for further evaluation, or medical management owing to poor candidacy for transplant surgery.
DONOR ALLOCATION AND EVALUATION ORGAN DEMAND VERSUS SUPPLY The disparity between organ demand and supply stresses the need for strict adherence to minimal listing criteria for transplantation. The availability of donor organs in the US is approximately 20 per million population.3 The rate is variable across Europe, with organ donation of 25 per million in Spain and less than 10 per million in Italy.3 The rate of supply of cadaveric donor livers has remained relatively steady for the last 5 years, despite numerous drives to increase donor awareness and the rapidly growing demand for transplantation.4
NEW TECHNIQUES TO IMPROVE THE DONOR POOL Several techniques have been adopted to expand the donor pool.4 These include the use of ‘marginal’ donors, cadaveric split liver transplantation, adult living donor liver transplantation (LDLT), domino transplantation, and xenotransplantation (Table 49-5).4 Domino liver transplantation may be an option in only a small percentage of patients.79 Xenotransplantation is an experimental tool but may become an option in the future.80
Marginal Donors The allocation of cadaveric livers based on the MELD score may result in exceedingly prolonged waiting periods for certain patient
Table 49-5. Current Approaches to Donor Pool Expansion Marginal donors Older donors (age > 55) Donors with fatty infiltration Anti-HBc (+) donors HCV-infected donors Split liver transplants, preferably in vivo technique Living donors Domino transplants Xenografts Adapted and reprinted with permission from: Wiesner RH, Rakela J, Ishitani MB, et al. Recent advances in liver transplantation. Mayo Clin Proc 2003;78:197–210.
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subgroups. The use of marginal cadaveric livers includes organs from donors older than 55 years, donor livers with significant fatty change, and livers from subjects with hepatitis B virus (HBV) or HCV infection. In the past, a higher risk of primary graft non-function was associated with the use of fatty livers and organs from older subjects.81,82 Measures that have improved allograft and host survival following transplant surgery include pretransplant evaluation of allografts by the surgeon, minimizing cold ischemia time, and improvement in post-transplant care.81,83 Allografts from donors with HBV and HCV infection can be considered for liver transplantation. Allografts from donors positive for anti-HBc but negative for HBsAg and anti-HBs are reasonable to consider for transplantation. The use of hepatitis B immunoglobulin plus lamivudine or lamivudine monotherapy can eliminate the risk of de novo post-transplant HBV infection from 72% in the absence of prophylaxis.84–86 Data from UNOS and individual transplant centers have demonstrated that patients with chronic HCV infection who received HCV-positive organs rather than HCVnegative donor organs have similar allograft survival rates.87–89
Split Liver Transplantation Split liver transplantation is a surgical technique that allows two patients to undergo liver transplantation by judicious use of a single donor allograft. A right trisegment (segments 4 to 8) can be used for an adult recipient and the left lateral segment (segments 2 and 3) can be utilized for a smaller adult or a child.90 The initial experience was associated with a higher risk of biliary complications requiring reoperation, with poor survival rates. In 1998, data from King’s College Hospital showed recipient and allograft survivals of 90% and 80%, respectively.91 These data were supported by experience with split liver transplantation using in vivo split grafts at the University of California at Los Angeles, with overall allograft and patient survival rates comparable to those with whole organs.92
Living Donor Liver Transplantation LDLT is a suitable option for patients who are unable to accrue sufficient MELD score based on the current allocation system and who may never receive a cadaveric liver. A typical example is a patient who carries the diagnosis of PSC with the associated risk of cholangiocarcinoma; LDLT offers the option of elective liver transplantation. Following LDLT, the transplanted liver segment requires a few weeks to regenerate into an adequate volume and provide for the recipient’s metabolic needs.93 The risks of partial hepatic resection must be discussed with the donor, in whom hepatic resection is associated with a postoperative morbidity of 15–20% and mortality of 0.1–0.5% (Tables 49-6 and 49-7). In addition to the routine pretransplant evaluation, an adequate hepatic volume for the recipient must be confirmed. The recommended graft-to-recipient body weight ratio (GBWR) is 0.8%.94 A compromise in GBWR is associated with higher risk of post-transplant allograft failure and host mortality. LDLT recipients are at an increased risk of biliary complications (Table 49-8).94 Adult-to-adult elective LDLT was first attempted in 1994.95 In adults, LDLT typically warrants right hepatic resection, but occasionally the right trisegment for larger recipients or the left hepatic lobe for smaller recipients.96,97 LDLT surgery has been shown to
Chapter 49 PRETRANSPLANT EVALUATION AND CARE
Table 49-6. Complications in Donors for Living Donor Liver Transplantation Complications Biliary tract – leaks, biloma, stenosis Hepatic encephalopathy Wound infection Pressure sores Pulmonary – atelectasis, pneumonia, embolus Bowel obstruction Phlebitis Incisional hernia Aborted donor Cholestasis Nerve palsy Portal vein thrombosis
Table 49-9. UNOS Transplant Listing Status for Patients > 18 Years of Age % 3–8 2 1.5 1.4 1 1 1 1 1 1 1 0.5
Adapted and reprinted with permission from: Shiffman ML, Brown RS Jr, Olthoff KM, et al. Living donor liver transplantation: summary of a conference at The National Institutes of Health. Liver Transpl 2002;8:174–188.
Table 49-7. Causes of Death in Recipients after Living Donor Liver Transplantation %
Sepsis Hemorrhage Vascular complications Recurrence of native disease Other
53 14 14 2–3 16
Adapted and reprinted with permission from: Shiffman ML, Brown RS Jr, Olthoff KM, et al. Living donor liver transplantation: summary of a conference at The National Institutes of Health. Liver Transpl 2002;8:174–188.
Table 49-8. Complications Reported in Recipients of Living Donor Liver Transplantation
Postoperative bleeding Biliary tract complications: leaks, strictures Hepatic artery thrombosis or stenosis Hepatic venous outflow obstruction Intrahepatic hemorrhage
Status 2A Hospitalized in the ICU for chronic liver failure with a CTP score of >10 and one of the following: Unresponsive active variceal hemorrhage Hepatorenal syndrome Refractory ascites/hepatohydrothorax Grade 3 or 4 encephalopathy unresponsive to medical therapy Status 2B CTP score >10 or presence of hepatocellular carcinoma or a CTP score > 7 and one of the following: Unresponsive active variceal hemorrhage Hepatorenal syndrome SBP Refractory ascites Status 3 Child–Pugh score >7 but not meeting the criteria for 2B
Causes of death
Complications
Status 1 Fulminant hepatic failure Primary graft dysfunction or hepatic artery thrombosis < 7 days of transplantation Acute decompensated Wilson’s disease
% 46 15–30 3–10 5 5
Adapted and reprinted with permission from: Shiffman ML, Brown RS Jr, Olthoff KM, et al. Living donor liver transplantation: summary of a conference at The National Institutes of Health. Liver Transpl 2002;8:174–188.
provide 60–70% 1-year post-transplant survivals in patients with UNOS statuses 1 and 2A.94 Adult-to-adult LDLT has not had a major impact in terms of reducing the donor shortage. The screening process for potential LDLT donors is strict, with only 15% of donors successfully completing the pretransplant evaluation.98
MELD SCORING SYSTEM FOR DONOR ALLOCATION The MELD system was formulated based on the natural history of cirrhosis and may be biased against patients with debilitating noncirrhotic liver conditions, such as metastatic neuroendocrine tumors to the liver or polycystic liver disease. Furthermore, patients with
Status 7 Temporarily inactive
end-stage liver disease may develop incapacitating manifestations, such as hepatic coma, which has no favorable impact on their MELD score and may predispose to a higher risk of complications. These patients should be considered for an appeal to a regionalized peer review board, which may or may not grant an exemption for priority listing with higher MELD score.
Previous UNOS Organ Allocation System: CTP Scoring System Recognizing the utility of the CTP criteria to predict 30 ml/h). Care is taken not to fluid-overload the donor as this produces edema within the organ. Catecholamine deficiency as a result of brain death may necessitate the use of vasopressors such as dopamine once sufficient central venous pressure (>10 mmHg) is obtained. The development
of diabetes insipidus may require the use of vasopressin and hypotonic fluids. Hormonal replacement therapy with steroids and triiodothyronine are also instituted. Coagulopathy is corrected with fresh frozen plasma, cryoprecipitate, or platelets. Severe anemia is also treated with the transfusion of packed red blood cells. Table 50-5 lists several donor factors that can be implicated with poor graft function postoperatively. Livers may be preserved up to 24 hours; however, the incidence of biliary strictures and graft dysfunction increases exponentially after 12 hours of preservation. Short cold preservation times (24 hours) Long warm ischemia time (>1 hour) Hepatic steatosis (>30%) Incompletely resuscitated donor (sodium >160 mg/dl) Hepatic dysfunction (AST or ALT > 4 ¥ normal, alkaline phosphate >2 ¥ normal, PT >2 ¥ normal, bilirubin >4.0 mg/dl) Metabolic acidosis AST, aspartate aminotransferase; ALT, alanine aminotransferase; PT, prothrombin time.
a
www.optn.org.
Table 50-6. Preservation Solutions for the Liver Used in Clinical and Experimental Transplantation Solution
Proprietary name
Supplier/reference
Basic composition
Pricea
Viscosityb
Bretschneiders-HTK Marshall’s (HOC) Celsior University of Wisconsin (UW) UW-HES Belzer-MPS
Custodiol Soltran Celsior Viaspan
Köhler Chemie, Alsbach, Germany Baxter Healthcare, Thetford, UK Sangstat, CA Barr Laboratories, New York
Low Low Medium High
Low Low Low High
N/A KPS-1
High High
Medium Medium
PBS140 Perfadex
N/A Perfadex
Reference28 Organ Recovery Systems, Chicago, IL Reference29 Vitrolife AB, Gothenburg, Sweden
Histidine-tryptophan-ketoglutarate Hypertonic citrate High sodium lactobionate High potassium actobionate; starch colloid UW without starch colloid High-sodium gluconate Phosphate-buffered sucrose Low-potassium, dextran 40 colloid; THAM/PGE, additive
Low High (including cost of additives)
Low Medium
a
High (> £100/l); medium (£100 > x > £80); low ( < £80). High (> 2.5 cP at 20°C); medium (2.5 > x > 1.5); low ( < 1.5 cP). Reproduced from Wilson CH, Stansby G, Haswell M, et al. Evaluation of eight preservation solutions for endothelial in situ preservation. Transplantation 2004;78:1008–1013, by permission of Lippincott. b
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occur in these accepted techniques.13 The surgical technique of conventional procurement involves in-situ dissection of individual organs. This technique is often used when the donor is physiologically stable. The sequence for removal of the abdominal organs is as follows: first, the liver, followed by the pancreas, and, lastly, the kidneys. The liver is placed in a plastic bag containing the preservation solution. The organ is then placed into two additional bags (three total) to avoid contamination. Finally, the liver is placed in an ice chest and covered with ice. The liver is now ready to be shipped to the recipient’s hospital. The iliac veins and arteries are then procured and placed in the preservation solution should the need arise for alternative methods of reconstruction for the hepatic artery and/or portal vein. Final preparations for implantation of the liver graft are usually performed at the recipient hospital. The en-bloc technique involves procurement of all the abdominal viscera and separation ex situ on the back table. This technique is carried out when the donor is physiologically unstable. The liver is removed from the abdomen en bloc with the abdominal viscera. Minimal dissection is carried out prior to the perfusion of the organs. Separation of the liver from the other abdominal viscera (pancreas, intestine, kidneys) is completed on the back table (ex situ). A comparison of the in-situ and the en-bloc techniques is listed in Table 50-7. Whichever technique is used, it must not jeopardize any of the organs being procured.
SPLIT LIVER PROCUREMENT The arterial and portal vein blood supply to the liver follows a segmental distribution allowing partial liver or cadaveric segmental transplantation (Figure 50-1). A liver allograft may be used for two recipients and is described in the literature as split-liver transplantation. The most common split is to use an extended right lobe (right lobe plus a segment of the left lobe or segments IV, V, VII, and VIII) for the adult recipient and the left lateral segment (a portion of the left lobe or segments II and III) for a child (Figure 50-2). Infrequently the liver has been split into right (segments V, VI, VII, and VIII) and left (segments I, II, III, and IV) lobes providing the opportunity to do a transplant in two adult recipients.14–16 The partition of the liver may be done in situ or ex situ. In the latter, the liver is procured as a whole organ and then divided at the back table. There are several tools the surgeon may use to divide the liver and create hemostasis. Several biological products based on fibrin materials to coat the raw surface of the liver are often used for hemostasis.
Right lobe
Left lobe
II VIII
Length of time for procurement Risk of vascular injury Length of warm dissection Risk of vasospasm Risk of donor instability Length of cold dissection Length of delay in procurement of thoracic viscera
Conventional technique
En-bloc techniquea
Longer Higher Longer Higher Higher Shorter Longer
Shorter Lower Shorter Lower Lower Longer Shorter
a
Required technique for donors after cardiac death.
Vena cava
V
950
I
III
Left lateral segment
VI
Figure 50-1. Anatomic segments of the liver based on its blood supply. The segmental blood supply to the liver along with its capacity for regeneration allows surgeons to conduct partial live or deceased liver transplantation.
Figure 50-2. Split-liver transplantation. Hepatic artery and bile duct are not shown but the concept of where to divide them is the same as that of the portal vein.
Left hepatic vein
To an adult
To a child
Right portal vein
IV
VII
Table 50-7. Comparison of Conventional Versus En-Bloc Technique of Organ Procurement
Left portal vein
Chapter 50 LIVER TRANSPLANTATION SURGERY
Unlike Europe, the split-liver transplant has not been totally embraced in the USA.16–18 The reason is based on initial reports showing a higher incidence of primary graft non-function after split compared to full-size transplantation. In contrast, the European data showed similar results in terms of patient and graft survival between split- and whole-liver transplantation. Thus, efforts should be made to maximize the number of split-liver transplants in the USA to alleviate, at least in part, the existing problem of organ shortage.19
LIVING-DONOR PROCUREMENT In live adult donor to an adult recipient, it is necessary to use the right lobe, which accounts for about 60–70% of the total liver volume.20–23 From an adult donor to a child, it is the left lateral segment (a portion of the left lobe) which accounts for approximately 20% of the liver volume. The true left lobe may be used in a small adult recipient or a larger child.24–26 The key issue is that the donor must be left with enough liver parenchyma to sustain life. This has been estimated to be at least 30% of the total liver volume. Likewise, the recipient needs enough liver mass, and a minimum of 40% of the estimated total liver volume is recommended.24 Another formula is to use the lean body weight of the recipient. The hepatic lobe must be at least 0.8% of the recipient’s lean body weight. For example, a 70-kg recipient will require at least 560 grams of liver parenchyma.25 In our institution we use a minimum of 1% to increase the margin of safety and to compensate for the potential risk of preservation-induced injury to the liver. The technique to divide the liver is similar to the technique described for the in-situ splitting of the allograft. The blood supply to the lobe (or segment) to be used for transplantation must be maintained to avoid anoxic injury to the liver (Figure 50-3). To minimize the blood loss, a transient Pringle maneuver, controlling the inflow to the liver, has recently been reported.27,28 The vascular structures in the hepatic hilum are intermittently cross-clamped for periods of 10–15 minutes, allowing flow through the liver for periods of 5 minutes in between the cross-clampings. This maneuver is helpful in minimizing the blood loss during the division of the
Right lobe
liver parenchyma. The goal is to avoid exposing the donor to transfusion with banked blood with the potential risk of transmission of viruses and transfusion reactions. Furthermore, living donors are required to donate 1 or 2 units of autologous blood prior to the operation. At the time of the operation, isovolemic hemodilution is also carried out, whereby another unit is removed from the donor and replaced with saline.29 If bleeding is significant enough to require blood transfusions, the blood drawn in the operating room is given first, followed by the autologous blood. Banked blood will be used in extenuating circumstances. The cell-saver is always used during the live donor hepatectomy. The advantages of living donation are having a physiologically stable and healthy donor, short preservation time, elective surgery, and the potential psychological benefit of helping a patient, who is usually a close relative. The disadvantages are the surgical and anesthetic risks to which the donors are exposed. This is problematic since the living donor is a healthy individual who is in no need of having major surgery.30 It is unlikely that surgeons would be doing this type of surgery if there were enough organs for transplantation. The morbidity among living donors is significant, with rates up to 65%.31–37 Table 50-8 lists major and minor complications among living donors reported in the literature.
Table 50-8. Reported Complications Among Living Liver Donors Death Liver failure requiring urgent transplantation Pulmonary embolism Perforated peptic ulcer Splenic injury requiring splenectomy Biliary strictures Portal vein thrombosis Arm paralysis Incisional hernia Wound infection Severe hypophosphatemia
Remaining left lobe
Figure 50-3. The sequence of steps following a live donor right lobectomy is as follows: division of the liver parenchyma, transection of the right hepatic duct, division of the right hepatic artery, division of the right portal vein and, finally, division of the right hepatic vein. The right lobe is flushed, ex situ, with preservation solution via the portal vein.
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Section VIII. Liver Transplantation
Figure 50-4. Incisions used for adult patients with a wide (A) or narrow (B) chest. A bilateral subcostal incision (C) is often enough for small children.
A
B
C
Table 50-9. Physiologic Changes According to Technique of Transplantation
1. No venous bypass 2. Venous bypass 3. Piggyback
Systemic venous return
Portal vein pressure
Renal vein pressure
Impaired
≠
≠
Slightly impaired Undisturbed
Ø ≠
Normal Normal
Diaphragm
Liver
RECIPIENT OPERATION The following description relates to the situation when the liver is placed in an orthotopic location. Three different techniques have been described for liver transplantation: (1) removal of the liver, including the retrohepatic cava on venous bypass; (2) removal of the liver, including the retrohepatic cava without venous bypass; and (3) removal of the liver without the vena cava and placing the liver in a piggyback position.38,39 The piggyback liver transplants may be done with or without venous bypass. The type of incision depends on the habitus of the patient. Incisions often used in liver transplantation are shown in Figure 50-4. The physiologic changes during the anhepatic state associated with each of these techniques are shown in Table 50-9. The decision as to which type of technique may be used depends on the physiologic condition of the patient at the time of the transplant. In hemodynamically stable patients who tolerate the cross-clamping of the vena cava, the hepatectomy and implantation of the liver can be done expeditiously since the removal of the liver requires less dissection than that of the piggyback transplant and also avoids the time needed for the initiation of the venous bypass (Figure 50-5). The disadvantage with this technique is that bleeding from the raw surfaces may be exacerbated because of the venous hypertension brought about by the crossclamping of the portal vein and vena cava. Prolonged crossclamping of the vena cava may result in postoperative renal dysfunction due to renal vein hypertension.40 The utilization of venous bypass may lengthen the operation, but the potential benefits of the bypass, such as decreased bleeding and protection of the renal function by avoiding venous hypertension, outweigh the extra time needed for setting up the equipment as well as the insertion of the cannulae. The insertion of the cannulae may be done through a venous cutdown or percutaneously (Figure 50-
952
Vena cava
Figure 50-5. View of a conventional hepatectomy with removal of the retrohepatic vena cava. The systemic venous return is impaired.
6). The potential complications of venous bypass are: embolism, hematoma formation at the site of cannulae insertion, lymphoceles, injury to brachial plexus in the axilla or femoral vessels or vein in the groin, and fibrinolysis.41,42 The piggyback transplantation is a more laborious procedure since the liver needs to be meticulously separated from the vena cava. There may be several tributaries from the liver to the vena cava and their dissection makes the procedure tedious. Once the liver is separated from the vena cava, a vascular clamp is placed across the confluence of the hepatic veins and the hepatectomy is completed (Figure 50-7). Since the systemic venous return is maintained intact, the hemodynamics change little compared to those procedures that require the cross-clamping of the vena cava. We have observed decreased blood transfusion requirements with the piggyback transplant compared to the conventional techniques.43 The potential complication is increased ascites during the perioperative period which may be the result of functional outlet obstruction.44 However,
Chapter 50 LIVER TRANSPLANTATION SURGERY
A
Subclavian vein
F
Inflow cannula
F D
B E
Portal vein cannula
Portal vein
C
F Inferior vena cava
Figure 50-8. Completion of a liver transplant. The anastomosis of the suprahepatic vena cava (A) is done first, followed by the anastomosis of the infrahepatic vena cava (B), portal vein (C), hepatic artery (D) and, lastly, common bile duct (E). (F) represents drains strategically placed near the anastomosis. Femoral vein cannula
Figure 50-6. Schematic drawing depicting the venous bypass during the anhepatic phase in liver transplantation. The arrows show the direction of blood flow.
Diaphragm Vena cava Confluence of hepatic veins
allograft vena cava and the recipient hepatic veins and one for the portal vein) are required. The diagnosis of portal vein thrombosis is not a contraindication to transplantation.45 This is dealt with at the time of transplantation by performing a thrombectomy or alternatively by using a “jump” graft from the recipient’s superior mesenteric vein to the portal vein of the allograft. Finally, the arterial reconstruction completes the revascularization of the liver. Arterial anomalies are often encountered and their reconstruction may need more innovative methods for their reconstruction.
BILIARY TRACT RECONSTRUCTION
Liver
Tributary veins Figure 50-7. The separation of the liver from the vena cava in piggyback transplantation. The clamp was placed across the confluence of the hepatic veins. The systemic venous return remains undisturbed.
this problem seems to be temporary. The piggyback technique may also be utilized during retransplantation.
VASCULAR RECONSTRUCTION In the conventional technique, a total of three venous anastomoses (two for the vena cava and one for the portal vein) are necessary. In piggyback transplantation only two anastomoses (one between the
Two methods of bile duct reconstruction may be used: (1) an endto-end anastomosis of the donor common bile duct to the recipient common bile duct with or without a T-tube or (2) end-to-side choledochojejunostomy to a Roux en Y with or without a stent. Fine absorbable sutures are utilized for these anastomoses. A choledochojejunostomy is done in patients whose bile duct is diseased or absent, such as in cases of primary sclerosing cholangitis and biliary atresia, respectively, as well as in those cases where a size mismatch or not enough length exists between the donor and recipient’s common bile ducts. Before the closure of the abdomen, two or three drains are placed in the right and left subhepatic spaces and adjacent to the bile duct reconstruction (Figure 50-8).
RESULTS Based on United Network for Organ Sharing data, the overall 1-, 3-, and 5-year patient actuarial survival is 87.6, 79.9, and 74.6%, respectively (Table 50-10). The actuarial graft survival at 1, 3, and 5 years is 82.5, 73.5, and 67.3%, respectively, as shown in Table 50-11.9 Gender of the recipient does not seem to influence patient or graft survival, as demonstrated in Tables 50-12 and 50-13.9
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Table 50-10. Organ Procurement and Transplantation Network (OPTN) Liver Kaplan–Meier Patient Survival Rates for Transplants Performed 1995–2002 Region USA USA USA USA USA USA
Transplant type
Years post-transplant
Number functioning/ alive
Survival rate
95% Confidence interval
Primary transplant Repeat transplant Primary transplant Repeat transplant Primary transplant Repeat transplant
1 year 1 year 3 years 3 years 5 years 5 years
11 362 875 11 409 939 8871 734
87.6 70.0 79.9 60.2 74.6 53.0
(87.0, 88.2) (67.5, 72.5) (79.3, 80.5) (57.9, 62.6) (73.8, 75.3) (50.6, 55.5)
Data subject to change based on future data submission or correction. 1-year survival based on 2000–2002 transplants, 3-year survival based on 1997–2000 transplants, 5-year survival based on 1995–1998 transplants. Patient survival was not computed due to n less than 10. Based on OPTN data as of March 4, 2005.
Table 50-11. Organ Procurement and Transplantation Network (OPTN) Liver Kaplan–Meier Graft Survival Rates for Transplants Performed 1995–2002 Region USA USA USA USA USA USA
Transplant type
Years post-transplant
Number functioning/ alive
Survival rate
95% Confidence interval
Primary transplant Repeat transplant Primary transplant Repeat transplant Primary transplant Repeat transplant
1 year 1 year 3 years 3 years 5 years 5 years
11 362 875 11 409 939 8871 734
82.5 62.8 73.5 51.5 67.3 42.9
(81.8, 83.1) (60.2, 65.3) (72.8, 74.2) (49.2, 53.7) (66.6, 68.1) (40.6, 45.2)
Data subject to change based on future data submission or correction. Graft survival was not computed due to n less than 10. 1-year survival based on 2000–2002 transplants; 3-year survival based on 1997–2000 transplants; 5-year survival based on 1995–1998 transplants. Based on OPTN data as of March 4, 2005.
Table 50-12. Organ Procurement and Transplantation Network (OPTN) Liver Kaplan–Meier Patient Survival Rates for Transplants Performed 1995–2002 Region USA USA USA USA USA USA
Recipient gender
Years post-transplant
Male Female Male Female Male Female
1 year 1 year 3 years 3 years 5 years 5 years
Number functioning/ alive 7717 4520 7374 4974 5498 4107
Survival rate
95% Confidence interval
86.4 85.4 77.9 78.0 71.4 73.4
(85.7, 87.1) (84.5, 86.4) (77.1, 78.7) (77.0, 79.0) (70.5, 72.4) (72.3, 74.6)
Data subject to change based on future data submission or correction. Patient survival was not computed due to n less than 10. 1-year survival based on 2000–2002 transplants; 3-year survival based on 1997–2000 transplants; 5-year survival based on 1995–1998 transplants. Based on OPTN data as of March 4, 2005.
Table 50-13. Organ Procurement and Transplantation Network (OPTN) Liver Kaplan–Meier Graft Survival Rates for Transplants Performed 1995–2002 Region USA USA USA USA USA USA
Recipient gender
Years post-transplant
Male Female Male Female Male Female
1 year 1 year 3 years 3 years 5 years 5 years
Number functioning/ alive 7717 4520 7374 4974 5498 4107
Survival rate
95% Confidence interval
81.1 79.9 71.3 70.9 63.7 65.8
(80.3, 81.9) (78.8, 81.0) (70.5, 72.2) (69.9, 72.0) (62.7, 64.6) (64.7, 67.0)
Data subject to change based on future data submission or correction. Graft survival was not computed due to n less than 10. 1-year survival based on 2000–2002 transplants; 3-year survival based on 1997–2000 transplants; 5-year survival based on 1995–1998 transplants. Based on OPTN data as of March 4, 2005.
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Chapter 50 LIVER TRANSPLANTATION SURGERY
SURGICAL COMPLICATIONS Major surgical complications associated with the liver transplant operation fall into broad categories and these are: graft dysfunction, biliary tract, and vascular complications.
Liver Transplant Patients with Hepatocellular Carcinoma Patient Survival 100 90 Probability
80 70 Survival (%)
Similarly, the type of donor (living versus deceased) bears no influence on the outcomes, as shown in Tables 50-14 and 50-15. In contrast, the analysis of age at the time of transplantation clearly shows that patient survival at 3 and 5 years is lower in the under-1-year of age and over 65 years of age groups, compared to the other groups (Table 50-16). The difference is more pronounced in the analyses of graft survival (Table 50-17). Tables 50-10 and 50-11 demonstrate that patient and graft survival is compromised by repeat transplantation. Other factors that may have an impact on patient and graft survival are the condition of the patient at the time of transplantation and the diagnostic indication for transplantation. Hepatocellular carcinoma is associated with the lowest patient survival (31.8% at 5 years) based on a retrospective analysis of 4000 cases at the University of Pittsburgh.5 However, the survival for this indication has improved greatly by following strict criteria for selection of candidates for transplantation. In the USA, patients with hepatocellular carcinoma considered suitable candidates for transplantation are those with a single lesion measuring less than 5 cm in diameter or with three or fewer lesions measuring no more than 5 cm in the aggregate. Following such criteria, the 5-year patient actuarial survival at Stanford University is 80% (Figure 50-9). In a comparison of the outcomes after hepatic transplantation for hepatitis C and B and alcoholic cirrhosis at Stanford University, a trend for lower patient and graft survival in the hepatitis C cohort was observed, but the differences did not reach statistical significance (Figure 50-10).
60 50 40 30 20 10 0 0
1
2
3
4
5
6
7
8
Years Post Transplant Figure 50-9. Actuarial patient survival at Stanford University, California, for patients who underwent hepatic replacement for hepatocellular carcinoma. No attrition was observed past 3 years after transplantation.
Table 50-14. Organ Procurement and Transplantation Network (OPTN) Liver Kaplan–Meier Patient Survival Rates for Transplants Performed 1995–2002 Region USA USA USA USA USA USA
Donor type
Years post-transplant
Number functioning/ alive
Survival rate
95% Confidence interval
Cadaveric Living Cadaveric Living Cadaveric Living
1 year 1 year 3 years 3 years 5 years 5 years
11 240 997 11 797 551 9433 172
85.8 89.1 77.8 80.2 72.1 80.0
(85.2, 86.3) (87.2, 90.9) (77.2, 78.4) (77.2, 83.2) (71.4, 72.9) (74.8, 85.1)
Data subject to change based on future data submission or correction. Patient survival was not computed due to n less than 10. 1-year survival based on 2000–2002 transplants; 3-year survival based on 1997–2000 transplants; 5-year survival based on 1995–1998 transplants. Based on OPTN data as of March 4, 2005.
Table 50-15. Organ Procurement and Transplantation Network (OPTN) Liver Kaplan–Meier Graft Survival Rates for Transplants Performed 1995–2002 Region USA USA USA USA USA USA
Donor type
Years post-transplant
Number functioning/ alive
Survival rate
95% Confidence interval
Cadaveric Living Cadaveric Living Cadaveric Living
1 year 1 year 3 years 3 years 5 years 5 years
11 240 997 11 797 551 9433 172
80.7 79.5 71.2 70.2 64.4 70.8
(80.1, 81.4) (77.2, 81.8) (70.5, 71.9) (67.0, 73.5) (63.7, 65.2) (65.3, 76.3)
Data subject to change based on future data submission or correction. Graft survival was not computed due to n less than 10. 1-year survival based on 2000–2002 transplants; 3-year survival based on 1997–2000 transplants; 5-year survival based on 1995–1998 transplants. Based on OPTN data as of March 4, 2005.
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Table 50-16. Organ Procurement and Transplantation Network (OPTN) Liver Kaplan–Meier Patient Survival Rates for Transplants Performed 1995–2002 Region USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA
Recipient age
Years post-transplant
Number functioning/ alive
Survival rate
95% Confidence interval
< 1 year 1–5 years 6–10 years 11–17 years 18–34 years 35–49 years 50–64 years 65 + < 1 year 1–5 years 6–10 years 11–17 years 18–34 years 35–49 years 50–64 years 65 + < 1 year 1–5 years 6–10 years 11–17 years 18–34 years 35–49 years 50–64 years 65 +
1 year 1 year 1 year 1 year 1 year 1 year 1 year 1 year 3 years 3 years 3 years 3 years 3 years 3 years 3 years 3 years 5 years 5 years 5 years 5 years 5 years 5 years 5 years 5 years
367 459 170 345 735 3991 5377 791 434 511 253 317 820 4450 4754 805 342 451 218 253 716 3546 3476 601
85.2 83.3 90.5 91.7 87.6 87.7 85.3 80.5 76.9 78.2 85.9 83.3 81.4 79.8 76.5 69.5 74.5 77.4 83.8 75.8 76.1 74.3 69.6 62.8
(81.7, 88.6) (80.1, 86.5) (85.9, 95.2) (88.7, 94.7) (85.4, 89.9) (86.7, 88.6) (84.4, 86.2) (78.0, 83.0) (73.5, 80.3) (75.1, 81.4) (81.8, 90.0) (79.6, 87.1) (79.0, 83.7) (78.8, 80.8) (75.5, 77.5) (66.8, 72.1) (70.7, 78.3) (74.2, 80.6) (79.3, 88.3) (71.5, 80.2) (73.4, 78.7) (73.1, 75.5) (68.4, 70.8) (59.9, 65.8)
Data subject to change based on future data submission or correction. Patient survival was not computed due to n less than 10. 1-year survival based on 2000–2002 transplants; 3-year survival based on 1997–2000 transplants; 5-year survival based on 1995–1998 transplants. Based on OPTN data as of March 4, 2005.
Table 50-17. Organ Procurement and Transplantation Network (OPTN) Liver Kaplan–Meier Graft Survival Rates for Transplants Performed 1995–2002 Region USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA
Recipient age
Years post-transplant
Number functioning/ alive
Survival rate
95% Confidence interval
< 1 year 1–5 years 6–10 years 11–17 years 18–34 years 35–49 years 50–64 years 65+ < 1 year 1–5 years 6–10 years 11–17 years 18–34 years 35–49 years 50–64 years 65+ < 1 year 1–5 years 6–10 years 11–17 years 18–34 years 35–49 years 50–64 years 65+
1 year 1 year 1 year 1 year 1 year 1 year 1 year 1 year 3 years 3 years 3 years 3 years 3 years 3 years 3 years 3 years 5 years 5 years 5 years 5 years 5 years 5 years 5 years 5 years
367 459 170 345 735 3991 5377 791 434 511 253 317 820 4450 4754 805 342 451 218 253 716 3546 3476 601
75.4 75.2 81.7 84.7 80.8 82.2 80.8 76.5 64.9 70.0 76.6 71.1 71.0 72.2 71.6 66.5 62.3 66.2 73.8 63.9 64.2 65.7 63.9 59.7
(71.5, 79.4) (71.6, 78.7) (76.1, 87.3) (81.0, 88.4) (78.1, 83.4) (81.1, 83.2) (79.8, 81.7) (73.9, 79.2) (61.3, 68.5) (66.7, 73.4) (71.9, 81.3) (66.8, 75.3) (68.4, 73.6) (71.1, 73.3) (70.5, 72.6) (63.8, 69.1) (58.4, 66.3) (62.8, 69.6) (68.8, 78.8) (59.4, 68.5) (61.5, 67.0) (64.5, 66.9) (62.7, 65.2) (56.7, 62.6)
Data subject to change based on future data submission or correction. Graft survival was not computed due to n less than 10. 1-year survival based on 2000–2002 transplants; 3-year survival based on 1997–2000 transplants; 5-year survival based on 1995–1998 transplants. Based on OPTN data as of March 4, 2005.
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Chapter 50 LIVER TRANSPLANTATION SURGERY
metabolic acidosis, hyperkalemia, and the onset of the adult respiratory distress syndrome. The treatment is urgent retransplantation. These patients deteriorate quickly, thus, the outcomes depend on the availability of an organ for a timely transplant. The reported 1-year actuarial patient survival following retransplantation for primary graft non-function ranges from 0 to 50%.49,50
Liver Transplant Patients with Hep B, Hep C, or Alcoholic Cirrhosis Patient Survival 100 90 80 Survival (%)
70 60
BILIARY TRACT COMPLICATIONS
50
The most common surgical complications observed in liver transplant recipients are biliary tract problems. In whole-organ liver transplantation an incidence from 6% to close to 20% has been reported.51,52 Among these, strictures account for two-thirds and bile leaks for the remaining third. Biliary strictures can occur at the level of the anastomosis with the cause likely being ischemia of the distal allograft common bile duct. Although some of these patients may respond to dilatation of the stricture and stent placement, this type of injury will require surgical excision of the stricture and reconstruction to a Roux en Y. Intrahepatic strictures are the result of an ischemic injury to the bile ducts which could be brought about by a prolonged preservation time (>12 hours), stenosis, or thrombosis of the hepatic artery.52 Clinical symptoms and radiological findings resemble those of primary sclerosing cholangitis. The patients may experience right upper quadrant pain, pruritus, and transient episodes of jaundice accompanied by abnormal elevation of the alkaline phosphatase and gammaglutamyl transpeptidase. Management of these patients is difficult, often requiring multiple interventions for percutaneous dilation of the strictures and placement of indwelling catheters for drainage of the biliary tree. Ultimately, these patients do end up needing retransplantation.53 Biliary leaks may originate from the anastomosis of the bile duct or from the raw surface of the liver in cases where split or live donor lobes are used.54 An anastomotic leak should raise the suspicion of hepatic artery thrombosis. Hepatic artery thrombosis results in necrosis of the extrahepatic biliary system leading to sepsis. Such a complication often requires an urgent retransplantation.55 On the other hand, leaks from the raw surface may be managed by drainage and observation. Reoperation for direct control is done for persistent leaks or when the leak results in the formation of a subphrenic abscess. In live donor liver transplantation, particularly among recipients of the right lobe, the rate of biliary tract complication has been as high as 49%. Causes implicated for this high complication rate are multiple ducts, ischemia, thermal injury to the ducts during the transection of the parenchyma, and leaks from the raw surface.54
40 30 20 10
Hep B
Hep C
Alcoholic Cirrhosis
0 0
1
A
2
3
4
5
Years Post Transplant
Liver Transplant Patients with Hep B, Hep C, or Alcoholic Cirrhosis Graft Survival 100 90 80 Survival (%)
70 60 50 40 30 20 10
Hep B
Hep C
Alcoholic Cirrhosis
0 0 B
1
2
3
4
5
Years Post Transplant
Figure 50-10. Comparison of patient survival (A) and graft survival (B) after transplantation for hepatitis B and C and alcoholic cirrhosis. The differences observed did not reach statistical significance.
GRAFT DYSFUNCTION Following transplantation, there is often some degree of graft dysfunction. The clinical presentation of graft dysfunction can be mild, characterized by a transient elevation of the international normalized ratio (INR) accompanied by abnormal levels of the liver enzymes, or severe, requiring urgent retransplantation. The latter is known as primary graft non-function and this complication has been observed in between 2 and 10% of the transplants. The incidence of graft failure increases with the use of non-heart-beating donors and donors with significant fatty infiltration.46–48 Between these two extremes, differing degrees of graft dysfunction may be observed. Suffice it to say that the more severe the graft dysfunction, the greater the potential for morbidity, particularly infections, which in turn lead to prolonged intensive care unit and hospital stays, driving up the costs of transplantation. The clinical manifestations of primary graft non-function are encephalopathy leading to coma, uncorrectable INR in spite of massive transfusions of fresh frozen plasma and cryoprecipitate,
VASCULAR COMPLICATIONS Vascular complications are associated with significant morbidity and mortality. The rate of hepatic thrombosis varies from 2% to 10% over the years.56–59 This complication rate has decreased because of improvements in the surgical technique and better solutions for organ preservation. The clinical presentation of hepatic artery thrombosis is variable. The majority of the patients develop necrosis of the extrahepatic biliary system, liver infarcts, and bilomas. Very few patients may not experience these problems but go on to develop intrahepatic biliary strictures. The definitive treatment for hepatic artery thrombosis is retransplantation. The 1-year actuarial survival following retransplantation for hepatic artery thrombosis is
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about 50%.50 Stenosis of the hepatic artery may result in the development of intrahepatic biliary strictures. Dilatation of the artery stenosis with stent placement has occasionally been successful.60 The ultrasonographic findings of dampening of the intrahepatic arterial wave or proximal dilation accompanied by turbulence suggest hepatic artery stenosis.61 In whole-liver transplantation, the incidence of portal vein thrombosis is about 2%.62,63 Portal vein thrombosis may occur immediately after transplantation or late. Factors implicated in thrombosis of the portal vein are: pretransplant thrombosis of the portal vein, congenital anomalies of the portal vein such as a preduodenal portal vein, or hypo- or aplasia of the portal vein, commonly seen in cases of extrahepatic biliary atresia.62,63 Segmental liver transplantation has been reported to be associated with a higher incidence of portal vein thrombosis.59 Clinical manifestations of portal vein thrombosis depend on the time of presentation. Early in the postoperative period the most common findings associated with portal vein thrombosis are a progressive increase of the INR value, an elevated serum ammonia, and gastrointestinal bleeding. The diagnosis is made by Doppler ultrasonography. Further studies such as a computed tomography or magnetic resonance imaging angiogram are unnecessary and further delay treatment. Unlike hepatic artery thrombosis, prompt exploration for thrombectomy and reconstruction of the portal veins anastomosis are frequently successful in salvaging the graft. Thrombotic complications of the vena cava in full-size liver transplantation are observed in fewer than 1% of the transplants.62,63 Among the complications of the vena cava reconstruction, outflow obstruction is the most common complication, resulting in symptoms similar to Budd–Chiari syndrome, such as intractable ascites, right pleural effusion, and abnormal levels of the hepatic enzymes. Recipients of the right lobe from living donors are at risk of developing outflow obstruction.64,65 Including the middle hepatic vein in addition to the right hepatic vein for drainage of living donor right lobes has eliminated this complication.66 Besides technical mishaps, hypercoagulable states in patients with malignancies, including myeloproliferative disorders and congenital anomalies, are other factors implicated as causes for thrombosis. Many of the patients with stenosis of the vena cava or vein in cases of living donor or split transplantation are successfully managed by transluminal dilatation of the stricture followed by stent placement.67,68 Once thrombosis ensues, the management becomes more difficult. Treatment with chronic anticoagulation may be indicated. Patients eventually develop collateral drainage through the azygos system with subsequent improvement of their symptoms. Retransplantation is rarely indicated.
RETRANSPLANTATION The rate of retransplantation varies from 10 to 30%.50,69–71 In decreasing order of frequency, the indications for retransplantation in children are: vascular complications with hepatic artery thrombosis being the most common, followed by primary graft nonfunction and, lastly, chronic rejection.71 Among adult patients, the indications in decreasing order of frequency are: primary graft nonfunction, recurrence of the underlying liver disease (particularly
958
Table 50-18. Causes for Retransplantation of the Liver and Patient Survival at the University of Pittsburgh Causes Primary non-function Hepatic artery thrombosis Chronic rejection Recurrent disease Acute rejection Biliary complications Technical Miscellaneous Unknown Total
Number (%)
Mean interval
Currently alive n (%)
249 (32.2) 214 (27.6) 113 (14.5) 44 (5.5) 38 (4.9) 22 (2.8) 12 (1.5) 48 (6.2) 34 (4.3) 774 (19.4)
0.3 + 0.4 5.5 + 14 25.1 + 28.3 31.9 + 32.2 5.4 + 20.4 16.8 + 24 34.9 + 35.2 16.4 + 28 28 + 47 10.7 + 24
97 (38.8) 85 (39.7) 48 (42.4) 15 (34.8) 13 (34.2) 8 (36.3) 6 (50) 12 (25) 14 (40) 298 (38.5)
Reproduced from Kashyap R, Jain A, Reyes J, et al. Causes of retransplantation after primary liver transplantation in 4000 consecutive patients: 2 to 19 years follow-up. Transplant Proc 2001;33:1486–1487, with permission.
Table 50-19. Indications for Use of Vascular Conduits During Primary or Secondary Liver Transplantation Aberrant vasculature in donor or recipient Injury to donor vessel during procurement Hepatic artery thrombosis Inadequate length of vessel for transplant, e.g. reduced size, split livers, thrombosed recipient celiac artery, large size discrepancy (adult donor vessel in pediatric recipient) Portal vein thrombosis Hypoplastic portal vein, e.g. biliary atresia
hepatitis C), chronic rejection and, lastly, vascular complications72 (Table 50-18). The outcomes after retransplantation are significantly worse than those of primary transplantation.70,71 Factors implicated in poor outcomes after retransplantation are the diagnostic indication, abnormal serum creatinine, prolonged INR, prolonged intensive care unit stay, and urgency for retransplantation.70–74 A difficult dilemma involves the decision-making for retransplantation for recurrence of hepatitis C following transplantation.75 The patient survival at 1 year following retransplantation for recurrence of hepatitis C varies from 33 to 50% compared to 65–75% for indications other than recurrence of hepatitis C.76,77 Interferon treatment for recurrence of hepatitis C after liver transplantation has been grossly ineffective.78 Further, the efficacy of pre-emptive treatment with interferon prior to retransplantation for recurrence of hepatitis C is unknown. The technique for retransplantation is roughly similar to primary transplantation; however, the degree of difficulty during retransplantation depends on the timing of the operation in relation to the primary transplant. For example, the operation is simple when retransplantation is carried out within a few days of the primary transplant, but the formation of dense adhesions may render the operation a formidable task should retransplantation be done several months or years after the first operation. In retransplantation, vascular conduits may be needed for reconstruction of the hepatic artery and portal vein (Table 50-19). A ductto-duct bile duct reconstruction may be done when the recipient’s
Chapter 50 LIVER TRANSPLANTATION SURGERY
bile duct appears healthy, but often this is not the case. Thus, a reconstruction to a Roux en Y will be the safest approach.
CONCLUSIONS During the last two decades, the techniques for liver transplantation have been refined and to a greater extent the outcomes following hepatic transplantation are related to the proper selection of donors and recipients. Nonetheless, the transplant surgeon must be familiar with the different surgical techniques to address adverse conditions at the time of the transplantation or retransplantation, such as the presence of portal vein thrombosis, arterial anomalies, and the presence of adhesions. Finally, the transplant surgeon must be aware of the ethical issues surrounding the utilization of living liver donors and must protect the potential living donors from coercion by the potential recipients and their relatives.
REFERENCES 1. Starzl TE. The saga of liver replacement, with particular reference to the reciprocal influence of liver and kidney transplantation (1955–1967). J Am Coll Surg 2002; 195:587–610. 2. Calne RY. Immunosuppression in liver transplantation. N Engl J Med 1994; 331:1154–1155. 3. Millard CE. The NIH Consensus Development Conference on liver transplantation. R I Med J 1984; 67:69–71. 4. Southard JH, Belzer FO. Organ preservation. Annu Rev Med 1995; 46:235–247. 5. Jain A, Reyes J, Kashyap R, et al. Long-term survival after liver transplantation in 4000 consecutive patients at a single center. Ann Surg 2000; 232:490–500. 6. Manzarbeitia CY, Ortiz JA, Rothstein KD, et al. Long-term outcome of controlled, non-heart-beating donor liver transplantation. Transplantation 2004; 78:211–215. 7. D’Alessandro AM, Fernandez LA, Chin LT, et al. Donation after cardiac death: the University of Wisconsin experience. Ann Transplant 2004; 9:68–71. 8. Busuttil RW, Tanaka K. The utility of marginal donors in liver transplantation. Liver Transpl 2003; 9:651–663. 9. http://www.optn.org/latestData/rptData.asp. 10. Janssen H, Janssen PH, Broelsch CE. Celsior solution compared with University of Wisconsin solution (UW) and histidinetryptophan-ketoglutarate solution (HTK) in the protection of human hepatocytes against ischemia-reperfusion injury. Transplant Int 2003; 16:515–522. 11. Pedotti P, Cardillo M, Rigotti P, et al. A comparative prospective study of two available solutions for kidney and liver preservation. Transplantation 2004; 77:1540–1545. 12. Wilson CH, Stansby G, Haswell M, et al. Evaluation of eight preservation solutions for endothelial in situ preservation. Transplantation 2004; 78:1008–1013. 13. Miller CM, Rapaport FT, Starzl TE. Organ procurement. In: Wilmore DW, Cheung LY, Harken AH, et al, eds. ACS surgery – principles and practice 2003. New York: WebMD, 2003:995–1007. 14. Kim JS, Broering DC, Tustas RY, et al. Split liver transplantation: past, present and future. Pediatr Transplant 2004; 8:644–648. 15. Merion RM, Rush SH, Dykstra DM, et al. Predicted lifetimes for adult and pediatric split liver versus adult whole liver transplant recipients. Am J Transplant 2004; 4:1792–1797. 16. Humar A, Khwaja K, Sielaff TD, et al. Split-liver transplants for two adult recipients: technique of preservation of the vena cava with the right lobe graft. Liver Transpl 2004; 10:153–155.
17. Adam R, McMaster P, O’Grady JG, et al. European Liver Transplant Association. Evolution of liver transplantation in Europe: report of the European Liver Transplant Registry. Liver Transpl 2003; 9:1231–1243. 18. Renz JF, Emond JC, Yersiz H, et al. Split-liver transplantation in the United States: outcomes of a national survey. Ann Surg 2004; 239:172–181. 19. Yersiz H, Renz JF, Farmer DG, et al. One hundred in situ splitliver transplantations: a single center experience. Ann Surg 2003; 238:496–505. 20. Marcos A, Fisher RA, Ham JM, et al. Right lobe living donor liver transplantation. Transplantation 1999; 68:798–803. 21. Bak T, Wachs M, Trotter J, et al. Adult-to-adult living donor liver transplantation using right-lobe grafts: results and lessons learned from a single-center experience. Liver Transpl 2001; 7:680–686. 22. Miller CM, Gondolesi GE, Florman S, et al. One hundred nine living donor liver transplants in adults and children: a singlecenter experience. Ann Surg 2001; 234:301–311. 23. Trotter JF, Wachs M, Everson GT, et al. Adult-to-adult transplantation of the right hepatic lobe from a living donor. N Engl J Med 2002; 346:1074–1082. 24. Fan ST, Lo CM, Liu CL, et al. Safety of donors in liver donor live transplantation using right lobe grafts. Arch Surg 2000; 135:336–340. 25. Sakamoto S, Uemoto S, Uryuhara K, et al. Graft size assessment and analysis of donors for living donor liver transplantation using right lobe. Transplantation 2001; 71:1407–1413. 26. Ben-Haim M, Emre S, Fishbein TM, et al. Critical graft size in adult-to-adult living donor liver transplantation: impact of the recipient’s disease. Liver Transpl 2001; 7:948–953. 27. Imamura H, Takayama T, Sugawara Y, et al. Pringle’s manoeuvre in living donors. Lancet 2002; 360:2049–2050. 28. Miller CM, Masetti M, Cautero N, et al. Intermittent inflow occlusion in living liver donors: impact on safety and remnant function. Liver Transpl 2004; 10:244–247. 29. Lutz JT, Valentin-Gamazo C, Gorlinger K, et al. Blood-transfusion requirements and blood salvage in donors undergoing right hepatectomy for living related liver transplantation. Anesth Analg 2003; 96:351–355. 30. Shaw BW Jr. Where monsters hide (editorial). Liver Transpl 2001; 7:928–932. 31. Trotter JF, Talamantes M, McClure M, et al. Right hepatic lobe donation for living donor liver transplantation: impact on donor quality of life. Liver Transpl 2001; 7:485–493. 32. Cronin DC, Millis JM, Siegler M. Transplantation of liver grafts from living donors into adults – too much, too soon. N Engl J Med 2001; 344:1633–1637. 33. Malagó M, Testa G, Marcos A, et al. Ethical considerations and rationale of adult-to-adult living donor liver transplantation. Liver Transpl 2001; 7:921–927. 34. Pomposelli JJ, Pomfret EA, Burns DL, et al. Life-threatening hypophosphatemia after right hepatic lobectomy for live donor adult liver transplantation. Liver Transpl 2001; 7:637–642. 35. Pomfret EA, Pomposelli JJ, Lewis D, et al. Live donor adult liver transplantation using right lobe grafts. Arch Surg 2001; 136:425–433. 36. Akabayashi A, Slingsby BT, Fujita M. The first donor death after living-related liver transplantation in Japan. Transplantation 2004; 77:634. 37. Broering DC, Wilms C, Bok P, et al. Evolution of donor morbidity in living related liver transplantation: a single-center analysis of 165 cases. Ann Surg 2004; 240:1013–1024. 38. Witkowski K, Piecuch J. Liver transplant without a venovenous bypass. Ann Transplant 2001; 6:16–17. 39. Reddy KS, Johnston TD, Putnam LA, et al. Piggyback technique and selective use of veno-venous bypass in adult orthotopic liver transplantation. Clin Transplant 2000; 14:370–374.
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40. Cabezuelo JB, Ramirez P, Acosta F, et al. Does the standard vs piggyback surgical technique affect the development of early acute renal failure after orthotopic liver transplantation? Transplant Proc 2003; 35:1913–1914. 41. Budd JM, Isaac JL, Bennett J, et al. Morbidity and mortality associated with large-bore percutaneous venovenous bypass cannulation for 312 orthotopic liver transplantations. Liver Transpl 2001; 7:359–362. 42. Scholz T, Solberg R, Okkenhaug C, et al. Veno-venous bypass in liver transplantation: heparin-coated perfusion circuits reduce the activation of humoral defense systems in an in vitro model. Perfusion 2001; 16:285–292. 43. Busque S, Esquivel CO, Concepcion W, et al. Experience with the piggyback technique without caval occlusion in adult orthotopic liver transplantation. Transplantation 1998; 65:77–82. 44. Arcari M, Phillips SD, Gibbs P, et al. An investigation into the risk of air embolus during veno-venous bypass in orthotopic liver transplantation. Transplantation 1999; 68:150–152. 45. Leonardi LS, Boin IF, Leonardi MI, et al. Ascites after liver transplantation and inferior vena cava reconstruction in the piggyback technique. Transplant Proc 2002; 34:3336–3338. 46. Bennett-Guerrero E, Feierman DE, Barclay GR, et al. Preoperative and intraoperative predictors of postoperative morbidity, poor graft function, and early rejection in 190 patients undergoing liver transplantation. Arch Surg 2001; 136:1177–1183. 47. Verran D, Kusyk T, Painter D, et al. Clinical experience gained from the use of 120 steatotic donor livers for orthotopic liver transplantation. Liver Transpl 2003; 9:500–505. 48. Abt PL, Desai NM, Crawford MD, et al. Survival following liver transplantation from non-heart-beating donors. Ann Surg 2004; 239:87–92. 49. Agnes S, Avolio AW, Magalini SC, et al. Should retransplantation still be considered for primary non-function after liver transplantation? Transpl Int 1992; 5 (Suppl 1):S170–S172. 50. Kashyap R, Jain A, Reyes J, et al. Causes of retransplantation after primary liver transplantation in 4000 consecutive patients: 2 to 19 years follow-up. Transplant Proc 2001; 33:1486–1487. 51. Roumilhac D, Poyet G, Sergent G, et al. Long-term results of percutaneous management for anastomotic biliary stricture after orthotopic liver transplantation. Liver Transpl 2003; 9:394–400. 52. Guichelaar MM, Benson JT, Malinchoc M, et al. Risk factors for and clinical course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant 2003; 3:885–890. 53. Sung RS, Campbell DA Jr., Rudich SM, et al. Long-term followup of percutaneous transhepatic balloon cholangioplasty in the management of biliary strictures after liver transplantation. Transplantation 2004; 77:110–115. 54. Gondolesi GE, Varotti G, Florman SS, et al. Biliary complications in 96 consecutive right lobe living donor transplant recipients. Transplantation 2004; 77:1842–1848. 55. Tachopoulou OA, Vogt DP, Henderson JM, et al. Hepatic abscess after liver transplantation: 1990–2000. Transplantation 2003; 75:79–83. 56. Settmacher U, Stange B, Haase R, et al. Arterial complications after liver transplantation. Transpl Int 2000; 13:372–378. 57. Cavallari A, Vivarelli M, Bellusci R, et al. Treatment of vascular complications following liver transplantation: multidisciplinary approach. Hepatogastroenterology 2001; 48:179–183. 58. Stringer MD, Marshall MM, Muiesan P, et al. Survival and outcome after hepatic artery thrombosis complicating pediatric liver transplantation. J Pediatr Surg 2001; 36:888–891.
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59. Sieders E, Peeters PM, TenVergert EM, et al. Early vascular complications after pediatric liver transplantation. Liver Transpl 2000; 6:326–332. 60. Raby N, Karani J, Thomas S, et al. Stenoses of vascular anastomoses after hepatic transplantation: treatment with balloon angioplasty. AJR Am J Roetgenol 1991; 157:167–171. 61. Stell D, Downey D, Marotta P, et al. Prospective evaluation of the role of quantitative Doppler ultrasound surveillance in liver transplantation. Liver Transpl 2004; 10:1183–1188. 62. Settmacher U, Nussler NC, Glanemann M, et al. Venous complications after orthotopic liver transplantation. Clin Transplant 2000; 14:235–241. 63. Buell JF, Funaki B, Cronin DC, et al. Long-term venous complications after full-size and segmental pediatric liver transplantation. Ann Surg 2002; 236:658–666. 64. Marcos A, Orloff M, Mieles L, et al. Functional venous anatomy for right-lobe grafting and techniques to optimize outflow. Liver Transplantation 2001; 7:845–852. 65. Ghobrial RM, Hsieh CB, Lerner S, et al. Technical challenges of hepatic venous outflow reconstruction in right lobe adult living donor liver transplantation. Liver Transpl 2001; 7:551–555. 66. Scatton O, Belghiti J, Dondero F, et al. Harvesting the middle hepatic vein with a right hepatectomy does not increase the risk for the donor. Liver Transpl 2004; 10:71–76. 67. Borsa JJ, Daly CP, Fontaine AB, et al. Treatment of inferior vena cava anastomotic stenoses with the Wallstent endoprosthesis after orthotopic liver transplantation. J Vasc Interv Radiol 1999; 10:17–22. 68. Yamagiwa K, Yokoi H, Isaji S, et al. Intrahepatic hepatic vein stenosis after living-related liver transplantation treated by insertion of an expandable metallic stent. Am J Transplant 2004; 4:1006–1009. 69. Biggins SW, Beldecos A, Rabkin JM, Rosen HR. Retransplantation for hepatic allograft failure: prognostic modeling and ethical considerations. Liver Transpl 2002; 8:313–322. 70. Azoulay D, Linhares M, Huguet E, et al. Decision for retransplantation of the liver – an experience- and cost-based analysis. Ann Surg 2002; 236:713–721. 71. Sieders E, Peeters PMJG, TenVergert EM, et al. Retransplantation of the liver in children. Transplantation 2001; 71:90–95. 72. Lerut J, Laterre PF, Roggen F, et al. Adult hepatic retransplantation. Acta Gastroenterol Belg 1999; 62:261–266. 73. Postma R, Haagsma EB, Peeters PM, et al. Retransplantation of the liver in adults: outcome and predictive factors for survival. Transpl Int 2004; 17:234–240. 74. Yao FY, Saab S, Bass NM, et al. Prediction of survival after liver retransplantation for late graft failure based on preoperative prognostic scores. Hepatology 2004; 39:230–238. 75. Burton JR Jr, Rosen HR. Retransplantation for hepatitis C: what do we really know? Liver Transpl 2004; 10:1504–1506. 76. Neff GW, O’Brien CB, Nery J, et al. Factors that identify survival after liver retransplantation for allograft failure caused by recurrent hepatitis C infection. Liver Transpl 2004; 10:1497–1503. 77. Berenguer M, Prieto M, Palau A, et al. Severe recurrent hepatitis C after liver retransplantation for hepatitis C virus-related graft cirrhosis. Liver Transpl 2003; 9:228–235. 78. Stravitz RT, Shiffman ML, Sanyal AJ, et al. Effects of interferon treatment on liver histology and allograft rejection in patients with recurrent hepatitis C following liver transplantation. Liver Transpl 2004; 10:850–858.
Section VIII: Liver Transplantation
51
POST LIVER TRANSPLANTATION MANAGEMENT AND COMPLICATIONS Paul J. Gaglio and Robert S. Brown, Jr Abbreviations ACR acute cellular rejection aLDLT adult-to-adult living donor liver transplantation ASHD atherosclerotic heart disease AZA azathioprine CMV cytomegalovirus CNI calcineurin inhibitor CYA ciclosporin DACD donor after cardiac death DD deceased donor
EBV EGD HAT HBV HCC HCV IMPDH INR MELD MMF
Epstein–Barr viral early graft dysfunction hepatic artery thrombosis hepatitis B virus hepatocellular carcinoma hepatitis C virus inosine monophosphate dehydrogenase international normalized ratio model for end-stage liver disease mycophenolate mofetil
INTRODUCTION Liver transplantation, once considered an experimental procedure, has now emerged as the treatment of choice for appropriately selected patients with end-stage liver disease. Based on recently published data by United Network for Organ Sharing (UNOS), 5671 liver transplants (5350 deceased donor (DD) and 321 living donor) were performed in 2004.1 The markedly improved graft and patient survival rates following liver transplantation observed over the last decade are derived from multiple factors. These include advances in surgical technique and immunosuppression, selection of appropriate donors, allografts, and recipients, and improved therapies to prevent and treat postoperative complications. Coincident with enhanced post-transplantation survival rates has been the emergence of complications associated with patient longevity, including non-hepatic disease, complications of immunosuppression, infections, neoplasia, and recurrence of the primary disease for which the liver transplantation was indicated. This chapter will delineate common issues related to post-transplantation management, describe short- and long-term post-transplant complications, and discuss therapies and strategies for prevention.
BACKGROUND Successful liver transplantation involves a complex interplay between the donor, allograft, and recipient. To appreciate fully strategies to enhance management and recognize complications following liver transplantation, donor, allograft, and recipient attributes
MRSA PCR PNF PTLD RAPA TAC US UNOS VRE
Methicillin-resistant Staphylococcus aureus polymerase chain reaction primary graft non-function post-transplant lymphoproliferative disease rapamycin tacrolimus ultrasound united network for organ sharing vancomycin-resistant enterococcus
that negatively affect post-transplantation outcome need to be recognized (Figure 51-1).
DONOR FACTORS It is well established that advanced age, medical comorbidities, and instability in the donor, including requirement for pressors to maintain blood pressure, prolonged hypotension, hypernatremia, and infection, may be associated with diminished recipient and graft survival post-transplantation.2,3 Clearly, when several of these factors are present in a potential donor, the negative impact may be cumulative, and thus, these donors are usually considered unacceptable.4 In stable donors, however, the definition of acceptable age has recently been expanded. Data from various animal models indicate that livers retain the ability to regenerate even in animals of advanced age. With this concept in mind, many transplant centers will accept appropriately selected donors up to age 80; data suggest that these grafts will function well without any negative impact on recipient outcomes.5,6 However, emerging evidence indicates that grafts from older donors should be used with caution in hepatitis C-positive recipients due to poor outcomes, including more severe histologic recurrence of hepatitis C and more rapid progression to fibrosis.7 Other important factors which impact recipient outcomes include donor type, specifically, if the allograft was obtained from a deceased, living, or donor after cardiac death (DACD). DD comprise the majority of liver donors. Either by self-identification while living, or after discussion with “next of kin” when donor brain death has been declared, individuals are acknowledged as potential organ
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Donor
Graft
Age Hypotension Hypernatremia Infection Medical co-morbidities Pressor requirements Deceased, Living, or Non-heartbeating
Preservation time Steatosis Fibrosis Intrinsic disease
Recipient
MELD score Immune-competence Co-morbid disease
Figure 51-1. Pre-, post-transplantation and donor variables affecting posttransplantation outcome. MELD: model for end-stage liver disease.
donors. Recent data from UNOS indicate that 1- and 3-year patient survival in recipients of DD liver transplant is 81 and 71% respectively. However, despite efforts to maximize utilization of organs acquired from DD, including the use of older donors, steatotic livers, and livers infected with hepatitis C or B, a growing disparity exists between the number of available livers and the number of individuals waiting for transplantation. This critical shortage of organs has resulted in both an increase in the waiting time for liver transplantation and death rate among patients on the waiting list. In response, and as a natural evolution of the pre-existing modality of using a left lateral segment graft for adult-to-child living donor liver transplantation, adult-to-adult living donor liver transplantation (aLDLT) has emerged as an alternative to DD liver transplantation.8,9 This procedure requires that the larger, right lobe of the liver (which accounts for approximately 50–60% of the hepatic mass) be removed from the donor, and implanted into the recipient. Rapid regeneration of the liver remnant in the donor and the partial allograft transplanted into the recipient occurs, resulting in restoration of appropriate liver volume within 1–2 months in both donor and recipient following surgery.10 As the recipient of an aLDLT receives a graft which over time must grow to an appropriate volume, selection of recipients best able to tolerate transplantation of a partial graft is necessary. Current data suggest that the 1-year survival of “sicker” patients formerly identified as UNOS status 2A, or with a model for end-stage liver disease (MELD) score (see below) greater than 25 is approximately 66% following LDLT, compared with 80–90% in less ill patients classified as status 2B or MELD less than 25.11 Thus, in appropriately selected recipients, 1-year graft and patient survival in individuals who undergo LDLT is similar to DD.1 Postoperative complications are similar when comparing DD to LDLT, although recipients of LDLT have a greater rate of biliary complications, including bile leaks and biliary strictures that occur in 15–32% of patients.11 Biliary complications following LDLT are
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usually managed through non-operative modalities, and do not affect post-transplantation outcomes. In addition, the “small-for-size syndrome,” manifested as prolonged post-transplantation cholestasis may occur following LDLT, particularly in recipients who receive a graft of inadequate size.12 Fortunately, the majority of patients who experience this syndrome recover without the requirement of retransplantation. Recently, significant interest in the utilization of DACD as another modality to increase the pool of available organs has emerged. In contradistinction to DD who are declared brain-dead, DACD are patients who are not declared brain-dead, but are critically ill, without any reasonable expectation of potential recovery, who based on previous stated or families’ wishes are removed from life support at the time of death. There are two types of DACD, controlled and uncontrolled. In the controlled DACD (Maastricht category 3, death anticipated) the patient is removed from life support and death occurs in the operating room. Once death has been declared, organs deemed suitable for transplantation are rapidly perfused with cold preservation solution and removed surgically. The uncontrolled DACD (Maastricht category 1 and 2, death not anticipated) is declared dead after cardiac arrest, rushed to the operating room, and organs are harvested. Uncontrolled DACD are not utilized for liver transplantation due to the high rate of primary non-function (defined below), usually due to prolonged ischemia of the graft.13 When utilizing controlled DACD for transplantation, emerging data indicate that recipient and graft survival are diminished when compared to deceased and LDLT, including a higher incidence of primary non-function, biliary injury, and retransplantation.14 However, several centers have reported acceptable outcomes when utilizing controlled DACD organs, particularly those without significant ischemia in well-selected recipients.15
GRAFT FACTORS Intrinsic to post-transplantation success is the quality of the donor organ. However, due to the rapidly expanding disparity between the number of individuals requiring liver transplantation and organ availability, the concept of “organ quality” is in a constant state of redefinition. By sheer necessity, liver transplant professionals have been required to reassess the limits of acceptable preservation time, degree of steatosis and fibrosis, and the impact of pre-existing disease in the donor related to recipient outcomes, in an attempt to achieve maximal use of available organs. In addition, within the limitations of the present organ allocation system, it is becoming increasingly apparent that appropriate matching of allograft and recipient will be associated with improved outcomes. Many stable liver transplant recipients will do well when transplanted with an allograft formerly defined as marginal, while hospitalized, critically ill patients will not thrive if graft quality is suboptimal.16 The ability to delay transplantation of a liver graft after it is harvested has been achieved by improvements in organ preservation solution. The current limitation in “cold” ischemic time, defined as the number of hours that donor grafts may be in preservation fluid prior to transplantation, is up to 14 hours. Recent data indicate that patient and graft outcomes begin to diminish if cold ischemic time exceeds this, particularly in livers with significant steatosis.17,18 Similar advances in understanding the appropriate degree of steatosis have allowed transplant professionals the ability to maximize use
Chapter 51 POST LIVER TRANSPLANTATION MANAGEMENT AND COMPLICATIONS
of available donor organs. It is now apparent that acceptable posttransplantation outcomes can be achieved when allografts with up to 60% steatosis are used.19 These organs were formerly deemed unacceptable. In addition, perceptions regarding acceptability of grafts with intrinsic disease are being revised, as satisfactory graft and patient survival have been achieved when hepatitis C-infected patients receive an appropriately selected graft from a hepatitis Cinfected donor,20 and when using isolated hepatitis B core antibodypositive, hepatitis B surface antigen-negative grafts.21
RECIPIENT FACTORS Given the relatively stable number of available donor organs in the setting of a rapidly expanding pool of potential recipients, the timing of transplantation is critical. Liver transplantation in a stable patient who is anticipated to do well for many years while waiting for an available organ may not be appropriate, while liver transplantation in a moribund patient with a low probability of post-transplantation survival is similarly inappropriate. Prior to 1997, prioritization for liver transplantation was based on the location where patients received their care (i.e., home, hospital, intensive care unit) and heavily dependent on waiting time. In 2002, UNOS instituted several policies in an attempt to produce a more equitable organ allocation scheme. Waiting time as well as location where patients received their care were eliminated as determinants of prioritization of organ allocation. The MELD score, a numerical calculation based on the recipient’s log transformed renal function (creatinine), total bilirubin, and INR (international normalized ratio for prothrombin time) which has been shown to predict transplantation mortality was adopted by UNOS as a mechanism to prioritize waiting-list candidates. MELD had been validated as a predictor of 3-month survival in diverse groups of patients with various etiologies and manifestations of liver disease.22 Thus, currently a patient’s position on the liver transplantation waiting list is now determined by the MELD score; patients with highest MELD scores are ranked highest on the list. Prospective analysis of the impact of MELD indicates improvement in the rate of transplantation, pretransplantation mortality, and short-term post-transplantation mortality rates.23 However, retrospective analysis has suggested that post-transplantation survival may be reduced in patients with very high pre-transplantation MELD score, particularly in hepatitis C-infected patients.24 Thus, it is clear that careful recipient selection, with attention to pressor and ventilatory requirements, need for dialysis and age are important factors in selecting appropriate candidates for liver transplantation. Another factor associated with post-transplantation outcomes is the innate immunocompetence of the recipient; this may modulate recognition and rejection of the newly transplanted liver. It is well established that the rate of acute cellular rejection (ACR) of the allograft is greater in patients who are “high immunologic responders,” i.e., patients with presumed autoimmune liver diseases such as primary biliary cirrhosis, sclerosing cholangitis, and autoimmune hepatitis.25 In addition, early immune-mediated injury may be a factor associated with primary non-function (see below) of a transplanted liver via mechanisms that are not clearly defined. Selection of immunosuppression as well as strategies to prevent both early and late cellular rejection are predicated on stratifying an individual patient’s risk for immunologic injury.
POST-TRANSPLANTATION MANAGEMENT IMMUNOSUPPRESSIVE MEDICATIONS A cornerstone to post-transplantation management is the ability to prevent rejection of the newly transplanted graft, which, when left untreated, can be associated with graft failure. Strategies to prevent both acute and chronic rejection are predicated on understanding how recognition of the newly engrafted liver as “foreign” occurs, how immune-mediated injury can be modulated, while at the same time avoiding overimmunosuppression, which places individuals at high risk of infection. The various immunosuppressive medications currently utilized in liver transplant recipients and their side effects are listed in Table 51-1 and portrayed schematically in Figure 51-2. Unfortunately, all immunosuppressive therapy is associated with undesired effects, the spectrum of which varies and in some cases overlap. In general, either two or three agents are utilized to prevent allograft rejection in the immediate post-transplant period, utilizing a combination of a calcineurin inhibitor (CNI) such as ciclosporin (CYA) or tacrolimus (TAC), a second agent such as mycophenolate mofetil (MMF) or azathioprine (AZA), and a glucocorticoid such as prednisone. As patients achieve adequate liver function and remain free from rejection beyond 6 months post-transplantation, satisfactory immunosuppression can be achieved in many patients with monotherapy, usually with a CNI.
CORTICOSTEROIDS The immune suppressive effects of corticosteroids and the ability of these agents to reduce the incidence of and treat allograft rejection and to prolong graft survival have been known for decades. Corticosteroids affect multiple aspects of immune function, achieved by the suppression of leukocyte, macrophage, and cytotoxic T-cell activity, and diminution of the effect of cytokines, prostaglandins, and leukotrienes.26 However, coincident with the immunosuppressive effects of corticosteroids is the potential for significant side effects, including hypertension, dyslipidemia, glucose intolerance, bone abnormalities, peptic ulcers, and psychiatric disorders. Thus, many transplant professionals adopt a strategy to taper and discontinue glucocorticoids within the first 6 months to a year following transplantation, while maintaining adequate levels of CNI. This strategy is often altered in patients who undergo liver transplantation secondary to an immunologic disorder such as autoimmune hepatitis, primary biliary cirrhosis, and sclerosing cholangitis due to an enhanced risk of ACR.25 In these patients, either long-term use of corticosteroids, with an attempt to minimize doses, is advocated, or chronic use of MMF or AZA in combination with a CNI is required.
T-CELL-DEPLETING AGENTS In the past, antilymphocyte agents (such as antilymphocyte globulin or antithymocyte globulin) or monoclonal antibody preparations (such as OKT3) directed against specific T-cell antigens have been utilized immediately after liver transplantation to induce rapidly an immune-suppressed state via rapid diminution of T cells.26 However, significant systemic side effects have been associated with the use of these agents, including fevers, allergic reactions, serum sickness,
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Table 51-1. Immunosuppressive Agents Mechanism of Action
Side effects
Prednisone
Suppression of leukocyte, macrophage, and cytotoxic T-cell activity Decrease cytokines, prostoglandins, and leukotrienes Depletes circulating lymphocytes
Hypertension Dyslipidemia
Antilymphocyte globulin Antithymocyte globulin OKT3
Depletes circulating T cells
Basiliximab daclizumab
IL-2 receptor blockade
Ciclosporin
Inactivates calcineurin, decreases IL-2 production, inhibits T-cell activation
Tacrolimus
Azathioprine
Mycophenolate mofetil
Sirolimus
Inactivates calcineurin, decreases IL-2 production, inhibits T-cell activation Inhibits adenosine and guanine production Inhibits DNA and RNA synthesis in rapidly proliferating T cells Inhibits inosine monophosphate dehydrogenase (IMPDH) Prevents T- and B-cell proliferation Inhibits mTOR (target of Rapamycin) Prevents T-cell replication
Glucose intolerance Bone abnormalities Peptic ulcers Psychiatric disorders Flu-like symptoms Anaphylaxis Lymphoproliferative disorders Flu-like symptoms Anaphylaxis Lymphoproliferative disorders Infections Gastrointestinal distress Pulmonary edema and bronchospasm (rare) Hypertension Renal insufficiency Neuropathy Hyperlipidemia Gingival hyperplasia Hirsutism Insulin resistance Hypertension Renal insufficiency Insulin resistance Neuropathy Hyperlipidemia Leukopenia Anemia Thrombocytopenia Pancreatitis Leukopenia Anemia Thrombocytopenia Gastrointestinal side effects Leukopenia Thrombocytopenia Hyperlipidemia Hepatic artery thrombosis (?) Inhibits wound-healing
Il-2, interleukin-2.
APC
IL-1
Corticosteroids
Cyclo TAC
T-CELL
IL-2
IL2 R
Agent
Sirolimus
Anti-IL2 R Nucleus
Replication MMF Azothiaprine
OKT3 ATG Figure 51-2. Mechanism of action of commonly used immunosuppressive therapies. APC, antigen-presenting cells; IL-1, interleukin-1; TAC, tacrolimus.
interleukin-2 (IL-2). Antibodies directed against the IL-2 receptor are effective for initial immunosuppression, as IL-2 receptor blockade down-regulates IL-2-mediated T-cell proliferation.26 Controlled trials have demonstrated that IL-2 receptor antagonists such as basiliximab and daclizumab, which are given intravenously at the time of transplant and during the first post-transplantation week, can reduce the incidence of acute liver graft rejection when utilized in combination with a CNI, although these agents may not be sufficient to prevent rejection when utilized alone. Side effects of IL-2 inhibitors may include infections, gastrointestinal distress, and rarely, pulmonary edema and bronchospasm. As these agents rarely induce renal dysfunction, many transplant programs utilize IL-2 receptor antibodies as induction therapy in individuals with renal insufficiency at the time of transplantation,28 in an attempt to delay initiation or diminish dose of CNIs which may exacerbate renal insufficiency.
CALCINEURIN INHIBITORS and thrombocytopenia. In addition, the long-term risk of lymphoproliferative disorders (see below) is increased in patients who receive these agents. As a result, at present, these preparations are utilized for the treatment of glucocorticoid-resistant rejection, or less commonly, in patients with renal insufficiency in an attempt to delay the use of either CYA or TAC which may be associated with worsening of renal function.27
IL-2 RECEPTOR BLOCKERS T-cell activation and proliferation following presentation of a foreign antigen requires the induction of several cytokines, including
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T-cell activation is modulated by multiple cytokines, including IL2. CYA and TAC bind to cytoplasmic receptors, forming complexes which inactivate calcineurin, a key enzyme in T-cell signaling. Multiple randomized controlled trials have demonstrated that both CYA and TAC are effective in reducing acute allograft rejection in liver transplant recipients (reviewed by Conti et al.26). The major adverse events associated with both CYA and TAC include hypertension, renal insufficiency, and neurologic complications. Various investigators have published data which indicate that obesity, hyperlipidemia, hirsutism, and gingival hyperplasia occur more commonly in patients who receive CYA, while a higher rate of
Chapter 51 POST LIVER TRANSPLANTATION MANAGEMENT AND COMPLICATIONS
insulin resistance is seen in patients who receive TAC. Several investigators have reported inconsistent absorption of standard CYA, which has largely been obviated by the use of a microemulsified formulation (e.g., Neoral) which allows more consistent blood levels.29 Despite these limitations, CNIs retain a central role in post-transplant immunosuppression.
ANTIPROLIFERATIVE AGENTS Antiproliferative agents such as AZA and MMF prevent the expansion of activated T cells and B cells and regulate immune-mediated injury. AZA, a purine analogue, is metabolized in the liver to its active compound, 6-mercaptopurine, which inhibits adenosine and guanine production, thus inhibiting DNA and RNA synthesis in rapidly proliferating T cells. MMF is a potent non-competitive inhibitor of inosine monophosphate dehydrogenase (IMPDH), an enzyme necessary for the synthesis of guanine, a purine nucleotide. Randomized controlled trials have demonstrated that MMF is more effective than AZA at reducing the incidence of ACR in solid organ transplant recipients.26 MMF may also reduce the incidence of chronic rejection by inhibiting the proliferation of B lymphocytes. The major toxicities associated with the use of either MMF or AZA are bone marrow suppression with leukopenia, anemia, and thrombocytopenia. MMF has been associated with a greater incidence of dyspepsia, peptic ulcers, and diarrhea when compared to AZA, while pancreatitis may occur in individuals prescribed AZA. These side effects usually abate by dose reduction or discontinuation. A newly released enteric formulation of MMF has been shown in renal transplant recipients to have equivalent efficacy when compared to standard MMF, with fewer gastrointestinal side effects, although this agent has not been well assessed in liver transplant recipients. The majority of transplant centers utilize a combination of a CNI with either MMF or, less commonly, AZA during the first 6 months post-transplantation. As both AZA and MMF are not associated with renal insufficiency, MMF can be utilized with a strategy toward minimizing or avoiding CNI use, particularly in patients with renal dysfunction.30
POST-TRANSPLANTATION COMPLICATIONS The complex nature of the surgical procedure utilized both to explant (remove) the diseased, cirrhotic liver and implant (transplant) the new allograft into the recipient makes it intuitive that the majority of the early complications following liver transplantation are technical and related to the surgical procedure itself. Figures 513 and 51-4 depict the surgical anastamoses required to perform a deceased as well as living donor liver transplantation. However, following the first postoperative days, and as patients progress to the first month post-transplantation and beyond, the nature and variety of complications change. Perhaps overly simplistic, a general approach to categorizing post-transplant complications is “the rule of twos,” i.e., understanding post-transplant complications which occur in the first 2 days, first 2 weeks, first 2 months, and beyond (Table 51-2).
Suprahepatic vena cava
Infrahepatic vena cava Portal vein Hepatic artery Bile duct Figure 51-3. Deceased donor liver transplantation.
OTHER IMMUNOSUPPRESSIVE AGENTS Sirolimus (Rapamycin: RAPA) and its derivative Everolimus represent a new class of compounds, which achieve their immuosuppressive effect by inhibiting mTOR (target of Rapamycin). Inhibition of mTOR diminishes intracellular signaling distal to the IL-2 receptor and prevents T-cell replication. As the lymphoproliferative pathways inhibited by RAPA and Everolimus are distinct from those affected by CNIs, investigators have utilized these agents in combination with CNIs to achieve synergistic effect.26 However, hepatic arterial thrombosis has been reported in patients who receive RAPA in the weeks immediately following transplantation.31 In addition, several investigators have noted problems with woundhealing in patients who receive RAPA, potentially due to impairment of granulation mediated by inhibition of transforming growth factorb. Leukopenia, thrombocytopenia, and hyperlipidemia are the principal toxicities associated with RAPA and Everolimus. A positive attribute of both RAPA and Everolimus is preservation of renal function; post-transplantation renal insufficiency can be reversed when RAPA is initiated and CNIs are withdrawn.32
Vena cava Hepatic vein
Hepatic artery Portal vein Bile duct
Intestine Figure 51-4. Living donor liver transplantation.
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Table 51-2. “Rule of Twos”: Complications During the first 2 Days, 2 Weeks, 2 Months, and Beyond Following Liver Transplantation Two Days Primary non-function Early graft dysfunction Hepatic artery thrombosis Hepatic and portal vein thrombosis Preservation injury Two Weeks Acute cellular rejection Bacterial and fungal infection Biliary complications Cytomegalovirus infection Two Months and Beyond Hypertension Hyperlipidemia Diabetes Obesity Cardiac disease Renal dysfunction Chronic rejection Fungal infection (Cryptococcus, Aspergillus) Cytomegalovirus Post-transplant lymphoproliferative disorder Malignancy Recurrence of primary disease
COMPLICATIONS IN THE FIRST 2 DAYS Primary Non-Function and Early Graft Dysfunction Primary graft non-function (PNF), defined as acidosis, rising INR, progressive elevation in liver transaminases and creatinine, and decreased mentation occur when the newly transplanted liver allograft fails to function normally. The mechanisms responsible for this phenomenon are incompletely understood but may relate to donor factors, inadequate preservation, prolonged ischemia, extensive steatosis of the graft, hepatic artery thrombosis (HAT: see below), or immune response to the implanted organ.33 In the setting of PNF, a rapid assessment of hepatic artery flow needs to occur, as immediate surgical repair of a thrombosed hepatic artery may reverse PNF. If PNF occurs in the absence of HAT, emergent retransplantation is required. In contrast to PNF, early graft dysfunction (EGD) is manifested by an early rise in serum transaminases to values greater than 2000–3000 IU/l, cholestasis with rising bilirubin levels, without concomitant impairment in mental status, coagulopathy, and renal function. EGD may be secondary to ischemic injury or steatosis in the graft, and typically occurs within the first 24–48 hours after the transplant. Unlike PNF, the manifestations of EGD usually improve, and emergency retransplantation is not necessary.
Hepatic Artery Thrombosis A potentially devastating post-transplantation complication is HAT. It is intuitive that HAT occurs more commonly in pediatric patients when compared to adults due to the technical difficulties associated with the anastomosis of smaller-size vessels.34 HAT in the immediate postoperative period may be associated with graft failure,
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massive elevation in serum liver transaminases, bile leak, hepatic necrosis, and sepsis. As the blood supply to the biliary tree is exclusively provided by the hepatic artery, it is not surprising that HAT is frequently associated with irreversible injury to the biliary tract.35 In individuals with HAT-associated biliary injury, bile duct necrosis, dehiscence of the bile duct or biliary-enteric anastomosis, and the development of diffuse intra- and extrahepatic biliary strictures may occur. These changes may induce chronic cholangitis, intrahepatic abscesses, and eventually secondary biliary cirrhosis and liver failure. For these reasons, HAT which occurs within the first 7 days after liver transplantation is an indication for emergent artery repair or retransplantation. Due to the potentially devastating consequences of HAT, most transplant centers utilize duplex-ultrasound (US) in the immediate post-transplant period as a mechanism to screen for this complication. If duplex-US suggests HAT, emergent angiography is usually performed to confirm the diagnosis prior to laparotomy and surgical revision of the hepatic artery. At specialized transplant centers with specific expertise in interventional radiology, emergency repair and stenting of a thrombosed hepatic artery may be effective in preventing the requirement for retransplantation.36 However, in the majority of cases, emergent surgical revision with an attempt to restore liver perfusion is required to prevent irreversible bile duct injury, and obviate the requirement for repeat liver transplantation. If this cannot be achieved, liver retransplantation may be necessary.
Portal and Hepatic Vein Thrombosis Thrombosis of portal and/or hepatic veins in the immediate posttransplant period is rare, but may be associated with significant consequences. Acute Budd–Chiari syndrome due to hepatic vein or vena cava thrombosis may occur, with abdominal pain, peripheral edema, and the threat of graft failure, as hepatic congestion in the newly transplanted allograft is poorly tolerated. In this circumstance, emergency surgical revision and repair of thrombosis are required. Acute portal vein occlusion may be associated with exacerbation of pre-existing portal hypertension, associated with gastrointestinal bleeding from portosystemic collateral vessels such as esophageal and gastric varices. Acute portal vein thrombosis is managed by surgical repair, while chronic portal vein thrombosis may be well tolerated. Over the last several years, interventional radiologic techniques have been utilized to successfully manage many vascular complications successfully following both deceased and living donor liver transplantation.37
Ischemic and Preservation Injury The newly transplanted liver can be subjected to multiple varieties of ischemic injury: cold (or hypothermic) and warm (or normothermic).38 Cold ischemia occurs prior to transplantation while the liver is cooled in preservation solution, awaiting implantation. Warm ischemia occurs during the transplantation procedure, when hepatic blood flow is interrupted to minimize blood loss during transplantation, or when the formerly cooled liver is subjected to room or body temperature during transplantation. Morphologically, cold ischemia induces injury to the sinusoidal endothelial cell, although this process is usually well tolerated. In contrast to cold ischemia,
Chapter 51 POST LIVER TRANSPLANTATION MANAGEMENT AND COMPLICATIONS
warm ischemia is tolerated poorly and rapidly leads to the death of hepatocytes, with resultant elevation in serum transaminases, apoptosis, and centrilobular necrosis. In general, ischemic injury is well tolerated, but, if significant, may be associated with graft failure. Several investigators have noted improvement in ischemic injury prior to liver transplantation by employing a technique described as ischemic preconditioning.39 Ischemic preconditioning consists of a brief period of ischemia followed by a short interval of reperfusion before the actual surgical procedure. During liver transplantation, hepatic inflow is occluded by placing a vascular clamp or a loop around the portal triad (i.e., portal vein, hepatic artery, and bile duct), rendering the whole organ ischemic for 10–15 minutes, after which the clamp is removed and the liver is reperfused for 10–15 minutes. This technique may be of particular benefit in allografts with significant steatosis.39
COMPLICATIONS IN THE FIRST 2 WEEKS Acute Cellular Rejection Liver allografts are relatively privileged immunologically, and thus, the incidence and consequences of ACR are diminished when compared to other solid organs utilized for transplantation. The reported incidence of ACR within the first post-transplant year is 30–50%, in most cases, usually occurring within the first 10–14 postoperative days. The clinical presentation is variable; ACR may be asymptomatic, or associated with fever or abdominal pain. Laboratory findings include elevation or failure of normalization of serum transaminases, concurrent with a rising alkaline phosphatase and/or bilirubin. The diagnosis of acute liver graft rejection is confirmed by liver biopsy and examination of liver histology.40 Conventional histologic criteria associated with ACR, now standardized in a numerical score called the BANFF criteria, include the presence of periportal lymphocytic infiltrate, as well as bile duct and vascular endothelial injury. The contribution of each component of rejection, i.e., degree of periportal infiltrate, bile duct injury, and endotheliitis, is tabulated and the degree of rejection, i.e., mild, moderate, or severe, is determined. Most cases of ACR respond to treatment with intravenous glucocorticoids. Approximately 10% of patients with ACR will not improve with intravenous glucocorticoids, requiring the administration of monoclonal or polyclonal anti-T cell antibody therapy. Mild and moderate ACR may also respond to either increasing the dose of the primary immunosuppressive agent, or switching to an alternate CNI. This approach has been used with increasing frequency, particularly in patients transplanted for hepatitis C virus (HCV) and hepatitis B virus (HBV) due to concerns regarding the adverse effects of overimmunosuppression on viral recurrence. It has been well established that HCV-induced graft failure, progression to advanced histologic injury, and cholestatic hepatitis occur more frequently in HCV-infected individuals who receive high-dose intravenous glucocorticoids and antilymphocyte preparations.41 Therefore, modulating immunosuppression in the setting of rejection by either increasing the dose or substituting a CNI, and/or reintroduction of mycophenolic acid may be preferred in the setting rather than the use of bolus glucocorticoids and/or anti-T-cell antibodies.
bidity and mortality in the immediate postoperative period. Both bacterial and fungal infection may be observed. Common sources of bacterial infections include the lungs, urogenital system, and the surgical wound. Unfortunately, infection with multidrug-resistant Gram-positive and Gram-negative organisms has proliferated at many transplant centers, likely coincident with the overall rise in severity of medical illness at the time of transplant, and the overuse of antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) are the most prevalent of these organisms, and may be associated with a prolonged intensive care unit course and hospital stay, as well as increased morbidity and mortality.42,43 In addition, the incidence of fungal infection is increasing, and appears to be more common in patients with advanced liver disease who have been treated with repeated courses of antibiotics prior to transplantation.44 As a mechanism to prevent post-transplant infection, many transplant centers have adopted a policy of instituting broad-spectrum antibiotics during the first several days to weeks following liver transplantation. Additionally, a short course of antifungal prophylaxis may be instituted, particularly in patients who have received multiple courses of antibiotics and/or perioperative transfusions, those requiring reoperation in the early period following transplantation, and in patients with renal failure.
Biliary Complications Bile leaks and strictures generally occur at the anastomosis of the donor and recipient bile ducts, recognized by a rise in serum bilirubin and/or alkaline phosphatase or by the presence of bile in surgical drains. The incidence of biliary complications is between 5 and 15% following DD liver transplantation. However, between 15 and 30% of patients who undergo living donor liver transplantation develop biliary complications, due to the number and complexity of the biliary anastamoses required, and due to bile leaks from small biliary radicles from the cut surface of the partial liver graft.45 In both deceased and living donor recipients, the majority of bile leaks resolve spontaneously. As the biliary tree receives the vast majority of its blood supply from the hepatic artery, the adequacy of hepatic artery blood flow needs to be evaluated in the setting of any biliary injury. If spontaneous resolution of the bile leak does not occur, endoscopic or radiologic placement of a biliary stent across the biliary anastamoses is often successful.36,46 In some cases surgical exploration and revision of the biliary anastamoses to a Roux en Y choledochojejunostomy may be required. Anastomotic biliary strictures require careful attention, as, if left untreated, cholangitis, graft dysfunction, and eventually secondary biliary cirrhosis may occur. Techniques for management include dilatation and stenting via biliary endoscopy or percutaneous transhepatic cholangiogram by an interventional radiologist. If these modalities are unsuccessful, surgical revision of the biliary anastamosis, achieved by the performance of a Roux en Y choledochojejunostomy in patients with a choledochocholodochostomy, may be required. In some cases, retransplantation may be necessary.
Cytomegalovirus (CMV) Infection Bacterial and Fungal Infections Infection is the most common complication following liver transplantation and is responsible for the majority of the short-term mor-
CMV infection after liver transplantation remains a significant cause of morbidity, and usually occurs within the first several weeks to months following transplantation. The incidence of CMV infection
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after liver transplantation ranges from 25% to 85%, although the vast majority of CMV infections remain asymptomatic. However, up to 5–15% of patients develop symptomatic CMV infection associated with fever, leukopenia, or thrombocytopenia, and a minority develops tissue-invasive disease. Tissue-invasive disease after orthotopic liver transplantation most frequently affects the liver graft, although pneumonitis, myocarditis, gastritis, and colitis have been reported.47 CMV infection and, especially, CMV hepatitis may have a role in enhancing chronic allograft rejection, although this remains controversial. In addition, CMV infection post liver transplantation has been implicated as a factor that adversely affects graft survival in HCV-infected patients.48 Risk for CMV infection post liver transplantation relates primarily to previous CMV exposure in both recipient and donor, with the lowest rates occurring when both donor and recipient are CMV IgG antibody-negative, while the highest rates occur when the donor is positive and recipient negative. Another important risk factor for acute CMV infection includes overimmunosuppression, as occurs during the treatment of rejection, particularly if anti-T-cell agents such as OKT3 or antithymocyte globulin are used. Thus, anti-CMV therapy is often initiated concurrently when treating ACR with these agents. The availability of intravenous as well as oral ganciclovir and the more bioavailable oral formulation (valganciclovir) for the prevention of CMV disease has dramatically changed the epidemiology and outcomes of CMV infection. In addition, newer diagnostic tests, including assays for CMV antigenemia and quantification of CMV DNA by polymerase chain reaction (PCR), have markedly improved the ability to diagnose CMV infection rapidly and accurately. All liver transplant programs provide prophylaxis to at least those patients at highest risk for developing active CMV infection. However, the precise treatment, duration of therapy, and patient groups offered extended prophylaxis vary considerably between transplant centers. In general, intravenous ganciclovir is administered for the first 7 days post-transplant, and is converted to oral ganciclovir or valganciclovir, either when the patient is able to take oral medications or after a predefined length of time. The duration of oral ganciclovir use also varies considerably between centers, but typically lasts several weeks to months. In the setting of invasive CMV disease, intravenous ganciclovir is initiated and continued for several weeks. Though it has been used by some centers and provides drug levels similar to intravenous administration, oral valganciclovir has not been approved for treatment of CMV. The risk of CMV relapse is reduced if treatment is continued until CMV-DNA can no longer be detected by a PCR assay.49 CMV-immunoglobulin is generally reserved for those patients who develop active CMV disease despite prophylaxis, or fail to respond to intravenous ganciclovir. Given the sensitivity of the current antigen and PCR techniques to detect viremia, several studies have advocated routine surveillance for CMV post-transplantation and pre-emptive treatment if and when the virus is detected.50
COMPLICATIONS BEYOND 2 MONTHS Improvements in the surgical techniques required to perform transplantation, the treatment of postoperative complications, and prevention of rejection have been associated with significant improvements in short-term morbidity and mortality following
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transplantation. Coincident with improvements in short-term outcomes has been a rise in long-term complications.
Cardiovascular Disease In general, patients with advanced cardiovascular disease, including atherosclerotic heart disease (ASHD), significant peripheral vascular disease, and poorly controlled hypertension, have been excluded from liver transplantation due to concerns regarding increased perioperative morbidity and mortality. Unfortunately, in patients without cardiovascular disease pretransplant, many of the risk factors for coronary artery disease will develop post liver transplantation and require long-term management to prevent complications.
Hypertension Hypertension is common following liver transplantation, and may be associated with multiple factors, including the direct effects of CNIs, renal insufficiency, and obesity. Pre-existing hypertension may be masked preoperatively due to low systemic vascular resistance associated with the hemodynamic milieu of cirrhosis, thus protecting the patient from developing elevated blood pressure. The incidence of post-transplant arterial hypertension in liver graft recipients ranges from 65% to 85% since CYA has been introduced as an immunosuppressant agent. Various mechanisms have been proposed to explain the hypertensive effect of CYA, including vasoconstriction of afferent renal arterioles with impairment of glomerular filtration rate and sodium excretion, increased sympathetic nervous system activation altering the renin–angiotensin system, increasing intracellular calcium concentration, or synthesis and release of endothelin-1.51 Although TAC may be associated with hypertension post liver transplantation, the effect may be less when compared to CYA.52 Unfortunately, as in non-transplanted patients, untreated hypertension may be associated with multiple complications, including end-organ disease and renal failure. The treatment of hypertension post liver transplantation proceeds in protocolized, stepwise fashion, including limiting salt intake, the use of calcium-channel blockers, and the addition or substitution of a beta-blocker or angiotensinconverting enzyme inhibitor in difficult-to-control patients. The dihyropyridine class of calcium-channel blockers, including nifedipine, israpidine, amlodipine, felodipine, and nicardipine, are preferred as first-line agents as they are long-acting, minimally interact with CNIs, and have limited side effects. Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers have also been utilized, although they may lead to hyperkalemia and renal insufficiency in liver transplant recipients and should be used with caution.
Hyperlipidemia Both increased cholesterol and triglyceride levels occur commonly post liver transplantation. It has been reported that 16–43% of patients have elevated serum cholesterol levels, and 40% have increased triglycerides. Risk factors for hyperlipidemia include the effects of commonly used pharmacologic agents after transplantation, such as CNIs, and intravenous and oral glucocorticoids, as well as RAPA. Many investigators have discussed the potential benefit of altering CNIs after liver transplantation in patients with hyperlipidemia; TAC has been reported by some to be associated with a
Chapter 51 POST LIVER TRANSPLANTATION MANAGEMENT AND COMPLICATIONS
decreased incidence of cholesterol and lipid abnormalities,53 while tapering glucocorticoid dose may also be of benefit. In addition, the use of statin cholesterol-lowering agents has been found to be safe and effective in decreasing serum total and low-density lipoprotein cholesterol, without changing CNI levels.54 Another potential, although as yet unproven, benefit of statin therapy following liver transplantation may be its effect on rejection. It has been observed that statins exhibit anti-inflammatory effects, modulate endothelial function, and may repress the induction of major histocompatibility complex class II complexes. Pravastatin has been shown to reduce acute rejection after cardiac and renal transplantation as well as reduce natural killer cell cytotoxicity.55 The short- and long-term effects of statins in the prevention of post-transplantation rejection are being evaluated in prospective clinical trials.
Diabetes The incidence of diabetes post liver transplantation ranges from 13 to 25%.56 Type 1 diabetes prior to or developing post liver transplantation is associated with a poor prognosis. A recent review of the UNOS database indicated that cryptogenic cirrhosis, hypertension, and coronary artery disease were significantly more common following liver transplantation in patients with type 1 and 2 diabetes compared to non-diabetics. Five-year patient and graft survivals by Kaplan–Meier analysis were significantly lower for type 1 compared with type 2 or non-diabetics.57 Despite these grim statistics, a small subset of patients with insulin-requiring diabetes pretransplant may lose their insulin requirement, likely due to a restoration of insulin sensitivity coincident with liver transplantation and loss of portal hypertension.58 Post-transplantation diabetes is multifactorial and may be related to immunosuppression, including the use of CNIs and glucocorticoids, treatment of rejection, and increased caloric intake concomitant with an enhanced feeling of well-being. Treatment of diabetes post-transplantation includes standard medical and nutritional therapy, including limiting caloric intake, an appropriate diet with weight loss, and agents that induce hypoglycemia, including oral hypoglycemics and/or insulin as necessary. Data is emerging which suggests that choice of immunosuppression may affect the incidence of diabetes. Patients treated with TAC appear to develop diabetes at a greater rate than those treated with CYA preparations, even when controlling for dose, exposure, and glucocorticoid use.59,60
Obesity The incidence of obesity, defined as a body mass index greater than 30 kg/m2, following liver transplantation has been reported to be as high as 30%.61 Various factors, including choice of immunosuppression, pretransplant obesity, and failure to return to work and healthy pretransplant activities following transplantation have been considered causative. The use of intravenous as well as prolonged oral glucocorticoid therapy contributes significantly to post-transplantation obesity. Despite the increasing incidence of obesity both pre- and post-transplantation, long-term outcomes in obese patients appear to be stratified by the degree of obesity. A recent review of the UNOS database found that, after adjusting for confounding variables, patient and graft survival at 1 month and 1, 2, and 5 years postoperatively were not significantly different when comparing obese to non-obese patients.62 However, when post-transplant sur-
vival in patients with morbid obesity was analyzed using the same database, primary graft non-function, as well as immediate, 1- and 2-year mortality was significantly higher in the morbidly obese group, and 5-year mortality was significantly higher in both severely and morbidly obese patients, mainly due to adverse cardiovascular events.63 Thus, morbid obesity appears to be an independent predictor of mortality. Many transplant centers either exclude patients with morbid obesity from consideration for transplantation or encourage weight loss prior to transplantation. Treatment of obesity post liver transplantation is unfortunately difficult: many centers adopt a policy of minimizing and/or rapidly tapering glucocorticoids as appropriate, and encouraging exercise and limiting caloric intake, with unfortunately limited success. Several investigators have studied the effect of changing immunosuppression (substituting TAC for CYA preparations) as there is evidence that rates of obesity post-transplantation are greater in patients treated with CYA preparations compared to TAC,60 even when controlling for effect of glucocorticoid dose.
Cardiac Disease As the incidence of obesity, diabetes, hypertension, and hypercholesterolemia is increased following liver transplantation, it is not surprising that ASHD post liver transplantation occurs commonly. Indeed, when excluding recurrent disease, graft loss due to technical complications, and de-novo malignancy, ASHD represents the most common cause of death post liver transplantation. Liver allograft recipients have a greater risk of cardiovascular deaths and ischemic events than an age- and sex-matched population of nontransplanted patients.64 Thus, as ASHD contributes significantly to post-transplantation morbidity and mortality, appropriately identifying and treating patients with ASHD before liver transplantation is vital. Patients with documented or major risk factors for ASHD should undergo extensive pretransplantation evaluation which includes a cardiac stress test and/or cardiac catheterization. Patients with advanced heart disease not amenable to interventional therapy or with poor cardiac function should be deemed non-candidates for liver transplantation. Aggressive therapies, including pharmacologic agents, and, if necessary, angiographic or surgical correction of cardiac vasculature, should be offered to appropriately selected patients with mild or moderate ASHD. An important issue that must be kept in mind when evaluating any patient prior to liver transplantation is the unique hemodynamic profile associated with portal hypertension. First described nearly half a century ago, the resultant increased cardiac output and reduced systemic vascular resistance may reduce afterload, and mask left ventricular dysfunction. Following liver transplantation, normalization of systemic vascular resistance may not be tolerated by a dysfunctional left ventricle.65 Thus, in addition to careful evaluation of ASHD, a complete evaluation of left and right ventricular function must occur as part of an appropriate pretransplantation cardiac assessment.
Renal Dysfunction A significant source of morbidity and mortality following liver transplantation is renal failure. Renal impairment has been well described in the immediate perioperative period due to hepatorenal syndrome, hypovolemia, and acute tubular necrosis. Although the majority of
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patients recover, up to 25% of patients develop significant renal dysfunction, defined as a glomerular filtration rate of < 40 ml/min by 5 years after transplantation, and approximately 10% develop endstage renal disease and dialysis dependence by 10 years after transplantation.66 Factors associated with the development of end-stage renal disease following liver transplantation include a higher preoperative serum creatinine level, hepatorenal syndrome, requirement for dialysis in the first 3 months postoperatively, and elevated serum creatinine at 1 year post-transplantation.67 Therapy of renal insufficiency following liver transplantation includes a thorough search for reversible factors, including hypovolemia and renal vascular injury, minimizing nephrotoxic medications such an aminoglycosides, non-steroidal anti-inflammatory medications, trimethoprim/sulfamethoxazole, controlling diabetes and hypertension, and appropriately adjusting CNI levels. It has been well established that CNIs such as CYA and TAC are associated with nephrotoxicity via multiple mechanisms, and are classified as acute or chronic: acute CNI nephrotoxicity occurs in the first weeks after transplantation, is associated with impaired renal blood flow, and is usually reversible with CNI dose reduction. In contrast, chronic CNI nephrotoxicity observed several months after transplantation is associated with parenchymal renal injury, and may occur even in the setting of appropriate CNI blood levels, and unfortunately, lead to permanent renal dysfunction. An alternative approach to diminishing CNI exposure is withdrawal and substitution of an alternative immunosuppresive agent. The effect of withdrawal of CNIs and substitution of MMF has been evaluated in prospective trials. This technique has resulted in a significant decrease in the mean values of serum creatinine 6, 12, and 18 months after beginning therapy with MMF, with no significant effect on graft function or rejection. Using this approach, the withdrawal of CNI may be achieved in the majority of patients by 12–18 months post-transplantation.68 It is important to remember however that immunosuppression with MMF monotherapy may be associated with a small but appreciable increased risk of ACR. Finally, conversion to RAPA, an immunosuppressant agent with minimal nephrotoxicity, has been associated with improvement in renal function in patients with CNI-associated renal insufficiency.32
Chronic Rejection Chronic allograft rejection, also termed vanishing bile duct syndrome, occurs rarely after liver transplantation. Diagnostic criteria for chronic rejection include bile duct atrophy affecting the majority of bile ducts, with or without bile duct loss, and obliterative arteriopathy and/or venopathy in large branches of the hepatic artery or portal vein.69 Risk factors for chronic liver rejection include transplantation for primary sclerosing cholangitis, primary biliary cirrhosis, human leukocyte antigen mismatch between donor and recipient, and CMV infection. Chronic rejection is a harbinger of poor outcomes; altering immunosuppression is rarely associated with improvement, often resulting in the requirement for retransplantation.
foreign, and avoidance of infectious complications associated with overimmunosuppression. Most liver transplant recipients are able to diminish rapidly the doses of immunosuppressants, to the extent that the majority of patients are on a single agent, usually a CNI, by 6–12 months post-transplant. Despite rapid tapering of immunosuppression, infectious complications continue to be observed.
FUNGAL INFECTIONS Invasive fungal infection, particularly with aspergillosis species has long been recognized as one of the most significant opportunistic infections in liver transplant recipients. Although uncommon, occurring in 1–6% of patients, the mortality rate for patients with invasive disease exceeds 90%. Most patients present with either pulmonary or central nervous system infections (such as brain abscess). The majority of Aspergillus infections occur within the first month after transplantation, and the incidence is correlated with the degree of illness of the recipient; the overwhelming majority of liver transplant recipients with invasive aspergillosis had evidence of significant hepatic and/or renal dysfunction prior to transplantation, particularly if they were dialysis-dependent. In addition, up to onefourth of all cases of invasive aspergillosis have occurred after retransplantation. Other factors associated with a greater risk of aspergillosis include concomitant CMV infection, treatment with OKT3, and neutropenia. Despite the historically significant death rate associated with aspergillosis infection, recent improvements in both recognition and treatment of infection have been associated with diminished death rates when compared to historical controls. A recently published manuscript indicates that Aspergillus infections in liver transplant recipients are occurring later in the posttransplantation period, with mortality rates decreasing from 90 to 60%.70
CRYPTOCOCCUS Although cryptococcal meningitis occurs rarely in a non-immunosuppressed patient population, organ transplantation remains one of the major factors for cryptococcosis in non-HIV-infected patients. Cryptoccoccal infection usually presents with central nervous system symptoms associated with meningeal irritation greater than 6 months post orthotopic liver transplantation, although cutaneous lesions in the absence of meningeal signs may occur. Decreased cerebrospinal fluid glucose as well as elevated protein and white blood cell count help to establish the diagnosis, while a positive India ink stain and cryptococcal antigen in cerebrospinal fluid and serum confirms the diagnosis. Treatment of cryptococcal infection usually requires a combination of amphotericin B with or without 5-flucytosine, followed by suppressive therapy with oral fluconazole for up to 6 months. Rarely, intrathecal administration of amphotericin B is required, usually in the setting of persistent symptoms of meningeal irritation, and elevated cryptococcal antigen titers in cerebrospinal fluid despite systemic antifungal therapy.71
CYTOMEGALOVIRUS INFECTION
INFECTIOUS COMPLICATIONS Ensuring long-term patient and graft survival requires an appropriate balance of suppression of immune recognition of the graft as
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Although the majority of CMV infections occur within the first several months post-transplantation, patients continue to remain at increased risk for CMV infection. The greatest risk factor associated with CMV infection includes overimmunosuppression. As in the
Chapter 51 POST LIVER TRANSPLANTATION MANAGEMENT AND COMPLICATIONS
immediate pretransplant period, this most commonly occurs when treating acute allograft rejection. The management of CMV infection and/or disease in the months to years following transplantation is similar to when CMV occurs in the short term postoperatively and is described in detail above.
POST-TRANSPLANT LYMPHOPROLIFERATIVE DISEASE Epstein–Barr viral (EBV) infection is rarely associated with clinical disease in patients who were previously exposed to EBV before liver transplantation. However, EBV-associated syndromes, including post-transplant lymphoproliferative disease (PTLD), are particularly important in children who experience a primary EBV infection after transplantation, and in adults who develop EBV reactivation. In general, clinical disease develops in the setting of impaired production of EBV-specific cytotoxic lymphocytes due to immunosuppression, with subsequent B-lymphocyte transformation and clonal expansion. Variants of PTLD exist where T cells may also be transformed. Clinical presentation is varied, ranging from a mononucleosis-like syndrome, localized lymphadenopathy, or systemic disease with multiorgan involvement. Therefore, unexplained fever, weight loss, lymphadenopathy, graft dysfunction, neurologic symptoms, diarrhea, or pulmonary symptoms following liver transplantation require a thorough investigation for PTLD.72 Evaluation of possible PTLD requires a biopsy of affected tissue and/or lymph nodes with assessment of cell type, gene rearrangement studies to determine B- versus T-cell origin, and a determination if the involved cell lines are monoclonal or polyclonal, as polyclonal disease has been reported to be more responsive to therapy. Treatment of PTLD includes the withdrawal of all immunosuppression (except low-dose prednisone), and in cases where significant lymphadenopathy or organ involvement is present, a multifaceted treatment approach, including antiviral agents (ganciclovir or aciclovir), anti-B-cell agents (anti-CD20 rituximab) and systemic chemotherapy in refractory cases.73 As primary infection with EBV is associated with PTLD, many pediatric transplant programs have initiated a surveillance program post-transplantation whereby EBV DNA is quantified, and immunosuppression is adjusted and/or EBV-directed antiviral therapy is initiated if titers increase.74
MALIGNANCY As long-term survival rates in patients who undergo liver transplantation have improved, a concomitant increase in late complications including malignancy has been noted. These may be de-novo neoplasia, or a recurrence of cancers detected prior to liver transplantation.
HEPATOCELLULAR CARCINOMA Early experience with liver transplantation in the setting of hepatocellular carcinoma (HCC) was associated with a recurrence rate of up to 80% and dismal long-term survival. This resulted in HCC being considered a contraindication to liver transplantation. However, based on work by Mazzaferro and others, selected patients with limited HCC (one solitary lesion < 5 cm or three lesions each
< 3 cm or or T1–2N0M0) were found to have excellent long-term outcomes, with a 5-year survival rate of 70% and a recurrence rate below 15%.75 These criteria are adopted by many transplantation programs to determine candidacy for transplantation in patients with HCC. As liver transplantation in patients with HCC is hampered by both a shortage of donor organs and increased waiting time, many transplant centers have adopted a strategy of considering living donor liver transplantation for these patients, and/or treating patients with ablative therapies such as hepatic artery chemoembolization or radiofrequency ablation prior to transplantation in an attempt to diminish tumor progression. In addition, the current organ allocation system using the MELD score has been modified to grant added priority to patients with HCC who meet Mazzaferro criteria. Unfortunately, HCC recurrence can occur post-transplantation, particularly in patients with large tumors, macrovascular invasion, and evidence of extrahepatic disease at the time of transplantation.
CUTANEOUS MALIGNANCY Skin cancer is the most common malignancy occurring after solid organ transplantation, with an incidence as high as 35–70% in areas of the world where sun exposure is common.76 The incidence of skin cancer after liver transplantation has been reported at 1.6–2.2%, but may be underreported as patients may be lost to follow-up to the transplant center and treated by local physicians. A recent study evaluated the incidence of cutaneous malignancies in 151 liver transplant recipients; 86 documented skin cancers were found in 34 patients, with the majority being squamous cell, followed by basal cell and melanomas. Predictors of malignancy included male gender, red hair, brown eyes, diagnosis of primary sclerosing cholangitis, and use of CYA.77 Thus, many transplant centers recommend an annual dermatologic evaluation in all patients who have undergone liver transplantation.
OTHER MALIGNANCIES An increased incidence of de-novo non-lymphoid malignancies has been shown in immunocompromised patients. To determine the incidence of malignancy in a post liver transplantation population, investigators from King’s College Hospital (London, UK) analyzed all patients who underwent liver transplantation between January 1988 and December 1999. Factors potentially related to risk of malignancy that were evaluated included etiology of liver disease, choice of immunosuppression, and number of rejection episodes. Of 1140 patients undergoing 1271 liver transplantations, 30 patients (2.6%) developed de-novo non-lymphoid malignancy after transplantation. As anticipated, skin cancer was the most common malignancy, followed by oropharyngeal carcinoma, bladder carcinoma, acute leukemia, breast carcinoma, and various other malignancies (sarcoma, seminoma, small-bowel, colon, renal, pancreas). The mean time of presentation of the malignancy after transplantation was 45.1 months (range, 6–133 months), and mean age at diagnosis was 55 years (range, 34–71 years). Interestingly, 1-, 3-, and 5-year survival was not different in patients with and without malignancy. Of note, the authors found that the incidence of de-novo malignancy was significantly greater in patients who underwent transplantation for alcoholic liver disease compared with other causes of liver disease.78
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Various investigators have also noted the more rapid progression of diverse premalignant conditions following liver transplantation. A greater incidence of colonic adenomas,79 progression of Barrett’s esophagus to high-grade dysplasia,80 and colonic dysplasia, particularly in patients with pre-existing inflammatory bowel disease,81 have been reported. Therefore, many transplant centers have adopted a policy of performing annual screening for malignancies, including skin exam, mammogram, Pap smear or prostate-specific antigen in the appropriate gender, and recommending colonoscopy every 3 years or more frequently if clinically indicated.
RECURRENCE OF PRIMARY DISEASE FOLLOWING LIVER TRANSPLANTATION A major challenge to the liver transplant community is recurrence of the primary disease which caused the patient’s liver to fail. Diseases that do not recur following liver transplantation include congenital anatomic anomalies (biliary atresia, polycystic liver disease, Caroli’s disease, Alagille’s syndrome, congenital hepatic fibrosis) and metabolic diseases (Wilson’s disease, a1-antitrypsin deficiency: Chapter 53). However, all other causes of liver disease, including primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune hepatitis, non-alcoholic fatty liver disease, hemochromatosis, and alcohol-associated liver disease, have been reported to recur after liver transplantation. In some cases this may lead to liver injury and graft failure.82–86 Disorders most commonly associated with recurrence include HBV and HCV (Chapter 52). Fortunately, recurrence of HBV after liver transplantation can be prevented by administering hepatitis B immune globulin at the time of transplantation and at regular intervals thereafter, with or without the use of antiviral agents such as lamivudine and adefovir.87 Unfortunately, HCV recurrence following liver transplantation represents a significant source of morbidity and mortality. In patients with active HCV replication prior to transplantation, reacquisition of viremia following transplantation is universal, and allograft hepatitis due to HCV occurs in up to 90% of patients followed for 5 years.41 Although histological injury in the allograft due to HCV is exceedingly common, disease progression after the development of hepatitis is variable, with some patients experiencing indolent disease and others rapidly progressing to cirrhosis and liver failure. In patents who develop HCV-associated cirrhosis post-transplantation, rapid decompensation is a common occurrence. It has been reported that up to 42% of patients with HCV-associated cirrhosis post-transplantation develop decompensation, manifested as ascites, encephalopathy, or hepatic hydrothorax, and less than 50% of patients survive more than 1 year after the development of decompensation.88 Thus, both prospective and retrospective data have established that the progression of HCV following orthotopic liver transplantation is accelerated when compared to non-transplanted patients. Several lines of anecdotal evidence suggest that HCV recurrence might be more severe in recipients of LDLT when compared to DD recipients. However, recent reports indicate that the overall incidence and time to HCV recurrence are not different when comparing DD with LDLT, and that severe sequelae of HCV recurrence – cholestatic hepatitis, grade III–IV inflammation, and/or HCVinduced graft failure requiring retransplantation – were also not different when comparing DD to LDLT recipients.89
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At present, both the optimal timing and therapy for the treatment of recurrent HCV following liver transplantation remain inadequately described. Theoretically, eradication of HCV prior to liver transplantation in patients with decompensated liver disease would be beneficial, although, in practice, this strategy has been associated with exacerbation of encephalopathy, infections, and other serious adverse events, particularly in patients treated with high-dose interferon and ribavirin.90 However, initial therapy with low-dose interferon and ribavirin with slow escalation in dose may be associated with improved tolerability and efficacy.91 Following liver transplantation, pre-emptive therapy prior to the development of histological injury or directed therapy after injury occurs have been attempted, with varying degrees of success. It is important to note however that post-transplantation, toxicities of interferon preparations with ribavirin are greater than for non-immunosuppressed patients and responses are lower (Chapter 50). Significant leukopenia and anemia are common and multifactorial, likely due to drug-induced bone marrow suppression and renal insufficiency potentiating ribavirininduced hemolysis.92
SUMMARY Liver transplantation is the treatment of choice for appropriately selected patients with end-stage liver disease. Advances in the past decade have greatly improved outcomes and enhanced survival rates. These advances include improvement in surgical technique and immunosuppression, appropriate selection of donors and recipients, and improvement in therapies to prevent and treat postoperative complications. Successful liver transplantation has led to the emergence of complications associated with patient longevity, including non-hepatic disease, complications of immunosuppression, infections, neoplasia, and recurrence of the primary disease for which the liver transplantation was indicated. It is thus clear that further advancements in patient and graft survival will be associated with enhanced recognition and treatment of long-term complications, particularly the effect of disease recurrence, and cardiovascular and renal complications.
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31. Trotter JF. Sirolimus in liver transplantation. Transplant Proc 2003; 35:193–200. 32. Nair S, Eason J, Loss G. Sirolimus monotherapy in nephrotoxicity due to calcineurin inhibitors in liver transplant recipients. Liver Transpl 2003; 9:126–129. 33. Schemmer P, Mehrabi A, Kraus T, et al. New aspects on reperfusion injury to liver – impact of organ harvest. Nephrol Dial Transplant 2004; 19:26–35. 34. Sieders E, Peeters PM, TenVergert EM, et al. Early vascular complications after pediatric liver transplantation. Liver Transpl 2000; 6:326–332. 35. Bhattacharjya S, Gunson BK, Mirza DF, et al. Delayed hepatic artery thrombosis in adult orthotopic liver transplantation – a 12-year experience. Transplantation 2001; 71:1592–1596. 36. Denys A, Chevallier P, Doenz F, et al. Interventional radiology in the management of complications after liver transplantation. Eur Radiol 2004; 14:431–439. 37. Vignali C, Cioni R, Petruzzi P, et al. Role of interventional radiology in the management of vascular complications after liver transplantation. Transplant Proc 2004; 36:552–554. 38. Selzner N, Rudiger H, Graf R, Clavien PA. Protective strategies against ischemic injury of the liver. Gastroenterology 2003; 125:917–936. 39. Clavien PA, Yadav S, Sindram D, Bentley RC. Protective effects of ischemic preconditioning for liver resection performed under inflow occlusion in humans. Ann Surg 2000; 232:155– 162. 40. Lefkowitch JH. Diagnostic issues in liver transplantation pathology. Clin Liver Dis 2002; 6:555–570. 41. Berenguer M. Natural history of recurrent hepatitis C. Liver Transpl 2002; 8:S14–S18. 42. Desai D, Desai N, Nightingale P, et al. Carriage of methicillinresistant Staphylococcus aureus is associated with an increased risk of infection after liver transplantation. Liver Transpl 2003; 9:754–759. 43. Singh N, Gayowski T, Rihs JD, et al. Evolving trends in multipleantibiotic-resistant bacteria in liver transplant recipients: a longitudinal study of antimicrobial susceptibility patterns. Liver Transpl 2001; 7:22–26. 44. Singh N. Fungal infections in the recipients of solid organ transplantation. Infect Dis Clin North Am 2003; 17:113–134. 45. Fondevila C, Ghobrial RM, Fuster J, et al. Biliary complications after adult living donor liver transplantation. Transplant Proc 2003; 35:1902–1903. 46. Gopal DV, Pfau PR, Lucey MR. Endoscopic management of biliary complications after orthotopic liver transplantation. Curr Treat Options Gastroenterol 2003; 6:509–515. 47. van der Bij W, Speich R. Management of cytomegalovirus infection and disease after solid-organ transplantation. Clin Infect Dis 2001; 33:S32–S37. 48. Razonable RR, Burak KW, van Cruijsen H, et al. The pathogenesis of hepatitis C virus is influenced by cytomegalovirus. Clin Infect Dis 2002; 35:974–981. 49. Sia IG, Wilson JA, Groettum CM, et al. Cytomegalovirus (CMV) DNA load predicts relapsing CMV infection after solid organ transplantation. J Infect Dis 2000; 181:717–720. 50. Norris S, Kosar Y, Donaldson N, et al. Cytomegalovirus infection after liver transplantation: viral load as a guide to treating clinical infection. Transplantation 2002; 74:527–531. 51. Textor SC, Taler SJ, Canznello VJ, et al. Post transplantation hypertension related to calcineurin inhibitors. Liver Transpl 2000; 6:521–530. 52. Risaliti A, Baccarani U, Vianello V, et al. Cardiovascular and metabolic complications after liver transplantation: Neoral- versus tacrolimus-based immunosuppression. Transplant Proc 2001; 33:3684–3685. 53. Charco R, Bilbao I, Chavez R, et al. Low incidence of hypercholesterolemia among liver transplant patients under
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tacrolimus monotherapy immunosuppression. Transplant Proc 2002; 34:1555–1556. Neal DA, Alexander GJ. Can the potential benefits of statins in general medical practice be extrapolated to liver transplantation? Liver Transpl 2001; 7:1009–1014. Mehra MR, Uber PA, Vivekananthan K, et al. Comparative beneficial effects of simvastatin and pravastatin on cardiac allograft rejection and survival. J Am Coll Cardiol 2002; 40:1609–1614. Sheiner PA, Magliocca JF, Bodian CA, et al. Long-term medical complications in patients surviving > 5 years after liver transplant. Transplantation 2000; 69:781–789. Yoo HY, Thuluvath PJ. The effect of insulin-dependent diabetes mellitus on outcome of liver transplantation. Transplantation 2002; 74:1007–1012. Stockmann M, Steinmuller T, Nolting S, Neuhaus P. Posttransplant diabetes mellitus after orthotopic liver transplantation. Transplant Proc 2002; 34:1571–1572. Risaliti A, Baccarani U, Vianello V, et al. Cardiovascular and metabolic complications after liver transplantation: Neoral- versus tacrolimus-based immunosuppression. Transplant Proc 2001; 33:3684–3685. Varo E, Padin E, Otero E, et al. Cardiovascular risk factors in liver allograft recipients: relationship with immunosuppressive therapy. Transplant Proc 2002; 34:1553–1554. Reuben A. Long-term management of the liver transplant patient: diabetes, hyperlipidemia, and obesity Liver Transpl 2001; 7:S13–S21. Yoo HY, Molmenti E, Thuluvath PJ. The effect of donor body mass index on primary graft nonfunction, retransplantation rate, and early graft and patient survival after liver transplantation. Liver Transpl 2003; 9:72–78. Nair S, Verma S, Thuluvath PJ. Obesity and its effect on survival in patients undergoing orthotopic liver transplantation in the United States. Hepatology 2002; 35:105–109. Johnston SD, Morris JK, Cramb R, et al. Cardiovascular morbidity and mortality after orthotopic liver transplantation. Transplantation 2002; 73:901–906. Moller S, Henriksen JH. Cirrhotic cardiomyopathy: a pathophysiological review of circulatory dysfunction in liver disease. Heart 2002; 87:9–15. Cohen AJ, Stegall MD, Rosen CB, et al. Chronic renal dysfunction late after liver transplantation. Liver Transpl 2002; 8:916–921. Fisher NC, Nightingale PG, Gunson BK, et al. Chronic renal failure following liver transplantation. Transplantation 1998; 66:59–66. Moreno JM, Rubio E, Pons F, et al. Usefulness of mycophenolate mofetil in patients with chronic renal insufficiency after liver transplantation. Transplant Proc 2003; 35:715–717. Demetris A, Adams D, Bellamy C, et al. Update of the international Banff schema for liver allograft rejection: working recommendations for the histopathologic staging and reporting of chronic rejection. Hepatology 2000; 31:792–799. Singh N, Avery RK, Munoz P, et al. Trends in risk profiles for and mortality associated with invasive aspergillosis among liver transplant recipients. Clin Infect Dis 2003; 36:46–52. Wu G, Vilchez RA, Eidelman B, et al. Cryptococcal meningitis: an analysis among 5521 consecutive organ transplant recipients. Transpl Infect Dis 2002; 4:183–188. Holmes RD, Sokol RJ. Epstein–Barr virus and post-transplant lymphoproliferative disease. Pediatr Transplant 2002; 6:456–464. Bueno J, Ramil C, Somoza I, et al. Treatment of monomorphic B-cell lymphoma with rituximab after liver transplantation in a child. Pediatr Transplant 2003; 7:153–156.
74. Smets F, Sokal EM. Epstein–Barr virus-related lymphoproliferation in children after liver transplant: role of immunity, diagnosis, and management. Pediatr Transplant 2002; 6:280–287. 75. Mazzaferro V, Regalia E, Doci R, et al. Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis. N Engl J Med 1996; 334:693–699. 76. Valero JM, Rubio E, Moreno JM, et al. De novo malignancies in liver transplantation. Transplant Proc 2003; 35:709–711. 77. Mithoefer AB, Supran S, Freeman RB. Risk factors associated with the development of skin cancer after liver transplantation. Liver Transpl 2002; 8:939–944. 78. Saigal S, Norris S, Muiesan P, et al. Evidence of differential risk for posttransplantation malignancy based on pretransplantation cause in patients undergoing liver transplantation. Liver Transpl 2002; 8:482–487. 79. Atassi T, Thuluvath PJ. Risk of colorectal adenoma in liver transplant recipients compared to immunocompetent control population undergoing routine screening colonoscopy. J Clin Gastroenterol 2003; 37:72–73. 80. Trotter JF, Brazer SR. Rapid progression to high-grade dysplasia in Barrett’s esophagus after liver transplantation. Liver Transpl Surg 1999; 5:332–333. 81. Loftus EV Jr, Aguilar HI, Sandborn WJ, et al. Risk of colorectal neoplasia in patients with primary sclerosing cholangitis and ulcerative colitis following orthotopic liver transplantation. Hepatology 1998; 27:685–690. 82. Neuberger J. Recurrent primary biliary cirrhosis. Best Pract Res Clin Gastroenterol 2000; 14:669–680. 83. Wiesner RH. Liver transplantation for primary sclerosing cholangitis: timing, outcome, impact of inflammatory bowel disease and recurrence of disease. Best Pract Res Clin Gastroenterol 2001; 15:667–680. 84. Molmenti EP, Netto GJ, Murray NG, et al. Incidence and recurrence of autoimmune/alloimmune hepatitis in liver transplant recipients. Liver Transpl 2002; 8:519–526. 85. Burke A, Lucey MR. Non-alcoholic fatty liver disease, nonalcoholic steatohepatitis and orthotopic liver transplantation. Am J Transplant 2004; 4:686–693. 86. Mackie J, Groves K, Hoyle A, et al. Orthotopic liver transplantation for alcoholic liver disease: a retrospective analysis of survival, recidivism, and risk factors predisposing to recidivism. Liver Transpl 2001; 7:418–427. 87. Marzan A, Salizzoni M, Debernardi-Venon W, et al. Prevention of hepatitis B virus recurrence after liver transplantation in cirrhotic patients treated with lamivudine and passive immunoprophylaxis. J Hepatol 2001; 34:903–910. 88. Berenguer M, Prieto M, Rayon J, et al. Natural history of clinically compensated hepatitis C virus related graft cirrhosis after liver transplantation. Hepatology 2000; 32:852–858. 89. Gaglio PJ, Malireddy S, Levitt BS, et al. Increased risk of cholestatic hepatitis C in recipients of grafts from living versus cadaveric liver donors. Liver Transpl 2003; 9:1028–1035. 90. Crippin JS, McCashland T, Terrault N, et al. A pilot study of the tolerability and efficacy of antiviral therapy in hepatitis C virusinfected patients awaiting liver transplantation. Liver Transpl 2002; 8:350–355. 91. Everson GT. Treatment of chronic hepatitis C in patients with decompensated cirrhosis. Rev Gastroenterol Disord 2004; 4:S31–S38. 92. Gane E. Treatment of recurrent hepatitis C. Liver Transpl 2002; 8:S28–S37.
Section VIII: Liver Transplantation
MANAGEMENT OF RECURRENT VIRAL HEPATITIS B AND C
52
Marina Berenguer and Teresa L. Wright Abbreviations antiantibody to HBV surface antigen HBsAg HBIg hepatitis B immunoglobulins HBsAg hepatitis B surface antigen HBV hepatitis B virus HCC hepatocellular carcinoma
HCIg HDV HIV HLA IFN LDLT
HCV immunoglobulin hepatitis delta virus human immunodeficiency virus human leukocyte antigen interferon living donor liver transplantation
INTRODUCTION With significant improvements in immunosuppressive therapy and surgical techniques over the past two decades, liver transplantation has become the definitive and effective therapy for patients with end-stage liver disease, with survival rates approaching 90–95% and 65–80% after 1 and 5 years of follow-up, respectively.1 Among several circumstances that may pose a threat to long-term survival, the greatest is likely the recurrence of the original liver disease.2,3 Viral hepatitis is the leading indication for liver transplantation in the majority of transplant centers. Post-transplantation outcome in these patients largely depends on the prevention of allograft reinfection. While hepatitis B recurrence has been effectively contained by the use of hepatitis B immunoglobulin and oral antivirals,2 recurrent hepatitis C is becoming an increasingly challenging problem to the transplant community.3 As patients survive longer and enter their second to third decade post-transplantation, it is likely that allograft failure related to recurrent hepatitis C will become an increasingly serious problem. In this chapter, we will summarize the current knowledge on recurrent viral diseases following liver transplantation, with particular emphasis on the natural history, pathogenesis, and treatment of these conditions.
RECURRENT HEPATITIS B VIRUS (HBV) INFECTION HBV-related liver disease represents 5–10% of liver transplantations in most series.1,2 Due to the recent improvements in the management of HBV infection, post-transplantation outcomes are now excellent, similar to those achieved by patients with cholestatic liver diseases.4,5 Historically, these patients had done poorly with transplantation. The 5-year survival rate was reported to be 50%, compared to 70–85% for patients with alcoholic or cholestatic liver diseases.2 This reduced survival was in large part related to the high rate of HBV recurrence in the absence of specific prophylactic therapies. Indeed, in an early study, the reinfection rate was 80%, resulting in graft loss in more than 70% of patients.2 Efforts to improve the outcome of these patients were predominantly focused upon
MELD MMF ORFs PBMC PEG
model for end-stage liver disease mycophenolate mofetil open reading frames peripheral blood mononuclear cells pegylated
strategies to prevent reinfection. In recent years, several new therapies have become available that have led to increased survival of this patient group, lending a sense of optimism to clinicians caring for patients with this disease. Interestingly, the number of publications related to “HBV and liver transplantation,” an indirect measure of the interest and scientific advances, has significantly increased in the last 5 years, from 30 publications reported in Medline from 1980 to 1991, to 429 in the period 1995–2003. Many of the questions of the early 1990s now have an evidence-based answer, so that the debate has currently shifted from whether liver transplantation is an option for this patient subgroup to selecting the best approaches to prevent reinfection, particularly in the long term.
INDICATIONS FOR LIVER TRANSPLANTATION The indications and contraindications for transplantation in these patients do not differ from those applied to other forms of liver diseases and typically include complications from portal hypertension, liver failure, or the development of hepatocellular carcinoma (HCC).1 Indeed, virally infected patients are at high risk for developing HCC. Cirrhosis itself is considered a premalignant state, irrespective of its etiology, with an annual incidence of HCC of 4–5%, ranging from 1 to 8%.6 This risk is 20 times greater in surface antigen-positive patients than in negative controls. Among HBV patients, the risk is minimal in carriers with normal enzymes, it is low in patients with chronic hepatitis, and is highest in those with cirrhosis. Other factors increasing the risk of HCC in HBV-infected patients include old age, male gender, long duration of infection, coinfection with delta or hepatitis C virus, aflatoxin exposure, and high levels of HBV replication. Although the rate of cancer recurrence after liver transplantation was very high in early series, more promising results have recently been obtained with accurate staging of tumors and improved patient selection.7 Careful expansion of traditional criteria can be attempted within experimental boundaries.
NATURAL HISTORY HBV infection post-transplantation typically results from the recurrence of an infection present prior to liver transplantation. In the
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absence of specific prophylactic measures, both type of disease and HBV DNA level before transplantation are the best predictors to assess the risk of recurrence, with the highest rates reported in hepatitis B surface antigen (HBsAg)-positive cirrhotic patients with evidence of active viral replication (HBV DNA and/or HBeAg-positive) and the lowest in those without detectable HBeAg or HBV DNA, those with fulminant hepatitis, or those coinfected with the delta virus (2-year actuarial risk: 75%, 67%, 17%, and 32%, respectively).2,4,5 Most cases of HBV reinfection occur during the first 3 years post-transplantation. Occasionally, HBV infection post-transplantation is a consequence of de novo infection, despite the use of strict screening measures in blood banks with exclusion of HBsAg-positive and also anti HB corepositive donations (Figure 52-1).8,9 The prevalence of de novo HBV hepatitis ranges from 2 to 8%, and is generally related to transmission from an HBsAg-negative anti-HBc-positive donor. The most significant factor associated with transmission is the serologic status of the recipient, so that the risk is almost null in patients who are antiHBs-positive, minor (@ 10%) in those who are anti-HBs-negative but anti-HBc-positive, and high (@ 50–70%) in those without markers of previous exposure to HBV.8,9 Although there have been reports of severe progression, the natural history of de novo hepatitis B is generally more benign than that described for recurrent hepatitis B. The natural history of recurrent hepatitis B is more aggressive than that observed in the immune-competent population. Two patterns of recurrence have been described. Typically, patients develop acute hepatitis after detection of HBsAg in serum. In these cases there is a significant increase in HBV DNA levels, together with a transaminase rise with mild bilirubin elevation, and features of acute lobular hepatitis on liver histology. Progression to chronic hepatitis and cirrhosis may occur within 2 years of transplantation.2 The second pattern of recurrence, fibrosing cholestatic hepatitis, is an entity initially described in these patients, and later, among HCV-infected recipients. It is characterized histologically by the presence of periportal and perisinusoidal fibrosis, ballooned hepatocytes with cell loss, pronounced cholestasis, and a paucity of inflammatory activ-
ity.2 Immunohistochemical stains show high cytoplasmic expression of viral antigens, which, in conjunction with the lack of inflammatory infiltrate, suggests a direct cytopathic effect of the virus. Clinically, patients present with jaundice, biochemical cholestasis with high bilirubin levels despite mild transaminase elevations, and extremely high levels of serum HBV DNA. The course is rapidly progressive with severe cholestasis, coagulopathy, and liver failure within weeks of onset. High early mortality rate occurs following liver retransplantation and among those surviving the postoperative period, an even more aggressive course of recurrent disease develops.2 Patients at risk for this syndrome include those with high levels of viremia pretransplantation and those infected with precore mutants. With currently available effective therapies, this syndrome is rarely, if ever, seen in patients undergoing liver transplantation for HBV disease.
PATHOGENESIS The mechanisms by which HBV leads to liver injury following liver transplantation are incompletely understood. Increased levels of HBV replication are typically observed, likely related to the use of immuosuppressive drugs, particularly corticosteroids. This enhanced replication with excess production of viral proteins in conjunction with the altered host immune responsiveness probably contributes to the pathogenesis of liver damage.2
PREVENTION OF HBV GRAFT REINFECTION (Figure 52-2) Early Prophylaxis Passive Immunization with High-Dose Hepatitis B Immunoglobulins (HBIg) HBIg consists of polyclonal antibodies directed against the viral envelope, and was originally derived from donors positive for antibody to HBV surface antigen (anti-HBsAg). The presumed mechanism of action of this antibody is to neutralize circulating virus by binding to the viral envelope. Empirical application of HBIg aimed at maintaining serum anti-HBs titers above 100 IU/l was initially shown to reduce the rate of viral recurrence. This was further con-
LT in the post-HBIg/ pre-lamivudine era risk. 25% Preemptive LT in the pre-HBIg/ pre-lamivudine era risk. 80%
LT with lamivudine monotherapy risk. 25%
Populations for treatment of post liver transplantation HBV LT in the post-HBIg/ post-lamivudine era risk. 5%
de novo infection: peri-LT acquisition post -LT reactivation
Figure 52-1. Populations for treatment of post-transplantation hepatitis B virus (HBV) disease. Recurrent HBV disease is uncommon, but five different groups with HBV disease of the allograft exist. Management of these populations depends on their prior treatment exposure and the presence of treatment-associated HBV variants. LT, liver transplantation.
976
Therapeutic
Before histologic recurrence
Before OLT
OLT
After histologic recurrence
Recurrent hepatitis
HBV Nucleoside analogues
HBIg 6 Lamivudine
Nucleoside analogs
Interferonribavirin
Interferonribavirin
Interferonribavirin
HCV Figure 52-2. Approaches to the prevention and treatment of HBV and HCV infection in the setting of liver transplantation. OLT, orthotopic liver transplantation.
Chapter 52 MANAGEMENT OF RECURRENT VIRAL HEPATITIS B AND C
firmed in a European multicenter study2 where the administration of HBIg for more than 6 months was shown to reduce dramatically the rate of HBV recurrence to a median rate of 20–30% after 2 years.2,4,5 Recurrence was 75% in patients receiving no or short-term HBIg versus 33% in those receiving long-term HBIg (P < 0.001).2 Long-term administration of HBIg reduced the rate of recurrence in patients with fulminant HBV hepatitis to less than 10%, in HDV coinfected patients to 10–15%, and in HBV DNA-negative cirrhotic patients to less than 30%. Yet, it did not reduce the rate of recurrence in patients with HBV DNA-positive HBV cirrhosis.2 More recent studies have further confirmed the efficacy of long-term HBIg in patients without active HBV replication, with recurrence ranging from 17 to 38% at 2 years when administered to reach titers over 100 UI/l, but reduced efficacy in those in whom HBV DNA is detected prior to transplantation by hybridization methods (>105–106 copies/ml) with rates of recurrence ranging from 70 to 96%.2,4,5 In a recent analysis of the long-term results of HBIg administration in 271 patients, the actuarial rate of HBV recurrence was 26.8%, 34.6%, 40%, and 41.6% at 1, 2, 5, and 10 years, respectively.5 Substantial differences were again observed between patients who were HBV DNA-positive and those who were negative pretransplantation with 5-year rates of 76.7% and 33%, respectively. The adverse prognostic characteristic of active viral replication pretransplantation may be overcome with more aggressive use of HBIg maintaining titers over 500 IU/l and, particularly, by the use of HBIg in combination with oral antivirals pre- and post-transplantation (see later).10 With the first alternative, at least during the first 6 months, recurrence in HBV DNA-positive patients may be reduced to approximately 16–35%. Various regimens have been described, with most including the administration of 10 000 IU HBIg intravenously during the anhepatic phase and 10 000 IU HBIg daily for the first week posttransplantation. The subsequent dosing is either given on a fixed schedule (generally on a monthly basis) or based on anti-HBs titers (readministration when antiHBs is less than 100–500 IU/l).2,10 Due to the high number of variables, including risk of recurrence, time from transplantation, and use of antivirals, the best target value for anti-HBs level and whether this target level can be reduced over
time in an individual patient are still a matter of debate. It is generally accepted though that HBIg should be given so as to obtain anti-HBs titers greater than 500 IU/l during the first week after transplantation, greater than 250 IU/l between days 8, and 90 and greater than 100 IU/l thereafter.2,10 Due to the good results, lifelong passive immunization with HBIg has been used in most transplant centers and considered, until very recently, the “standard of care.” However, HBIg has several drawbacks, including the cost, the need for parenteral and long-term administration, the need for close monitoring of anti-HBs levels, the issue of availability, the potential for breakthrough, and the reduced efficacy in patients with viral replication. Causes of breakthrough are multifactorial and include inadequate anti-HBs titers following transplantation, HBV overproduction coming from extrahepatic sites, and/or mutations in the region of the surface gene of the HBV genome which encodes the “a” determinant region, the putative region for antibody binding. While the first two circumstances appear to play a major role in the early post-transplant period, viral mutations are probably the major cause of HBIg failure in the long term.10 The most common mutation is a substitution of glycine for arginine at amino acid position 145. Discontinuation of HBIg results in reversion of the mutations to the wild-type virus in the majority of patients. One concerning limitation is the difficulty in discontinuing this product in the long term. Recurrent infection has been documented in patients stopping prophylaxis with HBIg after 1 year or more. Furthermore, HBV DNA has been detected by highly sensitive molecular techniques in the serum, liver, and peripheral blood mononuclear cells (PBMC) of HBsAg-negative patients on HBIg prophylaxis,5 suggesting that indefinite treatment is required.
Pretransplantation Therapy with Oral Antivirals Several alternatives are being evaluated to overcome the limitations of HBIg (Table 52-1). The first one is the use of antiviral treatment prior to transplantation to inhibit viral replication.10–12 Due to the risk of worsening hepatic decompensation and low tolerability of interferon in patients with decompensated liver disease, interferon is not recommended in this situation. Nucleoside analogues, though, have
Table 52-1. Prevention of Recurrent Hepatitis B: Alternatives to High Intravenous Doses of Hepatitis B Immunoglobulin (HBIg) Type and timing of intervention
End-point
Available drugs
Potential problems
Pretransplantation antiviral therapy
Decrease viral replication
• •
• •
Pre-emptive post-transplantation antiviral therapy Pre-emptive post-transplantation antiviral therapy in combination with: • High doses of HBIg • Low doses of HBIg HBIg ± oral antivirals in combination with post-transplantation vaccination against HBV
Prevent HBV recurrent infection Prevent HBV recurrent infection
Nucleos(t)ide analogues
Prevent HBV recurrent infection
Interferon ? Nucleos(t)ide analogues
HBIg in combination with nucleos(t)ide analogues
Double-dose vaccination following discontinuation (versus alternating) of HBIg ± oral antivirals
Tolerance Development of resistant mutants Development of resistant mutants/low efficacy • Same as with high-dose HBIg in monotherapy • Development of resistant mutants Failure of vaccination regimen
HBV, hepatitis B virus.
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Section VIII. Liver Transplantation
Figure 52-3. Pretransplantation therapy with lamivudine: development of lamivudine-resistant mutants. LAM, lamivudine; R, resistance. Mean follow-up 12 months.
100 Median follow-up: 12 months (3–29)
90 80
Villeneuve 00 (n=35) Perrillo 01 (n=30)
70
Yao 01 (n=23) 60 Seehofer 01 (n=17) 50
Marzano 01 (n=33)
40
Fontana 02 (n=162)
30
Andreone 02 (n=25)
20
Fontana 02 (n=154)
10 0 DNA (–) with LAM
Viral R
a potent antiviral effect, inducing a rapid suppression of HBV DNA in serum, are very well tolerated, orally administered, and, in contrast to interferon, do not precipitate worsening of liver function in patients with advanced disease. Recent experience with these drugs in cirrhotic patients awaiting liver transplantation is encouraging.13–19 With lamivudine (100 mg daily), HBV replication is decreased below detection of hybridization assays in 62.5–100% of treated cirrhotic patients, including both those infected with wild-type virus and the e-minus strain of HBV, allowing liver transplantation to be performed in conditions of low risk of recurrence (Figure 52-3). An additional benefit that may be obtained by some, but not all, patients with decompensated cirrhosis is an improvement in the hepatic synthetic function. Clinical improvement and stabilization of hepatic function are slow and gradual, being more apparent after 6 months of therapy. Although this clinical improvement may be achieved by a subgroup of cirrhotic patients, it is less likely in those with severe hepatic insufficiency, including those with increased serum bilirubin and creatinine levels and elevated Child–Pugh and model for end-stage liver disease (MELD) scores.20 Since progression of the disease and even death tend to occur early after the initiation of therapy, generally within the first 6 months, patients with the above characteristics, who most likely have presented late in their disease course, should be prioritized for urgent liver transplantation, irrespective of the antiviral response to lamivudine. Since viremia recurs in more than 80% of patients following treatment discontinuation, circumstances that may result in a flare of liver disease, treatment should be administered indefinitely in these patients. The major drawback of prolonged lamivudine therapy is the selection of drug-resistant mutants with HBV DNA reappearance (Figure 52-3). Mutations typically occur in the YMDD motif of the HBV DNA polymerase gene. This risk increases significantly after 6 months of therapy, reaching 27% after 1 year. While viremia may be lower in patients with YMDD mutations than before therapy because of the decreased replication fitness of the mutants, flares of liver disease with worsening of liver disease may occur.11,21 In addition, the selection of lamivudine-resistant mutants may increase the risk of HBV recurrence despite the use of high doses of HBIg plus lamivudine post-transplantation.22–25 Adefovir dipivoxil (10 mg daily,
978
with dose reductions based on creatinine clearance) is a potent nucleotide analogue that has been shown to suppress viral replication of the wild-type virus, the e-minus strain, and the lamivudineor famciclovir-resistant mutants. In cirrhotic patients who have failed lamivudine, adefovir leads to a significant reduction of HBV DNA levels and normalization of transaminase levels in 61% of the patients.11 It may either be used as salvage therapy or as a primary option. Recent data from 128 patients with decompensated cirrhosis treated with adefovir demonstrated that this drug is safe and effective in treating lamivudine-associated breakthrough, significantly suppressing serum HBV DNA levels. Clinical improvement was also achieved in a proportion of patients with improvement in serum bilirubin, albumin, and prothrombin time.26 The one concern with this drug is the potential for renal toxicity. Since resistance to adefovir is extremely low (2% after 2 years of continuous use), patients in whom a significant clinical improvement is achieved may even be removed from the waiting list. Tenofovir disoproxil fumarate, structurally similar to adefovir and approved for treatment of human immunodeficiency virus (HIV) is also effective in suppressing replication of YMDD mutants, and, interestingly, appears to have less renal toxicity than associated with adefovir. Data on this drug in the liver transplant setting are still lacking. The best posttransplantation prophylaxis in patients with lamivudine-resistant mutants is at present unknown, but probably should be based on triple therapy. Interestingly, adefovir-resistant variants are sensitive to lamivudine,27 providing scientific rationale for combination therapy with both of these agents in treating patients with advanced liver disease awaiting transplantation or those following transplantation who are infected with lamivudine-resistant strains.
Post-Transplantation Therapy with Oral Antivirals Once liver transplantation is performed, there are several alternatives to long-term HBIg (Table 52-1). The first is to continue the pre-emptive therapy with lamivudine which was begun prior to transplantation.28,29 Although this approach is initially effective and patient compliance is good given the few side effects of this drug, therapy is limited by the emergence of HBV mutants with prolonged treatment, required to avoid rebound of viral replication once
Chapter 52 MANAGEMENT OF RECURRENT VIRAL HEPATITIS B AND C
Table 52-2. Prevention of Recurrent Hepatitis B: Combination with Hepatitis B Immunoglobulin (HBIg) and Lamivudine % DNA + pre-OLT
Reference
Duration of pre-OLT treatment with lamivudine
Markowitz 199830 (n = 14) Yao 199931 (n = 10) McCaughan 1999 (n = 9) Yoshida 199932 (n = 7)
3 (0.7–7.8)
7%
8.6 (1–22)
20%
0
NA
Angus 200033 (n = 32) Marzano 200134 (n = 33) Rosenau 200124 (n = 21) Han 200135 (n = 59)
60% on lamivudine (duration NA) 3.2
0
50%
4.6 (0.6–14.1)
0
4.6 (0.06–14.1)
24%
NA
NA
HBIg regimen
Follow-up (months)
Standard high IV (10 000 day 0–7, then weekly during the first month, then monthly) 10 000 IU IV day 0 (7 days if DNA +), 1111 U i.m./weekly for 3 weeks, 1111 U i.m. every 3 weeks 4000 IU i.m. (daily first week, weekly for 3 weeks, then monthly) 2170 IU i.m. for 14 days, then twice weekly with progressive reduction until once monthly at 1 year post-OLT 400–800 IU i.m. daily the first week and then monthly
13
Recurrence rate 0
15.6
10%
15.6
0
17
0
18.5 (5–45)
3%
46 500 IU IV first month, 5000 IU IV monthly
30 ± 8
4%
45 000 IU IV first week then reinjection to maintain anti-HBs ≥ 500IU/l until day 14, then ≥ 200 IU/l Standard high IV (10 000 day 0–7, then monthly)
21 (2.4–49.1)
9.5%
15 (1–61.8)
0
OLT, orthotopic liver transplantation; HBIg, hepatitis B immunoglobulin; IV, intravenous; i.m., intramuscular.
therapy is stopped. Using this approach, the rate of recurrence is as high as 60%, mainly in patients with high levels of viral replication before treatment initiation.28 It may be sufficient though for nonreplicating patients. Due to the low risk of developing resistant mutants, primary adefovir monotherapy pre- and post-transplantation is an attractive alternative that is currently under evaluation. The second alternative, since treatment failures occur with both HBIg and with lamivudine monotherapy, is the use of combination therapy with HBIg and nucleoside analogues. It is the most promising alternative and is becoming the standard of care in most transplant programs.2,10,11 The advantages over a single agent are the following: (1) possibility of administering lower doses of HBIg (400–2000 IU/monthly) which then leads to a significant reduction in cost; (2) potential reduction of development of resistant mutants, which is a frequent event when each drug is given as a single agent; and (3) synergistic effect with failure rates lower than 10–12% in most series. The higher rates of recurrence are typically found in patients who have developed lamivudine resistance prior to transplantation. The best protocol is still unknown since doses, routes, type, and lengths of administrations vary substantially from center to center. In a preliminary report, lamivudine in combination with high intravenous doses of HBIg was shown to be safe and highly effective, but equally expensive.30 Subsequent studies have explored modifications of this approach using lower doses of HBIg, simpler routes of administration, and shorter durations of therapies31–36 (Table 52-2).
Long-Term Prophylaxis In the long term, two approaches have been investigated, particularly in patients at low spontaneous risk of HBV recurrence: HBIg discontinuation followed by monotherapy with oral antivirals and HBV vaccination. In a recent long-term study, it was shown that almost 91% of recurrences occurred within the first 2 years of transplantation and only 3% after the fifth year,5 hence raising the issue of HBIg discontinuation in the long term.
Active HBsAg Vaccination Post-transplantation vaccination against HBV may be attempted after an initial period of HBIg ± oral antivirals. In a pilot study, HBIg was discontinued in 17 low-risk selected liver transplant recipients after a median of 25 months (19–68 months) from transplantation and a double-dose recombinant HBV vaccine was administered at 0, 1, and 6 months.37 Seroconversion to anti-HBs was obtained in 82% of cases. In an update of this study, seroconversion rates to antiHBs (antiHBs titers higher than 10 IU/l) were less impressive, occurring in 64% of 22 patients.38 Disparate results were however reported in a second study,39 with a seroconversion rate of only 23% despite the use of a reinforced triple course of hepatitis B vaccination. The main differences between these two studies include the study population and the concomitant use of lamivudine following HBIg discontination (100% versus 20% in the first study). In fact, the use of lamivudine was found to be a predictive marker of failure to attain seroconversion in the first study (Table 52-3). More recently, a third group has reported the results of HBV vaccination using a more immunogenic vaccine with promising results.40 In that study, 20 patients received repeated doses of recombinant HBV vaccine (20 mg) in combination with a new adjuvant, monophosphoryl lipid A. Vaccination was performed at least 2 days before HBIg administration and titers of anti-HBs concentrations were determined before HBIg injections. HBIg was then discontinued whenever levels were >500 IU/l. Sixteen out of the 20 patients developed anti-HBs levels ranging from 721 to 83 121 IU/l, thus allowing HBIg withdrawal. After 13.5 months (range 6–22 months) of follow-up, all responders had serum anti-HBs concentrations >900 IU/l. If these results are further confirmed, HBV vaccination will enable a substantial proportion of patients now on HBIg to develop a sustained antibody response without the need for continuous passive immunoprophylaxis. This will have major impacts on costs and quality of life. However, several aspects need to be investigated further, including the best vaccine, the definition of protective anti-HBs titers, the amount of HBsAg in each dose, the number
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Table 52-3. Prevention of Hepatitis B Virus (HBV) Recurrence using HBV Vaccination Following Liver Transplantation Sanchez-Fueyo 200238 (n = 22)
Angelico 200239 (n = 17)
Bienzle 200340 (n = 20)
8/14
0/17
2/18
14/8
17/0
16/4
33 (18–76) months 1–4 weeks
48 (25–85) months 4.5 months
Lamivudine post HBIg D/C
20%
100%
Dose of HBV vaccine
Double at 0-1-6 months
Double at 0-1-2 months
Anti-HBs > 10 IU/l Anti-HBs > 100 IU/l Anti-HBs > 500 IU/l Maximum anti-HBs IU/l in responders HBV recurrence Follow-up after immunization
63% 23% 9% 47 (10–1000)
18% 12% 6% 258 (10–601)
78 (24–156) months Patients maintained on HBIg through HBV vaccination 4 patients also on oral antiviral therapy while vaccination More immunogenic vaccine at weeks 0, 2 , 4, 16, and 18 following HBIg administration 100% 100% 80% 25 344 (1255–83 121)
0% 41 (31–85) months
0% 66 (25–88) months
0% 13.5 (6–22) months
Cause of LT indication (acute/chronic) Immunosuppression (mono/combination) Time from LT to vaccination Time from HBIg D/C to vaccination
LT, liver transplantation; HBIg, hepatitis B immunoglobulin; D/C, discontinuation.
of doses, whether target titers should be the same for different subsets of patients or not, and finally, the necessity for boosting to maintain protective titers. Evaluation of this approach in high-risk patients should also be investigated.
HBIg Substitution with Lamivudine in the Long Term Successful results after a short follow-up have already been reported in low-risk patients.41,42 In the two reported series, only two recurrences were confirmed among 26 patients switched from HBIg ± lamivudine to lamivudine monotherapy after 1–6 months from transplantation. Longer follow-up is needed to determine the incidence of lamivudine-resistant mutants and the efficacy of this approach in high-risk patients. In these and other long-term studies, it has become apparent that, in a substantial subset of patients (up to 45% at 10 years), HBV DNA continues to be detected in serum, liver, or PBMC by polymerase chain reaction (PCR)-based methods.5 Yet the clinical significance of these findings in both the short and long term are unclear since patients are typically asymptomatic with normal liver enzymes and are HBsAg-negative in serum. These findings raise several issues, particularly the indefinite risk of graft reinfection, at least in some patients, and hence the need for the indefinite use of some type of prophylaxis. Determining in whom prophylaxis can be safely stopped may prove to be a difficult task, relying on sensitive PCR techniques to detect HBV DNA in serum, PBMC, and liver.
TREATMENT OF HBV DISEASE OF THE GRAFT Nucleoside analogues are the cornerstone of therapy due to their potent antiviral effect and lack of side effects.11,43 The need for continuous treatment and resistance remain the main limitations. The selection of the antiviral is likely dependent on the category of
980
patient (Figure 52-1). In those who have undergone liver transplantation in the pre-HBIg and/or lamivudine era or those with apparent de novo HBV acquisition, all known antivirals are potential good candidates. In contrast, for those who have undergone surgery in the post-HBIg/lamivudine era and who have broken through, new antivirals such as adefovir or tenofovir that have activity against resistant variants may be best options. Lamivudine is the most widely used nucleoside analogue with good tolerance, rapid loss of HBV DNA in serum in a substantial proportion of patients (60%), “e” seroconversion (30%), and histologic improvement. Adefovir has resulted in viral suppression of nucleoside analogue-resistant variants.11,44
EMERGENCE OF NUCLEOSIDE ANALOGUE RESISTANCE Monotherapy with lamivudine has resulted in the emergence of HBV-resistant variants. This resistance generally occurs after prolonged therapy (more than 6 months) and is associated with a rise in serum HBV DNA and alanine aminotransference levels, indicating a breakthrough in therapy. Molecular analysis has shown changes in the gene for the viral DNA polymerase. Because of the overlapping nature of the HBV open reading frames (ORFs), nucleotide changes in the polymerase may result in amino acid changes not only in the polymerase protein but also in the surface protein, which could in turn theoretically alter binding of HBIg.2,10 When lamivudine is stopped, the wild-type variant re-emerges as the dominant viral population, but retreatment is again associated with the development of resistant mutants at an accelerated rate. Although some cases of histological and clinical deterioration have been reported when drug-resistant mutants develop, these are not consistently associated with hepatic disease progression. The molecular mecha-
Chapter 52 MANAGEMENT OF RECURRENT VIRAL HEPATITIS B AND C
nisms associated with severe recurrence may be a drug-dependent enhanced replication of lamivudine-resistant HBV mutants.45
PREVENTION AND TREATMENT OF DE NOVO HBV INFECTION In order to avoid de novo HBV infection, two complementary approaches may be undertaken: 1. HBV vaccination prior to liver transplantation of all anti-HBsnegative candidates. Unfortunately, as with other immunosuppressed populations, results have been disappointing, with response rates barely reaching 40%;9 2. anti-HBc determination of the donor with use of organs from anti-HBc-positive donors only in recipients already infected with HBV. In order to obtain maximum benefit from these organs while at the same time reducing the risk of HBV transmission, these organs may be used in special circumstances in recipients not infected with HBV. Prophylaxis is then recommended if the risk of transmission is high, particularly in HBV-naive recipients, but may be obviated in those at low risk of transmission, such as those who are antiHBs/anti-HBcore-positive, antiHBs-positive/anti-HBc-negative, or antiHBs-negative/antiHBc-positive.8,9
LIVING RELATED LIVER TRANSPLANTATION Adequate timing of pretransplantation antiviral therapy is an advantage in this setting. Transplantation can thus be performed when HBV DNA has been cleared in serum but before the development of resistant mutants.
RETRANSPLANTATION The initial results on retransplantation for patients with graft failure due to recurrent hepatitis B were discouraging. Improved outcomes have been achieved with specific interventions, mainly with the use of aggressive immunoprophylaxis in combination with pre- and postretransplantation antiviral therapy.46
TRANSPLANTATION IN PATIENTS COINFECTED WITH HEPATITIS DELTA INFECTION Patients with HBV and hepatitis delta virus (HDV) infection are at low risk of viral recurrence.2,4,5 Delta infection is an uncommon indication for transplantation. In the absence of HBIg, both HBV and delta virus can infect the graft, but HDV is not pathogenic until HBV replication also occurs. Current recommendations for HBV apply to HBV–HDV-coinfected liver transplant candidates.
CONCLUSION HBV-related end-stage liver disease is an excellent indication for liver transplantation. Recurrence is effectively prevented with current therapies, and strategies can be tailored individually based on risk of recurrence. In low-risk patients, that is, cirrhotic patients with no evidence of HBV replication (HBV DNA-negative by sensitive hybridization assays), those with fulminant hepatitis, or those coinfected with the delta virus, oral antivirals are not required prior to transplantation, and HBIg in monotherapy may be sufficient initial prophylaxis. In the long term (> 6 months ?), HBIg may be switched to oral antiviral therapy ± HBV vaccination. In contrast, in
high-risk patients, that is, surface antigen-positive cirrhotic patients with evidence of active viral replication, antiviral therapy is needed before transplantation for at least 1 month, and early post-transplantation prophylaxis should be based on combination regimen with HBIg and antivirals. The best HBIg regimen in these patients is still controversial. Whether HBIg may be stopped in the long term is still a matter of debate. HBIg discontinuation could be proposed to patients with undetectable serum HBV DNA by PCR after at least 1–2 years of HBIg, with continuance of lamivudine. Whether adefovir or tenofovir should only be used as salvage therapy or whether these agents should be used as a primary option is also a matter of debate. Certainly, prior to liver transplantation in patients with HBV cirrhosis and detectable HBV DNA, combination therapy with lamivudine plus adefovir or tenofovir seems to make sense, in order to maximize pretransplantation viral suppression and minimize the development of resistance to either agent, resistance that could lead to life-threatening flares of liver disease.
RECURRENT HEPATITIS C VIRUS INFECTION More than 170 million people worldwide are chronically infected by HCV. HCV-associated end-stage liver disease with or without HCC has become the leading diagnosis in patients undergoing liver transplantation.1 In most centers, more than half of transplanted patients are infected with HCV prior to transplantation. It is likely that that this number will increase in future years, mainly as a consequence of the progressive nature of the disease, the inadequate detection of this largely asymptomatic infection, and the lack of effective treatments.47 Viral recurrence defined by the presence of HCV RNA in serum following transplantation occurs universally. Recurrence of chronic hepatitis C is a frequent event, with progression to allograft cirrhosis occurring in a substantial proportion of patients.3 The full consequences of HCV recurrence ultimately result in reduced graft and patient survival compared with patients transplanted for non-viral causes.48,49 There are several factors that may influence the outcome. In fact, it is likely that a different distribution of these factors accounts for the differences in outcome between centers.3,50,51 The need for an effective treatment derives from the potential seriousness of this disease. Unfortunately, to date, there is no suitable intervention to prevent HCV recurrence, and available treatments, interferon and ribavirin, have limited applicability and efficacy in the transplant setting.52 Due to the magnitude of the problem, a consensus conference was recently held to overview the state of the art concerning liver transplantation and HCV disease.53 The conference participants examined the definition of recurrent HCV, the natural history of HCV after liver transplantation, the potential clinical predictors of adverse outcomes, treatment and management strategies, the contribution of different immunosuppressive regimens to outcome, and the role of retransplantation for recurrent HCV in the face of an overall donor shortage.
INDICATION FOR LIVER TRANSPLANTATION Liver transplantation remains the most effective option in patients with decompensated HCV-related cirrhosis. In contrast, traditional
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medical management is indicated for compensated cirrhosis. A substantial proportion of HCV-infected candidates have a coincidental HCC.1,3,6 Their post-transplantation outcome does not differ from that of patients without the tumor if strict selection criteria are followed.6,7 In some early reports, the presence of HCC was the only variable associated with reduced patient and graft survival,54 possibly due to the high risk of tumor recurrence when no strict tumor criteria were applied. While indications and contraindications for transplantation in HCV-infected patients do not differ from those applied to other forms of liver diseases, post-transplantation outcome is frequently impaired by HCV recurrence. In these circumstances, decompensated patients and patients with HCC should be aggressively managed with traditional medical and/or surgical therapy in an effort to avoid the premature transplantation of some of these patients, even in the new era of living donor liver transplantation (LDLT). In that sense, alternative therapies of HCC including transarterial chemoembolization and hepatic resection should be explored before consideration of liver transplantation. Several series have reported promising results by using primary resection followed by “salvage transplantation.”6,7 However, resection can only be offered to a minority of patients, such as those with compensated cirrhosis without relevant portal hypertension. The addition of antiviral therapy in these patients may even lead to viral eradication and improvement of hepatic fibrosis, eliminating the future need for “salvage transplantation.”
NATURAL HISTORY (Figure 52-4) While recurrence of HCV infection is based on virological parameters, the recurrence of HCV disease requires protocol and/or clinically indicated liver biopsies that report both grade and stage of disease.53 Two patterns of HCV disease have been described, with differences in clinical presentation, prognosis, pathogenesis, and
therapeutic strategies.3,50,55 The commonest response to persistent HCV infection is the evolution over time to chronic hepatitis in a similar way to what has been described in the non-transplant patient but occurring at a viral set at least one log higher. Disease progression in these patients is typically accelerated compared to that observed in the immune-competent host.3,50,56 Progression in patients with this pattern of recurrence may follow two distinct pathways: (1) a linear rate of fibrosis progression;56 and (2) a delayed onset of rapid progression following an initial period of stabilization.57 Approximately two-thirds of patients with this pattern of recurrence develop an acute lobular hepatitis within the first 6 months post-transplantation. By the fifth postoperative year, over 80% of recipients will have developed chronic recurrent HCV disease, characterized histologically by portal and periportal inflammation with or without the presence of portal and/or periportal fibrosis. Lobular hepatitis can also be part of the pathological picture. Progression to chronic hepatitis and cirrhosis is linear in a substantial proportion of patients with a median rate of fibrosis progression of 0.3, ranging from 0.6 to 0.8 stage/year.3,50,56,59 In a subgroup of patients with initial benign recurrence (@ 30%), delayed hepatitis C-related severe liver damage may occur.57 In these patients, progression to severe disease is not linear and patients develop a sudden acceleration in fibrosis following an initial and sometimes prolonged period of stabilization. The presence of some degree of fibrosis and elevated liver enzymes at 3 years post-transplantation may predict this sudden change in the natural history of recurrent hepatitis C, with 70% of patients with these predictive factors developing this acceleration as opposed to 5% of those without these factors. Cholestatic hepatitis is an infrequent but extremely severe pattern of recurrence that leads to graft failure in 50% of patients within a few months of onset.3,50,55 Clinically, it is characterized by progressive jaundice and biochemical cholestasis, usually beginning
Figure 52-4. Risk of recurrent hepatitis C virus infection and variables associated with post-transplantation liver disease progression. FCH, fibrosing cholestatic hepatitis; LT, liver transplantation.
Viral factors Viral load Genotype Quasispecies HCV RNA (–) 2%
FCH 2–6%
Donor factors Age, sex, steatosis Surgical factors Warming ischemic time
LT
99% HCV RNA +
25–45% acute hepatitis
50–98% chronic hepatitis
8–44% graft cirrhosis in 5–7 years
Minimal injury 30–50%
Host factors HLA, race, gender, age, immune genetic background, immune system
Delayed injury 30%
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42% decompensated 1 year
External factors Immunosuppression, alcohol, viral coinfection, antiviral therapy
Chapter 52
Fibrosis stage (fitted line, 95% CI)
MANAGEMENT OF RECURRENT VIRAL HEPATITIS B AND C
1996–97
4
1994–95
1992–93
1990–91
1985–89
Table 52-4. Transplant Recipient at High Risk of Severe Hepatitis C Virus (HCV)-Related Liver Disease Following Liver Transplantation
• • • • • • • •
3
2
1
0 0
1
2
3
4
5
6
7
8
Years from transplantation to fibrosis score measurement Figure 52-5. Fibrosis progression after liver transplantation. Effect of year of transplantation. Reproduced with permission from M Berenguer.58
in the first trimester post-transplantation after high levels of immunosuppression. Cholestatic HCV disease can occur as the initial manifestation of recurrent HCV disease or can emerge in the setting of chronic HCV disease. The following criteria need to be fulfilled to define fibrosing cholestatic hepatitis: 1 month posttransplant, serum bilirubin >100 mmol/l, serum alkaline phosphatase, and g-glutamyltransferase >5 times the upper limits of normal, characteristic histology of central ballooning (not necrosis or fallout), a paucity of inflammation ± cholangiolar proliferation without bile duct loss, very high serum HCV RNA levels, and absence of surgical biliary complications or hepatic artery thrombosis. A cytopathic mechanism of allograft damage is thought to be involved given the concurrence of extremely high viral burdens, reduced immune response with intrahepatic non-specific Th2 cytokine response, and unusual histology characterized by little inflammation, and severe centrizonal hepatocyte ballooning. Regardless of the pattern of recurrence, the hepatitis C-driven fibrosis response in the allograft leads to the development of graft cirrhosis in 8–30% of cases after a follow-up of 5–10 years.3,48,50,53–55,59–62 In addition, concerning data suggest that the rate of fibrosis progression, and thus the rate of development of graft cirrhosis due to recurrent hepatitis C is accelerated in patients transplanted in recent years (Figure 52-5).48,56,61,63 While short- and medium-term survivals are unaffected by the HCV status, recent data have confirmed that HCV infection is detrimental for long-term survival, with survival rates approaching 75–80% at 5 years in uninfected patients, but only 60–70% in those who are HCV-positive.48,49 The natural history of post-transplantation hepatitis C is, however, highly variable and, while some patients develop cirrhosis in less than 1 year due to recurrent infection, others show minimal or no injury in their protocol liver biopsies during years of followup, even in the presence of high levels of viremia3,50,51 (Figure 524). Factors influencing this variability are poorly understood, and while there are enough data to support the role of some variables such as viral load pretransplantation and donor age, the data regarding other variables such as genotype are controversial.
• • • •
Genotype 1 b Viral load pretransplantation >1 mmol/ml Viral load at 4 months post-transplantation >10 mmol/ml Donor age >50 years Rewarming ischemic time >60 minutes Early histologic recurrence (3 at 4 months or >8 at 1 year • Fibrosis stage >2 at 1 year Rejection episodes >2 Over-immunosuppression Treatment with antilymphocyte globulin Rejection treatment with >3 g methylprednisolone
VARIABLES ASSOCIATED WITH DISEASE SEVERITY AND/OR DISEASE PROGRESSION Pretransplant or early post-transplant recognition of patients with high risk of severe outcome post-transplantation (Table 52-4) is desirable since these patients can be targeted for intervention, or potentially even denied transplantation if outcomes are considered unacceptably poor. In contrast to HBV, where the availability of new therapies has obviated the need for selection of patients based on predictive factors, the elucidation of these predictive factors remains of paramount importance in HCV-infected patients, given the absence of effective prophylactic or therapeutic agents. Factors influencing the rate of progression relate to the virus, the host, or environmental and/or iatrogenic influences on the infected individual (Figure 52-4, Table 52-4). The high-risk patient likely derives from the interaction between these factors, particularly between the virus, the quality of the graft, and the immune system.
Host-Related Variables Immunosuppression Recent data have implicated the immune system in the pathogenesis of liver injury due to HCV.55 In fact, it is likely that immunosuppression is the most powerful determinant of HCV progression. Several findings highlight the deleterious effect of immunosuppression. These include: 1. a higher rate of fibrogenesis in immunosuppressed patients, both liver transplant recipients and HIV-coinfected patients compared to that observed in immune-competent patients;3,55,56 2. a shortened course to allograft cirrhosis, direct consequence of the above observation. While the timeframe between infection and development of cirrhosis is calculated in decades in the immune-competent individual, this time is reduced to a median of approximately 10 years in liver transplant immunosuppressed patients; 3. a greater risk of clinical decompensation following the establishment of compensated cirrhosis in liver transplant recipients compared to non-transplant patients. Indeed, the rate of decompensation is 42% at 1 year and 63% at 3 years in liver transplant recipients with recurrent HCV cirrhosis compared
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Section VIII. Liver Transplantation
4.
5.
6.
7.
to 3% by 1 year and 18% at 5 years in immune-competent patients with HCV cirrhosis;64 a worse outcome following decompensation for liver transplant recipients compared with non-transplant patients with decompensated HCV cirrhosis with a 3-year survival less than 10% in the former population compared with 60% in the latter;64 an accelerated course of recurrent hepatitis C in more recent years at a time in which more potent immunosuppressive agents are being utilized for both induction and maintenance immunosuppression;48,56,61,63 an adverse effect of cytomegalovirus infection, a virus with inmunosuppressive properties, on HCV-related disease outcome;63 the development of fibrosing cholestatic hepatitis only in the setting of significant immunosuppression, including after solid organ and bone marrow transplantation as well as in HIV infection.55
While the above indirect findings clearly suggest that immune suppression is strongly linked to the severity of recurrent HCV disease, the effect of specific immunosuppressive drugs on both viral replication and HCV-related fibrosis progression is less evident (Table 52-5).3,65–67 Several studies have demonstrated that the use of corticosteroid boluses to treat acute cellular rejection is harmful to HCV-infected recipients as a result of their association with an increased frequency of acute hepatitis, earlier time to recurrence, and higher risk of progressing to graft cirrhosis or developing cholestatic hepatitis.3,65–66 Corticosteroid boluses are associated with an increase in serum HCV RNA concentrations of four- to 100-fold. In fact, the absence of corticosteroids from induction immunosuppression therapy was found in one study to be associated with a
delay in the increase of HCV viral load following transplantation.68 Similarly, the use of OKT3 to treat rejection is associated with a greater risk of recurrence of hepatitis C, but mainly greater risk of post-transplant fibrosis progression and graft loss.3,66,67 While the adverse effect of additional immunosuppression for the treatment of rejection on hepatitis C is demonstrated, the effect of agents used for induction or maintenance immunosuppression is less clear (Table 52-5).3,66,67 For example, there are data that suggest that complete avoidance of corticosteroids may be beneficial but there is also evidence that abrupt and early withdrawal of corticosteroids may be harmful to these patients. Similarly, the data with mycophenolate mofetil (MMF) and antibody induction are also confusing. In fact, results of the potential association between the type of administered immunosuppression and disease severity are difficult to prove due to the multiplicity of immunosuppressive regimens together with the changes in immunosuppressive drugs in individual patients over time. In addition, most studies are single-center studies, based on small sample sizes, retrospective in nature and hence unable to control for selection biases or for the presence of confounding variables. Data on calcineurin inhibitors are less confusing and overall show similar results with tacrolimus and cyclosporine. It has been suggested that the outcome is probably not related to the use of a specific drug but rather with the dose and drug level achieved, reflecting overall immunosuppression, and the way the drug is modified. It is interesting to speculate that a threshold on immunosuppression may exist that warrants absence of rejection while keeping the immune pressure to HCV at a significant level.
Genetic Background It is likely that it is the interplay between the immune system and the virus, modulated by the immunogenetic background, such as the
Table 52-5. Effect of Different Immunosuppressive Agents on Viral Replication and Disease Progression
Pulse steroids Maintenance steroidsa Ciclosporinb Tacrolimusb Azathioprine OKT3 Anti-IL2 receptor antibodies Sirolimus Mycophenolate mofetil MMF + IL-2 receptor antibodies
Mechanism of action
Effect on viral load
Effect on HCV-disease severity/ progression
Global anti-inflammatory and immunosuppressive actions Global anti-inflammatory and immunosuppressive actions Inhibition of early T-cell signal pathways and IL-2 production and release Same as ciclosporin (more potent) Inhibition of adenosine monophosphate production Antilymphocyte antibodies Inhibition of lymphocyte activation and clonal expansion Inhibition of lymphocyte proliferation, fibrosis and fibroblast proliferation Inhibition of inosine monophosphate dehydrogenase Addition of the two compound effects
Increase
Negative
Increase
Controversial
Decrease
Controversial
Unknown Unknown
Controversial Controversial
Unknown Unknown
Controversial Controversial
Unknown
Unknown
Increase
Controversial
Increase
Negative
HCV, hepatitis C virus; MMF, mycophenolate mofetil; IL-2, interleukin-2. a Greater doses or prolonged exposure to steroids appear to worsen recurrence in some studies. However, some authors have suggested that early abrupt steroid withdrawal may lead to immune-reconstitution, and in doing so, induce worsen graft injury through an immune-mediated attack on the virus. b Most studies on calcineurin inhibitors suggest that the risk of severe hepatitis C is not necessarily related to type of drug, but rather the dose, level, and overall level of immunosuppression.
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Chapter 52 MANAGEMENT OF RECURRENT VIRAL HEPATITIS B AND C
human leukocyte antigen (HLA) system, that shapes the outcome post-transplantation. In that sense, specific HLA class II alleles, such as HLA B14 and HLA DRB1*04 have emerged as possible modulators of disease severity3 and disease severity has been linked with HLA-B-sharing between the donor and the recipient in some, but not all, studies.3,51 In addition, preliminary reports highlight the potential importance of the immunogenetic background of the donor.51
Race Race has recently been found to influence outcome in patients with recurrent HCV infection, with non-Caucasians doing worse than Caucasians.56,62 However this association deserves further analysis.
Viral-Related Variables Post-Transplantation HCV RNA Levels Circulating HCV RNA levels following liver transplantation are typically 10–20-fold higher than levels prior to transplantation,65 without a clear association between levels of viremia and disease severity. High levels of viremia, however, have been described in the setting of fibrosing cholestatic hepatitis55 as well as during the acute phase of recurrent hepatitis C. These observations suggest that, while in many circumstances liver damage is immune-mediated, in some instances it is due to a direct cytopathic effect of HCV.
Pretransplantation HCV RNA Levels Several studies have shown that, as described for HBV, level of viremia pretransplantation predicts the occurrence and/or severity of recurrent hepatitis C.56,62
HCV Genotype The effect of the infecting genotype on the severity of liver disease post-transplantation is unclear.3,51 Some, but not all, studies have implicated genotype 1b in a more severe post-transplantation disease compared to non-1b genotype. Furthermore, a fast HCVrelated disease progression has been observed in centers with a high prevalence of genotype 1b, thus indirectly implicating this genotype in a more aggressive course of the disease. Preliminary data suggest that different strains belonging to genotype 1b may be involved in the pathogenesis of severe liver injury.69
HCV Diversity HCV heterogeneity may play a role in the pathogenesis of progressive HCV disease. The hypervariable region (HVR)-1, a putative target for neutralizing antibodies, is the most common region analyzed. Results from the few studies published to date are however inconclusive and somewhat discrepant, and may be related to the small number of patients included, the different methodologies applied to assess HCV heterogeneity, the type of end-point chosen, the region of the genome evaluated, and the definition of disease severity.3,55 Overall, severe fibrosis seems to be correlated with changes in HCV quasispecies composition. In one study, immunosuppressed HCV transplant recipients had fewer quasispecies and fewer amino acid sequence changes than untreated HCV patients, and the two patients with the least HCV variation died of HCV graft infection. Published data suggest that preferential replication of few genetically divergent quasispecies is enhanced by routine
immunosuppression, particularly in patients with fibrosing cholestatic hepatitis. The reduction in the number of HCV variants can be interpreted as indicative of a decreased immune pressure to the virus due to overall immunosuppression. In contrast, patients with mild recurrence show more HCV variants in the HVR1, but less genetic divergence. A recent study showed an association between HCV clearance post-transplantation and an increase in HCV HVR1 variants following steroid-tapering.55
Donor-Related Variables The age of the donor has been found to be independently associated with disease severity, disease progression, and survival.3,48,51,61,70,71 In one series, only 14% of the recipients who received an organ from a donor younger than 30 developed recurrent HCV-related cirrhosis. In contrast, 45% and 52% of those receiving the organ from donors aged 31–59 or older than 59 developed graft cirrhosis, respectively (P < 0.0001).48 The changing organ donor profile with increasing age of the donors in recent years may explain the observed worsening in outcomes over the same time period. This observation has important implications for donor liver allocation, in that older donors might be more appropriate for HCVnegative recipients in whom the adverse effects of donor age appear to be less deleterious. In addition, the effect of donor age correlates with data from the immunocompetent population where age at the time of infection is an important and powerful determinant of fibrosis development. Age-related changes in liver response may be the key factor that determines the increased susceptibility of the older liver to HCV-related fibrosis.
Coinfection with Other Viruses Patients who develop cytomegalovirus viremia may be at increased risk of severe HCV recurrence.63 The reasons for this association are unknown but likely relate to induction of immune deficiencies. Coinfection with other hepatotropic viruses such as HBV may influence histologic disease severity, but results are conflicting.3,51
Other Variables Prolonged rewarming time during allograft implantation has been associated with severe recurrent disease.3 If these data were confirmed, special emphasis should be paid to minimizing rewarming time. A lack of association has been found between the rate of fibrosis progression prior to transplantation and that observed after transplantation, suggesting that variables present at the time of transplantation and those related to post-transplantation management are more important in influencing disease progression than genetic or viral variables unique to the individual.56
HCV KINETICS AND PATHOGENESIS A rapid and sharp decline in viral load occurs immediately after removal of the infected liver.65,68 Following reperfusion, HCV RNA levels typically decrease further at a rate that exceeds the decrease observed during the previous anhepatic phase. HCV binding to and/or uptake by hepatocytes may contribute to this early posttransplant decrease in viremia. Following this initial decline, HCV RNA levels either increase exponentially, reaching pretransplantation levels as soon as day 4, or continue to decline in the first post-
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Section VIII. Liver Transplantation
transplant week to increase thereafter, peaking by the fourth postoperative month. These differences in kinetics appear to be related to the use of corticosteroids, so that HCV RNA levels increase rapidly in patients receiving corticosteroids as part of the immunosuppressive regime, while they continue to decrease in those not receiving this drug. At 1 year post-transplantation, the levels are 10–20-fold higher than pretransplantation. The mechanisms by which HCV leads to liver injury are incompletely understood. Several lines of indirect evidence support a role for the cellular immune response in shaping outcome following transplantation. It is likely that the increased levels of HCV replication in conjunction with the altered host immune responsiveness contribute to the pathogenesis of liver damage, particularly to the severe course of disease of the grafted liver.55 In addition, there is a spectrum of severity of recurrent HCV disease that may differ in pathogenesis and clearly differs in outcome. Recurrent chronic hepatitis C disease, which can lead to cirrhosis, should be distinguished from recurrent cholestatic HCV disease. In the former, it is possible that the relative antiviral control by the immune response prevents cytopathic injury while perpetuating chronic liver injury. In contrast, a direct cytopathic mechanism of liver injury appears to predominate in patients with severe cholestatic hepatitis.
HISTOLOGIC CHANGES: IMPORTANCE OF PROTOCOL LIVER BIOPSIES Liver function tests are not correlated with either viremia or with histologic disease severity3,51,72,73 and protocol liver biopsies are generally needed to identify progression to severe forms of chronic hepatitis. In addition, histological findings may be helpful in selecting the patients at high risk of disease progression. In particular, the degree of activity and fibrosis staging observed in the liver biopsy at 1 year are associated with the subsequent risk of developing cirrhosis, with only 3–10% of those with mild hepatitis developing cirrhosis compared to 30–60% in those with moderate to severe activity in their first-year liver biopsy. 3,50,60 Some specific histological features, including the presence of significant steatosis, ballooning degeneration, cholestasis, and confluent necrosis, are also helpful in predicting progression of disease. Finally, the presence of some degree of fibrosis at 3 years post-transplantation predicts the delayed onset of severe liver damage.57
LIVE-DONOR LIVER TRANSPLANTATION FOR HEPATITIS C-INFECTED PATIENTS Despite all the measures to improve the outcome of HCV-infected patients, the increased organ shortage has led to a dramatic increase in the number of patients on the waiting list and in those dying while waiting. The implementation of LDLT was believed to be a potential solution to this problem. The main unanswered question relates to the outcomes obtained with this new technique, and whether they are the same as achieved in recipients of deceased donors. To date, the question remains unanswered since results between centers are conflicting.74 While some studies have suggested that HCV recurs earlier and is associated with more severe hepatitis, other studies have not confirmed these data. However, the number of HCV-infected LDLT recipients to date is still limited, the posttransplant follow-up interval is short, and the available reports lack, in general, protocol liver biopsies. As a result, the data regarding the
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impact of LDLT on severity of HCV recurrence are inconclusive, as reviewed by the consensus group.53
TREATMENT Given the increasing number of patients progressing to HCV-related graft cirrhosis and the shortage of organ donors, development of strategies to improve the outcome of these recipients is mandatory. These strategies include an adequate timing of liver transplantation in patients with HCV-related end-stage liver disease, antiviral therapy, and the use of specific immunosuppression regimens. In terms of antiviral therapy, three potential alternative and/or complementary approaches may be attempted (Figure 52-2): (1) pre-emptive antiviral therapy as the patient is awaiting the availability of an organ donor; (2) early post-transplant antiviral therapy before histological damage has occurred; and (3) treatment of disease when and if it occurs. The goals of treatment and end-points for success of therapy may be different in these situations. The major end-point of therapy in patients awaiting transplantation may be the stabilization and/or improvement of hepatic function so that the need for surgery is delayed or even obviated. Alternatively, viral eradication or at least viral suppression is also a relevant goal, so that the risk of post-transplantation HCV recurrence and/or aggressive recurrent HCV disease is reduced. The major goal of pre-emptive post-transplantation therapy is to prevent reinfection of the graft, and in doing so, to reduce the incidence and potentially the severity of recurrent disease. Finally, the primary end-point in patients with established disease is viral eradication, since a sustained clearance of HCV RNA is associated with improvement in liver histology in most patients. Indeed, two long-term studies have shown that loss of HCV after treatment is durable (90–100% after 2–3 years of follow-up), and that the durability of the response is associated with improvement in hepatic inflammation (50 and 60% after 2 and 5 years of follow-up) and regression of fibrosis up to 67% after 3–5 years of follow-up.75,76 While the timing and aim of these alternatives are firmly established, their efficacy and tolerance are less clear. It is well known that current antiviral therapy based on interferon with ribavirin is poorly tolerated in both the pre- and post-transplant setting, therefore limiting its general application.
Prevention of Reinfection and/or HCV-Related Recurrent Disease Currently, there is no available intervention to prevent predictably HCV recurrence. In one study, polyclonal immunoglobulin containing anti-HCV was shown to decrease the incidence of recurrent HCV viremia measured 1 year post-transplantation.77 However, preliminary data analyzing the efficacy of anti-HCV immunoglobulin (HCIg) for prevention of HCV recurrence did not demonstrate clinical or virological benefits. Whether this negative study is a function of the dose of HCIg used, the timing of administration, or the type of preparation used (non-neutralizing antibodies) is unknown. It is also possible – if not likely – that the diverse quasispecies nature of HCV makes this virus inherently more difficult to neutralize than the more stable HBV virus populations present in any individual.
Pretransplantation Antiviral Therapy In theory, the rationale for treating patients with decompensated HCV-related cirrhosis is the same as for hepatitis B, that is: (1) to
Chapter 52 MANAGEMENT OF RECURRENT VIRAL HEPATITIS B AND C
slow down clinical disease progression and improve hepatic synthetic function, and in doing so, to reverse the complications of liver disease and obviate or delay the need for liver transplantation; and (2) to achieve an improvement in post-transplantation outcome by clearing the virus prior to transplantation. While the first aim is achievable in some patients with HBV-related decompensated cirrhosis, it is rarely achieved in patients infected with HCV once hepatic decompensation has occurred. In contrast, clearance of HCV prior to transplantation may prevent viral recurrence, and reduction of HCV RNA levels could potentially improve post-transplantation disease progression. Patients waiting for liver transplantation due to HCV-related liver disease typically include two types of patients: (1) those with compensated cirrhosis and HCC; and (2) those with decompensated cirrhosis. While in the former group it is likely that a complete course of antiviral therapy may be achieved with currently available drugs, it is less so in those with advanced hepatic insufficiency. Treatment of Compensated Cirrhosis Therapy of chronic hepatitis C has improved in recent years with the addition of ribavirin and development of pegylated (PEG) forms of interferon (IFN). Sustained virological responses are achieved in 54–56% of patients compared to 44–47% of those treated with standard interferon and ribavirin. Although tolerance is adequate, the response rate to these therapies appears to be lower in patients with compensated cirrhosis or transition to cirrhosis than in patients with less advanced liver disease (43–50% versus 57–65% in non-cirrhotic patients). Tolerability and side effects are similar to those observed with standard interferon, with the exception of cytopenias, particularly neutropenia, that are more frequently seen with the PEG-IFNs. These side effects may become a limitation when treating cirrhotic patients with marginal counts. The use of growth factors (i.e., erythropoietin and neutrophil-stimulating factor) may be helpful in some cases and avoid the reduction and/or discontinuation of antiviral drugs. Combination therapy with PEG-IFN and ribavirin is recommended in patients with cirrhosis provided that no contraindications are present.78 For genotype 1-infected patients, the optimal length of therapy and dose of ribavirin are probably 48 weeks and 1000–1200 mg; in contrast, for genotypes 2 and 3-infected patients, 24 weeks and 800 mg of ribavirin are probably sufficient. Treatment of Patients with Decompensated Cirrhosis (Table 52-6) Data regarding this alternative are limited to small uncontrolled case series.79 These studies emphasize the advantages and disadvantages of this approach. Although antiviral therapy may be successful in some cases, it should be administered with extreme caution due to
the increased risk of infectious complications and hepatic decompensation. In the first study, HCV-infected patients at or near the top of their respective waiting lists were randomly assigned to one of three treatment arms, two involving therapy with interferon in monotherapy, and one in combination with ribavirin. Less than half the patients screened met entry criteria, with thrombocytopenia and leukopenia being the most common reasons for exclusion. Eventually, only 15 patients from five large transplant centers were treated. Nine patients received interferon monotherapy while six received combination therapy with ribavirin (400 mg twice daily). Most patients had advanced liver disease with a mean Child–Pugh score of 12. While on treatment, loss of detectable HCV RNA was seen in 33%, but recurrence was seen in two transplanted patients. In addition, a significant number of adverse effects occurred (n = 23), many of which were considered severe. While thrombocytopenia was the most frequent adverse event, infection was the most severe one, leading to death in one patient. These side effects, particularly life-threatening infections, ultimately led to the early termination of the study. Several conclusions were drawn from this study: 1. a large proportion of patients awaiting transplantation will not benefit from this approach due to the presence of contraindications, particularly thrombocytopenia and neutropenia; 2. awareness of the potential complications should be kept in mind when treating the small proportion who meet initiation criteria. In the second study, 101 patients, 70% of whom were infected with HCV genotype 1, were treated with low doses of interferon (1.5 mU thrice weekly) and ribavirin (600 mg daily), with slow increases in dose of both drugs every 2 weeks as tolerated. Growth factors were administered as needed. Patients were relatively well compensated (50% were Child’s class A), with most having a low Child–Pugh score of less than 7–8. On treatment virological responses occurred in 38% and sustained virological response in 22% of patients. As for previous studies, sustained responses were more common in patients infected with genotypes other than 1. Interestingly, recurrent infection, which was observed in all patients with detectable HCV RNA at the time of transplantation, was prevented in the eight patients who were HCV RNA-negative at the time of transplantation. Although overall rates of severe adverse events were not reported, 28% of the patients had treatment discontinued because of the development of side effects, and serious complications occurred in 8% of the patients. Results from this study are more optimistic than those of the first published report, the
Table 52-6. Pretransplantation Antiviral Therapy with Interferon or Interferon + Ribavirin Author, year (number of patients/% genotype 1)
Child A (%)
Eligibility
VRa
Adverse events
Treatment D/C (%)
Prevention of recurrence
Crippin 200280 (n = 15/67%) Everson 200281 (n = 91/77%) Forns 200382 (n = 30/83%)
0%* 50% 50%
47% NA 62%
33% 38% 30%
87% NA 63%
100% 28% 20%
No 100% 67%
a
All patients had advanced liver disease (Child C). NA, not available; VR, hepatitis C virus RNA undetectable by polymerase chain reaction; D/C, discontinuation.
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Section VIII. Liver Transplantation
difference possibly related to the best hepatic function of most patients included in the latter study. In addition, lower doses were used at initiation. In the most recent study, patients on the waiting list for liver transplantation were considered for antiviral therapy if the expected time on the waiting list was shorter than 4 months, if there was not evidence of renal failure, and if patients met minimal blood count criteria. Of 50 patients evaluated, only 62% met entry criteria. The majority of these patients were infected with HCV genotype 1. At the time of inclusion, half of the patients were Child–Pugh A while the other half were Child B/C. The regimen used consisted of interferon-alpha-2b 3 MU/daily and ribavirin 800 mg/day. A virologic response was achieved in 30% of patients. Pretreatment viral load was significantly lower in responders than in non-responders (3 ¥ 105 versus 6.5 ¥ 105 IU/ml). Of the nine patients who were HCV RNA-negative at the time of transplantation, six remain free of infection after a median follow-up of 46 weeks. Although side effects and dose reductions were frequent, particularly due to cytopenias, no patients died while on therapy. However, treatment had to be discontinued in six patients due to thrombocytopenia (n = 4) and sepsis (n = 2). An association was found between viral load at 4 weeks of initiation of therapy and virologic response, an association that could be used in the clinical practice to guide discontinuations of therapy in those with minimal chances of obtaining viral clearance. The positive and negative predictive values of an early decrease in viral load (≥2 logs) were 82 and 100%, respectively. In summary, prevention of recurrence may be achieved in 20% of treated patients on the waiting list. The recommended dose is still unclear. Results were similar in terms of virological response in the three studies regardless of the regime used. Indeed, in two studies, therapy was started with low doses of interferon ± ribavirin, and doses were increased slowly as tolerated. In contrast, complete doses of both drugs were used in the third.
Post-Transplantation Antiviral Therapy Post-transplantation therapy may be started early following surgery or, alternatively, at a later stage, when disease is established. The major goal of pre-emptive post-transplantation therapy is to prevent reinfection of the graft, and, in so doing, to reduce the rate and/or severity of recurrent disease. In contrast, the major goal of treatment of established disease is to eradicate viremia, and, in so doing, to improve histology. Post-Transplantation Pre-emptive Therapy. Interferon alone or in combination with ribavirin has been attempted within the first 2
weeks after liver transplantation.83 The rate of sustained virological response achieved with interferon monotherapy ranges from 0 to 17%. Slightly better results may be achieved with interferon in combination with ribavirin (11–33%). However, side effects are common, leading to dose modifications or drug discontinuation in 28–50% and 47–85% of those treated with interferon or interferon–ribavirin, respectively. PEG-IFNs have also been attempted in this setting. In a recent multicenter US study, 54 transplant recipients were randomized within the first 3 weeks to receive 48 weeks of PEG-IFN-a2a or no treatment. Therapy was tolerated reasonably well, with only 8% of the patients discontinuing PEG-IFN due to side effects. However, efficacy was dismal, with a sustained virological response of only 8% in the treated group as opposed to 0% in the untreated arm.84 The applicability of this approach is rather limited since a substantial proportion of patients (40–60%) will not meet minimal criteria in the first 2–4 weeks post-transplantation to receive therapy with interferon ± ribavirin.
Treatment of Patients with Recurrent Hepatitis C Monotherapy has resulted in extremely disappointing results, with biochemical, virological, and/or histological response being rarely achieved with interferon or ribavirin as single agents.52,79 Although combination therapy probably yields the greatest potential benefit, results are still far from satisfactory (Table 52-7). Studies on therapy of recurrent hepatitis C are scarce, non-randomized, and generally based on small sample sizes.52,79,85,86 Sustained virological response achieved in studies of standard interferon with ribavirin ranges from 9% to 33%. Both dose adjustments (up to 78%) and drug discontinuations (30–50%), mainly due to ribavirin toxicity, are frequent. Although less common, severe adverse effects, particularly hepatic decompensation, may occur in treated patients (5% in previous studies). Treatment has also resulted in frequent hospital admissions, blood transfusions, as well as use of growth factors. The optimal duration of therapy is still unknown. Six versus 12 months of combination therapy were compared in 57 transplant patients.85 A sustained virological response was achieved in six of 27 patients treated for 6 months (22%) and in five of 30 patients treated for 12 months (17%) (P = 0.4). Response was better in those infected with genotype non-1 than in genotype 1, after both 6 and 12 months of therapy (43% versus 15% and 43% versus 9%, respectively), although the numbers of patients studied were too small to draw definitive conclusions regarding treatment duration.
Table 52-7. Combination Therapy with Interferon-Alpha or Pegylated Interferon and Ribavirin in Patients with Established Recurrent Hepatitis C Treatment regimen IFN + ribavirin Pegylated IFN Pegylated IFN + ribavirin
End-treatment virological response (%)
Sustained virological response (%)a
Histological improvement
Treatment reduction (%)
D/C (%)
35 35 37
22 19 25–30
70% 60% 50%
65 75 75
45 15 45
IFN, interferon; D/C, discontinuation. a Prognostic factors: sustained virological response significantly higher in hepatitis C virus genotype 2–3-infected patients, in those with low levels of viremia, and in those without advanced liver damage.
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Chapter 52 MANAGEMENT OF RECURRENT VIRAL HEPATITIS B AND C
In terms of dosage, some authors have suggested that starting therapy with low doses with subsequent increases as tolerated may yield better results in terms of patient tolerance and compliance than starting with full doses, although such an approach would be predicted to compromise an already low virological response. Regardless of the protocol used, tolerance remains poor in this setting, with 80% of the patients requiring dose reductions of one or both drugs. Many patients have renal insufficiency secondary to immunosuppressive agents, and because of impaired renal clearance of ribavirin, drug-associated hemolysis can be profound.86 Thus if ribavirin is initiated as part of combination therapy, the dose should be 600–800 mg, depending on renal function and presence of anemia. Given that anemia is the major side effect of therapy posttransplantation, erythropoietin would be predicted to improve the tolerability of ribavirin. In contrast to non-transplant patients, the natural history of recurrent hepatitis C may not necessarily improve after successful viral eradication.75,76 The cause of persistent hepatitis and absence of fibrosis regression in many transplant patients who achieve a sustained viral clearance remains speculative, and includes infections with cryptic viruses, persistence of HCV RNA in liver tissue,88 and chronic allograft rejection. Indeed, although problems with interferon-induced rejection are less evident with combination therapy than with monotherapy,89,90 there are preliminary data suggesting an increased risk of rejection following a successful course of antiviral therapy, possibly as a consequence of low levels of immunosuppression. It is suggested that antiviral therapy improves hepatocyte microsomal function which leads to decreased immunosuppression levels. Persistence of elevated liver enzymes despite viral clearance warrants routine follow-up liver biopsy in order to rule out posttreatment rejection. In summary, response to standard interferon and ribavirin is generally lower following liver transplantation than in immunecompetent patients. Reasons for this low response rate likely include all of the following: (1) high levels of viremia; (2) high prevalence of HCV genotype 1; (3) low tolerability of interferon and ribavirin leading to frequent dose reductions; (4) previous non-response to interferon; and (5) lower responses to HCV therapy in patients with impaired immune function. Indeed, preliminary data from kinetics studies have shown that viral decay in the first 24 hours after the injection of interferon is significantly lower in transplant patients compared to those who are immune-competent. In addition, the efficacy of therapy appears to be reduced in recipients with advanced recurrent hepatitis and reversal of fibrosing cholestatic hepatitis is highly unusual.91 With combination PEG-IFN–ribavirin, the rate of sustained eradication has slightly increased to 26–45%.88,92,93 Response is generally associated with improvement in histology, particularly necroinflammation and, to a lesser extent, fibrosis. The main predictors of response include genotype and the completion of the treatment course. Tolerance remains an issue, with up to 90% requiring dose reductions and 24–49% drug discontinuations. PEGIFN may yield a higher risk of inducing acute cellular rejection than standard interferon. Pegylation properties, including extended half-life and increased serum concentrations of interferon, could make it more likely to increase HLA expression, and therefore risk of rejection. Rejection associated with PEG-IFN therapy can
lead to serious consequences such as graft loss from resistant rejection.89 There are still many aspects that need to be addressed, such as the optimal dose and duration of therapy, whether ribavirin maintenance is required following interferon discontinuation, the potential benefit of using growth factors, and whether fibrosis progression may be slowed down by continuing interferon therapy in those who do not achieve a sustained virological response. In summary, each of the strategies has advantages and disadvantages, but all remain highly unsatisfactory (Table 52-6). Prophylactic therapy while the patient is awaiting transplantation is the best theoretical approach. It is however limited by the low applicability and low tolerance in patients with advanced liver disease. Preemptive post-transplantation therapy is another attractive approach from a theoretical point of view. Although effective in some patients and apparently not associated with a substantial increased rate of rejection, tolerance is problematic and benefits are low. In addition, with these two strategies treatment is offered to all patients while only a proportion will develop serious complications from recurrent infection. With the available drugs, treatment of the established disease is probably the most attractive option.94 Although still limited by a low efficacy, tolerance appears to be better than when these drugs are given pre-emptively. Treatment should be offered preferentially to patients who develop histological progressive liver disease. Protocol liver biopsies perhaps at yearly intervals will identify early histologic changes that herald a progressive disease. A major barrier to improved response is the frequent occurrence of untoward effects requiring dose reduction or even cessation of therapy. In conclusion, pretransplantation antiviral therapy with PEG-IFN and ribavirin may be tried in patients with Child A cirrhosis, especially in those infected with genotypes 2 and 3. In the early posttransplantation period, pre-emptive treatment before development of disease cannot be advocated because PEG-IFN plus ribavirin is very poorly tolerated. In patients with established disease, treatment should be initiated, if no contraindications are present, once portal fibrosis and/or moderate necroinflammation is detected. Protocol liver biopsies at intervals may be necessary to identify such patients. Optimal dose and duration of therapy are unknown. While we wait for studies to define the best regimen, most clinicians follow the same guidelines that are used in immune-competent patients. In transplant patients, though, therapy may need to be indefinite in those with the most severe forms of recurrent hepatitis.91
ALTERNATIVE APPROACHES Since the efficacy of antiviral therapy is limited in HCV-infected recipients, selection of patients who will be at low risk for severe recurrence and optimal management of long-term immune suppression will likely be important in improving long-term outcomes. Some authors have developed predictive models of severe outcome based on simple variables, including age of the donor and type of immunosuppression.61 Validation of these models is however required before they can be generalized. Given the deleterious effect of intense immune suppression on the progression of recurrent HCV disease, an adequate management of immunosuppression appears mandatory in these patients. As a general rule, the optimal strategy should be to achieve a balance
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Section VIII. Liver Transplantation
between prevention of acute and chronic rejection while minimizing the adverse effects of immunosuppression on recurrent hepatitis C. Based on the available information, the following recommendations were made at the recent consensus conference:53,66,67 (1) induction immunosuppression should be performed with two drugs or reduced doses of one or more agents when using triple-drug therapy; and (2) when rejection is diagnosed histologically, the first approach should be to increase the dose(s) of the agents used for maintenance immunosuppression while avoiding bolus corticosteroids or T-cell-depleting agents. Additionally, when doubts exist between rejection and hepatitis C because of overlapping histological findings, serial biopsies should be performed to clarify the clinical picture, rather than therapeutic trials of steroids. Features more suggestive of HCV infection include lymphoid aggregates, fatty changes, and sinusoidal dilatation, while those more suggestive of rejection include endotheliitis, bile duct necrosis, and a mixed portal inflammatory infiltrate (eosinophils and neutrophils as well as mononuclear cells).
insufficiency, and as a result, most patients with recurrent allograft failure due to hepatitis C will only receive an organ at a point when they are unlikely to survive retransplantation. Thus, while retransplantation may be a reasonable option in low-risk patients, it is unlikely to be a feasible option unless a live donor is available (see below). Whether retransplantation is justified in patients with several variables associated with poor outcome needs carefully consideration. If retransplantation is performed, therapeutic strategies, including different immunosuppressive protocols and use of antivirals, should be implemented despite the frequent coexistence of comorbidities and development of side effects. Additional important aspects to be considered and that were recommended at the consensus conference53 are: (1) the presence of cholestatic HCV disease should preclude retransplantation other than in exceptional circumstances; (2) additional transplantation beyond the second graft for recurrent hepatitis should be discouraged; (3) there are minimal data concerning the role of retransplantation in living donor recipients with recurrent HCV disease and no recommendations could be made.
RETRANSPLANTATION Retransplantation is the last option for patients with failing grafts due to recurrent disease. The results of retransplantation are inferior to those reported for first transplants. As predicted from natural history studies, the prevalence of HCV infection in patients undergoing retransplantation has progressively increased in most transplant centers (from 6.5% in 1990 to 38.4% in 1995), reaching a plateau thereafter.95 It has thus become imperative to determine whether all patients with graft failure due to recurrent HCV disease are candidates for further transplantation, or whether there is a subset in whom the outcomes would be so poor that retransplantation should not be undertaken.96 Early reports have suggested poor outcomes and with increasing shortage of organ donors, retransplantation is likely not the most beneficial use of a limited resource. In addition, the severity of recurrent HCV disease in the first graft may predict the severity of recurrence in the second graft.101 While we wait for a consensus, it has become apparent that this procedure is becoming less common at many centers. The fear with retransplantation, particularly in those with early severe recurrence, is related to four major aspects: 1. early reports suggesting a worse outcome following retransplantation in HCV-infected recipients than in those uninfected; 2. uncertainty regarding the natural history of recurrent hepatitis C in the second graft; 3. frequent comorbidities in these patients who generally have an advanced age by the time they require retransplantation; 4. increased organ shortage. Most series have shown that the outcome is generally poor, significantly worse than that obtained with retransplantation for other causes of late graft loss.95,96–100 Most cases of death occur in the first 6 months and are due to sepsis. However, it has also been shown that the outcome may be improved if performed before significant renal impairment and hepatic failure develop,98–101 and with the use of younger donors.98 Unfortunately, under the current MELD organ allocation system, patients have no realistic hope of receiving an organ until they have developed significant coagulopathy and renal
990
CONCLUSION Viral hepatitis is the leading indication for liver transplantation in the majority of transplant centers. Post-transplantation outcome in these patients largely depends on the prevention of allograft reinfection. In contrast to hepatitis B, where excellent results have been achieved following the implementation of effective interventions to prevent and to treat disease, recurrent hepatitis C is an increasing problem facing liver transplant hepatologists and surgeons. Currently no effective prophylactic therapy is available for hepatitis C so that recurrent hepatitis C occurs invariably. Progression to severe allograft fibrosis is often rapid. Current antivirals, including PEGIFNs, carry with them substantial toxicities that compromise efficacy. Hence, it is not surprising that, although some improvements have been made in the treatment of recurrent hepatitis C, a substantial proportion of HCV-infected patients develop recurrent allograft end-stage liver disease, leading to decreased graft survival, increased need for retransplantation, and ultimately, decreased patient survival. Only therapies that are not yet available are likely to change this picture.
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recurrence after liver transplantation. J Hepatol 2003;38:811–817. Berenguer M, Wright TL. Treatment of recurrence of hepatitis B in transplant patients. J Hepatol 2003; 39: S190–S193. Perrillo R, Schiff E, Yoshida E, et al. Adefovir dipivoxil for the treatment of lamivudine-resistant hepatitis B mutants. Hepatology 2000; 32:129–134. Bock CT, Tillmann HL, Torresi J, et al. Selection of hepatitis B virus polymerase mutants with enhanced replication by lamivudine treatment after liver transplantation. Gastroenterology 2002; 122:264–273. Roche B, Samuel D, Feray C, et al. Retransplantation of the liver for recurrent hepatitis B virus infection: the Paul Brousse experience. Liver Transpl Surg 1999; 5:166–174. Davis GL, Albright JE, Cook SF, Rosenberg DM. Projecting future complications of chronic hepatitis C in the United States. Liver Transpl 2003; 9:331–338. Berenguer M, Prieto M, San Juan F, et al. Contribution of donor age to the recent decrease in patient survival among HCV-infected liver transplant recipients. Hepatology 2002; 36:202–210. Forman LM, Lewis JD, Berlin JA, et al. The association between hepatitis C infection and survival after orthotopic liver transplantation. Gastroenterology 2002; 122:889–896. Gane E. The natural history and outcome of liver transplantation in hepatitis C virus-infected recipients. Liver Transpl 2003; 9 (Suppl 3):S28–S34. Berenguer M. What determines the natural history of recurrent hepatitis C? J Hepatol 2005; 42:448–479. Berenguer M, Wright LT. Treatment strategies for hepatitis C: intervention prior to liver transplant, pre-emptively or after established disease. Clin Liver Dis 2003; 7:631–650. Wiesner RH, Sorrell M, Villamil F. Report of the First International Liver Transplant Society consensus conference on liver transplantation and hepatitis C. Liver Transpl 2003; 9 (Suppl 3):S1–S9. Feray C, Caccamo L, Alexander GJM, et al. European collaborative study on factors influencing the outcome after liver transplantation for hepatitis C. Gastroenterology 1999; 117:619–622. McCaughan GW, Zekry A. Mechanisms of HCV reinfection and allograft damage after liver transplantation. J Hepatol 2004; 40:368–374. Berenguer M, Ferrell L, Watson J, et al. HCV-related fibrosis progression following liver transplantation: increase in recent years. J Hepatol 2000; 32:673–684. Berenguer M, Aguilera V, Prieto M, et al. Delayed onset of severe hepatitis C-related liver damage following liver transplantation: a matter of concern? Liver Transpl 2003; 9:1152–1158. Berenguer M, Ferrell L, Watson J, et al. HCV-related fibrosis progression following liver transplantation: increase in recent years. J Hepatol 2000; 3:673–684. Neumann UP, Berg T, Bahra M, et al. Long-term outcome of liver transplants for chronic hepatitis C: a 10-year follow-up. Transplantation 2004; 77:226–231. Prieto M, Berenguer M, Rayón M, et al. High incidence of allograft cirrhosis in hepatitis C virus genotype 1b infection following transplantation: relationship with rejection episodes. Hepatology 1999; 29:250–256. Berenguer M, Crippin J, Gish R, et al. A model to predict severe HCV-related disease following liver transplantation. Hepatology 2003; 38:34–41. Charlton M, Seaberg E, Wiesner R, et al. Predictors of patient and graft survival following liver transplantation for hepatitis C. Hepatology 1998; 28:823–830. Burak KW, Kremers WK, Batts KP, et al. Impact of cytomegalovirus infection, year of transplantation, and donor age on outcomes after liver transplantation for hepatitis C. Liver Transpl 2002; 8:362–369.
64. Berenguer M, Prieto M, Rayón JM, et al. Natural history of clinically compensated HCV-related graft cirrhosis following liver transplantation. Hepatology 2000; 32:852–858. 65. Charlton M. Liver biopsy, viral kinetics, and the impact of viremia on severity of hepatitis C virus recurrence. Liver Transpl 2003; 9 (Suppl 3):S58–S62. 66. McCaughan GW, Zekry A. Impact of immunosuppression on immunopathogenesis of liver damage in hepatitis C virusinfected recipients following liver transplantation. Liver Transpl 2003; 9 (Suppl 3):S21–S27. 67. Lake JR. The role of immunosuppression in recurrence of hepatitis C. Liver Transpl 2003; 9:S63–S66. 68. Garcia-Retortillo M, Forns X, Feliu A, et al. Hepatitis C virus kinetics during and immediately after liver transplantation. Hepatology 2002; 35:680–687. 69. Lopez-Labrador FX, Berenguer M, Sempere A, et al. Genetic variability of hepatitis C virus NS3 protein in human leukocyte antigen-A2 liver transplant recipients with recurrent hepatitis C. Liver Transpl 2004; 10:217–227. 70. Wali M, Harrison RF, Gow PJ, Mutimer D. Advancing donor liver age and rapid fibrosis progression following transplantation for hepatitis C. Gut 2002; 51:248–252. 71. Machicao VI, Bonatti H, Krishna M, et al. Donor age affects fibrosis progression and graft survival after liver transplantation for hepatitis C. Transplantation 2004; 77:84–92. 72. Berenguer M, Rayón M, Prieto M, et al. Are post-transplantation protocol liver biopsies useful in the long-term? Liver Transpl 2001; 7:790–796. 73. Sebagh M, Rifai K, Feray C, et al. All liver recipients benefit from the protocol 10-year liver biopsies. Hepatology 2003; 37:1293–1301. 74. Sugawara Y, Makuuchi M. Should living donor liver transplantation be offered to patients with hepatitis C virus cirrhosis? Second forum in liver transplantation. J Hepatol 2005; 42. 75. Bizollon T, Admed SNS, Radenne S, et al. Long-term histologic improvement and clearance of intrahepatic hepatitis C virus RNA following sustained response to interferon-ribavirin combination therapy in liver transplant patients with hepatitis C recurrence. Gut 2003; 52:283–287. 76. Abdelmalek MF, Firpi RJ, Soldevila-Pico C, et al. Sustained viral response to interferon and ribavirin in liver transplant recipients with recurrent hepatitis C. Liver Transpl 2004; 10:199–207. 77. Feray C, Gigou M, Samuel D, et al. Incidence of hepatitis C in patients receiving different preparations of hepatitis B immunoglobulins after liver transplantation. Ann Intern Med 1998; 128:810–816. 78. Wright TL. Treatment of patients with hepatitis C and cirrhosis. Hepatology 2002; 36 (Suppl 1):S185–S194. 79. Garcia-Retortillo M, Forns X. Prevention and treatment of hepatitis C virus recurrence after liver transplantation. J Hepatol 2004; 41:2–10. 80. Crippin JS, Sheiner P, Terrault NA, et al. A pilot study of the tolerability and efficacy of antiviral therapy in patients awaiting liver transplantation for hepatitis C. Liver Transpl 2002; 8: 350–355. 81. Everson GT. Treatment of patients with hepatitis C virus on the waiting list. Liver Transpl 2003; 9 (11):S90–S94. 82. Forns X, Garcia-Retortillo M, Serrano T, et al. Antiviral therapy of patients with decompensated cirrhosis to prevent recurrence of hepatitis C after liver transplantation. J Hepatol 2003; 39: 389–396. 83. Terrault N. Prophylactic and preemptive therapies for hepatitis C-virus infected patients undergoing liver transplantation. Liver Transpl 2003; 9 (Suppl 3):S95–S100. 84. Chalasani N, Manzarbeitia C, Teperman L, et al. Peginterferon alfa 2a for recurrence of hepatitis C after liver transplantation: two randomised controlled trials. Hepatology 2005; 41:289–298.
Chapter 52 MANAGEMENT OF RECURRENT VIRAL HEPATITIS B AND C
85. Lavezzo B, Franchello A, Smedile A, et al. Treatment of recurrent hepatitis C in liver transplants: efficacy of a six versus twelve month course of interferon alfa 2b with ribavirin. J Hepatol 2002; 37:247–252. 86. Samuel D, Bizollon T, Feray C, et al. Interferon-alpha 2b plus ribavirin in patients with chronic hepatitis C after liver transplantation: a randomized study. Gastroenterology 2003; 124:642–650. 87. Jain AB, Eghtesad B, Venkataramanan R, et al. Ribavirin dose modification based on renal function is necessary to reduce hemolysis in liver transplant patients with hepatitis C virus infection. Liver Transpl 2002; 8:1007–1013. 88. Neff GW, O’Brien CB, Cirocco R, et al. Prediction of sustained virological response in liver transplant recipients with recurrent hepatitis C virus following combination pegylated interferon alfa2b and ribavirin therapy using tissue hepatitis C virus reverse transcriptase polymerase chain reaction testing. Liver Transpl 2004; 10:595–598. 89. Saab S, Kalmaz D, Gajjar NA, et al. Outcomes of acute rejection after interferon therapy in liver transplant recipients. Liver Transpl 2004; 10:859–867. 90. Todd-Stravitz R, Shiffman ML, Sanyal AJ, et al. Effects of interferon treatment on liver histology and allograft rejection in patients with recurrent hepatitis C following liver transplantation. Liver Transpl 2004; 10:850–858. 91. Gopal DV, Rosen HR. Duration of antiviral therapy for cholestatic HCV recurrence may need to be indefinite. Liver Transpl 2003; 9:348–353. 92. Dumortier J, Scoaxec JY, Chevallier P, Boillot O. Treatment of recurrent hepatitis C after liver transplantation: a pilot study of
93.
94.
95.
96.
97.
98. 99.
100.
101.
peginterferon alfa 2b and ribavirin combination. J Hepatol 2004; 40:669–674. Rodríguez-Luna H, Khatib A, Sharma P, et al. Treatment of recurrent hepatitis C infection after liver transplantation with combination of pegylated interferon alfa 2b and ribavirin: an open label series. Transplantation 2004; 77:190–194. Saab S, Ly D, Han SB, et al. Is it cost-effective to treat recurrent hepatitis C infection in orthotopic liver transplantation patients? Liver Transpl 2002; 8:449–457. Watt KD, Lyden ER, McCashland TM. Poor survival after liver retransplantation: is hepatitis C to blame? Liver Transpl 2003; 9:1019–1024. Biggins SW, Beldecos A, Rabkin JM, Rosen HR. Retransplantation for hepatic allograft failure: prognostic modeling and ethical considerations. Liver Transpl 2002; 8:313–322. Berenguer M, Prieto M, Palau A, et al. Severe recurrent hepatitis C following liver retransplantation for HCV-related graft failure. Liver Transpl 2003; 9:228–235. Roayaie S, Schiano TD, Thung SN, et al. Results of retransplantation for recurrent hepatitis C. Hepatology 2003; 38:1428–1436. Facciuto M, Heidt D, Guarrera J, et al. Retransplantation for late liver graft failure: predictors of mortality. Liver Transpl 2000; 6:174–179. Ghobrial RM. Retransplantation for recurrent hepatitis C in the model for end-stage liver disease era: how should we or shouldn’t we? Liver Transpl 2003; 9:1025–1027. Rosen H, Prieto M, Casanovas-Taltavull T, et al. Validation and refinement of survival models for liver retransplantation. Hepatology 2003; 38:460–469.
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MANAGEMENT OF RECURRENT NON-VIRAL CONDITIONS
53
James Neuberger Abbreviations AIH autoimmune hepatitis AMA antimitochondrial antibodies ERC endoscopic retrograde cholangiography
HLA MRC NAFLD
human leukocyte antigen magnetic resonance cholangiography non-alcoholic fatty liver disease
INTRODUCTION Recognition that the allograft may be affected by the same disease process that resulted in the failure of the native liver is of both clinical and academic importance. The recipient needs to be counseled about the possibility of recurrence and the possible impact on graft function and survival; the clinician needs to be aware of the potential of recurrence, to interpret the clinical, laboratory, radiological, and histological findings appropriately and, where appropriate, alter management. Finally, understanding which conditions recur in the allograft and those factors that are associated with recurrence may shed light on the pathogenesis of the disease. In this chapter, recurrence of non-viral and non-malignant diseases will be discussed, together with diagnosis and management.
DIAGNOSIS OF RECURRENT DISEASE IN THE ALLOGRAFT The criteria for the diagnosis of disease in the native liver may not be applicable in the allograft. The difference between the liver and host human leukocyte antigen (HLA) and other antigens and the effects of immunosuppression may modify the pattern of recurrent disease. Furthermore, the clinical, serological, and histological features of recurrent disease may be mimicked by other causes of graft damage. Thus the diagnosis of recurrent disease is often not straightforward. It is important that stringent criteria for the diagnosis of recurrent disease are agreed and followed (Table 53-1).
AUTOIMMUNE DISEASES Primary Biliary Cirrhosis (PBC) Diagnosis The criteria for diagnosis of recurrent PBC are shown in Table 532. Thus, the diagnosis is made primarily on the basis of histology. As with PBC in the native liver, the diagnostic histologic features of recurrent PBC may be found in the presence of normal liver tests. It is important to recognize that antimitochondrial antibodies (AMA), which, in the native liver, may antedate the clinical, biochemical, and histological features of PBC, will persist after liver
PBC PSC UDCA
primary biliary cirrhosis primary sclerosing cholangitis ursodeoxycholic acid
transplantation. The aberrant distribution of the dihydrolipoamide acetyl transferase (the antigen recognized by the AMA) in the biliary epithelial cells that is characteristic of PBC in the native liver may be seen in the graft as early as 7 days after transplantation. Titers of AMA may show a transient fall and then return to or exceed levels seen pretransplantation. Similarly, serum immunoglobulins may be elevated, especially the serum immunoglobulin M, and again this does not correlate with recurrence.1
Incidence The reported incidence of recurrent PBC varies in the literature; this variation is in part dependent on the criteria used to define PBC and whether protocol biopsies are used in the center. Histological features of recurrent PBC may be found in the presence of normal liver tests. Our own data (Figure 53-1) show that PBC recurs in 17% of patients2 at an overall median time to detection of recurrence of about 3 years.3 The prevalence of recurrent PBC is between 8 and 20% at 5 years and 20–30% at 10 years.2,4,5 Our recent studies suggest that immunosuppression with ciclosporin is associated with a longer time to recurrence compared with tacrolimus (median time to diagnosis of recurrence was 62 months for those on tacrolimus and 123 months on ciclosporin).6
Treatment There is no definitive treatment for those with recurrent PBC. However, most centers offer treatment with ursodeoxycholic acid (UDCA) 10–15 mg/kg per day as this bile acid is recommended for treatment in those with PBC in the native liver. There is no evidence whether this alters the natural history of recurrent disease. UDCA is well tolerated and may affect absorption of other medication; this has not been a significant problem in practice. Given the observation that recurrence is more rapid in those on tacrolimus compared to those on ciclosporin medication, it may be that switching between calcineurin inhibitors may be of benefit. Likewise, both azathioprine and mycophenolate have been suggested to be of benefit in the native liver in slowing progression, so modification of the immunosuppressive regime may be of benefit.
Outcome In the short to medium term, recurrent disease does not affect patient or graft survival. In our own series of 400 patients, only two developed graft failure over a 10-year follow-up.
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Table 53-1. Some causes of graft damage that may complicate the diagnosis of recurrent disease in the allograft
0.8 Recurrence of PBC
Infection Drug toxicity Graft damage Immune-mediated Acute rejection Chronic rejection De novo autoimmune disease Non-immune-mediated Ischemia Reperfusion Infection Viral Bacterial Mycobacterial
1.0
0.6 0.4 0.2 0.0 –0.2 –20
0
20
40
60
80
100
120
Months Table 53-2. Criteria for the diagnosis of recurrent primary biliary cirrhosis (PBC) Transplantation for PBC Characteristic histologic features of PBC Mononuclear inflammatory infiltrates Lymphoid aggregates Epithelioid granulomas Bile duct damage Persistence of antimitochondrial antibodies Elevated immunoglobulins Exclusion of other causes of graft damage Note: normal liver tests do not exclude the diagnosis of recurrent PBC
Autoimmune Hepatitis (AIH) Diagnosis The criteria for the diagnosis of recurrent AIH are shown in Table 53-3.7 It should be noted that all criteria should be met to establish the diagnosis. None of the features of recurrent AIH is specific to the diagnosis and all may be found in other conditions. Few studies have been done to look at the target antigens in recurrent AIH but one study suggested that the immune response was directed to a graft rather than host antigen,8 raising the possibility that this is, in fact, rejection rather than a true autoimmune recurrence. Application of the scoring systems developed to define AIH in the native liver to the transplant situation is not validated and therefore this system should not be used in the diagnosis of recurrent AIH.
Incidence The incidence of recurrent AIH is difficult to determine as few series have used stringent criteria to make the diagnosis.9–12 Our own series of 93 patients suggests that 13 have developed recurrent disease and of these, three have developed graft failure as a consequence.13
Treatment The approach to treatment is generally to increase immunosuppression. The first approach is to add corticosteroids (such as prednisolone 20 mg/day). If this does not result in resolution of the serological and histological features, then, for those on ciclosporinbased treatment, switching to tacrolimus should be considered.14
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Figure 53-1. Risk of survival free of recurrence of primary biliary cirrhosis confirmed histologically in patient receiving ciclosporin and tacrolimus (data from Birmingham, UK).
Table 53-3. Criteria for the diagnosis of recurrent autoimmune hepatitis (after Manns18) Liver transplant for autoimmune hepatitis Autoantibodies in significant titer (1.2 ¥ 109 gEq/ml) HCV RNA-positive HCV RNA-positive and HIV positive HBsAg-positive and HDV-positive Active infection at time of birth
> 85%
1. Immune serum globulin and hepatitis A vaccine after delivery 2. Hepatitis A vaccine repeated at 5–6 months of age 1. HBIG and vaccine at birth 2. Vaccine repeated at 1 and 6 months of life 1. HBIG and vaccine as above 2. Consider lamivudine for mother before delivery None None Same as hepatitis B ? Immune serum globulin
Hepatitis C Hepatitis D Hepatitis E
8.5% Up to 30% Similar to hepatitis B 50–100%
HBIG, hepatitis B immunoglobulin; HCV, hepatitis C virus; HVD, hepatitis D virus.
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ALCOHOL AND PREGNANCY The unique aspect of alcohol ingestion during pregnancy is fetal involvement and the fetal alcohol syndrome. This syndrome generally includes facial abnormalities, congenital malformations, growth retardation, and central nervous system dysfunction. Liver involvement has also been found in some affected infants,72–74 including hepatomegaly and elevated serum levels of transaminases and alkaline phosphatase. Liver histology is usually abnormal, with varying degrees of fatty infiltration, centrizonal hepatocyte degeneration, perivenular sclerosis, portal and perisinusoidal fibrosis, and proliferation of bile ducts. One affected child exhibited cirrhosis and esophageal varices by the age of 8.74 These reports suggest that maternal ethanol intake can cause chronic liver disease in offspring. Analysis of additional cases will be needed to define the spectrum of liver pathology.
CHRONIC LIVER DISEASE Impact of Chronic Liver Disease on Pregnancy Women with well-controlled mild chronic hepatitis and normal liver function appear to have normal fertility and to tolerate pregnancy well without adverse fetal or maternal outcomes.75 However, women with active liver disease, significant liver dysfunction, and/or cirrhosis exhibit decreased fertility76–78 and may experience liver deterioration during pregnancy and have higher rates of spontaneous abortion, premature birth, and perinatal death. Indeed, women with alcoholic liver disease often exhibit severe and irreversible gonadal failure, amenorrhea, and infertility, and rarely become pregnant. Infants born alive, however, are generally normal and do well, although mothers with clinically significant liver disease are more likely to die before their children reach adulthood. Contraceptive options for women with chronic liver disease include sterilization, barrier methods, and progestin-containing contraceptives.79 Pregnancy-related issues for specific liver diseases are outlined below.
Autoimmune Hepatitis Women with autoimmune hepatitis treated with immunosuppressive therapy are surviving for longer periods of time and on therapy many regain fertility and some become pregnant. In general, women with well-controlled autoimmune hepatitis receiving immunosuppressive therapy appear to tolerate pregnancy fairly well.76,80–84 Modest deteriorations in liver tests, particularly the serum bilirubin and alkaline phosphatase, may occur. These changes in tests usually return to the patient’s previous baseline values after delivery and most likely represent the imposed cholestatic effects of pregnancy. Reports of severe flares, liver failure, and even death in women who stopped immunosuppressive therapy during pregnancy or who were not on therapy during pregnancy indicate that successful therapy should not be stopped during pregnancy and that patients need continued monitoring.76,81,84 Whether these flares are due to pregnancy per se is not known, and remission during pregnancy has also been reported.81–83,85 Autoimmune hepatitis is associated with increased fetal morbidity and mortality: 15 spontaneous abortions and 9 perinatal deaths were reported in 128 pregnancies.76,80–85 The infants born alive were healthy and did well. With use of immunosuppressive therapy, including azathioprine, it appears that women with autoimmune hepatitis can conceive and deliver healthy
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children with relative safety.81,82 Postpartum, women should be monitored closely as disease flares may also occur in the first few months after delivery, likely related to immune reactivation.81,82,84,85 Finally, autoimmune hepatitis may present in the early postpartum period.86
Wilson’s Disease Chelation therapy has allowed patients with Wilson’s disease to survive in good health into and through the reproductive years. Many such patients become pregnant and bear children. Amenorrhea, infertility, and spontaneous abortions are common in symptomatic untreated women (due in part to high tissue copper levels as well as to the effects of liver dysfunction), but therapy restores fertility and allows a normal reproductive life.87–89 Some women with Wilson’s disease, although satisfactorily treated with chelators, have liver disease, including cirrhosis, that antedates initiation of therapy. These women have increased fetal and maternal morbidity and mortality. In both normal women and women with Wilson’s disease, concentrations of copper and ceruloplasmin in serum and urine increase during pregnancy or use of estrogens.15,87,90 In women with Wilson’s disease, ceruloplasmin and copper concentrations in sera may double by the third trimester of pregnancy. The former may increase into the low-normal range. Currently patients are treated lifelong with D-penicillamine, trientine (triethylene tetramine dihydrochloride), and/or zinc.87,89,91 Discontinuing therapy with D-penicillamine during pregnancy has been associated with symptomatic, and occasionally fatal, flare-ups of disease activity.89,92 Although D-penicillamine is potentially teratogenic, in 153 babies born to 111 mothers receiving the drug for Wilson’s disease, there were only 2 miscarriages, 3 premature births, 1 baby with a chromosomal defect, and 1 with cleft palate.89 Trientine and zinc appear to be similarly well tolerated during pregnancy.87,89,91 It is recommended that treatment with D-penicillamine or trientine (0.75–1 g/day during the first two trimesters and 0.5 g/day during the third trimester) or zinc be continued throughout pregnancy.87,89,91,93 Also, because of the antipyridoxine effects of D-penicillamine, oral supplementation with pyridoxine is recommended.
Cirrhosis Women with cirrhosis can and occasionally do become pregnant, although pregnancy in these women is uncommon.77,78 Reports of at least 156 pregnancies in 125 women with cirrhosis of varying etiology have been published.80,94–107 Evaluating the actual risk of hepatic complications during pregnancy is difficult, however, as only one study94 identified a potential control group of non-pregnant, cirrhotic women. Similarly, few authors have compared rates of obstetric complications in women with cirrhosis to rates in women without liver disease. During the course of pregnancy, liver tests (most commonly serum bilirubin and alkaline phosphatase activity) were reported to deteriorate in 30–40% of cirrhotic women,80,95–97 but in two-thirds of these cases, postpartum tests returned to baseline values. Much of this apparent deterioration may, in fact, reflect the cholestatic effect of pregnancy.
Chapter 54 THE LIVER IN PREGNANCY
Maternal morbidity and mortality are high during pregnancy (10.5% mortality in the 115 reported cases). Development of jaundice, ascites, hepatic encephalopathy, and postpartum hemorrhage are also common (Table 54-4). Maternal deaths are primarily due to gastrointestinal hemorrhage from varices, with liver failure accounting for many of the remaining deaths (Table 54-4). This degree of morbidity and mortality may not differ greatly from the natural history of cirrhosis in these women. Borhanmanesh and Haghighi94 noted, over a 40-month period, 2 deaths among 9 pregnant cirrhotic women and 3 deaths among 12 age-matched, non-pregnant, cirrhotic women. Bleeding from esophageal varices occurs in 18–32% of pregnant women with cirrhosis, but in up to 50% of women known to have portal hypertension.97,108 Patients with a past history of variceal bleeding may or may not bleed again during pregnancy97,99–101 and the risk is reduced after successful portosystemic shunting (Table 54-4). During or before pregnancy it is reasonable to screen for varices to estimate the risks of bleeding and, if varices are large, consider use of beta-blockers or of prophylactic banding. However, there are no data to indicate whether prophylactic treatment of varices with propranolol, sclerotherapy, or banding reduces bleeding and mortality during pregnancy. Variceal bleeding can be treated with octreotide infusion, sclerotherapy, banding, placement of a transjugular intrahepatic portosystemic shunt (TIPS) or by surgical portosystemic shunting.95,97,99–102,105,106,108 Balloon tamponade may also be used. Finally, although elective delivery by cesarean section has been recommended to avoid the strain of labor and risk of precipitating variceal hemorrhage, there is no evidence that vaginal deliveries precipitate hemorrhage and large intra-abdominal collateral veins may complicate surgical delivery.97,107 The effects of cirrhosis on the fetus are varied. First, before or during pregnancy, risks of maternal medications on the fetus should be considered. Spironolactone, which can cause genital malformations, should be stopped; propranolol, which might impair fetal growth, should be considered; and the need for other medications should be reassessed. Second, the rates of spontaneous abortion,
Table 54-4. Pregnancy and Cirrhosisa Features Variceal hemorrhage Maternal death Percentage of deaths from: Gastrointestinal hemorrhage Liver failure Other Complications Jaundice Ascites Hepatic encephalopathy Spontaneous abortions Premature births Prenatal deaths Postpartum hemorrhage a
Shunted (29 women)
Not shunted (90 women)
0 4%
18–32% 13%
0 100% 0
40–70% 15–25% 10–35%
NA NA NA 3% NA 17% 24%
28% 17% 4% 17% 23% 20% 9%
Data compiled from case reports and series published from 1968 to 1999 (representative references: 94,97,101,103). NA, not available.
premature birth, and perinatal death are all greater than expected in women with cirrhosis (Table 54-4). Infants born alive, however, are generally normal and do well. Third, fetal distress and perinatal mortality may be due, in part, to maternal hepatic decompensation and its attendant metabolic abnormalities. For example, severe maternal hyperbilirubinemia (16 and 33 mg/dl) has been reported109,110 to result in severely jaundiced infants due to maternal-to-infant placental transfer of unconjugated bilirubin, fetal distress in utero, and many postnatal complications, including kernicterus.109 It would seem prudent to monitor the fetuses of cirrhotic women and to consider early delivery when fetal distress and/or severe maternal hyperbilirubinemia is detected. Non-cirrhotic portal hypertension, associated with normal liver function, is present in 0.1% of pregnant women in India111 and does not appear to alter fertility to any significant degree. These women are reported to be at high risk (35%) of variceal bleeds during pregnancy. Although these episodes can usually be treated successfully, fetal loss during bleeding is reported to be as high as 40%.111
Viral Hepatitis B Chronic hepatitis B infection per se does not appear to alter fertility, conception, or pregnancy beyond the effects of liver dysfunction or cirrhosis itself.112 Vertical transmission of hepatitis B virus (HBV) from chronically infected mothers to offspring occurs during pregnancy or at delivery and prevention is an important clinical issue, as > 90% of infected infants become chronically infected.57,113–115 The risk of vertical transmission is related to the HBV viral load and replication rate, being over 85% in infants of women who exhibit HBeAg or detectable HBV DNA by qualitative assays.57,113,114 Therefore the American College of Obstetrics and Gynecology and the Centers for Disease Control recommend universal screening for HBsAg of all pregnant women during the third trimester.69,70 Infants of HBsAg-positive women should be treated immediately after birth (within 24 hours) with a single intramuscular injection of hepatitis B immunoglobulin and one injection of hepatitis B vaccine (Table 54-3).69,70,112,116,117 Further doses of vaccine should be given at 1 and 6 months of age. This immunoprophylaxis is highly effective in preventing > 80–90% of vertically transmitted HBV infection and provides future protection against horizontal transmission of both HBV and hepatitis D.112,115–118 However, for women with very high HBV DNA levels (>1.2 ¥ 109 gEq/ml), vaccine and HBIG prophylaxis fail in 28% of cases. Addition of maternal lamivudine treatment in the last month of pregnancy reduces this failure rate by half.119
Viral Hepatitis C Most women with chronic hepatitis C have mild disease and normal liver function and experience uncomplicated pregnancies. However, during pregnancy many women exhibit normalization of serum transaminase levels (AST, ALT), often associated with an increase in the hepatitis C virus (HCV) viral load, that reverses after delivery (Figure 54-2).120,121 These changes may be related to subtle immunologic shifts during pregnancy. The long-term consequences, if any, are not known, but a preliminary report122 suggests that after pregnancy HCV-infected women may experience worsening of necroinflammatory and fibrosis changes on liver biopsy. Further, an epidemiologic study of over 16 000 pregnant women identified a
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Section IX. Liver Affected by Other Organs (Non-Hepatic Conditions)
100
Serum HCV RNA Serum ALT
150 10
100 50
Normal
0 Prepregnant
1st
2nd
3rd
After delivery
Serum HCV RNA (105 viral particles/ml)
Serum ALT (IU/mI)
200
1
Figure 54-2. Reciprocal changes in serum alanine aminotransferase (ALT) and in hepatitis C virus (HCV) RNA levels before, during, and after pregnancy in women with chronic hepatitis C infection. Data represent the median values of 26 patients. (Modified from Gervais A, Bacq Y, Bernuau J, et al. Decrease in serum ALT and increase in serum HCV RNA during pregnancy in women with chronic hepatitis C. J Hepatol 2000; 32: 293–299, with permission from EASL.)
greater rate of cholestasis of pregnancy in HCV-positive women (15.9%) compared to HCV-negative women (0.8%), although the mechanism for this association is unclear.123 Hepatitis C can be transmitted to babies at or around the time of birth from infected or viremic mothers.124 Vertical transmission of hepatitis C does not appear to be related to the method of delivery or to breast-feeding.120,125,126–128 In 1800 pregnancies from 11 studies (most from Italy, where HCV infection is prevalent), 8.5% of babies were infected as determined by HCV RNA testing.120,125–127,129–136 The transmission rate appears to be up to two- to threefold higher, up to 30%, if mothers are also infected with human immunodeficiency virus (HIV),120,125,126,128,131 although antiretroviral therapy may decrease this risk.120,125,126,128 No prophylaxis is available (Table 54-3). Infants may exhibit HCV RNA early after delivery or not until 3–6 months of life.120,126,137 Up to 20% of infants will spontaneously eliminate the virus138 and maternal anti-HCV antibody may persist in uninfected infants for up to 18 months.120,126 Thus, efficient detection of true vertical transmission is best achieved by testing babies for HCV RNA and ALT at 3 and 6 months of age. Negative infants should be tested for HCV antibody once more at 18 months of age and positive values confirmed by HCV RNA testing.137,138 Outcome of children infected at birth is not well understood. One group of 62 asymptomatic children, 93% of whom exhibited abnormal ALT values, were followed for a mean of 5 years (range 2–11 years). By the end of follow-up, 81% were still viremic.138 High ALT levels and genotype 3 were more common in those infants who spontaneously cleared the virus. The only other data involve a 20year follow-up of children infected with HCV through blood transfusions for cardiac surgery. Only 1 patient developed cirrhosis in that time period (2.7%), and all other patients had normal liver tests.139 There are few data on the efficacy, side effects, or long-term benefits of treating children with hepatitis C,140 although treatment has been successful in some children.141
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As HCV-positive women cannot be reliably identified by history or examination, broad-based prenatal screening for HCV RNA has been proposed to counsel infected women and provide for long-term follow-up and eventual treatment of these women and any infected offspring. The safety and efficacy of treating pregnant HCV-positive women with interferon-based regimens are not known. A few case reports suggest treatment is safe;142 however, interferon is an abortifacient in some animals.
Primary Biliary Cirrhosis and Primary Sclerosing Cholangitis Cholestasis and pruritus in these disorders may be exacerbated or spontaneously improve during pregnancy and may respond to ursodeoxycholic acid (UDCA) therapy.143–146 Pregnancy outcome is more dependent on liver function and portal hypertension than the disease per se.
Liver Masses A wide variety of liver masses may be identified coincidentally during pregnancy, including liver cysts, intrahepatic pregnancy, hepatic hemangioma, liver cell adenoma, and hepatocellular carcinoma.147–150 Simple liver cysts are benign, are not affected by estrogens or pregnancy, and require no treatment. Intrahepatic pregnancy149 is extremely rare and may require surgical intervention. Other liver masses may be estrogen-responsive, may be affected by pregnancy, and are discussed later in this chapter.
Dubin–Johnson Syndrome Pregnancy or use of oral contraceptives in women with the Dubin–Johnson syndrome causes a reversible 2–2.5-fold increase in plasma concentrations of bilirubin.151,152 Plasma concentrations of bile acids remain normal.151 Affected women may be deeply jaundiced during pregnancy, but pruritus and other signs of generalized cholestasis are not seen. This transient exacerbation of the Dubin–Johnson syndrome is related, presumably, to the cholestatic effects of estrogens superimposed upon a liver with markedly impaired capacity for canalicular excretion of conjugated bilirubin.
PREGNANCY AFTER LIVER TRANSPLANTATION Although women with cirrhosis and severe liver dysfunction are usually amenorrheic and infertile,77,78 premenopausal women usually regain menstrual function and fertility after successful liver transplantation,77,78,153 most by 7 months after surgery. Pregnancy has been reported as early as 3 weeks post-transplantation,78 therefore contraceptive methods should be discussed soon after transplantation.77–79 Immunosuppressive drugs must be continued throughout pregnancy and blood levels monitored as changes in drug metabolism, especially of ciclosporin, may occur.78 Although azathioprine is teratogenic in animals and immunosuppressive drugs other than corticosteroids have not been adequately tested for safety in pregnancy,77,154 to date there is no evidence of increased fetal malformations in offspring of mothers with liver or kidney trans-
Chapter 54 THE LIVER IN PREGNANCY
plants.78,153–156 Many of these drugs are probably present in breast milk and the effects of breast-feeding are unknown. Pregnancy after liver transplantation is high-risk, with ~18% spontaneous abortions, 2% stillbirths, 36% premature births, 31% low-birth-weights, and 25% neonatal complications in 136 pregnancies in 130 women.154 Only 70% of pregnancies resulted in a live birth; however, these babies all did well. Mothers experience a variety of medical problems, including hypertension (40%), pre-eclampsia (25%), infections (30%, including cytomegalovirus infections that may adversely affect the fetus), and acute rejection (10%).154 Complications of pregnancy may be increased in women with pre-existing decreased renal function. Maternal mortality is low and related to recurrent liver disease and renal failure rather than to pregnancy per se. Liver transplantation has been performed during pregnancy, usually but not always resulting in fetal loss.
LIVER DISORDERS PROBABLY RELATED TO PREGNANCY BILIARY TRACT DISEASE Gallstone Formation Women develop cholesterol gallstones and clinical symptoms related to gallstones more frequently than do men.157,158 The increased incidence of gallstone formation begins at puberty, is related to the number of pregnancies, and tapers off after menopause, suggesting that sex hormones may be important etiologic factors.159 Compared with men, women exhibit increased saturation of bile with cholesterol and have a smaller pool of bile acids.157 Use of oral contraceptives14,157,160–162 or pregnancy163,164 increases the concentration of cholesterol and its total output in hepatic and gallbladder bile. The pool of bile acids is also increased but more of the bile acid pool is sequestered in the gallbladder and intestine due to decreased motility.164 As a result, there is little change or even a decrease in bile acids secreted into bile, and the enterohepatic cycling of bile acids is decreased in pregnancy, as is the proportion of chenodeoxycholic acid relative to cholic acid. All of these changes predispose to precipitation of cholesterol. Further, development of gallstones is promoted by pregnancy and oral contraceptive-induced decreases in gallbladder contractility.162–167 During pregnancy, biliary sludge develops in about one-third of women and by the time of delivery 10–12% of women exhibit gallstones on ultrasonographic examination.168–171 During pregnancy, biliary colic occurs in approximately one-third of those with existing stones, but not in those with sludge or a normal gallbladder.168,169 Biliary pain in most women responds to conservative medical management. Postpartum, biliary sludge disappears in virtually all women, but only about one-third of small stones disappear. Epidemiologic studies from pregnant women, women taking oral contraceptives, postmenopausal women receiving estrogens, men treated with diethylstilbestrol, and young women undergoing cholecystectomy all suggest that estrogen exposure may accelerate the development of symptoms in patients with pre-existing gallstones.172–175 Thus pregnancy may predispose not only to formation of gallstones but to presentation with clinical symptoms.
Biliary Tract Disease During Pregnancy Acute cholecystitis is second to appendicitis as the most common cause of non-obstetric surgery during pregnancy, accounting for 1–8 cases per 10 000 pregnancies.176 Furthermore, common duct stones are a common cause of jaundice during pregnancy.16 Diagnosis of biliary tract disease, with modern ultrasound, is straightforward,176–178 and, if necessary, magnetic resonance imaging may be safe.179 99Technetium-labeled hydroxyiminodiacetic acid (99TcHIDA) and other nuclear medicine scans are probably best avoided during pregnancy. Therapy for symptomatic disease during pregnancy is often conservative. About 55–85% of pregnant women with biliary colic, acute cholecystitis, or gallstone pancreatitis respond to general medical management and surgery may be postponed until after delivery.176,180,181 Patients with recurrent or worsening symptoms or common bile duct obstruction may require treatment during pregnancy. This can be accomplished with relatively little maternal or fetal mortality, even with open cholecystectomy.176,180,181 Laparoscopic cholecystectomy may be even safer during pregnancy as over 180 such operations have been performed with no maternal mortality, 1.7% spontaneous abortions, and 3.9% premature deliveries.182,183 Endoscopic management of gallstone pancreatitis or biliary obstruction during pregnancy also appears to be safe and effective in the few cases reported.184
HERPES SIMPLEX VIRUS HEPATITIS Although herpes simplex virus hepatitis (types I or II) is rare in previously healthy adults, about half the cases reported have occurred in association with pregnancy, and the mortality rate is about 40%.185–189 Patients generally present with a 4–14-day history of fever, systemic viral-type symptoms, and abdominal or right upper quadrant pain. Hepatitis is characterized by very high aminotransferase levels (> 1000 units), an increased prothrombin time, and a low bilirubin level, typically less than 3 mg/dl. Liver biopsy may be diagnostic, showing areas of focal or confluent hemorrhagic and coagulative necrosis, relatively little inflammatory infiltrate, and “ground-glass” nuclear inclusions or Cowdry type A inclusions at the periphery of areas of necrosis that are positive on immunohistochemical stain (Figure 54-3).187 Liver, vaginal, cervical, or throat cultures are often positive. Since therapy with agents such as aciclovir has been successful in salvaging both mothers and infants,185,186,189 aggressive evaluation of pregnant women who exhibit fever, a viral syndrome, and elevated transaminase values with a modestly elevated or low serum bilirubin should be initiated followed by immediate institution of antiviral therapy. Although vertical transmission of herpes simplex to infants, either in utero or at the time of delivery, is not inevitable, infants born to these women should be closely observed and treated as appropriate.
ESTROGEN-RESPONSIVE HEPATIC NEOPLASMS AND PREGNANCY The liver is an estrogen-responsive organ. Estrogens, either endogenous or exogenous, are thought to be involved in several hepatic vascular and neoplastic processes. These include hepatic sinusoidal dilatation, focal nodular hyperplasia, hepatocellular adenoma, and, possibly, some cases of hepatocellular carcinoma.147,190,191 Circum-
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A
B
Figure 54-3. Herpes simplex hepatitis during pregnancy. (A) Large area of confluent hepatocellular necrosis (double-headed arrow); (B) infected hepatocytes with intranuclear eosinophilic Cowdry type A inclusions (arrows). Hematoxylin and eosin. (Courtesy of Dr. H. Appelman.)
stantial epidemiological evidence links some of these disorders to use of oral contraceptives; the association between pregnancy and these abnormalities is based on case reports and by analogy to the effects of oral contraceptive use. With the widespread availability of high-quality ultrasonography and magnetic resonance imaging, hepatic mass lesions may be safely identified and monitored throughout pregnancy.179 Hepatic sinusoidal dilatation with associated hepatomegaly and abdominal pain has been reported in a few women receiving oral contraceptives. The lesion has been noted in livers that contained oral contraceptive-associated adenomas.190,192–194 Improvement follows discontinuing oral contraceptives. The prognosis is benign.192–194 Focal nodular hyperplasia (FNH) is a benign lesion, consisting of normal liver elements disposed around a central stellate scar that is often found incidentally. The lesion occurs almost exclusively in women.147,195 An association with oral contraceptive use is suggested by some,195–197 but not all, studies.198 FNH has not been reported to rupture during pregnancy or delivery, thus surgery could be considered only in those women with proven estrogen-responsive lesions who desire to bear children. Indeed, 22 women with FNH of
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4–13 cm tolerated 25 pregnancies without complications or an increase in tumor size.198,199 Hemangiomas also are very common and rarely grow during pregnancy or exhibit a clear estrogen-sensitivity.147 Rupture of even large hemangiomas is very rare, with only 2 cases occurring during pregnancy. Pregnancy and vaginal delivery are well tolerated, even in women with very large hemangiomas and prophylactic treatment does not seem to be indicated. Hepatocellular adenomas are benign hepatocellular tumors linked causally to estrogens and use of oral contraceptives.147,195,196,200,201 It is not known whether estrogens initiate the adenomas, but they appear to promote growth202 and the development of clinical symptoms, such as a mass lesion, abdominal pain, acute hemorrhage, necrosis, or rupture.147 Estrogens and/or pregnancy have also been suggested to promote progress to hepatocellular carcinoma. Conversely, many adenomas regress after removal of estrogens.203 Pregnancy has not been associated with an increased incidence of adenomas, but pregnancy is associated with growth of adenomas, development of symptoms (nausea, vomiting, right upper quadrant pain), and a risk that large adenomas (>6.5 cm) may rupture.147,199,200 Women with adenomas of 5 cm or greater in size should be monitored with ultrasound during pregnancy and resection considered for rapidly enlarging adenomas. In non-pregnant women, surgical excision should be considered for large, symptomatic tumors, for those that do not shrink after stopping oral contraceptives, and for tumors >5 cm in women who desire to bear children.147,204 Resection should be performed immediately for ruptured adenomas in both pregnant and non-pregnant women. Indeed, surgical resection of large adenomas has been successfully carried out during pregnancy. Hepatocellular carcinoma is extremely rare in pregnancy, but may be aggressive, perhaps related to the high levels of estrogen and subtle immunosuppression of pregnancy.147,150 Management options are few. Overall evaluation and management of this, like other liver masses detected during pregnancy, must be individualized with consideration of risks to both the mother and the fetus.
BUDD–CHIARI SYNDROME Budd–Chiari syndrome associated with use of oral contraceptives was noted as early as 1966, and the association has been well documented.205–208 Development of hepatic vein thrombosis is attributed to an oral contraceptive-induced increase in clotting factors plus a generalized propensity to venous thrombosis. Budd–Chiari syndrome associated with pregnancy is much less common. Thirtyone cases have been reported.209–212 The predisposing factors for hepatic vein occlusion are thought to be the estrogen-related increases in clotting factors and decreases in the activity of plasma antithrombin III likely associated with an underlying inherited thrombophilia.211,213 In some women, hepatic vein occlusion is associated with widespread venous thrombosis and may represent local propagation of clot originating in the iliac veins and inferior vena cava. Another syndrome that clinically resembles the Budd–Chiari syndrome, hepatic veno-occlusive disease, has been reported in 3 women postpartum214 and in 1 woman receiving an oral progestational agent for contraceptive purposes.215 Clinical symptoms of the Budd–Chiari syndrome frequently begin postpartum or immediately after an abortion rather than during the pregnancy itself. Management is the same as in the non-pregnant patient, although the fetus
Chapter 54 THE LIVER IN PREGNANCY
may be at high risk of intrauterine death if maternal liver function is poor.
Table 54-5. Intrahepatic Cholestasis of Pregnancy:a Clinical Features Incidence in pregnancy
LIVER DISORDERS UNIQUE TO PREGNANCY Four unique syndromes of liver dysfunction have been identified during pregnancy: (1) hepatic involvement in hyperemesis gravidarum; (2) intrahepatic cholestasis of pregnancy; (3) acute fatty liver of pregnancy (AFLP); and (4) pre-eclampsia/eclampsia-related liver disease.
HEPATIC INVOLVEMENT IN HYPEREMESIS GRAVIDARUM Hyperemesis gravidarum is not a liver disease, but liver dysfunction occurs in severe cases. For example, among women affected severely enough to require hospitalization for dehydration and weight loss, liver dysfunction and jaundice were noted in 13–33% and 10–13% of cases, respectively.16,216,217 Liver dysfunction usually presents in the first trimester, within 1–3 weeks after the onset of severe vomiting. Jaundice, dark urine, and occasionally pruritus are the major hepatic manifestations.16,42,216–218 Mild hyperbilirubinemia is the most frequently noted laboratory abnormality (mean value 1–7 mg/dl). Moderate increases in serum transaminase activities (2–3 times normal) occur in slightly more than half the patients and, rarely, values up to 800 IU/l have been noted.216 Alkaline phosphatase activities are elevated in a minority of patients. Autopsy specimens from 19 women who died of hyperemesis exhibited excess pigment in centrilobular areas and some fat, but no necrosis.219 Cholestasis has been seen in liver biopsies from some affected women,218 but most biopsies are normal. The etiology of hepatic dysfunction is unknown, but may be related to dehydration and malnutrition as similar liver findings are seen in patients with kwashiorkor and prolonged fasting. Hepatic dysfunction in hyperemesis gravidarum is a relatively benign process with little clinical consequence. Women who have died of hyperemesis in the past did so from starvation and dehydration, not from liver failure. If vomiting is controlled the hepatic dysfunction rapidly resolves, usually within a few days, although it may recur in subsequent pregnancies.16,42,216,218
INTRAHEPATIC CHOLESTASIS OF PREGNANCY Intrahepatic cholestasis of pregnancy (ICP) is a relatively benign cholestatic disorder that generally commences late in pregnancy, disappears abruptly after delivery, and frequently recurs with subsequent pregnancies. The main clinical manifestations are pruritus and jaundice. The term pruritus gravidarum is frequently applied to women with pruritus and biochemical cholestasis, whereas the terms cholestatic jaundice of pregnancy or cholestasis of pregnancy are often applied to those women who also develop clinically apparent jaundice.
Incidence ICP is identified in less than 2% of pregnancies in the USA and Europe (Table 54-5).220–241 The disorder appears to be more frequent
Onset Onset of pruritus Onset of jaundice Recurrence in subsequent pregnancies Signs and symptoms Pruritus Jaundice Nausea, vomiting Abdominal pain Skin excoriations
USA and Europe: 0.5–1.7% India: 0.8%–1.4% Scandinavia: 0.5–3% Chile: 4.7–6.5% 70% in the third trimester 30% before the third trimester Average: 28–30 weeks; range: 7–40 weeks 1–4 weeks after pruritus 21–70% 100% 10–25% 5–75% 9–24% Common
a
Data derived from over 1000 patients in case reports and series published from 1967 to 2003 (representative references: 219–240).
in Scandinavia and in Chile, being reported in 1–6% of all pregnancies there.220–231 It accounts for 20–50% of all causes of jaundice in pregnancy in series reported from Scandinavia;16,42,220 however, the overall incidence appears to exhibit seasonal fluctuations and to have decreased in the past two decades.225,232,233,240,241 In an Italian population, ICP was diagnosed more commonly (16%) in women who also had hepatitis C than in those who did not (0.8%), although the basis for this association is not known.123
Etiology The etiology and pathogenesis of ICP remain poorly defined but ICP appears to be the same disorder as oral contraceptive-induced cholestasis. Indeed, after the clinical introduction of oral contraceptives (containing high doses of estrogen), a number of women developed cholestasis which resembled ICP.242–244 Further, as many as 50% of women who experience ICP also develop pruritus and cholestasis when given oral contraceptives, and vice versa.245,246 It is likely that both genetic and hormonal factors are important in the development of ICP and/or contraceptive-induced cholestasis. The best hypothesis is that these disorders reflect a genetic sensitivity to the cholestatic effects of estrogens, although ICP has also been associated with altered plasma levels of selenium, zinc, and copper, as well as changes in biliary secretion of sulfated progesterones.247,248 Estrogens, especially ethinylestradiol and its 17b-glucuronide metabolite, reproducibly cause mild cholestasis in both humans and animals, as does pregnancy in animals, likely through inhibition of the hepatocyte basolateral bile-salt transporter NTCP as well as the canalicular BSEP (ABC B11).33–35,39–41,249,250 Estrogens and pregnancy also markedly impair the canalicular bilirubin transporter MRP2 (ABC C2),34,35,250 further promoting development of jaundice. Thus pregnancy and/or use of oral contraceptives can be considered to be states of mild and usually asymptomatic cholestasis. However clinical cholestasis (ICP) develops in the minority of these women due to underlying, presumably inheritable factors. Indeed, ICP frequently affects female relatives of index cases,222,225 including up to three generations of women in some families.229,251,252 ICP and famil-
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ial benign recurrent intrahepatic cholestasis have been observed in the same family.253 ICP and/or estrogen-related cholestasis are identified in 10–15% of both mothers and sisters of women who develop cholestasis while receiving oral contraceptives.254,255 Further, ethinylestradiol administration impairs biliary excretion of the organic anion BSP (a substrate for the canalicular bilirubin transporter MRP2) in both men and women, and the effect is much more marked both in women with a history of ICP and in women and men with a family history of ICP.256 Thus, it is likely that ICP results from a combination of high estrogen levels in women with mild mutations in one or more genes involved in bile acid transport and/or bile formation. Mutations in the canalicular phospholipid transporter MDR3 (ABC B4) have been described in several families in which heterozygously affected women have developed ICP.257–259 Homozygous children in these families have developed progressive familial intrahepatic cholestasis (PFIC) type 3. In these families, cholestasis is characteristically accompanied by high levels of GGTP. However, ICP must be genetically heterogeneous with involvement of other genes and/or other ABC B4 mutations as only 6/389 other women with ICP have been found to carry known ABC B4 mutations:260–266 sequencing of the genes for MDR3 (ABC B4) and BSEP (ABC B11) in 21 patients with ICP and in 40 control woman identified a further 10 new ICP-related mutations in MDR3 in 9 of the 21 patients.267 In this study,267 no ICP-related mutations in BSEP were identified, although analysis of single nucleotide polymorphisms in 57 women with ICP suggested associations with the gene for the canalicular bile salt transporter BSEP (ABC B11).268 At this time genetic testing is not generally available or clinically useful. However research laboratories may be able to test selected women with ICP who also have high GGTP levels and/or a family history of ICP, PFIC, or other cholestatic liver diseases.
Clinical Features The clinical and laboratory features of ICP233,240,269 are summarized in Tables 54-5 and 54-6,37,220–228,230–235,237–240,261,270–272 and Figure 54-
4. In addition, lipoprotein X may be identified in plasma, and gallbladder size and residual volume are often increased.273 Serum transaminase activities in a few patients with ICP are high enough to overlap with those typical of hepatocellular disorders such as acute viral hepatitis. Serologic tests for hepatitis viruses A and B, and the clinical course of the disease, particularly after delivery, may be helpful in the differential diagnosis. Liver biopsy is generally unnecessary for diagnosis. Liver failure and hepatic encephalopathy are not reported in ICP and their appearance indicates another etiology for the liver disease. The pruritus can be disabling, and in exceptional cases, can be so severe as to mandate termination of pregnancy. Fat malabsorption and vitamin K deficiency223 may develop in severe cases and may be responsible for some instances of maternal postpartum hemorrhage.269
Pathology The histopathology of ICP is that of intrahepatic cholestasis (Figure 54-5). Typical findings include centrilobular cholestasis, canaliculi containing bile plugs, and bile pigment in hepatocytes.16,274 Cholestasis may be patchy and subtle. Inflammation and hepatocellular necrosis are usually absent. Portal tracts and interlobular bile ducts are normal. Electron microscopic examination shows dilated bile canaliculi with loss of microvilli and occasional abnormal mitochondria.274,275 Histologic changes typically disappear after delivery and resolution of clinical symptoms.16,274
Natural History and Prognosis The cholestasis of ICP generally progresses until the time of delivery or termination of pregnancy.233,240,269 The severity of cholestasis and of laboratory abnormalities can be quite variable however, both during one pregnancy and between different pregnancies, with recurrence rates of 21–70%.224,233,237,240 For example, serum transaminase activities and even bilirubin concentrations may fluctuate and even temporarily normalize as pregnancy progresses.224,233,239,276 Pru-
Table 54-6. Intrahepatic Cholestasis of Pregnancy:a Laboratory Findings Test
Patients (n)
% of Women with abnormal values
Average value reported
Range of values reported
Normal valuesb
0.4–8.4
£ 1.1 mg/dl
nl–750
£ 60 KU/l
nl–1127 nl–1734 nl–430 nl–148 10–33 —
£ 40 IU/l £ 40 IU/l £ 6.5 mmol/l £ 5 mmol/l £ 12% —
2–31 1–10
£ 7 g/24 h £ 7 g/24 h
Bilirubin
991
12–27
Alkaline phosphatase
753
AST ALT Serum bile salts Serum cholic acid BSP retention at 45 minutes Prothrombin time Fecal fat: Patients with jaundice Patients with pruritus
837 749 576 91 77 177
73 (at least 2.4-fold upper limit of normal) 64 62 96–100 79 97–100 14c
1.0 (all patients) 2.9 (jaundiced patients) 146 ± 66 (up to 12.5-fold upper limit of normal) 115 160 35 24 23 —
— —
14.0 4.0
12 11
AST, aspartate aminotransferase; ALT, alanine aminotransferase; BSP, bromosulfophthalein; nl, normal. a Data derived from 1052 patients in case reports and series published from 1967 to 2002 (representative references: 37,219–227,229–234,236–239,260,268–270). b For non-pregnant individuals. c All corrected with vitamin K.
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Chapter 54 THE LIVER IN PREGNANCY
17
CA
ALAT 110
15 90 13 70 Enzyme activity (U/l)
11
Bile acid concentration (mmol/l)
9 7 5
50 30 10 90
ASAT
3 70 1 50
CDCA 7
30
5
10
3
16
20
30 32 34 36 38 40 2 4 35–60
Weeks of pregnancy
1 B
Days after delivery
DCA 3 1 16
20
30 32 34 36 38 40 2 4 35–60
Weeks of pregnancy A
Days after delivery
Figure 54-5. Intrahepatic cholestasis of pregnancy showing canalicular bile plugs (arrows) with well-preserved hepatocytes containing yellow pigment. Hematoxylin and eosin. (Courtesy of Dr. H. Appelman.)
Figure 54-4. Serum bile acid concentrations (A) and liver tests (B), during pregnancy in control women (shaded areas; mean ± 2 SD) and in 8 women who developed intrahepatic cholestasis of pregnancy (line; mean values). CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase. (Modified from Heikkinen J. Serum bile acids in the early diagnosis of intrahepatic cholestasis of pregnancy. Obstet Gynecol Scand 1975; 54:437.)
ritus, however, rarely improves before delivery. After delivery, pruritus quickly disappears, usually within 24–48 hours. Biochemical abnormalities and histologic findings resolve over the following weeks to months.31 Rarely, symptoms may persist for several weeks postpartum and respond to a short course of prednisone.277 Cholestasis may recur during treatment with oral contraceptives, although this appears to be less common with low-dose estrogen contraceptives.233 During long-term (up to 15-year) follow-up, prognosis for women who have had typical ICP is excellent, aside from a higher incidence (1.4–2.3-fold) of cholelithiasis and gallbladder disease.42,221,278 Chronic cholestatic liver disease, however, has developed in rare familial cases, probably due to specific and uncommon gene mutations.279 The prognosis for the fetus is not as benign. Problems include increases in premature labor (4–20%), intrauterine growth retardation (8–10%), and neonatal death (0.6–2.5%).37,220–222,225,226,230,231,234,237,238,241,268,272,280–284 Fetal monitoring has documented high rates of premature labor and delivery,237 fetal distress during labor (19–60%), and meconium staining
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Section IX. Liver Affected by Other Organs (Non-Hepatic Conditions)
(15–45%).228,231,237,238,241,280,283,284 The true incidence of fetal death due to ICP is not known; however, when several large series are combined, there is a trend towards increased fetal complications and death related to ICP (1.75% mortality in 679 infants born to women with ICP compared to 1.01% mortality in 710 infants born to unaffected women), especially in older series without aggressive obstetric management.31,37,221,222,237 The mechanism(s) of premature labor, fetal death, and meconium staining are not known but these events are attributed to elevated bile acids leading to increased uterine and fetal colonic muscle contractions, meconium passage, changes in fetal cardiac and vascular function, umbilical vein constriction, and acute fetal anoxia.229,238,269,285,286 These findings have prompted experienced physicians to recommend aggressive obstetric management.283,287,288 It is recommended that all women with ICP be closely followed from 34 weeks or the onset of jaundice, possibly including weekly fetal non-stress tests. Aggressive monitoring is thought to be especially important if onset of ICP is before 32 weeks, if there is a twin pregnancy, if jaundice occurs, or if there is a past history of a fetal death. Babies should be delivered promptly if any signs of fetal distress or meconium passage are found but even in the absence of fetal distress, labor should be induced, once fetal lung maturity has been documented, at 37 weeks for mild ICP and at 36 weeks for women who are jaundiced.222,223,226,231,237,240,241,269,283,287,288 Some have advocated even more aggressive care, including hospitalization and fetal non-stress tests at least once a day.231,238,284,289 Although the management plan outlined here appears to be associated with fewer sudden fetal deaths, fetuses still die in utero within hours or days of normal stress tests.237,238,283,289
Therapy In the past women were treated symptomatically for pruritus with cholestyramine,269,290 phenobarbital,290 and/or hypnotics. Success was variable and these agents did not improve fetal outcome. Fetal hemorrhage has been reported due to vitamin K deficiency from ICP and cholestyramine usage269 and thus vitamin K supplementation should be given near term to all women with jaundice and/or prolonged cholestasis. UDCA, a hydrophilic bile acid that improves other cholestatic liver diseases, possibly by stimulating biliary excretion of other, potentially toxic, bile acids or sulfated progesterones, is the most promising treatment for ICP.240,291 UDCA appears to be safe as no adverse outcomes have been reported in over 180 pregnancies.239,270–272,276,282,292–296a During both open-label and placebocontrolled treatment trials (especially when doses of > 1 gram (or 15 mg/kg) per day were used), most women receiving UDCA exhibit substantial improvement in pruritus, liver tests, and serum bile acid levels239,270,271,276,292–300 and better placental bile acid transport.271 Pregnancies were allowed to go further preceding delivery as well.276,292,294,296,296a,298 It is not clear whether UDCA actually improves fetal outcome when pregnancies are also aggressively managed, although anecdotal reports suggest such might be the case.282,293 Other less well studied therapeutic options include S-adenosyl-Lmethionine (SAME), which methylates phospholipids and improves experimental cholestasis301,302 and dexamethasone.303 SAME, which must be given parenterally and is not widely available, has been
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tested in four trials239,295,304,305 with either no295,304 or slight239,305 benefits for the mother. Overall, UDCA would appear to be the treatment of first choice.
ACUTE FATTY LIVER OF PREGNANCY AFLP is a rare and potentially fatal idiopathic disorder that appears during the third trimester of pregnancy.306,307 Its principal and distinctive histologic feature is infiltration of hepatocytes with microvesicular fat, histology strikingly similar to that in Reye’s syndrome, Jamaican vomiting sickness, valproic acid hepatotoxicity, tetracycline hepatotoxicity, and medium- and long-chain acyl coenzyme A (CoA) dehydrogenase deficiency.308–312 This group of disorders collectively have been termed the hepatic microvesicular steatoses, and they also share many clinical and laboratory features, suggesting a common abnormality in lipid metabolism and, perhaps, related etiology.
Incidence The frequency of AFLP appears to be 1 in 900 to 1 in 6660 pregnancies,235,306 including mild cases identified and treated early. AFLP accounts for 16–70% of cases of severe liver disease during pregnancy and, consequently, a significant number of maternal and fetal deaths.235,313,314 AFLP is more common in primagravidas, in twin pregnancies, and in those with male fetus (Table 54-7),235,306,311,313–324 although it can occur with any pregnancy.
Etiology AFLP, like other microvesicular steatoses, may be due to a combination of increased flux of triglycerides and fatty acids from adipose tissue to liver and defects in mitochondrial b-oxidation of fatty acids.307,310,317,325–327 During late pregnancy, increased plasma levels of triglycerides and fatty acids as well as mildly impaired boxidation have been documented in experimental animals and humans, possibly due to the effects of estrogens, progesterones, and fetal metabolic demands.307,312,325,328,329 In women who develop AFLP it is thought that additional factors further impair boxidation, leading to steatosis, decreased ATP generation, lipid peroxidation, elevated free fatty acids to toxic levels, inhibition of gluconeogenesis and, ultimately, liver failure.307,309,325,327 Potential triggering factors may include drugs, like aspirin or non-steroidal anti-inflammatory drugs that are known to impair b-oxida-
Table 54-7. Acute Fatty Liver of Pregnancy: Clinical Features and Presentationa Feature
Frequency
Incidence Age Primagravidas Twin gestations Male fetus Recurrences Onset Pre-eclampsia
1/875–1/6660 deliveries 28 years (16–40 years) 46% 10% 71% 3/22 35.5 weeks (26–40 weeks) 28%
a
Data derived from 202 patients in case reports and series published from 1955 to 2002 (representative references: 234,304,309,311–322).
Chapter 54 THE LIVER IN PREGNANCY
tion,309,312,330 inflammatory cytokines,325 or pre-eclampsia, which is also associated with increased fatty acid fluxes.328 Although most cases of AFLP appear to be sporadic, some cases, perhaps up to 20%,307,325,326,331,332 are likely due to a specific mutation in the gene for long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), which catalyzes an essential step in mitochondrial boxidation of fatty acids.307,310,325,327,333,334 Indeed, heterozygosity for the common LCHAD mutations occurs in European-derived populations at rates of 1/150–1/680.323,325 Children homozygous for LCHAD mutations usually present early in life with rapidly fatal hypoketotic hypoglycemia, fatty liver, a Reye’s-like syndrome and skeletal or cardiac myopathies. The mothers of these children appear to be at high risk of AFLP: during 82 pregnancies in which 59 mothers heterozygous for LCHAD mutations carried homozygous affected fetuses, AFLP occurred in 43% and pre-eclampsia occurred in 22%.323,326,333–337 These same women had 74 pregnancies with normal or heterozygous fetuses and AFLP (2.7%) and preeclampsia (1.4%) were uncommon. Therefore, in women heterozygous for LCHAD deficiency, AFLP likely develops because of the combination of maternal genetic and pregnancy-related defects in liver mitochondrial b-oxidation of fatty acids exacerbated by placental defects in fatty acid metabolism,338,339 by unknown toxic factors generated by a homozygous affected fetus and, possibly, by concurrent pre-eclampsia.325,328 AFLP may be expected to recur in such women in future pregnancies if the fetus is homozygous for LCHAD deficiency. Prenatal testing for families with identified LCHAD mutations has been accomplished by research laboratories.340,341 Most infants born to women with AFLP, however, do not exhibit evidence of clinically significant defects in fatty acid metabolism. Thus, some combination of more subtle maternal and/or fetal genetic307,326,331,332,342 and environmental factors affecting maternal fatty acid oxidation may account for sporadic AFLP in the remaining 80% of cases. Based on these findings it is recommended that, in all cases of AFLP, both baby and mother be tested for at least the most common
(Glu474Gln) LCHAD mutation, that babies be screened for abnormal organic acids, acyl CoAs and acyl carnitines, and that babies be kept on a high-carbohydrate diet with frequent feedings until metabolic abnormalities have been excluded.307,325,331,332,334,340–342 Testing for the common LCHAD mutation is available on a fee-for-service basis through several laboratories that can be located through www.genetests.org. Determination of other mutations would likely require analysis by research laboratories.
Clinical Features The clinical and laboratory features of AFLP are summarized in Tables 54-7 to 54-10. Illness usually begins in the third trimester, around 35 weeks of gestation, although onset may be as early as 26 weeks or as late as the immediate postpartum period. The earliest manifestations are non-specific and include nausea, fatigue, malaise, vomiting, and abdominal distress (right upper quadrant or epigastric
Table 54-8. Acute Fatty Liver of Pregnancy: Signs and Symptomsa Feature
No. of patients for which data are available
Frequency (%)
198 202
77 54
195 202 199 88
56 86 44 32
132 158 77
32 84 13
Nausea, vomiting Abdominal pain; epigastric distress Encephalopathy Jaundice Hypoglycemia Ascites Extrahepatic manifestations Gastrointestinal hemorrhage Renal impairment Pancreatitis a
Data derived from 202 patients in case reports and series published from 1955 to 2002 (representative references: 304,309,311–321).
Table 54-9. Acute Fatty Liver of Pregnancy: Laboratory Abnormalitiesa Test
No. of patients
Frequency of abnormal results (%)
Abnormal values Average
Range
b
RBC smear White blood count Platelet count Prothrombin time Antithrombin III level Bilirubin Alkaline phosphatase
38 142 167 182 30 202 141
50 93 73 92 100 95 90
AST (nl £ 40) ALT (nl £ 40) Serum uric acid Serum creatinine
100 108 76 144
99 95 89 87
— — 22 s 11% of normal 12.2 mg% 4.4-fold upper limit of normal 218 IU/l 366 IU/l — 3.0 mg/dl
12 000–46 000 5000–121 000 nl–78 s 1.8–36 mg% nl to 10-fold upper limit nl–1300 nl–3670 up to 18.5 mg% up to 6.6 mg/dl
RBC, red blood cell; AST, aspartate aminotransferase; ALT, alanine aminotransferase; nl, normal. a Data derived from 202 patients in case reports and series published from 1955 to 2002 (representative references: 304,309,311–322). b Nucleated RBCs seen frequently; RBC fragments in those with disseminated intravascular coagulation.
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Section IX. Liver Affected by Other Organs (Non-Hepatic Conditions)
Table 54-10. Acute Fatty Liver of Pregnancy: Outcome Maternal mortality All reportsa Reports from 1991 to 2002 in which delivery was initiated promptlyb
26% 5%
(59/224 cases) (5/101 cases)
Infant mortality All reportsa Reports from 1991 to 2002 in which delivery was initiated promptlyb
32% 9%
(80/249 cases) (10/108 cases)
a
Data derived from 224 patients in case reports and series published from 1955 to 2002 (representative references: 304,312,313,318–321,331). b Data derived from 101 patients in case reports and series published from 1991 to 2002 (representative references: 304,312,318–321,324,331).
pain). Fever, headache, diarrhea, back pain suggestive of pancreatitis, and myalgias are reported in some patients. Clinical signs of liver dysfunction and even frank liver failure, such as jaundice, hepatic encephalopathy, or bleeding, may ensue 1–2 weeks later. Mild cases may exhibit few, if any, signs or symptoms. Physical findings are often minimal, especially early in the disease. Right upper quadrant tenderness may be the only abnormality found. The liver is generally small and not palpable. As the disease progresses, jaundice, changes in mental status, edema, and ascites may appear. Signs and symptoms of pre-eclampsia and its hepatic complications are seen in 28% or more of patients.306,314,320,334,343 This is a much greater incidence than in normal pregnancies and may reflect roles for abnormalities in triglyceride and fatty acid metabolism in both AFLP and pre-eclampsia.325,328 Acute fatty liver of pregnancy is associated with a number of abnormal laboratory findings (Table 54-9). The blood smear frequently contains nucleated red blood cells and, in those patients with disseminated intravascular coagulopathy (DIC), fragmented erythrocytes, and Burr cells. Leukocytosis is frequent, even in women with no evidence of infection. Coagulation disorders are common, occasionally progressing to frank DIC. The abnormalities in clotting factors probably represent impaired hepatic synthesis as well as accelerated consumption. Indeed, a profound decrease in plasma antithrombin III activity has been found in all tested patients, even before the onset of AFLP.324 Liver test abnormalities include hyperbilirubinemia, increases in alkaline phosphatase activity, and modest elevations of serum transaminase activities (usually 40 kg/m2
BMI >35 kg/m2
Bariatric surgery; mean BMI 47 kg/m2
Abnormal LFTs
Yes
>45
≥50
Yes
Yes
Yes
Yes
Yes
≥30 kg/m
Age Gender BMI
Male Yes
Centripetal obesity Type 2 diabetes mellitus
Female 2
≥28 kg/m
Male
Harrison et al.284
Female ≥28 kg/m2
2
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes >5.2%
HbA1c Insulin resistance
Yes
C-peptide
Yes
ALT elevation
Yes
AST/ALT ratio
>1
2 ¥ ULN
Yes >0.8
Hypertension
Yes
Triglycerides
>1.7 mmol/l
Ferritin
No
No
BMI, Body mass index; LFT, liver function test; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ULN, upper limit of normal. Independent risk factors identified by multivariate analysis noted in bold type.
small bowel during the absorptive phase of digestion. The liver also synthesizes free fatty acids, a metabolic step that is the major fate of excess circulating glucose not needed as an immediate energy source. The caveat is that a low-fat diet may do little to decrease the amount of fat delivered directly to the liver, yet a diet characterized by excessive carbohydrates may rapidly increase the accumulation of fat in the liver through de novo synthesis. A minor source of triglyceride accumulation in the liver is the hepatocellular endocytosis of lipoprotein remnants, specifically lowdensity lipoprotein (LDL) and chylomicron remnants. These lipoproteins originate from very-low-density lipoprotein (VLDL) and chylomicrons secreted into the circulation by the liver and gut respectively, and the remnants containing a small amount of triglyceride are taken up by the liver once most of their triglyceride content has been removed by muscle and adipose tissue. Free fatty acids in the liver have two major fates (Figure 55-3). They can be shuttled into mitochondria by the carnitine cycle where they serve as a source of energy (adenosine triphosphate: ATP) or they can be esterified into triglyceride, packaged as VLDL, and secreted by exocytosis into the circulation. A relatively minor pathway in terms of its overall consumption of free fatty acids is their oxidation in peroxisomes. Peroxisomes are small hepatocellular organelles that metabolize very-long-chain fatty acids and dicarboxylic acids by oxidation.
1036
Microvesicular Steatosis Hepatocellular triglyceride accumulation in NAFLD is typically macrovesicular or a mix of microvesicular and macrovesicular steatosis. Microvesicular steatosis is defined as lipid droplets in hepatocytes less than 1 μm in diameter, or roughly less than the diameter of hepatocyte nuclei. Pure microvesicular steatosis can sometimes be difficult to identify without specific fat stains such as the oil-red O. It is often associated with life-threatening acute liver dysfunction and is almost uniformly associated with disorders of mitochondrial dysfunction, such as drug toxicity,34 acute fatty liver of pregnancy, Reye’s syndrome, or toxins such as methylenecyclopropylalanine (MCPA, hypoglycin A) in unripe ackee fruit (Blighia sapida). Eating unripe ackee is responsible for Jamaican vomiting sickness and death from liver failure. Why triglyceride accumulates as small droplets in some disorders and large droplets that displace cell contents peripherally in other disorders such as NAFLD is not known. The rate of fat accumulation may be one explanation. In most cases of microvesicular steatosis, the fat accumulates over a relatively short period, whereas macrovesicular steatosis is commonly associated with more prolonged metabolic disturbances. Inconsistent with this explanation is the development of macrovesicular steatosis very early in the course of alcohol ingestion, as shown in an early study by Rubin and Lieber.35 In general, disorders of purely microvesicular fat accumu-
Chapter 55 NASH
Table 55-7. Multiple Components of Very-Low-Density Lipoprotein (VLDL)
VLDL
Triglyceride Peroxisomal oxidation
Mitochondrial -oxidation Free fatty acids
Lipoprotein remnants Free fatty acids bound to albumin
Excess glucose
Lipolysis in adipose tissue
Figure 55-3. Uptake and disposal of fat by the liver. Two major sources contribute to fat in the liver: delivery from peripheral adipose tissue as free fatty acids bound to albumin in the circulation or de novo synthesis within the liver from excess carbohydrate. Uptake of lipoprotein remnants delivers triglyceride to the liver but probably accounts for a relatively minor contribution. Free fatty acids in the liver have two major fates: mitochondrial b-oxidation or esterification to triglyceride and export as very-low-density lipoprotein (VLDL). Peroxisomal oxidation has a quantitatively minor role in the disposition of fat, although it probably plays an important metabolic role. Fat accumulates as triglyceride to present as non-alcoholic fatty liver disease when the delivery or de novo synthesis exceeds oxidation or secretion.
lation are not classified as NAFLD or NASH, even with associated necroinflammatory changes.
VLDL Synthesis and Secretion (Figure 55-3) In the postprandial state, ingested triglycerides are hydrolyzed to free fatty acids in the lumen of the small bowel (and a small amount is hydrolyzed in the mouth by lingual lipase), and the released free fatty acids are absorbed by enterocytes. Only short-chain fatty acids are then delivered from enterocytes directly into the blood, whereas most are esterified back into triglycerides within enterocytes and secreted into the lymphatics as chylomicrons. Adipose tissue and muscle remove free fatty acids from chylomicrons and store them as triglyceride or use them immediately as a source of energy. Also in the postprandial state, the liver converts unneeded carbohydrates to free fatty acids that are esterified and secreted into the blood as VLDL. The synthesis and secretion of VLDL require multiple intact metabolic pathways. Seemingly disparate dietary deficiencies and metabolic abnormalities comprising any one of these pathways can cause the accumulation of fat in the liver, i.e., NAFLD (Table 55-7). Few data are available on rates of VLDL production in NASH, although one study suggested synthesis is impaired.36 Impaired secretion of triglyceride from hepatocytes is likely the most common metabolic defect contributing to the development of NAFLD and thus warrants particular attention.37 VLDL synthesis requires the protein apoB100, a 550-kDa protein translated from a liver-specific splice form of the APOB transcription product. An
Essential for VLDL formation
Function
Functional apolipoprotein B100 gene Minor apolipoproteins Amino acid availability
Key component285,286
Protein synthesis Functional microsomal triglyceride transfer protein Choline/betaine/SAMe Essential fatty acids Vesicle trafficking/intact cytoskeleton
Key component of VLDL in circulation38 Necessary for lipoprotein synthesis (e.g., NAFLD associated with kwashiorkor) Necessary for lipoprotein synthesis Necessary for apoB100 lipidation38,40,132
Methyl donors necessary to form phosphatidyl choline (lecithin)255 Arachidonic acid necessary to form phosphatidyl choline (lecithin)255 Necessary for secretion from hepatocytes
NAFLD, non-alcoholic fatty liver disease; SAMe, S-adenosylmethionine.
alternate splice form of the same gene is used by enterocytes to form chylomicrons. Apolipoprotein E (apoE) is also an important component of circulating VLDL and polymorphisms of apoE such as apoE3-Leiden are associated with hepatic steatosis in mice.38 Whether apoE is incorporated in VLDL in hepatocytes or in the circulation has not been firmly established. In the liver, newly formed apoB co-translationally inserts into the endoplasmic reticulum where it undergoes essential disulfide bond formation and lipidation by triglyceride. Two proteins, protein disulfide isomerase and microsomal triglyceride transfer protein (MTTP) are needed for lipidation.39 Impaired MTTP expression is a cause of abetalipoproteinemia, a condition associated with NASH and cirrhosis. Less severe defects cause hypobetalipoproteinemia and have been linked to an increased risk of hepatic steatosis in patients with diabetes40 and increased liver injury after exposure to endotoxin.41 Whereas abetalipoproteinemia presents in infancy with severe fat malabsorption and early death, hypobetalipoproteinemia is found in 1/500 to 1/1000 people and is associated with NAFLD in adults. A clinical clue to the presence of this rare abnormality is an unexpectedly low serum cholesterol level (below 150 mg/dl).42,43
Mitochondrial b-Oxidation
Fat that is not secreted from hepatocytes as VLDL undergoes b-oxidation, either in mitochondria or the peroxisomes. Mitochondrial boxidation converts the energy stored in fat to the metabolically usable forms of nicotinamide-adenine dinucleotide (NADH) and ATP. It is also the source of the ketone bodies acetoacetate, acetone, and D-3-hydroxybutyrate. These are essential metabolic fuel sources for peripheral tissues, especially neurons, muscle, and brain when glucose is in short supply because of the inability of these tissues to use free fatty acids as an energy source. A number of drugs, toxins, and genetic abnormalities of mitochondrial function have been described, often in association with NALFD.34,44 Alcohol causes significant disruption of mitochondrial function and thus may share this mechanism of injury with NAFLD. Morphological abnormalities in mitochondria, characterized as crystalline inclusions, have also been observed in patients with NASH,
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Section IX. Liver Affected by Other Organs (Non-Hepatic Conditions)
Peroxisomal Oxidation Peroxisomal b-oxidation of fatty acids may have a significant role in the pathogenesis of NASH. Peroxisomes metabolize long-chain free fatty acids (22 carbons and greater) to shorter fatty acids that can be further metabolized by mitochondrial b-oxidation.48 About 10% of short- and medium-chain free fatty acids are also metabolized by peroxisomes. The enzymes of the peroxisomal compartment are induced by the fibrates and a high-fat diet.49 An obligate product of peroxisomal b-oxidation is the production of hydrogen peroxide. Although peroxisomes are well endowed with the enzyme catalase to facilitate the destruction of this reactive oxygen molecule, peroxisomal metabolism of fatty acids remains a potential source of oxidant stress. While this might suggest that increased activity of the peroxisomal pathway could participate in the development of NASH, the data are conflicting. The peroxisomal knockout mouse develops morphological features of steatohepatitis50–52 but upregulation of peroxisomal b-oxidation with a peroxisome proliferator-activated receptor-a (PPARa) ligand prevents fat accumulation and NASH in the methionine- and choline-deficient diet mouse model53 and a mouse model of lipoatrophy.54
Insulin Resistance, Hyperinsulinemia, and Hepatocellular Triglyceride Accumulation In the fed state, circulating insulin levels rise and signal muscle and adipose tissue to take up glucose from the blood for metabolism or storage (as glycogen). Insulin also signals adipose tissue to stop the production of free fatty acids from triglyceride and signals the liver to stop the de novo synthesis of glucose needed for survival during fasting. Down-regulating both of these metabolic steps in the fed state is logical since circulating free fatty acids are not needed by muscle and the liver as a source of energy and blood levels of glucose are sufficient such that the liver does not need to make glucose to keep the central nervous system functioning. When the responses of muscle, adipose tissue, and the liver to insulin are impaired, blood glucose levels rise and insulin secretion by the pancreas increases to compensate and keep glucose levels within a narrow range. Such an abnormal metabolic state is called insulin resistance with compensatory hyperinsulinemia. When the compensatory hyperinsulinemia becomes insufficient to facilitate normal glucose disposal, glucose levels rise and diabetes results. At some point, after years to decades of insulin overproduction, the pancreatic beta-cell population begins to diminish by attrition in response to the excessive demand, and diabetes progresses to the point where even fasting blood glucose levels become elevated. Most patients with NAFLD and NASH are now recognized as having underlying insulin resistance (Figure 55-4).14,17,18,55–57 The role of insulin resistance and its compensatory hyperinsulinemia in causing fatty liver is demonstrated by epidemiological studies, anatomical observations, and well-described biochemical pathways.23,57–60 If fat accumulation in the liver is a common metabolic
1038
6 P= 0.007 5 Insulin (ng/ml)
although their functional significance is unknown.16,45,46 Although assessment of mitochondrial respiratory chain components shows fairly global impairment in NASH patients,47 the overall role of mitochondrial dysfunction in the pathogenesis of NASH has not been determined.
4 3 2 1 NASH
Control
0 0
30
60
90
120
Minutes Figure 55-4. Fasting and postprandial hyperinsulinemia in non-diabetic patients with non-alcoholic steatohepatitis (NASH). Eighteen subjects with NASH and 18 controls matched for body mass index, age, and gender were given a 500-calorie meal; fasting insulin levels were elevated in the NASH group and the homeostasis model assessment method for insulin resistance (HOMAIR) was 6.5 ± 5.0 in the NASH group compared to 3.2 ± 3.0 in controls (P < 0.001). (Reproduced from Chalasani N, Deeg MA, Persohn S, Crabb DW. Metabolic and anthropometric evaluation of insulin resistance in nondiabetic patients with nonalcoholic steatohepatitis. Am J Gastro 2003; 98:1849–1855, with permission.)
consequence of hyperinsulinemia, then it is not surprising that emerging evidence suggests that NAFLD may be a very early indicator of insulin resistance.23 Insulin resistance is also likely the major underlying disorder in children with NAFLD.61 As is the case in adults, obesity is the primary cause of insulin resistance in children,62,63 especially centripetal adiposity.64 Epidemiological evidence has also shown that alanine aminotransferase (ALT) elevations, a surrogate for NAFLD, correlate strongly with insulin resistance in children.65 The complexities of obesity in children are underscored by the suggestion that intrauterine imprinting occurs, in which birth weight and being underweight in the first 2 years predispose to later weight gain and diabetes.66,67
MECHANISMS OF INSULIN RESISTANCE Postreceptor Signal Transduction and Insulin Resistance Understanding the common mechanisms of insulin resistance is essential to identify potentially effective treatments for most patients with NAFLD. Abnormalities of the insulin receptor have been associated with severe insulin but are quite rare. Most patients with insulin resistance have acquired defects of postreceptor intracellular signaling. The insulin receptor is a tyrosine kinase that autophosphorylates itself in response to insulin binding. The activated receptor then phosphorylates other intracellular signaling mediators, the most prevalent of which are the insulin receptor substrates 1 and 2 (IRS-1 and IRS-2, Figure 55-5). The net outcome is increased glucose disposal, decreased gluconeogenesis, increased lipogenesis, decreased lipolysis, and stimulation of cell growth.
Chapter 55 NASH
Insulin binding to insulin receptor
TNF␣
IRS-1
Free fatty acids
PI3 kinase
IRS-2
Normal effects of insulin: Increased glucose disposal Increased lipogenesis Decreased gluconeogenesis Decreased lipolysis Trophic effects
Figure 55-5. Intracellular signaling by insulin. Tumor necrosis factor-a (TNF-a) and high levels of intracellular free fatty acids both impair signal transduction after insulin binds its receptor, especially in target tissues such as muscle and adipose tissue. The result is a loss of the downstream effects of insulin with impaired glucose disposal and a failure to down-regulate hepatic gluconeogenesis and peripheral lipolysis. These effects of TNF-a and free fatty acids may be mediated by increased activation of the intracellular kinase JNK1.
Other
Impaired activation of phosphatidyl inositol-3 kinase (PI-3 kinase) by IRS-1 has been identified as a common site for impaired insulin signaling. Tumor necrosis factor-a (TNF-a) and increased free fatty acids both impair this pathway.68–70 In response, glucose disposal is impaired and serum glucose levels rise, which signals the pancreas to increase insulin output. A state of relative hyperinsulinemia thus follows. If the compensatory increases in insulin levels are sufficient to bring glucose levels under control, patients simply have insulin resistance without diabetes. When the elevated insulin levels are inadequate to achieve normal glucose disposal, then impaired glucose tolerance and diabetes develop. The relative roles played by the liver, muscle, and adipose tissue in modulating insulin sensitivity and predisposing to NAFLD have not been clarified. Data obtained primarily in animal studies have also shown that signaling defects in the liver play an important role in the development of insulin resistance.71–74 For example, liverspecific knockout of the insulin receptor causes severe insulin resistance in mice, manifested by glucose intolerance and hyperinsulinemia.75 On the other hand, knockout of adipose tissue glucose transporter GLUT4 also causes insulin resistance.76
TNF-a and Free Fatty Acids as Common Mediators of Insulin Resistance The most common mediators of impaired insulin signaling are elevated intracellular levels of free fatty acids and increased circulating levels of the cytokine TNF-a (Figure 55-5). A key role of TNF-a in mediating insulin resistance has been demonstrated in TNF-a knockout mice which fail to develop insulin resistance when fed to the point of obesity70 or in mice treated with antibodies to TNFa.77 Excessive intracellular free fatty acids stimulate the serinethreonine kinase JNK-1, which, through serine phosphorylation of key members of the insulin signal transduction pathway such as IRS1, prevents normal insulin signaling.78 TNF-a probably also impairs insulin signaling by the same mechanism of JNK1 activation, placing JNK1 activation in a central role in the development of insulin resistance. Further evidence is provided by the JNK1 knockout mouse that also fails to develop obesity-induced insulin resistance.78 Therefore, modulating JNK1 activity is being examined as a treatment option for diabetes and may be a reasonable focus for future studies in NASH.
Liver Disease as a Cause of Insulin Resistance The preponderance of data indicates that the liver is the target of too much insulin and suffers the consequences of hyperinsulinemia. However, the presence of liver disease can also contribute to the underlying state of insulin resistance. For example, cirrhosis from any cause is a risk factor for the development of insulin resistance.79,80 Additionally, studies in rats with NAFLD caused by tetracycline develop insulin resistance. Nonetheless, the observations that insulin resistance and hyperinsulinemia appear well before the development of significant liver dysfunction in patients with NASH suggest that the insulin resistance comes first and NAFLD follows as a consequence in most patients.
Iron and Insulin Resistance The relationship between hepatic iron stores and insulin resistance is complex and poorly understood. Current data indicate that the relationship may be bidirectional: excess iron stores can contribute to insulin resistance81,82 and yet insulin resistance appears to increase iron accumulation.83 The mechanisms underlying this mutually synergistic effect are the subject of ongoing investigation. Perhaps both components of this positive feedback mechanism may not exist commonly in the same patients or much more iron accumulation associated with insulin resistance would be expected.84–86
Lipodystrophies and Insulin Resistance Adipose tissue in the right place and in the right amount may be desirable because adipocytes can serve as a depot for fat, thus sparing other tissues from inappropriate fat deposition. Disorders of peripheral adipose tissue development and survival, the lipodystrophies, are defined by partial or complete inability to form adipose tissue. NAFLD and cirrhosis are known sequelae of these disorders, with the degree of hepatic steatosis being proportional to the extent of adipose tissue loss.87 For example, congenital generalized lipodystrophy is a rare disorder characterized by nearly absent peripheral fat, severe hepatic steatosis, and a significant risk of cirrhosis.88 Mutations associated with partial lipodystrophies include abnormalities of gene encoding PPAR-g, PPARG,89 and the nuclear envelope protein lamin A, LMNA.90 Lamin A may participate in regulating
1039
Section IX. Liver Affected by Other Organs (Non-Hepatic Conditions)
Increased hepatocellular triglyceride content
Mitochondrial dysfunction
Substrate for lipid peroxidation
Cellular ATP depletion
Increased gut-derived endotoxin
Increased circulating free fatty acids
Cellular membrane dysfunction
Figure 55-6. Mechanisms of hepatocellular injury in non-alchoholic fatty liver disease (NAFLD). Several mechanisms of hepatocellular injury have been proposed to explain the necroinflammatory changes characteristic of non-alcoholic steatohepatitis (NASH). The mechanisms are not mutually exclusive and probably each contributes to the development of NASH in varying degrees.
Oxidant stress
Hepatocellular necrosis, apoptosis, release of inflammatory cytokines
sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and -2). These transcription factors modulate insulin signaling and lipid synthesis in hepatocytes. In animal studies, overexpression of SREBP-1 is associated with severe hepatic steatosis91 whereas loss of SREBP-1 makes mice resistant to the development of fatty liver.92
Hepatocellular Injury in the Triglyceride-Loaded Hepatocyte Much more is known about how triglyceride accumulates in hepatocytes than why fat-laden hepatocytes are prone to injury. Oxidant stress in the setting of ample substrate for lipid peroxidation has been proposed as one link between the accumulation of fat and subsequent injury.93 Alternative explanations of cellular injury in NASH include toxicity of free fatty acids and impaired mitochondrial ATP production (Figure 55-6). These mechanisms are not mutually exclusive and a combination of these or yet to be discovered additional mechanisms of injury is probably important. One challenging idea proposed in the 1970s is that the underlying abnormality in patients with NASH causes both the injury and the fat to accumulate such that the presence of fat is simply a marker of the disease process.94
Oxidant Stress and Lipid Peroxidation Oxidant stress is defined as the production of reactive oxygen species in excess of what can be handled by endogenous antioxidant mechanisms leading to altered cellular physiology. Day and James proposed a “two-hit” hypothesis for the pathogenesis of NASH that states that the accumulation of fat is the primary abnormality and secondary oxidant stress leads to the inflammation, injury, and fibrogenesis that characterize NASH.95 The evidence that oxidant stress plays a role in the pathogenesis of NASH is largely derived from animal studies. For example, ferrets with diet-induced steatosis have higher liver markers of oxidant stress than control animals without steatosis 96 An animal model that recapitulates many of the abnormalities of NASH, the methionine- and choline-deficient diet, also causes increased oxidant stress in the liver.97 Rats fed this diet develop steatosis in 2 weeks, necroinflammatory changes in 5 weeks,
1040
and fibrosis in 12–17 weeks.98 Additionally, they had lower levels of the endogenous antioxidant glutathione, suggesting demands on defense mechanisms. Other studies in animals have also found upregulated antioxidant pathways, suggesting that the liver is responding to oxidant stress. Despite these findings in animal models, limited data in patients with NASH have confirmed the role of oxidant stress in NASH. While some studies confirm increased markers of oxidant stress,99 few have shown that antioxidants prevent NASH (see Treatment, below).
Possible Sources of Oxidant Stress in NAFLD Multiple sources of oxidant stress in the fatty liver have been identified and include cytochrome P450,100 peroxisomal b-oxidation, mitochondrial electron leak, and recruited inflammatory cells. The presence of excess fat in the liver provides ample substrate for lipid peroxidation and reactive lipid peroxidation products such as 4hydroxynonenal can further amplify the oxidant stress that led to their initial formation.93 Electron transport initiated by cytochrome P450 enzymes can cause oxidant stress101 and these enzymes are up-regulated in patients with NASH102,103 and in animal models of steatohepatitis.104 Definitive proof of the role of one key P450 isoenzyme, CYP2E1, was sought by feeding CYP2E1 knockout mice the methionine and choline-deficient diet. Interestingly, mice with this model of steatohepatitis diet still developed steatohepatitis, indicating that 2E1 is not necessary for liver injury in this model. One explanation is that another isoenzyme, CYP4A, was up-regulated and in vitro inhibition with an antibody to this enzyme decreased oxidant stress.97,100 Increased flux of free fatty acids through the liver may be the major stimulus for the induction of P450 activity in NASH patients. The reversibility of this process has been shown by the downregulation of CYP2E1 associated with weight loss following bariatric surgery.105 Finally, the peroxisomal b-oxidation pathway has been examined as a source of oxidant stress. If this metabolic pathway is responsible for injury, then its down-regulation should be beneficial. Paradoxically, the pathway is up-regulated by PPARa ligands
Chapter 55 NASH
(fibrates) and these agents may be beneficial in NASH (see Treatment, below). Moreover, the peroxisomal knockout mouse is not protected against the development of steatohepatitis,50,51 suggesting that PPARa-mediated disposal of fat may be an important fate of fat.53
Iron as a Contributor to Oxidant Stress and Insulin Resistance Excessive hepatic iron accumulation plays an important role in causing oxidative stress and chronic liver injury in patients with genetic hemochromatosis. Lesser degrees of iron accumulation are commonly present in other forms of liver disease, especially alcoholic liver disease and NAFLD. Whether this iron contributes to the liver disease and its likelihood to progress to cirrhosis and hepatocellular carcinoma (HCC) is less certain.106,107 If excess iron is important in the pathogenesis of NASH, then abnormalities of the HFE gene associated with the iron overload of classical hemochromatosis might be overrepresented in patients with NASH. One study in Australia of 51 subjects with NASH found that 31% had an abnormal HFE genotype compared to a normal prevalence of 13% in the general population.108 In this study, HFE mutations were associated with increased iron staining on biopsy, increased transferrin saturation, and increased fibrosis. In contrast, another study conducted in Australia found that HFE mutations and stainable iron were did not correlate with fibrosis.109 Detecting excess hepatic iron in patients with NAFLD generally requires a liver biopsy with an iron stain or even iron quantitation. Serum ferritin is unreliable because it can be elevated by the lowgrade necroinflammatory injury in the liver. In patients with NASH, serum ferritin did not correlate with either biopsy-detectable iron or abnormal HFE genotypes.108 Some studies have suggested that ferritin correlates better with insulin resistance and the presence of NAFLD rather than true iron overload.110 In summary, the two-hit theory of the pathogenesis of NASH is appealing but finding evidence that confirms a causal role of oxidant stress in the pathogenesis of liver injury in humans with NASH has been difficult. Markers of oxidant stress are increased, in both the liver and the serum. However, treatment trials of various antioxidants have generally been disappointing. This could be explained by a lack of efficacy of currently used antioxidants or that oxidant stress is not critical to the pathogenesis of NASH. Hopefully, continued trials with antioxidants and further work into the pathogenesis of NASH will provide clarity and guidance for therapy.
Free Fatty Acid Toxicity The increased flux of free fatty acids through the liver in states of increased peripheral lipolysis not only promotes insulin resistance,12 but could play a direct role in hepatocellular injury. One of the difficulties in examining the role of free fatty acids in hepatocellular injury is the lack of reliable methods to measure their intracellular levels. However, if free fatty acids are agents of cellular injury in the pathogenesis of NAFLD, abundant and overlapping protective mechanisms against this toxicity would be expected and could provide indirect evidence of their toxicity. Indeed, such mechanisms exist. Hepatocytes in particular are well endowed with mechanisms to bind, transform, catabolize, and export excess free fatty acids
through the combined actions of fatty acid-binding proteins, triglyceride synthesis, secretion as VLDL, mitochondrial b-oxidation, and enzymatic removal of lipid peroxidation products. One example is up-regulation of the nuclear receptor PPARa, which plays a central role in sensing excess free fatty acids and up-regulating the genetic program of fatty acid disposal.
Impaired Hepatocellular ATP Production Mitochondrial dysfunction in the liver may occur in NAFLD and contribute to the hepatocellular injury that leads to NASH.16,45,111–113 Hepatocellular mitochondria oxidize free fatty acids as an energy source and use this energy to generate ATP. Without normal mitochondrial function, hepatocytes become depleted of ATP and normal cellular functions become crippled, leading to cellular injury and death. Cells can become similarly impaired by oxygen deprivation and thus metabolic mitochondrial dysfunction shares mechanistic features with hypoxic injury. One provocative finding is that dietary constituents can increase demands on mitochondrial function. Fructose requires ATP to initiate its catabolism and an oral fructose load was found to cause more depletion of hepatic ATP in patients with NAFLD than normal.114,115 Whether the increased oral fructose consumption associated with the common use of high-fructose corn syrup in soft drinks and other foods contributes to liver injury in NAFLD is unknown. Some data also suggest that muscle mitochondrial dysfunction plays a significant role in the development of insulin resistance. A study of healthy but insulin-resistant children of diabetics demonstrated that their muscle is insulin-resistant and exhibits impaired ATP production.116 Although the functional significance of this is uncertain, insulin resistance increases with aging, in conjunction with a decrease in muscle mitochondrial function. Thus, progressive deterioration of muscle mitochondrial function probably occurs with aging and is associated with progressively worsening insulin resistance independent of obesity.117
Inflammation and Cytokines NASH, by definition, is an inflammatory condition. Understanding the roles of intrahepatic and extrahepatic cytokines and their respective receptors in mediating the inflammatory process is in its infancy.63 TNF-a is a potent proinflammatory cytokine that has been examined for a possible role in NAFLD and NASH. Several studies have measured circulating levels of TNF-a and found that they are increased in patients with NASH.118–120 Since visceral or mesenteric adipose tissue is a major source of TNF-a production and the liver is immediately downstream in its venous drainage, the liver may be a primary target of this cytokine. Because not all patients with NAFLD develop significant necroinflammatory changes in response to the hepatocellular fat accumulation, there may be genetic polymorphisms in proinflammatory cytokines and their receptors that predispose to unbridled inflammation in a subset of patients.121 Evidence also suggests that a TNFa receptor polymorphism is overexpressed in patients with NASH and a similar observation has been made in patients with alcoholic hepatitis. On the other hand, blocking the effect of TNF-a does not prevent NAFL in a diabetic mouse model, suggesting that TNF-a may not be central to the pathogenesis of NAFL.122
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Section IX. Liver Affected by Other Organs (Non-Hepatic Conditions)
Endotoxin
AMIODARONE
One stimulus for an inflammatory state in NASH may be the abnormal production and absorption of bacterial endotoxin from the gut.93,119 This phenomenon has been the leading explanation for the severe and rapidly progressive fatty liver disease that developed in patients who underwent jejunoileal bypass several decades ago. Following this procedure, the surgically created blind loop served as a reservoir for bacterial overgrowth and the production of endotoxin. Even without surgically altered small-bowel anatomy, abnormalities such as small-bowel diverticula can be a source of bacterial overgrowth and may contribute to the development of NASH in selected patients. Animal models have provided additional evidence that gutderived endotoxin may play a role in fatty liver disease. The obese leptin-deficient ob/ob mouse develops fatty liver and the extent of liver injury, as measured by serum ALT levels and hepatic NFkB activation is modulated by gut-derived endotoxin.77,123
Amiodarone may be one of the most common causes of NASH induced by currently available drugs. Amiodarone use is associated with multiple histologic abnormalities; it causes phospholipidosis, or the accumulation of whorls of lysosomal membrane, that requires electron microscopy to identify and it can also cause the histological abnormalities typically associated with NASH. Specifically, macrovesicular and microvesicular steatosis, hepatocellular ballooning, Mallory’s hyaline, fibrosis, acidophil bodies and glycogen nuclei can be features of amiodarone hepatotoxicity. These abnormalities occur in 1–3% of patients treated with the drug and have been rarely associated with severe progressive liver disease.133
HEPATIC FIBROGENESIS IN NASH An imbalance of extracellular matrix production in the liver in response to chronic injury is probably the cause of cirrhosis in NASH as it is in all other forms of chronic liver disease. Why some individuals develop fibrosis and some do not has not been established, but roughly a third of patients with any form of chronic liver disease appear to develop fibrosis and are therefore at risk for progressing to cirrhosis. Genetic predisposition related to a variety of polymorphisms may play a key role.124,125 Stellate cells are the primary source of hepatic extracellular matrix production and these cells have been shown to be in an activated state in NASH.126,127 Especially relevant to NASH is the finding that insulin may stimulate activation of hepatic stellate cells to a more fibrogenic phenotype.128 A direct role for hyperinsulinemia that accompanies insulin resistance may also explain the increased risk of fibrosis in patients with diabetes.15,129,130
DRUGS THAT CAUSE NAFLD AND NASH Compared to insulin resistance, drugs are a relatively uncommon cause of NAFLD and NASH. A number of drugs have been associated with NASH (Table 55-2), many of which are identified in case reports. Such single cases or small case series of drug-induced NASH cannot establish with certainty that NASH was not a pre-existing disorder before the drug or if the drug in question exacerbated underlying insulin resistance, leading to the development of NASH by mechanisms common to most patients.131 Several mechanisms are important in drug-induced NAFLD and NASH. Drugs may increase the burden of free fatty acids delivered to the liver (e.g., corticosteroid-induced peripheral lipolysis). Perhaps more commonly, some drugs impair the removal of fat from the liver by impairing mitochondrial b-oxidation34 or compromising the normal formation and secretion of VLDL.132 Two drugs, amiodarone and tamoxifen, are particularly relevant to current clinical practice because of the difficult decisions required for optimal patient management if drug-induced NASH develops.
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TAMOXIFEN Tamoxifen has been clearly implicated as a cause of NAFLD and even NASH in several studies.134–136 In some patients there may be irresolvable issues as to whether tamoxifen caused NASH or the NASH was a pre-existing condition. Nonetheless, in a case series of 66 patients undergoing serial computed tomography (CT) imaging for tumor surveillance, 24 developed NAFLD and 11 of those developed liver enzyme elevations while being treated with tamoxifen.137 Onset of NAFLD occurred 1–44 months after beginning tamoxifen and resolution occurred 1–14 months after stopping tamoxifen. Some studies have shown that being overweight predisposes to developing NAFLD on tamoxifen, suggesting that, mechanistically, tamoxifen may cause NASH by exacerbating underlying metabolic abnormalities.138–140 The mechanisms of tamoxifen-induced NAFLD and NASH have been debated. While commonly used as an estrogen antagonist, tamoxifen also exerts estrogen agonist effects in a tissue-dependent fashion. On the other hand, estrogens may be important for normal lipid metabolism141 and the antagonism of estrogen effects or the absence of estrogens may also contribute to the development of NAFLD.140,142,143 The management of tamoxifen-induced NASH is challenging (see Special issues, below). Comparatively few data are available for alternative agents such as raloxifene and toremifene, but early reports have suggested the development of NASH while on toremifene.138,144 Initial reports of a benefit of the PPARa ligand bezafibrate in patients with tamoxifen-induced NASH have suggested that this approach needs further study.138,140
CLINICAL FEATURES OF NAFLD AND NASH (Table 55-8) DIAGNOSIS The presence of NAFLD is often first suspected based on the results of imaging studies such as the finding of a diffusely echogenic liver on ultrasound or a relatively hypodense liver on CT. A diagnosis of NAFLD can be confirmed by liver biopsy, although the current practice of most clinicians is not to pursue the diagnostic impression further if liver enzymes are normal. The potential pitfalls of this clinical algorithm are discussed further below. NASH, the subset of NAFLD characterized by significant necroinflammatory changes, can only be diagnosed by biopsy because there are currently no symp-
Chapter 55 NASH
toms, signs, or useful non-invasive markers of the features of NASH that set it apart from the broader inclusive diagnosis of NAFLD.
SYMPTOMS NASH is most commonly asymptomatic.25 This is in contrast to patients with alcoholic liver disease, who tend to be more sympto-
matic with similar degree of fatty liver.145,146 Many patients with NASH note fatigue and poor exercise tolerance. Case series have also highlighted right upper quadrant abdominal pain as an associated symptom. Few data exist on the prevalence and nature of this pain. It may be more common than is currently perceived and the symptoms can be difficult to discern from symptoms of cholelithiasis in some patients.
Table 55-8. Clinical Features of Non-Alcoholic Steatohepatitis (NASH)
ALCOHOL HISTORY
Symptoms and physical findings • Fatigue (correlates poorly with histological stage) • Vague aching right upper quadrant pain (usually mild but may be mistaken for gallstone disease) • Hepatomegaly • Increased waist circumference indicates central adiposity220 • Acanthosis nigricans (especially in children206) • Lipodystrophy Laboratory • Aminotransferase elevations (rarely more than 10-fold above upper reference range; aminotransferases can be in the normal range with NASH or cirrhosis266) • ALT typically greater than AST (AST > ALT suggests occult alcohol abuse or significant fibrosis • Elevated insulin ¥ glucose product (basis of the HOMA-IR and QUICKI) • Hypertriglyceridemia • Positive antinuclear antibody in about one-third of patients287 • Abnormal iron indices Imaging • No imaging modality can reliably identify fibrosis and stage the disease167 • Ultrasound demonstrates a “bright” liver but is insensitive (cannot detect steatosis less than 25–30%) and non-specific (increased echogenicity of fibrosis or cirrhosis can be mistaken for steatosis) • CT allows accurate quantification of fat but at increased cost • MR imaging and spectroscopy allow measurement of fat content and possibly ATP levels in fatty liver114
Excluding significant alcohol use in a patient with steatohepatitis is essential for establishing a diagnosis of NASH. How much alcohol constitutes enough to contribute to liver disease is debated. A large population study in Italy, the Dionysos study, suggested that alcohol consumption less than 30 grams daily is not associated with adverse sequelae.10 This seems quite generous to many, and an upper limit of alcohol consumption for most studies of NAFLD is set at 20 grams daily to exclude fully the possibility that alcohol could be playing a role. Certainly, when consumption reaches an amount over 60 grams daily, then it most likely plays a role in the development of hepatic steatosis. For an amount between 20 and 60 grams daily, the role of alcohol is uncertain. Quantifying alcohol consumption can be at best a rough approximation in the clinical setting. For the sake of clinical studies, daily alcohol consumption is typically described as grams of ethanol consumed daily. This of course begs the question of exactly how much beer, wine, or liquor delivers a specified amount of alcohol. A commonly used conversion is that 10 grams of alcohol is roughly the alcohol content of one beer, one glass of wine, or one standard drink containing distilled spirits. In reality, these drinks typically contain anywhere from 10 to 20 grams of ethanol, depending on the type of beer or wine, what constitutes a “glass of wine” and the volume of distilled spirits actually used to prepare a drink. Shown in Table 55-9 is a compilation of the alcohol content of various common beverages to assist in the accurate estimation of daily alcohol consumption.
ALT, alanine aminotransferase; AST, aspartate aminotransferase; HOMA-IR, homeostasis model assessment method for insulin resistance; QUICKI, quantitative insulin sensitivity check index; CT, computed tomography; MR, magnetic resonance; ATP, adenosine triphosphate.
Table 55-9. Alcohol Content of Beverages Commonly Consumed in the USA Beverage
Nominal alcohol content (vol/vol)a Typical
Domestic beers Domestic light beers Malt liquors, ales, stoutsb Wine “Fortified” winec Distilled spirits
Range
Alcohol content (g/dl)
Volume (ml) for 10 g alcohol
Typical
Range
Typical
Range
“Standard drink” volume
Grams alcohol per standard drink Typical
Range
4.66% (e.g., Budweiser) 3.43% (e.g., Bud Light)
4–7% (3.2–5.5% wt/vol) 3.1–4.3% (2.5–3.4% wt/vol)
3.7 2.7
3.2–5.5 2.5–3.4
270 370
180–320 300–410
12 oz (355 ml) 12 oz (355 ml)
13 10
11–20 8.7–12
5.89% (e.g., Colt 45)
5–12% (4–9% wt/vol)
4.7
4.0–9.5
215
110–260
12 oz (355 ml)
16.5
14–32
14% 20% 40%
12–15% 18–20% 40–75% (80–151 proof )
11.1 15.8 32
9.5–11.9 14.2–15.8 32–60
90 60 32
85–105 60–70 17–32
4 oz (115 ml) 4 oz (115 ml) 1 oz (30 ml)
13 18 9.5
11–14 16–18 9.5–18
a
By federal law, the alcohol content of beer in the USA is measured in wt% (g/dl). All other alcoholic beverages are measured in vol% (ml/dl). The conversion is g/dl ¥ 1.27 = ml/dl, or ml/dl ¥ 0.79 = g/dl. Since 1935, federal law has prohibited mention of alcohol content on beer labels (to avoid using it to market a product) unless otherwise required by state law. This law was successfully challenged in a 1992 federal court case. b In California, a product labeled “beer” must have an alcohol content of less than 4% wt/vol (5% vol/vol). Products with higher alcohol content are identified by other names, such as malt liquor, ale, or stout. c Fortified wines are produced by adding distilled alcohol after fermentation is complete to increase the alcohol content.
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Section IX. Liver Affected by Other Organs (Non-Hepatic Conditions)
EXAMINATION FINDINGS There are no specific physical examination findings that reliably indicate the presence of NAFLD or NASH. Because insulin resistance is an underlying abnormality in most patients with NAFLD, centripetal obesity, hypertension, and possibly acanthosis nigricans may be seen in association with NAFLD. Hepatomegaly is present in up to 75% of patients,25 but can be difficult to identify on physical exam in patients with marked visceral adiposity. An atypical distribution of body fat may suggest a lipodystrophy, but subtle forms of lipodystrophy can be difficult to distinguish from the spectrum fat distribution observed in the normal population.147
LABORATORY TESTS Clues to the presence or absence of NAFLD can be obtained from laboratory testing, although the presence of NASH in the setting of NAFLD cannot be established without a liver biopsy. Laboratory testing also serves to exclude the presence of other diseases or identify diseases such as hepatitis C infection that may coexist with NAFLD.
Aminotransferases Elevated serum levels of the aminotransferases ALT and aspartate aminotransferase (AST) are commonly used as a screen for unsuspected liver diseases such as NAFLD. Once other causes of liver disease have been excluded, NASH is the most common cause of elevated aminotransferases.148 On the other hand, the prevalence of elevated serum ALT and AST levels in patients with NAFLD or NASH is unknown.
Disease Despite “Normal” Aminotransferases Data obtained from morbidly obese subjects undergoing bariatric surgery have demonstrated that the entire spectrum of NAFLD from minor amounts of fat to aggressive NASH and cirrhosis can be found, even with normal aminotransferases.22,149,150 Although aminotransferases lack specificity and sensitivity for the detection of NAFLD,22 better tests short of liver biopsy are not available.
Defining Normal Reference Ranges for Serum Aminotransferases A contributing factor to poor sensitivity of aminotransferases in detecting NAFLD or other liver diseases such as hepatitis C is how the upper limit of the reference range (commonly called the upper limit of normal, or ULN) is defined.151 By convention, normal ranges are defined by the results of any test in a disease-free population. Since imaging and liver biopsy are needed to exclude NAFLD and NASH, clinical laboratories cannot exclude these in the reference population used to define normal. Surrogate markers such as body mass index (BMI) and other clinical markers of insulin resistance that correlate with an elevated ALT in population studies are not used to exclude patients from the reference population by clinical laboratories.22,152 This has resulted in a wide range of upper reference range values used by different clinical laboratories, each with its own reference population. Although some variability is introduced by the use of different analyzers in clinical laboratories, the major contributor to the variability is variability of reference population chosen (B. Neuschwan-
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der-Tetri, unpublished data). One study based on a carefully screened population suggested that the upper reference range for serum ALT activity for men and women should be 30 and 19 U/l respectively.153 This compares to an upper reference range among laboratories that varies from 30 to 75 U/l. One disadvantage to lowering the upper reference range to increase the sensitivity of the ALT is the loss of specificity. This could lead to unnecessary testing until a new paradigm for the clinical management of mildly elevated levels (e.g., 35 U/l) emerges.154
Usefulness of ALT:AST Ratio One of the ways in which measuring the ALT and AST can contribute to the diagnosis of NAFLD is to calculate the ratio of one to the other. An AST greater than the ALT is highly suggestive of alcoholic liver disease whereas the ALT is typically greater than the AST in the non-cirrhotic NAFLD patient.155 However, with the development of significant fibrosis or cirrhosis in patients with NASH, the AST can exceed the ALT. One series of 70 patients with NASH found AST:ALT ratios of 0.7, 0.9, and 1.4 with no fibrosis, mild fibrosis, or cirrhosis respectively.156
Cholestatic Enzymes NAFLD is primarily a hepatocellular disease with only occasional biliary tract abnormalities identified on biopsy. Nonetheless, g-glutamyltranspeptidase (GGT) can be elevated in NAFLD in proportion to the degree of fat accumulation149 and elevated serum alkaline phosphatase levels have been reported in obese patients.157 A pilot study of an insulin-sensitizing agent (see below) that caused improvement in aminotransferases also found corresponding decreases in both the serum alkaline phosphatase and GGT.158
Other Laboratory Tests The role of other laboratory tests in the diagnosis of NAFLD is primarily to exclude other causes of liver disease or to identify concomitant diseases. Antibodies associated with autoimmune hepatitis are sometimes found in low to moderate titers.159 High titers provide an additional impetus to perform a liver biopsy to exclude fully this treatable disease. Identifying insulin resistance by laboratory testing can suggest a diagnosis of NAFLD (see below). Serum lipid levels can provide important information regarding other correlates of the metabolic syndrome that require appropriate management. Identifying the relatively rare patient with hypobetalipoproteinemia as a cause of NAFLD can be facilitated by an astute observation of an unexpectedly low serum cholesterol. Wilson’s disease must be fully excluded as an underlying disorder in children and adolescents with NAFLD by measuring the serum ceruloplasmin and other testing as needed (Chapter 66).
LIVER BIOPSY The diagnosis of NASH can only be established with certainty by performing a liver biopsy (see review of histological findings in Chapter 13).4,160 An unresolved issue requiring further studies before definitive recommendations can be established is the appropriate selection of patients who would benefit from a liver biopsy. The decision to perform a liver biopsy in a patient suspected of having NAFLD should take into account the inherent risks associ-
Chapter 55 NASH
ated with a biopsy balanced against the benefits.161,162 An uninterested patient, substantial comorbidities such as advanced vascular disease, and a variety of other clinical circumstances lessen the enthusiasm for performing a liver biopsy. Despite the known reasons not to perform a liver biopsy, studies have suggested that significant benefit can be gained from the information obtained. For example, at least a third of patients suspected of having NAFLD on clinical grounds were found to have another cause of liver enzyme elevations when a biopsy was performed.163 Historically, biopsies have changed management in a fraction of patients, but the need to consider treatment will likely increase as new therapeutic alternatives for NAFLD emerge.155,163,164 Establishing the diagnosis of NASH and distinguishing it from simple steatosis, or non-NASH NAFLD, relies on finding steatosis, characteristic inflammation, and evidence of cellular injury (Table 55-1).4 The extent of necroinflammatory changes and fibrosis (if any) needed to establish the diagnosis of NASH continues to be refined. An occasional finding is a component of microvesicular steatosis along with macrovesicular steatosis. Histologically, the hepatocellular triglyceride accumulation in NAFLD is commonly a mixture of microvesicular and macrovesicular fat droplets. Microvesicular fat is identified as droplets smaller than the nucleus that do not displace the nucleus whereas macrovesicular fat displaces cell contents, including the nucleus to the cell periphery. Whether the differences between the two patterns of fat accumulation are related to different pathophysiological processes or the rate at which fat accumulates is unknown.
IMAGING STUDIES Imaging studies often provide the first evidence that a patient has otherwise unsuspected NAFLD. Unfortunately, a rational and costeffective approach of what to do next has not been developed. A reasonable approach includes estimating insulin sensitivity, excluding other causes of liver disease, and using this information to determine whether a liver biopsy should be pursued (Figure 55-7). The characteristics of commonly used imaging studies in NAFLD are discussed below. Although each has its strengths and weaknesses, imaging variably identifies steatosis and cirrhosis. No imaging study can assess the necroinflammatory changes or fibrosis that distinguish NASH from less worrisome forms of NAFLD.165–167
Ultrasound Ultrasound examination of the liver may be the least costly imaging method, but it lacks both specificity and sensitivity. Fat in the liver confers a “bright” appearance on ultrasound, but significant fibrosis without fat can have a similar appearance.165 Methods have been proposed to increase the sensitivity of ultrasound using quantitative techniques, but these have not been widely accepted or established in clinical practice. Despite these shortcomings, ultrasound remains a commonly used method of identifying the NAFLD as a cause of unexplained liver enzyme elevations.
Computed Tomography CT imaging of the liver is more sensitive than ultrasound for detecting NAFLD, but at a greater cost.165 Like ultrasound, CT cannot
Incidental fatty liver on imaging (ultrasound, CT, MRI)
ALT > 40 U/L
Evaluate for other causes of liver disease •Alcohol •Viral •Drug •Autoimmune •Other metabolic (e.g., Wilson's disease, alpha-1 antitrypsin deficiency) Assess insulin sensitivity: •Non-diabetic: measure fasting insulin and glucose •Type 2 diabetic: assume insulin resistance
If NAFLD or NASH suspected:
Consider biopsy
Consider empiric therapy Lifestyle modification (exercise, dietary modification)
Figure 55-7. A simple diagnostic algorithm for non-alchoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Most patients come to medical attention because of unexplained serum alanine aminotransferase (ALT) elevations or evidence of NAFLD on imaging studies. One approach is to evaluate other possible causes of enzyme elevations or fatty liver and then assess insulin sensitivity. If NAFLD or NASH is suspected based on the results of this testing, then a decision whether to perform a liver biopsy or simply recommend empiric therapy needs to be made. Factors favoring a biopsy include risks for advanced disease (higher age, diabetes, obesity, hypertension), level of patient concern, and level of confidence that other diseases have been excluded, especially treatable diseases such as autoimmune hepatitis and hemochromatosis. The degree of ALT elevation undoubtedly influences the decision of most clinicians, although this probably lacks a rational basis because advanced NASH can be found with normal liver enzymes.
identify necroinflammatory changes or early fibrosis that signify the presence of NASH. Radiographic tissue density is estimated using Hounsfield units. The fatty liver has a lower density than normal and the presence of fat can be calculated by comparing the liver density to spleen or paraspinal muscle density (Figure 55-8). Common formulas include either subtracting the liver density from the spleen density or calculating the liver-to-spleen ratio. A difference between liver and spleen of > 10 Hounsfield units indicates liver fat,168 as does a liver-to-spleen ratio of less than 1.
Magnetic Resonance (MR) MR imaging and MR spectroscopy are the most sensitive means of detecting NAFLD, with the tradeoff of also being the most costly.165 In fact, the specificity and sensitivity of MR techniques may exceed the accuracy of the subjective interpretation of a limited sample of tissue obtained by liver biopsy. A number of MR techniques have been proposed for optimizing the detection of fat169 and spectroscopy techniques have also proved useful in detecting disruption of ATP production in experimental studies.115
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Section IX. Liver Affected by Other Organs (Non-Hepatic Conditions)
Figure 55-8. Low-density liver on abdominal computed tomographic imaging of a patient with nonalcoholic steatohepatitis (NASH). Whereas the liver density should be roughly equivalent to the spleen, in this typical example, the liver has a mean density of 26 Hounsfield units (HU) compared to a spleen density of 48 HU, resulting in a liver-to-spleen density ratio of 0.54. By definition, water has a density of 0 HU and air a density of –1000 HU.
Nuclear Medicine Improvements in ultrasound, CT, and MR have led to general abandonment of nuclear medicine studies as a means of detecting hepatic abnormalities in general. One very sensitive means of estimating liver fat content is the use of 133Xe washout, a technique reported in the past but no longer in clinical use.
Focal Fat and Focal Sparing The common use of abdominal imaging to evaluate suspected liver abnormalities or other intra-abdominal processes has led to increasing discovery of incidental focal lesions in the liver caused by focal sparing in an otherwise fatty liver or focal fat within an otherwise normal liver. Focal fat may be caused by aberrant venous drainage of insulin-enriched pancreatic blood into a specific region of the liver such as the posterior aspect of segment IV near the porta hepatis (Figure 55-9).60 Focal sparing may be related to variations in the blood supply as well. Focal sparing adjacent to the gallbladder is common and can be explained by a localized blood supply originating from the gallbladder. In 290 patients with hepatic steatosis, 78% of those with a gallbladder had focal sparing in this area.170 In rare circumstances (about 1% of patients), focal fat or focal sparing can occur adjacent to the falciform ligament and may be difficult to distinguish from tumor.171 Focal sparing in this area may be related to non-insulin-enriched blood draining directly into the liver from a gastric vein that fails to join the portal vein before it enters the liver.60
CLINICAL ESTIMATION OF INSULIN RESISTANCE The ability to estimate insulin resistance and thus predict the presence of complications associated with this disorder has become
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necessary in the management of patients with NAFLD. The presence of insulin resistance suggests that therapeutic options aimed at improving insulin sensitivity may be beneficial, whereas the occasional patient with NAFLD but normal insulin sensitivity should be further evaluated for uncommon or unrecognized causes such as other metabolic abnormalities or covert alcohol abuse. Unfortunately, how best to define and measure insulin resistance is a source of continued debate. Perhaps it is no coincidence that the difficulties encountered in defining and measuring insulin resistance are no different from those with defining NAFLD and NASH,6 namely the difficulty in drawing a distinction between what is normal and what constitutes disease. This becomes impossible when patients present within a continuum from normal to overtly lifethreatening disease yet measures are sought that dichotomize between the presence and absence of a disorder. Some of the commonly used methods to assess insulin sensitivity and the practical issues related to reliably measuring serum insulin concentrations have been reviewed.172 Despite potential problems, biochemical assessment of insulin resistance commonly relies on measuring serum insulin levels, and two measures, the homeostasis model assessment method for insulin resistance (HOMA-IR) and the quantitative insulin sensitivity check index (QUICKI), use the fasting insulin level multiplied by the fasting glucose level.173 Although HOMA-IR has been widely used, Katz et al. suggest that the QUICKI is a reasonable estimate of the glucose clamp-derived index of insulin sensitivity (SIClamp) because the QUICKI is linearly related to the SIClamp.173–175 This may be due to the fact that both the QUICKI and the SIClamp calculations use log-transformed values. Nonetheless, the QUICKI and the HOMA-IR are both based on the product of the fasting insulin multiplied by the fasting glucose (Figure 55-10), a point missed by many authors when they calcu-
Chapter 55 NASH Figure 55-9. Focal fat caused by aberrant pancreatic venous drainage. (A) Focal fat in this patient in posterior segment IV (arrow) was attributed to pancreatic venous drainage into a focal area of the liver. This image was obtained during the portal venous phase after injection of contrast into the superior mesenteric artery, showing that the area of focal fat was not perfused by portal blood flow. (B) Selective injection of the superior pancreaticoduodenal artery resulted in venous filling of the region of focal fat, indicating that the insulin-enriched pancreatic blood flow was directed to the region with focal fat. (Reproduced with permission from Fukukura Y, Fujiyoshi F, Inoue H, et al. Focal fatty infiltration in the posterior aspect of hepatic segment IV: relationship to pancreaticoduodenal venous drainage. Am J Gastro 2000; 95:3590–3595.)
A
B
late that the two are found to be highly correlated in a given study. Because they are derived from the same number (glucose ¥ insulin), the HOMA-IR and QUICKI are potentially interchangeable in their utility.173 Because the QUICKI is log-transformed, the arithmetic mean of HOMA-IR values from a study cohort may not be equivalent to the arithmetic mean of the corresponding QUICKI values. The relevance of this difference in evaluating study data has not been determined. Because insulin sensitivity varies over a range and there are intermediate values associated with disease in some people and not in
others, defining a cutoff between normal and abnormal has proven difficult. Additionally, the absence of standardization of insulin measurements may prevent direct comparison of results among different study sites. A lower limit of normal QUICKI in the range of 0.357–0.382 is often reported with values less than this indicating insulin resistance (typically in the range of 0.25–0.35 for NASH patients). An upper limit of normal HOMA-IR is in the range of 1.0–1.5 with higher values signifying insulin resistance. By calculation, the QUICKI values of 0.25–0.35 typical of NASH patients equate to HOMA-IR values of 25–1.8 respectively. A potentially
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Section IX. Liver Affected by Other Organs (Non-Hepatic Conditions)
useful and expedient clinical pearl is that based on these normal limits, values for the glucose ¥ insulin product exceeding 700 when one multiplies insulin (mU/l) ¥ glucose (mg/dl) may correspond to insulin resistance (equivalent of QUICKI < 0.351 and HOMA-IR > 1.73). Why this simple product is not used in place of more complicated formulas is not justified in the literature. Although a simple mathematical relationship exists between the two indices of insulin sensitivity, the HOMA-IR has a greater spread between values in patients with insulin resistance and little spread between values of patients with normal or near-normal insulin sensitivity. The opposite is true for the QUICKI, in which patients with normal insulin sensitivity have little variation in their values. The clinical relevance of this significant difference is yet to be established, although a treatment that causes a modest improvement in insulin sensitivity in patients with severe insulin resistance will cause a large change in the HOMA-IR and a small change in the QUICKI. Obtaining postprandial insulin levels after an oral glucose challenge may prove to be an even better means of assessing insulin resistance,158,176 but this too will need further study.
HOMA-IR =
glucose[mM] x insulin[mU/L] 22.5
(or) HOMA-IR =
glucose[mg/dL] x insulin[mU/L] 405 1
QUICKI =
log(glucose[mg/dL] x insulin[mU/L]) Figure 55-10. Clinical estimation of insulin sensitivity. Two easily used measures of insulin sensitivity are the homeostasis model assessment of insulin resistance (HOMA-IR) and the quantitative insulin sensitivity check index (QUICKI). Both indices are derived from the fasting insulin level multiplied by a simultaneously obtained fasting glucose level and are therefore mathematically directly related to each other. The QUICKI is log-transformed, which makes it linearly related to the SIClamp, a more accurate measure of insulin sensitivity based on the insulin clamp technique.
PREDICTORS OF NASH AND FIBROSIS Appropriate management of patients with suspected NASH requires the ability to predict which patients with elevated liver enzymes or NAFLD detected by imaging are at risk for progressive disease and thus warrant more aggressive evaluation and treatment (Tables 55-5 and 55-6). One study of severely obese patients undergoing bariatric surgery (BMI > 35 kg/m2) found that hypertension, ALT elevation, and insulin resistance predicted the presence of NASH in patients with NAFLD.15 In fact, three-quarters of these severely obese patients with both hypertension and diabetes had NASH whereas 7% with neither condition had NASH (Figure 5511). Diabetes and hypertension were also predictive of advanced fibrosis. All patients with advanced fibrosis in this study had at least diabetes or hypertension. Another study established the importance of age; significant fibrosis (stage 3 or 4) was present in only 4% of NASH patients under the age of 45 yet it was present in 40% of those 45 years and older.177 The available data can be summarized by observing that the greatest risk for significant fibrosis on a liver biopsy is the presence of obesity and diabetes in a patient over the age of 45 years with an AST:ALT ratio > 1. Many studies evaluating risk factors for the presence of fibrosis identify the degree of steatosis and necroinflammatory changes on the liver biopsy as predictive.150 While this association is informative with respect to the pathogenesis of fibrosis, it offers no clinical utility. The association of one biopsy finding to another obviously does not help in deciding whom to biopsy. Moreover, the association between necroinflammatory changes and fibrosis on a given biopsy has not been shown to predict who will develop fibrosis with time. The ability to predict who will develop fibrosis based on findings in a non-fibrotic biopsy would be a major contribution to clinical management.
DIFFERENTIAL DIAGNOSIS Because the diagnosis of NASH is based on histological findings, alcoholic hepatitis must be excluded from the outset. Other causes of steatohepatitis that must also be excluded with appropriate clinical and laboratory evaluation are Wilson’s disease and drug-induced steatohepatitis. Whether these entities share pathophysiologic
Figure 55-11. Type 2 diabetes and hypertension (HTN) as major risk factors for non-alcoholic steatohepatitis (NASH) in obese patients. The presence of both diabetes and HTN confers the greatest risk for having NASH in severely obese patients undergoing bariatric surgery, whereas the risk of having NASH is low in the absence of both risk factors. (Reproduced from Dixon JB, Bhathal PS, O’Brien PE. Nonalcoholic fatty liver disease: predictors of nonalcoholic steatohepatitis and liver fibrosis in the severely obese. Gastroenterology 2001; 121:91–100, with permission.)
Percentage of patients with NASH
80 70 60 50 40 30 20 10 0 Neither diabetes nor HTN (n=57)
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Hypertension alone (n=29)
Diabetes alone (n=8)
Both diabetes and HTN (n=11)
Chapter 55 NASH
mechanisms with NASH is yet to be defined and therefore whether they cause NASH or must be excluded to establish a diagnosis of NASH is a source of ongoing debate.
CONCOMITANT DISEASES NASH was once thought of as a diagnosis of exclusion, implying that the diagnosis could not be reached unless all other causes of liver disease were excluded. As the histologic criteria have been refined and underlying pathophysiologic mechanisms better understood, the coexistence of NASH with other causes of liver disease is increasingly recognized. For example, 5.5% of biopsies with steatohepatitis were found to have concomitant other diseases in one series.9 In this series, chronic hepatitis C was the most common; conversely, NASH was found in about 4% of biopsies obtained for hepatitis C. Another series identified alpha1-antitrypsin defects as the most common concomitant disease process in NAFLD, occurring in about 8% of biopsies.178
NAFLD and Chronic Hepatitis C Infection NAFLD and hepatitis C are two common forms of liver disease and overlap between the two is not surprising.9,178,179 However, the presence of NAFLD has been found to be more common in patients infected with hepatitis C than the general population in both adults and children. For example, a series of 148 hepatitis C virus (HCV) patients in Australia identified NAFLD in 61% compared to the noninfected adult prevalence of about 20%.180 Studies in humans and animals have shed some light on the interaction between NAFLD and HCV infection to show that obesity and insulin resistance are the major factors predisposing to NAFLD in HCV patients (Figure 55-12).181,182 HCV infection does contribute to insulin resistance and could promote the development of NAFLD by this mechanism.183,184 Although obesity and insulin resistance are the underlying causes of NAFLD in most patients with HCV, HCV genotype 3 infection can directly cause NAFLD.181,185–187 Patients with genotype 3 infection often have lower circulating lipids than patients with other genotypes, indicat-
HCV infection
+
+
Insulin resistance
NAFLD/NASH
(genotype 3) +
+
Fibrosis
Cirrhosis/HCC
Figure 55-12. The interaction between non-alcoholic fatty liver disease (NAFLD) and hepatitis C virus (HCV) infection. The primary contributor to NAFLD in most patients with HCV infection is insulin resistance caused by obesity, sedentary lifestyle and probably genetic factors. However, HCV infection contributes to the progression of NAFLD by increasing the degree of insulin resistance, accelerating fibrogenesis, and contributing to the risk of developing hepatocellular carcinoma (HCC). In the case of genotype 3 infection, HCV directly promotes the accumulation of fat in the liver.
ing impaired hepatic secretion of triglyceride as VLDL.8,186,188,189 Moreover, serum lipids typically increase190 and the NAFLD in these patients improves with successful treatment of the HCV infection.191 Significantly impaired secretion of fat from the liver is rare with other genotypes. Studies in animals and cultured cells have explored the basis for the interaction between viral proteins and hepatocellular fat trafficking.192 HCV core and NS-5 proteins can impair triglyceride export as VLDL from hepatocytes. Viral proteins associate with nascent lipid droplets, apolipoprotein A, or microsomal triglyceride transfer protein. This interaction and increased oxidant stress have been proposed as mechanisms of virally impaired hepatocellular triglyceride handling.193–196 Paradoxically, in vitro studies reported to date have used only genotype 1 virus, yet the clinical association between HCV infection and NAFLD is strongest in genotype 3 infected patients. The coexistence of the two disorders is important because the presence of NAFLD accelerates the progression of liver disease associated with hepatitis C. Liver fibrosis in HCV patients correlated with the presence of NAFLD independent of age and obesity in one study,197 Another study from Italy found that NAFLD doubled the rate of fibrosis progression from approximately one Scheuer stage every 8 years in HCV without NAFLD to one stage every 4 years in HCV with coexisting NAFLD.198 Similar findings of accelerated progression in studies from around the world have been reported in patients with NAFLD or its surrogate, obesity.8,178,187,199,200 Just as it increases the rate of fibrosis, the presence of NAFLD may also increase the risk of developing HCC in HCV patients.201 The presence of NAFLD also appears to reduce significantly the likelihood of obtaining a sustained virologic response to treatment with interferon and ribavirin, even with appropriate weight-based drug doses. Analysis of 1428 patients treated with interferon and ribavirin in a study that excluded NASH demonstrated that patients without steatosis achieved a sustained virologic response rate (SVR) of 66% whereas those with simple steatosis had a lower average SVR of 50%.8 A negative impact on response to treatment has been reported in other studies as well.178,187 Obesity, a surrogate marker for insulin resistance and NAFLD, has also been associated with a lower response rate despite weight-based dosing.202,203 The optimal management of patients with both NAFLD and HCV infection is not known. Some studies suggest that insulin resistance can directly promote fibrogenesis,204 possibly through the expression of TNF-a and cytochrome P450 2E1.205 This observation may provide further evidence of the need to assess insulin sensitivity in patients with HCV infection and direct initial management towards improving insulin sensitivity. The one notable exception is genotype 3 patients who may have NAFLD as a direct outcome of the viral infection and the NAFLD improves with treatment of the infection.
DISTINGUISHING ALCOHOLIC STEATOHEPATITIS FROM NASH Excluding alcohol as a cause of steatohepatitis can be challenging in a subset of patients. Contributing to this challenge is the likely, but unproved, possibility that some patients may have both excessive alcohol consumption and insulin resistance as underlying causes of
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steatohepatitis. Identifying such patients will require a better understanding of the pathogenesis of both diseases and the development of specific disease markers. In addition, some authors have concluded that NASH and ASH are indistinguishable and, indeed, the landmark paper by Ludwig that popularized the term NASH did so to establish that the previously described “alcohol-like” liver disease was a separate entity but with remarkably similar histologic findings to alcoholic liver disease. The published data and growing experience of clinicians suggest that a distinction can often be established from clinical and pathological findings.145 Specifically, the features below can be used to help distinguish alcoholic steatohepatitis (ASH) from NASH (Table 55-10).
Clinical and Historical Findings Compared to ASH, NASH is more commonly asymptomatic. Vague right upper quadrant abdominal pain is increasingly recognized in patients with NASH, but significant symptoms such as anorexia, fever, jaundice, and weight loss are characteristics of patients with severe alcoholic liver disease.145 These symptoms tend to be found in patients hospitalized with severe ASH while the patient with covert alcohol abuse and less severe ASH may present with minimal symptoms similar to patients with NASH.146 NASH tends to be
Table 55-10. Features Suggestive of Either Non-Alcoholic Steatohepatitis (NASH) or Alcoholic Steatohepatitis (ASH) Features associated with NASH Clinical/historical Alcohol AST (unless cirrhotic) Normal bilirubin
Liver biopsy Poorly formed or undetectable Mallory bodies Steatosis always present (except in cirrhosis) Glycogenated nuclei
Anorexia Leukocytosis, fever
Jaundice
AST > ALT Elevated bilirubin Hypoalbuminemia Peripheral leukocytosis Well-formed, numerous Mallory bodies Steatosis may be less prominent Sclerosing hyaline necrosis and central vein injury
ALT, alanine aminotransferase; AST, aspartate aminotransferase.
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more associated with features of the metabolic syndrome, such as diabetes, hypertension, obesity, dysmenorrhea, and possibly polycystic ovary syndrome (PCOS).145 Patients with severe ASH have a mortality that approaches 50% related to liver failure per se whereas patients with NASH are primarily at risk for the gradual development of cirrhosis and its complications.
For the first two decades after NASH was described, case series identified associated conditions such as obesity, diabetes, hyperten-
Chapter 55 NASH
sion, and hyperlipidemia in an effort to understand better the causes and identify treatments for NASH. With recent advances in the understanding of NASH, especially with respect to the role of insulin resistance, the importance of identifying associated conditions has evolved. As the importance of using associated conditions to identify pathophysiologic mechanisms has diminished, the role of identifying associated conditions has changed to identifying patients at risk for having NASH. The associated conditions identified in earlier studies relate mostly to insulin-resistance: obesity as an underlying cause of insulin resistance and diabetes, hypertension, and hyperlipidemia as common manifestations of insulin resistance. Additionally, acanthosis nigricans, the dermatologic abnormality associated with insulin resistance, may be associated with NASH, especially in children. Anecdotal reports suggest that PCOS may also be overrepresented in patients with NASH. PCOS is the ovarian response to hyperinsulinemia and is characterized by ovarian testosterone production, leading to menstrual irregularity, hirsutism, and acne.210 Interestingly, ovarian cysts are no longer required for the diagnosis.
DISEASE COMPLICATIONS The complication of NAFLD that is the primary cause of concern is its risk of causing progressive fibrosis, cirrhosis, and the complications of cirrhosis, including HCC and death. Our current understanding of how often this occurs and the clinical risk factors associated with progression to cirrhosis are discussed in the review of natural history. NAFLD is not recognized to be a direct cause of extrahepatic complications. In general, the associated conditions described above are thought to arise from the underlying metabolic abnormalities that lead to fatty liver and are not a direct consequence of fatty liver.
TREATMENT No effective therapies or preventive measures have been established by rigorous clinical studies for NASH. A number of agents and interventions have been examined in small trials and case series, often with encouraging findings initially, but few randomized controlled trials have been conducted in patients with NASH (Table 55-11).211 One exception was a well-designed and appropriately powered trial of ursodeoxycholic acid (UDCA) that followed an initial promising pilot study.212 Unfortunately, this trial determined that UDCA was no better than placebo (see below).213 As more complex studies are designed and executed, a therapeutic approach to NASH may emerge that combines a multidisciplinary approach to obesity, insulin resistance, sedentary lifestyle, dietary imbalances, and genetic variations. Current understanding of the pathogenesis of NASH raises the possibility that addressing insulin resistance may be an effective preventive measure, as well as a valuable treatment option.
IMPROVING INSULIN SENSITIVITY Exercise and Lifestyle Modifications Experimental data from animals and humans convincingly demonstrate that exercise reverses insulin resistance,214,215 the most
Table 55-11. Treatment Options for Non-Alcoholic Steatohepatitis (NASH) Target
Possible intervention(s)
Comments
Obesity
Caloric restriction Exercise Drugs
Insulin resistance
Exercise
Several case series suggest a benefit in NASH Necessary for sustained weight loss Insufficient data with antiobesity drugs Most effective means of improving insulin sensitivity known;288 effect on NASH to be established Most effective in conjunction with exercise for improving insulin sensitivity Pilot studies suggest a benefit of metformin and thiazolidinediones Needs confirmatory data
Weight loss
Drugs Abnormal hepatic fat metabolism Oxidant stress
Betaine
Vitamin E
Hepatocellular injury
Other antioxidants Cytoprotective agents
Fibrogenesis
Antifibrotics
Beneficial in one small pilot study in children No controlled trials Rationale for ursodeoxycholic acid and silymarin; ursodeoxycholic acid proven to be ineffective Effective antifibrotics for liver disease yet to be found
common cause of NAFLD. Perhaps the best rationale for promoting a non-sedentary lifestyle as a treatment for NAFLD is provided by the results of the Diabetes Prevention Program (DPP) trial.216 Subjects at risk for developing diabetes who adhered to a modified lifestyle that included increased regular physical activity experienced better improvement in their insulin sensitivity than similar subjects treated with the insulin-sensitizing agent metformin. Additionally, an observational study of over 50 000 nurses also found that less television-watching and more physical activity prevented the onset of diabetes, a disease that represents the final stages of insulin resistance.217 Despite the preponderance of evidence that exercise reduces mortality from cardiovascular causes, data supporting a similar salutary effect in patients with NASH are lacking.211 However, several small trials have shown that exercise may be an effective means of treating fatty liver.218 One 12-week trial of diet and exercise resulted in improved liver enzymes but histopathology was not evaluated.120 How much exercise is needed to normalize insulin sensitivity continues to be debated. Any activity is better than none and this is the approach taken by the National Cholesterol Education Program (NCEP) in recommending a minimum of 30 minutes daily of brisk walking or other moderate activities for the 70% of adults who are sedentary or minimally active. Although such a modest step represents a large accomplishment for many patients, more intense exercise provides a further benefit for those physically able and willing to pursue such lifestyle changes.219
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Weight Reduction The difficulties in achieving and maintaining weight loss are abundantly evident to health care providers and the public alike,220,221 yet the adverse health outcomes of obesity may soon outpace smoking as a preventable cause of illness and death.222 Current treatment approaches to obesity include exercise,223 dietary modifications,224 surgery,225 and drugs.226 Weight loss to treat NAFLD and NASH has been the subject of a number of small trials and case series.221 These trials each have their weaknesses and no firm conclusions can be drawn. In trials that included liver biopsies, a concern has been raised that inflammation or fibrosis could worsen. However, the histological criteria used to assess NASH were those used to evaluate chronic viral hepatitis. The changes associated with improvement of NASH may be different from the resolution of viral hepatitis. For example, a trial of rosiglitazone found that, as lobular inflammation diminished, periportal inflammation increased.227 If a histologic grading scheme is used that assesses only periportal inflammation, then such changes could be interpreted as worsening rather than what may turn out to be a step towards resolution of NASH. A current trend in the dietary management of obesity is to favor low-carbohydrate diets, eschewing the carbohydrate-based food pyramid. Although low-carbohydrate diets may be beneficial in the short term, long-term benefits have not been found in the absence of sustained lifestyle modifications that include increased physical activity.223,228,229
Bariatric Surgery Frustration by patients and their physicians with the failure of standard treatment modalities has led to increasing reliance on obesity surgery which, at a price in terms of risks and side effects, can improve insulin sensitivity and its complications.225,230,231 The current enthusiasm for surgery is fueled in part by the relative safety of current procedures compared to the jejunoileal bypass operation performed three decades ago. The older procedure left a blind loop of small bowel that may have caused or exacerbated a NASH-like form of aggressive liver disease. The procedures as they are currently performed have not been associated with progressive liver disease and death from liver failure. In fact, preliminary data suggest that the NASH associated with severe obesity may improve following the Roux en Y gastric bypass.149,232–235 A multicenter study group sponsored by the National Institutes of Health is currently gathering data that should provide definitive information about the safety of current bariatric surgery and whether it is as effective in treating NASH as it is in treating the other manifestations of insulin resistance.
Insulin-Sensitizing Agents Using pharmacological agents to improve insulin sensitivity can provide additional evidence for the role of insulin resistance in NAFLD and these agents may be an important adjunct to therapy in certain patients such as those unable to increase physical activity or lose weight. Two classes of drugs are currently available to improve insulin sensitivity: the thiazolidinediones (TZDs) and the biguanides.
Thiazolidinediones The TZD PPAR-g nuclear receptor ligands were developed as therapeutic agents for the treatment of type 2 diabetes by improving
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insulin sensitivity.236 Experimentally, these agents lead to diminished fat accumulation in the liver and muscle of diabetic animals and humans,237 suggesting that they could be beneficial in patients with NASH. Three clinically available agents, troglitazone, rosiglitazone, and pioglitazone, have been evaluated in NASH but only in small pilot studies. In a series of 7 subjects treated for 3–6 months, troglitazone was associated with improved liver enzymes,46 although it has been withdrawn from clinical use because of its idiosyncratic hepatotoxicity. Similar toxicity has not been observed with pioglitazone or rosiglitazone, possibly because troglitazone is metabolized by cytochrome P450 CYP3A4 and this is not a major pathway for the other two. Nonetheless, occasional adverse reactions have been observed with both pioglitazone and rosiglitazone which justify caution with their use. Weight gain, typically caused by peripheral fat accumulation rather than increased visceral adiposity, is a common side effect of the TZDs and can be disheartening to patients with NASH. Preliminary studies with pioglitazone and rosiglitazone indicate that both agents improved liver enzymes and the histological features of NASH.227,238,239 However, 1 patient treated with rosiglitazone experienced a precipitous rise in aminotransferases that correlated with the concomitant use of corticosteroids.158 This observation underscores the need to examine the utility of these agents within the structure of clinical trials. Whether these agents improve NASH by improving insulin sensitivity or through a recently recognized anti-inflammatory mechanism is unknown. Studies of human240 and mouse241 monocytes and macrophages have demonstrated down-regulation of cytokine production in response to PPAR-g ligands. The clinical utility of PPARg ligands as anti-inflammatory agents was further supported by their beneficial effect in patients with ulcerative colitis. The TZDs may also be beneficial because of a direct antifibrotic effect. Experimental evidence has demonstrated the ability of PPAR-g ligands to inhibit collagen production by hepatic stellate cells, the cell type responsible for abnormal fibrogenesis in the liver.242,243
Metformin Metformin, the only clinically available biguanide, is increasingly used to normalize hepatic gluconeogenesis and improve insulin sensitivity in patients with type 2 diabetes.244 It activates hepatic AMPactivated protein kinase (AMPK), shifting metabolism of free fatty acids away from esterification into triglyceride and towards mitochondrial b-oxidation.245 It also decreases hepatocellular SREBP-1 expression which down-regulates the hepatic synthesis of fatty acids. AMPK may also be important in the beneficial effect of metformin of increasing skeletal muscle glucose uptake.246 How these beneficial metabolic changes in diabetes might be helpful in NASH is uncertain. One pilot study of 20 patients with NASH treated with 1.5 gram metformin daily for 4 months found improved liver enzymes, insulin sensitivity, and liver volume, but the effect on histopathology was not determined.247 Metformin improved histopathologic findings of NAFLD in the leptin-deficient mouse,248 yet it did not reduce liver fat content in a study of diabetics.237 Like the TZDs, metformin has been established as a good first-line therapy for patients with diabetes, but both need further evaluation in clinical trials as treatments for NASH.
Chapter 55 NASH
PPARa LIGANDS
Betaine
Unlike the PPAR-g nuclear receptor ligands which promote adipocyte differentiation, PPARa ligands promote the disposal of fat through metabolic oxidation in the liver and other tissues.236 Mouse models of NAFLD have shown that PPARa ligands can prevent fat accumulation53,54 and that genetically modified mice lacking PPARa are predisposed to NAFLD.53 The clinically available PPARa ligands, the fibrates, have been examined in small studies of patients with NASH. Clofibrate was found to have no effect on liver enzymes or histopathology in a pilot study of 16 patients.212 A 4-week treatment of 23 patients with gemfibrozil demonstrated improved liver enzymes but the effects on histopathology were not ascertained.249 Limited experience with bezafibrate, a PPARa ligand not available in the USA, suggests that it might reduce the NASH associated with tamoxifen use.138,140
In a pilot study of patients with NASH, administration of betaine improved both the biochemical and histological abnormalities of NASH.254 Betaine, pronounced either bee-tane or beta-een, was first isolated from extracts of the common sugar beet (Beta vulgaris) and synthesized well over a century ago. Chemically, betaine is simply trimethylglycine. Endogenous betaine, once considered an inert metabolic byproduct, is derived from oxidation of choline or from dietary intake. Studies in the 1940s established that betaine can prevent lipid accumulation in the liver, and as such it was identified as a lipotropic agent. Betaine can substitute for choline and function as an effective dietary methyl donor when dietary methionine and choline are inadequate to meet metabolic needs. A major function of choline is to serve as a substrate for the production of phosphatidyl choline (lecithin), a component of cell membranes and an obligate component of VLDL. Because VLDL synthesis and export are the only mechanism by which fat can be exported from hepatocytes, insufficient phosphatidyl choline can prevent the synthesis of functional VLDL in the liver.255 Betaine also functions as a methyl donor for the conversion of homocysteine to methionine and thus alleviates defects in the methylation pathway caused by deficiencies of the folate cycle and vitamin B12. Homocysteine accumulation can impair insulin signaling,256 suggesting that betaine-mediated metabolism of homocysteine could improve insulin sensitivity, although this has not been examined experimentally.
STATINS AND OTHER HYPOLIPIDEMIC AGENTS Because NAFLD is associated with hyperlipidemia, one therapeutic approach has been to treat the hyperlipidemia. This approach has met with little success and the published experience fails to record the results of negative pilot studies, an unfortunate but common phenomenon.250 The hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, or statins, were developed to impair cholesterol synthesis, reduce circulating cholesterol levels, and reduce the risk of vascular disease and its complications. Based on the original mechanism of action, there is little theoretical rationale for using the statins as therapeutic agents to treat NAFLD. However, newly recognized but important anti-inflammatory effects of these drugs have rekindled interest in their use in liver disease. The hypolipidemic agent probucol has antioxidant properties that could be beneficial in NASH. An open-label pilot study with probucol in 17 patients led to improved liver enzymes but liver biopsies were not examined.251
OTHER DRUGS Vitamin E If oxidant stress is a requisite pathophysiological process in the development of NASH, then antioxidants should prove to be effective therapies. Unfortunately, this has not been the case. Admittedly, the absence of data demonstrating a benefit of antioxidants could be due to the lack of available and effective pharmacological antioxidants or alternatively because of insufficiently powered studies to identify a benefit. On the other hand, the detection of oxidant stress by various means in patients with NASH may be documenting an epiphenomenon unrelated to the pathophysiology of the disease. One pilot study did show that vitamin E at daily doses ranging from 400 to 1200 IU improved liver enzymes in children and may also have improved histopathological changes in some.252 However a small pilot study in adults showed that vitamin E supplementation did not offer significant benefits beyond those achieved with exercise.120 One study in adults suggested that a combination of vitamin E and vitamin C was beneficial, yet the control group demonstrated a similar benefit.253
Ursodeoxycholic Acid UDCA is a minor component of human bile and its pharmacologic supplementation replaces more toxic bile acids within the bile acid pool. Because of its potential to reduce injury in the liver, UDCA has been investigated as a therapy for NASH. A widely quoted but uncontrolled pilot study of 24 patients treated for 12 months showed improvements in the serum ALT level and a small improvement in the degree of steatosis.212 A subsequent double-blind placebo-controlled study for 2 years in 126 adults treated with UDCA 13–15 mg/kg found that treatment was not different from placebo (Figure 55-13).213 This well-conducted clinical trial not only demonstrated the inefficacy of UDCA in NASH, it also highlights the need to include placebo-controlled treatment groups in studies of NASH because subtle lifestyle changes that may accompany the diagnosis and subsequent management could lead to improvement in the control group. Additional negative data with UDCA include a trial in 31 children with NASH that showed weight loss was effective in normalizing enzymes and sonographically detected NAFLD whereas UDCA was not.257
LIVER TRANSPLANTATION When liver disease caused by NAFLD progresses to decompensated cirrhosis, liver transplantation can be an option. About 2% of transplants are performed for a known diagnosis of NASH and a much larger fraction are performed for cryptogenic cirrhosis, a condition that is mostly caused by NASH, as described below. One of the biggest challenges often facing these patients and the transplant team coordinating their care is the presence of significant obesity.
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60 UDCA Percentage of subjects
Placebo 40
20
0 –2
–1
0
1
2
Steatosis change Figure 55-13. Ursodeoxycholic acid (UDCA) as a treatment for non-alcoholic steatohepatitis (NASH). UDCA, 13–15 mg/kg, given for 24 months did not cause significant improvement in hepatic steatosis (shown above), necroinflammatory changes, or serum alanine aminotransferase (ALT) levels compared to controls. Although significant improvement was found in each of these parameters when compared to baseline values, the improvement was the same in UDCA and placebo-treated groups. (Data from Lindor KD, Kowdley KV, Heathcote EJ, et al. Ursodeoxycholic acid for treatment of nonalcoholic steatohepatitis: results of a randomized trial. Hepatology 2004; 39:770–778.)
Also, because NASH develops in the setting of insulin resistance, the underlying metabolic abnormality most likely remains after transplantation. Recurrent NASH after transplant occurs in patients with prior diagnoses of both NASH and cryptogenic cirrhosis.258
SPECIAL ISSUES USE OF HMG-COA REDUCTASE INHIBITORS (“STATINS”) IN PATIENTS WITH NASH As a class, the HMG-CoA reductase inhibitors are known to cause occasional increases in the serum aminotransferase levels and the manufacturers of these drugs warn against their use in patients with known liver disease. This warning appears to be based on hypothetical concerns, as there are no data to suggest that patients with pre-existing chronic liver disease are any more susceptible to increased liver enzymes or serious liver injury than patients with normal enzymes. In fact, a survey of one large university practice found that patients with elevated liver enzymes before starting a statin may have experienced overall improvement in their liver enzymes.259 Therefore, a reasonable approach is to use this class of drugs as they are intended to treat hypercholesterolemia with appropriate routine monitoring of liver enzymes as recommended in patients with normal liver enzymes.
TAMOXIFEN USE The use of the antiestrogen agent tamoxifen after resection of estrogen receptor-positive breast cancer provides a clear survival benefit. Reports of women found to have NAFLD, NASH, and cirrhosis while taking tamoxifen have raised the question of whether tamoxifen use is causally implicated or whether the liver disease may have
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pre-existed but was unrecognized before treatment. Early reports established the presence of significant NAFLD in patients treated with tamoxifen,135–137,260–262 and subsequent reports showed that, at least in some patients, NAFLD developed while being treated with tamoxifen and thus provide evidence for causality.134,139,140,263 The approach to a patient with NASH while taking tamoxifen is challenging. Some studies have shown that overweight and obesity are risk factors for the development of NAFLD after starting tamoxifen.138–140 This is important because it suggests that improving body weight might diminish the risk of developing NAFLD or possibly lead to improvement after it has developed. Alternative antiestrogen agents might also be considered. However, one study found that the use of an alternative antiestrogen agent, toremifene, was also associated with the development of NASH, suggesting that this approach may be of little benefit.144 Additional studies are needed of other antiestrogen agents and also the fibrates as concomitant treatment to prevent NASH in this setting.138,140 The best advice is to approach each situation on a case-by-case basis, weighing the potential risks and benefits of discontinuing tamoxifen. Available data suggest that overweight patients should be counseled on the benefits of weight reduction.
HEPATITIS A VACCINATION Severe hepatitis A is rare, but pre-existing liver disease may increase the likelihood of developing acute liver failure leading to death or liver transplant after hepatitis A infection. For this reason, the Centers for Disease Control and the Advisory Committee on Immunization Practices recommend that all patients with chronic liver disease should be vaccinated against hepatitis A. The vaccine, given as two injections 6 months apart, is highly effective and well tolerated. Most patients with NAFLD have not been vaccinated for hepatitis A and it is reasonable to consider recommending the vaccine to patients when a diagnosis of NAFLD or any other form of chronic liver disease is established.
HOW MUCH ALCOHOL IS ALLOWABLE? No uniformly accepted recommendation for the allowable amount of alcohol ingestion exists for patients with chronic liver disease, with the exception of abstinence for patients with alcoholic liver disease. Most clinicians recommend allowable alcohol consumption that ranges from complete abstinence to one drink (10–20 grams ethanol; see Table 55-9) weekly. The Italian Dionysos population study did not find adverse consequences of consumption < 30 grams daily, but few clinicians are comfortable making recommendations to patients allowing this much regular alcohol consumption. On the other hand, there are no data to indicate that the occasional (less frequent than weekly) single drink poses a danger to the liver. Decisions must be made based on the patient’s desires, expectations, and severity of underlying liver disease.
PROGNOSIS AND NATURAL HISTORY Accurately describing the natural history of NAFLD is difficult because the existing data are compromised by variable definitions of
Chapter 55 NASH
NAFLD and NASH, the possible unrecognized coexistence of HCV in earlier studies, the prolonged duration of study needed to identify progression, and probably a failure to recognize cirrhosis as a contributor to death from the known complications of cardiovascular disease.
PROGRESSION TO CIRRHOSIS The prognosis of NAFLD is directly related to its likelihood to progress to cirrhosis. Early studies of small cohorts followed for relatively short durations suggested that NASH was a relatively benign disease. Despite more recent data indicating that NASH may have become the most common form of chronic liver disease leading to cirrhosis, HCC, and death,25,264 a nihilistic diagnostic and therapeutic approach to these patients persists. The absence of hard figures to describe the prevalence of cirrhosis caused by NASH undoubtedly contributes to a non-aggressive diagnostic approach. However, as awareness increases, more patients may be recognized as having significant silent liver disease while being treated for the complications of insulin resistance.
A
CRYPTOGENIC CIRRHOSIS If NASH now has a prevalence of 2–3% of the adult population and a significant proportion of these patients will have progressive fibrosis and be at risk for cirrhosis, then a key question is why more patients succumbing from NASH cirrhosis have not been identified in death or transplant statistics. One factor may be that cirrhosis has been a silent and unrecognized contributor to death from obesity, cardiovascular disease, and diabetes.265 The presence of such silent cirrhosis has been well documented in morbidly obese patients undergoing bariatric surgery.266 The second factor contributing to a lack of accounting of NASH as a cause of cirrhosis is the curious loss of the typical histological features of NASH after cirrhosis develops. When this happens, a liver biopsy may reveal relatively bland cirrhosis and, because of the absence of markers for other liver disease, the cirrhosis is classified as cryptogenic (Figure 55-14).32,33 The recognition that “burned-out” NASH may explain many patients with cryptogenic cirrhosis is based on the following two observations: (1) diabetes and obesity are overrepresented in patients with cryptogenic cirrhosis;267 and (2) NASH often occurs after liver transplantation for cryptogenic cirrhosis as it does in patients with known NASH.268–270 About 12% of liver transplants are currently performed for a diagnosis of cryptogenic cirrhosis. If most of these patients had NASH as a cause of cirrhosis, then these patients plus the 2% transplanted for known NASH add up to 12–14% of liver transplants being performed for cirrhosis caused by NASH. This percentage is unlikely to represent the total fraction of the population with cirrhosis caused by NASH. Because end-stage liver disease caused by NASH is often associated with complicated diabetes or massive obesity, many of these patients will not be candidates for liver transplant and succumb to the complications of their liver disease or other manifestations of insulin resistance.
HEPATOCELLULAR CARCINOMA Once considered primarily a complication of chronic hepatitis B and C and a few less common forms of liver disease, hepatocellular car-
B
Figure 55-14. Evolution of non-alcoholic steatohepatitis (NASH) to cirrhosis. (A) A liver biopsy performed to evaluate elevated liver enzymes showed necroinflammatory changes, hepatocellular ballooning, and some hepatocytes containing Mallory’s hyaline, findings diagnostic of NASH (top panel, 40¥). (B) Twelve years later, a second biopsy showed none of the necroinflammatory changes of NASH but showed regenerative nodules among dense bands of fibrosis consistent with cirrhosis. Without the prior biopsy, this case would fulfill criteria for “cryptogenic” cirrhosis, and demonstrates the loss of active lesions of steatohepatitis with progression to cirrhosis. (Courtesy of E.M. Brunt.)
cinoma (HCC, Figure 55-15) is now recognized to be a complication of cirrhosis from any cause. The development of HCC in patients with cirrhosis caused by NASH occurs with the same frequency as in patients with chronic hepatitis C.271–275 Additionally, evidence suggests that the surrogates of NAFLD, obesity and diabetes,272,274 are associated with an increased risk of HCC and synergistically contribute to the risk of HCC in patients with coexisting viral hepatitis.276 Because of the comparatively high prevalence of HCV and NAFLD compared to other forms of chronic liver disease, these two diseases are now the most common underlying causes of HCC.273 As reviewed above, most patients diagnosed with cryptogenic cirrhosis had prior NASH as the cause of their liver disease. Given this fact, HCC should be associated with cryptogenic cirrhosis just as it is with NASH cirrhosis. Indeed, one survey of patients with HCC identified it as a complication of cryptogenic cirrhosis and, in other studies, patients with cryptogenic cirrhosis had risk factors for prior NASH as a cause of their liver disease.277
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Figure 55-15. Hepatocellular carcinoma developing in a patient with non-alcoholic steatohepatitis (NASH) cirrhosis. An obese non-alcoholic 58-yearold male presented with ascites and a liver biopsy demonstrated cirrhosis with steatohepatitis. One year later, a surveillance computed tomography scan of the liver demonstrated a 6-cm mass in the right lobe. The patient subsequently underwent transarterial chemoembolization; the lipiodolenhanced image used to direct therapy is shown. (Courtesy of A.M. Di Bisceglie.)
Table 55-12. Important Areas for Future Research
• • • • •
Identify the pathogenesis of hepatocellular injury in patients with NAFLD that leads to the development of NASH Identify non-invasive predictors of NASH in patients with NAFLD or elevated liver enzymes Identify non-invasive predictors of fibrosis (e.g., demographics, comorbidities, blood tests, genomic polymorphism analysis, imaging) Define the spectrum and prevalence of liver disease associated with insulin resistance Determine the role of exercise, weight loss, dietary changes, antioxidants, insulin-sensitizing agents and antifibrotic agents in the prevention and treatment of NASH
NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis.
CONCLUSIONS More than two decades of active research into the causes and consequences of NAFLD have advanced the field significantly. NAFLD is now recognized as a remarkably common disorder, affecting 1 in 5 adults. NASH, which was once thought to be an uncommon disorder affecting obese diabetic women, is now recognized as a relatively common cause of liver-related morbidity. It is associated with insulin resistance, affects both genders, and is a cause of significant liver disease before diabetes develops. Much work is still needed to identify the causes of NASH at the molecular level, establish better diagnostic tools, and identify effec-
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tive therapeutic strategies. Major areas needing active investigation that will build on the current foundation are listed in Table 55-12.20
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aminotransferases, hepatic steatosis and body weight in patients with non-alcoholic steatohepatitis. J Hepatol 2003; 38:434–440. Loria P, Lonardo A, Leonardi F, et al. Non-organ-specific autoantibodies in nonalcoholic fatty liver disease: prevalence and correlates. Dig Dis Sci 2003; 48:2173–2181. Bianchi L. Liver biopsy in elevated liver functions tests? An old question revisited. J Hepatol 2001; 35:290–294. Mathiesen UL, Franzén LE, Frydén A, et al. The clinical significance of slightly to moderately increased liver transaminase values in asymptomatic patients. Scand J Gastro 1999; 34:85–91. Pratt DS, Kaplan MM. Evaluation of abnormal liver-enzyme results in asymptomatic patients. N Engl J Med 2000; 342:1266–1271. Skelly MM, James PD, Ryder SD. Findings on liver biopsy to investigate abnormal liver function tests in the absence of diagnostic serology. J Hepatol 2001; 35:195–199. Sorbi D, McGill DB, Thistle JL, et al. An assessment of the role of liver biopsies in asymptomatic patients with chronic liver test abnormalities. Am J Gastro 2000; 95:3206–3210. Siegelman ES, Rosen MA. Imaging of hepatic steatosis. Semin Liver Dis 2001; 21:71–80. Mortele KJ, Ros PR. Imaging of diffuse liver disease. Semin Liver Dis 2001; 21:195–212. Saadeh S, Younossi ZM, Remer EM, et al. The utility of radiological imaging in nonalcoholic fatty liver disease. Gastroenterology 2002; 123:745–750. Jacobs JE, Birnbaum BA, Shapiro MA, et al. Diagnostic criteria for fatty infiltration of the liver on contrast-enhanced helical CT. Am J Roentgenol 1998; 171:659–664. Fishbein MH, Stevens WR. Rapid MRI using a modified Dixon technique: a non-invasive and effective method for detection and monitoring of fatty metamorphosis of the liver. Pediatr Radiol 2001; 31:806–809. Aubin B, Denys A, Lafortune M, et al. Focal sparing of liver parenchyma in steatosis: role of the gallbladder and its vessels. J Ultrasound Med 1995; 14:77–80. Soyer P, Devine N, Somveille E, et al. Hepatic pseudolesion around the falciform ligament: prevalence on CT examination. Abdom Imag 1996; 21:324–328. Wallace TM, Matthews DR. The assessment of insulin resistance in man. Diabet Med 2002; 19:527–534. Mather KJ, Hunt AE, Steinberg HO, et al. Repeatability characteristics of simple indices of insulin resistance: implications for research applications. J Clin Endo Metab 2001; 86:5457–5464. Katz A, Nambi SS, Mather K, et al. Quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans. J Clin Endo Metab 2000; 85:2402–2410. Abbasi F, Reaven GM. Evaluation of the quantitative insulin sensitivity check index as an estimate of insulin sensitivity in humans. Metab Clin Exp 2002; 51:235–237. Ikai E, Ishizaki M, Suzuki Y, et al. Association between hepatic steatosis, insulin resistance and hyperinsulinaemia as related to hypertension in alcohol consumers and obese people. J Hum Hypertens 1995; 9:101–105. Angulo P, Keach JC, Batts KP, Lindor KD. Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis. Hepatology 1999; 30:1356–1362. Sanyal AJ, Contos MJ, Sterling RK, et al. Nonalcoholic fatty liver disease in patients with hepatitis C is associated with features of the metabolic syndrome. Am J Gastro 2003; 98:2064–2071. Lonardo A, Adinolfi LE, Loria P, et al. Steatosis and hepatitis C virus: mechanisms and significance for hepatic and extrahepatic disease. Gastroenterology 2004; 126:586–597.
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180. Hourigan LF, Macdonald GA, Purdie D, et al. Fibrosis in chronic hepatitis C correlates significantly with body mass index and steatosis. Hepatology 1999; 29:1215–1219. 181. Monto A, Alonzo J, Watson JJ, et al. Steatosis in chronic hepatitis C: relative contributions of obesity, diabetes mellitus, and alcohol. Hepatology 2002; 36:729–736. 182. Colloredo G, Sonzogni A, Rubbia-Brandt L, Negro F. Hepatitis C virus genotype 1 associated with massive steatosis of the liver and hypo-b-lipoproteinemia (letter). J Hepatol 2004; 40:562–563. 183. Aytug S, Reich D, Sapiro LE, et al. Impaired IRS-1/PI3-kinase signaling in patients with HCV: a mechanism for increased prevalence of type 2 diabetes. Hepatology 2003; 38:1384– 1392. 184. Shintani Y, Fujie H, Miyoshi H, et al. Hepatitis C virus infection and diabetes: direct involvement of the virus in the development of insulin resistance. Gastroenterology 2004; 126:840–848. 185. Mihm S, Fayyazi A, Hartmann H, Ramadori G. Analysis of histopathological manifestations of chronic hepatitis C virus infection with respect to virus genotype. Hepatology 1997; 25:735–739. 186. Hofer H, Bankl HC, Wrba F, et al. Hepatocellular fat accumulation and low serum cholesterol in patients infected with HCV-3a. Am J Gastro 2002; 97:2880–2885. 187. Patton HM, Patel K, Behling C, et al. The impact of steatosis on disease progression and early and sustained treatment response in chronic hepatitis C patients. J Hepatol 2004; 40:484–490. 188. Petit JM, Benichou M, Duvillard L, et al. Hepatitis C virusassociated hypobetalipoproteinemia is correlated with plasma viral load, steatosis, and liver fibrosis. Am J Gastro 2003; 98:1150–1154. 189. Negro F. Hepatitis C virus and liver steatosis: when fat is not beautiful. J Hepatol 2004; 40:533–535. 190. Serfaty L, Andreani T, Giral P, et al. Hepatitis C virus induced hypobetalipoproteinemia: a possible mechanism for steatosis in chronic hepatitis C. J Hepatol 2001; 34:428–434. 191. Kumar D, Farrell GC, Fung C, George J. Hepatitis C virus genotype 3 is cytopathic to hepatocytes: reversal of hepatic steatosis after sustained therapeutic response. Hepatology 2002; 36:1266–1272. 192. Lai MM. Hepatitis C virus proteins: direct link to hepatic oxidative stress, steatosis, carcinogenesis and more. Gastroenterology 2002; 122:568–571. 193. Lerat H, Honda M, Beard MR, et al. Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus. Gastroenterology 2002; 122:352–365. 194. Perlemuter G, Sabile A, Lettéron P, et al. Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viralrelated steatosis. FASEB J 2002; 16:185–194. 195. Shi ST, Polyak SJ, Tu H, et al. Hepatitis C virus NS5A colocalizes with the core protein on lipid droplets and interacts with apolipoproteins. Virology 2002; 292:198–210. 196. Okuda M, Li K, Beard MR, et al. Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein. Gastroenterology 2002; 122:366–375. 197. Clouston AD, Jonsson JR, Purdie DM, et al. Steatosis and chronic hepatitis C: analysis of fibrosis and stellate cell activation. J Hepatol 2001; 34:314–320. 198. Adinolfi LE, Gambardella M, Andreana A, et al. Steatosis accelerates the progression of liver damage of chronic hepatitis C patients and correlates with specific HCV genotype and visceral obesity. Hepatology 2001; 33:1358–1364. 199. Castéra L, Hézode C, Roudot-Thoraval F, et al. Worsening of steatosis is an independent factor of fibrosis progression in untreated patients with chronic hepatitis C and paired liver biopsies. Gut 2003; 52:288–292.
200. Hu K-Q, Kyulo NL, Esrailian E, et al. Overweight and obesity, hepatic steatosis, and progression of chronic hepatitis C: a retrospective study on a large cohort of patients in the United States. J Hepatol 2004; 40:147–154. 201. Ohata K, Hamasaki K, Toriyama K, et al. Hepatic steatosis is a risk factor for hepatocellular carcinoma in patients with chronic hepatitis C virus infection. Cancer 2003; 97:3036–3043. 202. Bressler BL, Guindi M, Tomlinson G, Heathcote EJ. High body mass index is an independent risk factor for nonresponse to antiviral treatment in chronic hepatitis C. Hepatology 2003; 38:639–644. 203. McCullough A. Obesity and its nurturing effect on hepatitis C. Hepatology 2003; 38:557–559. 204. Hickman IJ, Powell EE, Prins JB, et al. In overweight patients with chronic hepatitis C, circulating insulin is associated with hepatic fibrosis: implications for therapy. J Hepatol 2003; 39. 205. Gochee PA, Jonsson JR, Clouston AD, et al. Steatosis in chronic hepatitis C: association with increased messenger RNA expression of collagen I, tumor necrosis factor-alpha and cytochrome P450 2E1. J Gastroenterol Hepatol 2003; 18:386–392. 206. Rashid M, Roberts EA. Nonalcoholic steatohepatitis in children. J Pediatr Gastroenterol Nutr 2000; 30:48–53. 207. Brunt EM. Alcoholic and nonalcoholic steatohepatitis. Clin Liver Dis 2002; 6:399–420. 208. Itoh S, Yougel T, Kawagoe K. Comparison between nonalcoholic steatohepatitis and alcoholic hepatitis. Am J Gastro 1987; 82:650–654. 209. Goodman ZD, Ishak KG. Occlusive venous lesions in alcoholic liver disease: a study of 200 cases. Gastroenterology 1982; 83:786–796. 210. Legro RS. Polycystic ovary syndrome: the new millennium. Mol Cell Endocrinol 2001; 184:87–93. 211. Angulo P, Lindor KD. Treatment of nonalcoholic fatty liver: present and emerging therapies. Semin Liver Dis 2001; 21:81–88. 212. Laurin J, Lindor KD, Crippin JS, et al. Ursodeoxycholic acid or clofibrate in the treatment of non-alcohol-induced steatohepatitis: a pilot study. Hepatology 1996; 23:1464–1467. 213. Lindor KD, Kowdley KV, Heathcote EJ et al. Ursodeoxycholic acid for treatment of nonalcoholic steatohepatitis: results of a randomized trial. Hepatology 2004; 39:770–778. 214. Tuomilehto J, Lindstrom J, Eriksson JG, et al. Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance. N Engl J Med 2001; 344:1343–1350. 215. Center for Disease Control and Prevention Primary Prevention Working Group. Primary prevention of type 2 diabetes mellitus by lifestyle intervention: implications for health policy. Ann Intern Med 2004; 140:951–957. 216. Diabetes Prevention Program Research Group. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002; 346:393–403. 217. Hu FB, Li TY, Colditz GA, et al. Television watching and other sedentary behaviours in relation to risk of obesity and type 2 diabetes mellitus in women. JAMA 2003; 289:1785–1791. 218. Gauthier MS, Couturier K, Latour JG, Lavoie JM. Concurrent exercise prevents high-fat-diet-induced macrovesicular hepatic steatosis. J Appl Physiol 2003; 94:2127–2134. 219. Slentz CA, Duscha BD, Johnson JL, et al. Effects of the amount of exercise on body weight, body composition, and measures of central obesity. Arch Intern Med 2004; 164:31–39. 220. Klein S, Wadden T, Sugerman HJ. AGA technical review on obesity. Gastroenterology 2002; 123:882–932. 221. Wang RT, Koretz RL, Yee HF, Jr. Is weight reduction an effective therapy for nonalcoholic fatty liver? A systematic review. Am J Med 2003; 115:554–559.
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222. Mokdad AH, Marks JS, Stroup DF, Gerberding JL. Actual causes of death in the United States, 2000. JAMA 2004; 291:1238–1245. 223. Bonow RO, Eckel RH. Diet, obesity, and cardiovascular risk. N Engl J Med 2003; 348:2057–2058. 224. Klein S. Outcome success in obesity. Obesity Res 2001; 9:354S–358S. 225. Mun EC, Blackburn GL, Matthews JB. Current status of medical and surgical therapy for obesity. Gastroenterology 2001; 120:669–681. 226. Yanovski SZ, Yonovski JA. Obesity. N Engl J Med 2002; 346:591–602. 227. Neuschwander-Tetri BA, Brunt EM, Wehmeier KR, et al. Improved nonalcoholic steatohepatitis after 48 weeks of treatment with the PPAR-g ligand rosiglitazone. Hepatology 2003; 38:1008–1017. 228. Foster GD, Wyatt HR, Hill JO, et al. A randomized trial of a low-carbohydrate diet for obesity. N Engl J Med 2003; 348:2082–2090. 229. Samaha FF, Iqbal N, Seshadri P, et al. A low-carbohydrate as compared with a low-fat diet in severe obesity. N Engl J Med 2003; 348:2074–2081. 230. Polyzogopoulou EV, Kalfarentzos F, Vagenakis AG, Alexandrides TK. Restoration of euglycemia and normal acute insulin response to glucose in obese subjects with type 2 diabetes following bariatric surgery. Diabetes 2003; 52:1098–1103. 231. Steinbrook R. Surgery for severe obesity. N Engl J Med 2004; 350:1075–1079. 232. Ranløv I, Hardt F. Regression of liver steatosis following gastroplasty or gastric bypass for morbid obesity. Digestion 1990; 47:208–214. 233. Marceau P, Hould FS, Lebel S, et al. Malabsorptive obesity surgery. Surg Clin North Am 2001; 81:1113–1127. 234. Duchini A, Brunson ME. Roux-en-Y gastric bypass for recurrent nonalcoholic steatohepatitis in liver transplant recipients with morbid obesity. Transplantation 2001; 72:156–171. 235. Kral JG, Thung SN, Biron S, et al. Effects of surgical treatment of the metabolic syndrome on liver fibrosis and cirrhosis. Surgery 2004; 135:48–58. 236. Yki-Järvinen H. Thiazolidinediones. N Engl J Med 2004; 351:1106–1118. 237. Tiikkainen M, Häkkinen A-M, Korsheninnikova E, et al. Effects of rosiglitazone and metformin on liver fat content, hepatic insulin resistance, insulin clearance and gene expression in adipose tissue in patients with type 2 diabetes. Diabetes 2004; 53:2169–2176. 238. Shadid S, Jensen MD. Effect of pioglitazone on biochemical indices of non-alchoholic fatty liver disease in upper body obesity. Clin Gastroenterol Hepatol 2003; 1:384–387. 239. Promrat K, Lutchman G, Uwaifo GI, et al. A pilot study of pioglitazone treatment for nonalcoholic steatohepatitis. Hepatology 2004; 39:188–196. 240. Ricote M, Li AC, Willson TM, et al. The peroxisome proliferator-activated receptor-g is a negative regulator of macrophage activation. Nature 1998; 391:79–82. 241. Jiang C, Ting AT, Seed B. PPAR-g agonists inhibit production of monocyte inflammatory cytokines. Nature 1998; 391:82–86. 242. Miyahara T, Schrum L, Rippe R, et al. Peroxisome proliferatoractivated receptors and hepatic stellate cell activation. J Biol Chem 2000; 46:35715–35722. 243. Galli A, Crabb DW, Ceni E, et al. Antidiabetic thiazolidinediones inhibit collagen synthesis and hepatic stellate cell activation in vivo and in vitro. Gastroenterology 2002; 122:1924–1940. 244. Kirpichnikov D, McFarlane SI, Sowers JR. Metformin: an update. Ann Intern Med 2002; 137:25–33. 245. Fryer LGD, Parbu-Patel A, Carling D. The anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein
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279. Klain J, Fraser D, Goldstein J, et al. Liver histology abnormalities in the morbidly obese. Hepatology 1989; 10:873–876. 280. Silverman JF, O’Brien KF, Long S, et al. Liver pathology in morbidly obese patients with and without diabetes. Am J Gastro 1990; 85:1349–1355. 281. Silverman EM, Sapala JA, Appelman HD. Regression of hepatic steatosis in morbidly obese persons after gastric bypass. Am J Clin Pathol 1995; 104:23–31. 282. Ratziu V, Giral P, Charlotte F, et al. Liver fibrosis in overweight patients. Gastroenterology 2000; 118:1117–1123. 283. Crespo J, Fernandez-Gil P, Hernandez-Guerra M, et al. Are there predictive factors of severe liver fibrosis in morbidly obese patients with non-alcoholic steatohepatitis? Obesity Surg 2001; 11:254–257. 284. Harrison SA, Oliver DA, Torgerson S, et al. NASH: clinical assessment of 501 patients from two separate academic medical centers with validation of a clinical scoring system for advanced hepatic fibrosis (abstract). Hepatology 2003; 34 (suppl 1):511A. 285. Tarugi P, Lonardo A, Ballarini G, et al. A study of fatty liver disease and plasma lipoproteins in a kindred with familial hypobetalipoproteinemia due to a novel truncated form of apolipoprotein B (APO B-54.5). J Hepatol 2000; 33:361–370. 286. Tarugi P, Lonardo A, Gabelli C, et al. Phenotypic expression of familial hypobetalipoproteinemia in three kindreds with mutations of apolipoprotein B gene. J Lipid Res 2001; 42:1552–1561. 287. Tajiri K, Takenawa H, Yamaoka K, et al. Nonalcoholic steatohepatitis masquerading as autoimmune hepatitis. J Clin Gastroenterol 1997; 25:538–540. 288. Ross R, Dagnone D, Jones PJH, et al. Reduction in obesity and related comorbid conditions after diet-induced weight loss or exercise-induced weight loss in men. Ann Intern Med 2000; 133:92–103.
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56
THE LIVER IN SYSTEMIC ILLNESS Naga Chalasani and Oscar W. Cummings Abbreviations ALT alanine aminotransferase APS antiphospholipid syndrome AST aspartate aminotransferase CDC centers for disease control CPAP continuous positive airway pressure CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia
FAP FMF HBV HCV HPS IL-6 LDH
familial amyloidotic polyneuropathy familial mediterranean fever hepatitis B virus hepatitis C virus hemophagocytic syndrome interleukin-6 lactate dehydrogenase
INTRODUCTION When patients with abnormal liver tests are encountered, it is often assumed that the liver is the primary culprit in the disease process. However, numerous systemic illnesses and diseases of other organs can produce signs and symptoms that are indistinguishable from primary liver diseases. The hepatic manifestations in these disorders may range from mild enzyme abnormalities to significant liver injury and liver failure. In this chapter, we will review liver involvement in selected systemic disorders such as heart disease, pulmonary disease, amyloidosis, connective tissue disorders, Reye’s syndrome, jejunoileal bypass, and hematological disorders. Other systemic disorders with hepatic involvement, such as sarcoidosis, cystic fibrosis, and sepsis, are covered elsewhere in this textbook.
HEART DISEASE Liver involvement occurs in patients with both acute and chronic heart disease and its spectrum ranges from asymptomatic increases in liver biochemistries to fulminant liver failure. The liver receives a significant portion of the cardiac output and therefore any condition that decreases cardiac output will lead to a fall in hepatic perfusion. The liver is able to compensate for changes in hepatic blood flow via vasoactive mechanisms and by increasing oxygen extraction during periods of hepatic hypoperfusion.1 However, when critical levels of left or right heart failure are reached, hepatic injury may occur. In right-sided heart failure, it has been suggested that this damage is caused by elevation in right atrial pressure, leading to elevation in hepatic venous pressure that causes distention of hepatic sinusoids and liver cell hypoxia. In left-sided heart failure, decreased cardiac output results in diminished hepatic perfusion, which leads to hepatic hypoxia. In general terms, liver involvement in right heart failure is referred to as congestive hepatopathy, whereas liver injury resulting from left heart failure is known as ischemic hepatitis. However, these two phenomena often coexist and may be indistin-
NRSSS OSA OTC PBC SARS SCoV SLE
national reye syndrome surveillance system obstructive sleep apnea ornithine transcarbamylase primary biliary cirrhosis severe acute respiratory syndrome SARS corona virus systemic lupus erythematosus
guishable in clinical practice. The final common pathway for liver damage appears to be centrilobular (zone III) hepatocellular necrosis. This portion of the liver lobule is the most vulnerable to hypoxic injury due to the organization of the liver acinus. Highly oxygenated blood enters the hepatic lobule via branches of the hepatic artery and portal vein in the periportal region. As it passes through the hepatic sinusoids toward the terminal hepatic venule, oxygen is extracted and hepatocytes in the centrilobular area are perfused by blood that is the least well oxygenated.1
LIVER IN RIGHT HEART FAILURE (CONGESTIVE HEPATOPATHY) Liver abnormalities in patients with right heart failure are common. Right heart failure can be isolated (due to cor pulmonale or primary pulmonary hypertension) or, more likely, a consequence of left ventricular failure. In a large study of 175 patients with both acute and chronic right heart failure,2 hepatomegaly was present by physical examination in over 90% and splenomegaly in 20% of these patients.2 Other findings of right heart failure, such as peripheral edema, pleural effusion, and ascites, were also frequently present (Table 56-1). Ascites is more prominent in patients with chronic right heart failure than in acute right heart failure.2 Characteristic changes in histology are seen in the liver of patients with congestive heart failure. On gross inspection, the congested liver appears enlarged and purple with blunt edges.3 The classically described “nutmeg” appearance is caused by alternative areas of pale, more normal-appearing parenchyma contrasting with congested, hemorrhagic areas that correspond to the centrilobular regions of the liver (Figure 56-1). Microscopically, central veins and sinusoids in the centrilobular region become dilated and engorged with erythrocytes. Inflammation is noticeably absent (Figure 56-2). Adjacent hepatocytes may become compressed and atrophied. With long-standing hepatic congestion, fibrosis and cirrhosis may develop (cardiac cirrhosis) (Figure 56-3). Hepatic congestion due to the right heart failure results in numerous biochemical abnormalities (Table 56-2). In chronic congestive
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Table 56-1. Symptoms and Signs of Congested Livers in 175 Patents with Right-Sided Heart Failure
Table 56-2. Liver Tests of 175 Patients with Right-Sided Heart Failure
Symptom/sign
Liver tests
Hepatomegaly Peripheral edema Pleural effusion Splenomegaly Ascites
Acute heart failure (%)
Chronic heart failure (%)
99 77 25 20 7
95 71 17 22 20
Adapted from Richman SM, Delman AJ, Grob D. Alterations in indices of liver function in congestive heart failure with particular reference to serum enzymes. Am J Med 1961; 30:211.
Acute heart failure
Bilirubin BSP retention Alkaline phosphatase Aspartate aminotransferase Alanine aminotransferase Globulins Prothrombin time Albumin Cholesterol
Chronic heart failure
n
Abnormal(%)
n
Abnormal (%)
86 71 80 67 53 100 68 100 87
37 87 10 48 15 60 84 32 49
57 55 55 37 29 67 43 67 60
21 71 9 5 3 37 74 27 42
BSP, Bromosulfophthalein. Adapted from Richman SM, Delman AJ, Grob D. Alterations in indices of liver function in congestive heart failure with particular reference to serum enzymes. Am J Med 1961; 30:211.
Figure 56-1. Nutmeg liver. In chronic passive congestion of the liver, red cells pool and distend the sinuses around the central vein. These regions develop a darker red-violet color, in contrast to the surrounding tan liver parenchyma. This color stippling is reminiscent of the cut surface of a nutmeg.
Figure 56-2. Liver congestion. The sinuses around the central vein are distended by normal red cells. As the severity of this lesion increases, the adjacent hepatocytes become atrophic. (Hematoxylin and eosin.)
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Chapter 56 THE LIVER IN SYSTEMIC ILLNESS Figure 56-3. Cardiac cirrhosis. Dense fibrous bands emanate from the central veins (arrows) to surround a nodule of regenerating hepatocytes. (Trichrome.)
heart failure, hyperbilirubinema occurs in over 20% of patients.2 The elevation is generally mild, less than 3 mg/dl, and composed predominantly of unconjugated bilirubin. Serum aminotransferase levels are usually normal or minimally elevated in compensated, chronic congestive heart failure, but may become elevated during exacerbations of heart failure. Prothrombin time is prolonged in the majority of patients with hepatic congestion from right heart failure. Patients anticoagulated with warfarin sodium (warfarin) for dilated cardiomyopathy may have decreased warfarin requirements during exacerbations of congestive heart failure and this effect, if not appreciated, could result in dangerously prolonged prothrombin times. Hepatic biochemical abnormalities generally improve with improvement in cardiac function. Several signs and symptoms of congestive heart failure (e.g., ascites, pedal edema, mild hyperbilirubinemia) are also seen in patients with decompensated hepatic cirrhosis and distinguishing these two entities may be difficult. In some patients, it is particularly difficult to distinguish cardiac ascites from cirrhotic ascites clinically. In such cases, characterization of ascitic fluid or measurement of hepatic venous pressure gradient may be of assistance. In a prospective study of 13 patients with cardiac ascites, the serum ascites albumin concentration gradient was 1.1 g/dl and the total protein was 2.5 g/dl.3 Additionally, cardiac ascites had significantly more ascitic fluid red cell counts and higher levels of lactate dehydrogenase.3
LEFT HEART FAILURE AND ISCHEMIC HEPATITIS Ischemic hepatitis can be defined as hepatocellular necrosis associated with a decrease in hepatic perfusion.4–6 It is relatively infrequent, with a reported incidence of 0.16–1.5% of hospitalized patients. It can affect any age group, although it is most frequently reported in the older population. This undoubtedly reflects the increased risk of underlying cardiovascular disease in older people. When seen in children, it is often associated with congeni-
tal heart disease or overwhelming sepsis.7 The term ischemic hepatitis is a misnomer because ischemic liver injury is characterized by centrilobular necrosis in the absence of inflammation. The diagnosis of ischemic hepatitis should be considered in any patient with elevations of liver enzymes (aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH)) in the setting of documented or suspected systemic hypotension.
Causes and Pathogenesis Liver is a highly vascular organ, receiving approximately 25% of cardiac output. Seventy percent of the hepatic blood flow is derived from the portal system. The other 30% is delivered by the hepatic artery and liver arterial perfusion expresses a linear relation between blood pressure and blood flow.8 The liver can maintain normal oxygen uptake by increasing oxygen extraction, with as much as 95% of the oxygen from the blood being extracted in a single pass through the liver.6 This remarkable compensatory mechanism most likely accounts for the low incidence of liver damage in shock (resistance to ischemia). Nevertheless, these compensatory mechanisms are overwhelmed in some patients with severely diminished hepatic perfusion leading to ischemic liver injury. Cardiogenic shock from any cause (e.g., myocardial infarction, tamponade) is the most commonly reported risk factor for the development of ischemic hepatitis. Transiently decreased cardiac output seen in patients with arrhythmia or valvular heart disease may also result in hepatic injury even in the absence of bona fide hypotension (? relative hypotension).9 Episodes of hypotension resulting in ischemic hepatitis may be very brief and sometimes there may not be any documented episodes of hypotension. Although diminished hepatic perfusion from systemic hypotension (either absolute or relative) is essential, a recent study suggested that systemic hypotension or shock alone is insufficient to cause ischemic hepatitis.10 In this study, 31 patients with ischemic hepatitis were compared to a control group consisting of 31 previously
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Section IX. Liver Affected by Other Organs (Non-Hepatic Conditions)
healthy subjects with major non-hepatic trauma and marked hypotension (mean systolic pressure of 54 ± 22 mmHg lasting for a mean of 19 ± 14 minutes). All 31 patients with ischemic hepatitis had organic heart disease and more than 90% had demonstrable right heart failure. None of the subjects in the control group developed ischemic hepatitis despite profound hypotension. These findings suggest that right heart failure with passive hepatic congestion renders the liver susceptible to ischemic liver injury during transient systemic hypotension.10 Henrion et al. recently published a paper that described clinical and hemodynamic features of 142 episodes of ischemic hepatitis. When ischemic hepatitis occurred in patients with congestive heart failure or acute heart failure, the hepatic hypoxia resulted primarily from the decreased hepatic blood flow and venous congestion. However, when ischemic hepatitis occurred in patients with toxic or septic shock, oxygen delivery to liver was decreased but oxygen needs were increased, leading to hepatic hypoxia.11 Non-cardiogenic causes of ischemic hepatitis include hypovolemic shock from hemorrhage or dehydration, heat stroke, and septic shock.12–14 Rare episodes of ischemic hepatitis have been reported in patients who ingest vasoactive medication (ergotamine overdose) and after protracted seizures in children.15,16
Clinical Syndrome The clinical picture is usually dominated by the cardiovascular, septic, or hemorrhagic illness that precipitated the hepatic hypoperfusion. A distinctive biochemical pattern is characteristic of
this disorder.17,18 Serum aminotransferase levels rise rapidly after an ischemic episode and peak within 1–3 days (Figure 56-4). With treatment of the underlying illness, serum aminotransferases usually return to near-normal within 7–10 days of the initial insult. Persistent elevation of serum aminotransferase levels beyond this period implies a poor prognosis due to continued hepatic hypoperfusion. Serum ALT and AST activity are strikingly elevated and may exceed 200 times the upper limits of normal (Figure 56-4). Less marked elevation (500 slight ≠
slight ≠ slight ≠ ≠≠
No relationship (cardiac disease) Biliary tract and stomach
Uncommon Common
1–4 days Days–weeks
>500 200–300
Slight ≠(LDH) ≠≠
ALT, Alanine aminotransferase; AP, alkaline phosphatase; LDH, lactate dehydrogenase; ≠, increase; ≠≠, greater increase.
in multiple organ system failure in that acute liver failure (encephalopathy with a coagulopathy) is not the cause of death.
BILE DUCT OBSTRUCTION A common concern is the development of extrahepatic bile duct obstruction in the postoperative patient who becomes icteric. Coincidental choledocholithiasis after surgery is rare. A far more common occurrence is bile duct injury after biliary tract or gastric surgery. Bile duct injury after laparoscopic cholecystectomy is an increasingly common problem and frequently goes unrecognized during the cholecystectomy.69–71 The patient develops clinical jaundice with or without signs of cholangitis days to weeks after the initial surgery. Diagnosis is made by endoscopic retrograde cholangiopancreatography or transhepatic cholangiography (see Chapters 16 and 64). Postoperative pancreatitis may also cause bile duct obstruction because of edema of the head of the pancreas. The diagnosis is made by finding an elevated serum level of amylase and a computed tomography scan of the abdomen showing edema of the pancreas and bile duct dilation. The jaundice resolves as the patient recovers from the pancreatitis.72 In the postoperative jaundiced patient who has not undergone biliary or gastric surgery and who does not have evidence of pancreatitis, biliary tract disease is uncommon and other causes of jaundice should be considered initially. Acute cholecystitis (calculous or acalculous) may occur postoperatively and can be associated with abnormal liver tests and jaundice.73 The presence of right upper quadrant abdominal pain and fever suggests the diagnosis, with the ultrasound findings of pericholecystic fluid, thickening of the gallbladder wall, and perhaps stones supporting the clinical suspicion.74 Gangrene, perforation, and empyema of the gallbladder are common in the postoperative patient and associated with a high mortality.73 Abnormal liver tests are frequently observed in patients receiving total parenteral nutrition, which is discussed in detail in Chapter 57. Fatty liver with mild elevations of the serum aminotransferases and alkaline phosphatase is commonly observed.75 Less common, but of greater concern, especially in children, is the development of jaundice. The abnormal liver tests develop days to weeks after the institution of therapy.75 The liver biopsy findings are non-specific and the diagnosis is one of excluding the other causes of postoperative hepatic dysfunction. The cause of the disorder remains poorly understood.
EVALUATION OF THE PATIENT WITH POSTOPERATIVE LIVER DYSFUNCTION If the patient is within the first 2 weeks of surgery and has a hepatitis-like injury, anesthetic-related hepatitis or ischemic hepatitis is of major concern (Table 58-2). Injury by a direct hepatotoxin such as acetaminophen should also be considered. The development of cholestasis in the immediate postoperative period in a patient who has undergone biliary or gastric surgery suggests bile duct injury. If the patient has undergone major cardiac or abdominal surgery and is infected or has received multiple blood transfusions, benign postoperative cholestasis should be the initial diagnosis. If the abnormal liver tests develop more than 2 weeks after surgery, drug or total parenteral nutrition-induced liver injury should be considered, as should bile duct injury if gallbladder surgery had been performed. Postoperative cholecystitis is associated with abdominal pain and fever, which are unusual features of the other types of injury, and abdominal ultrasonography should be performed in this situation. Hepatitis C should be considered in the transfused patient who develops elevated AST/ALT levels more than 3 weeks after exposure to blood products but is very rare unless the donor was incubating the virus when the blood was donated. Antibody tests may be negative during the acute illness and identification of viral ribonucleic acid in the serum by polymerase chain reaction may be required (Chapter 32). Tests for acute hepatitis A and B are not usually necessary because they infrequently cause post-transfusion hepatitis.
REFERENCES 1. Jackson FC, Christophersen ER, Peternel WW. Preoperative management of patients with liver disease. Surg Clin North Am 1968; 48:907. 2. LaMont JT. Postoperative jaundice. Surg Clin North Am 1974; 54:637. 3. Probst A, Probst T, Zengerl G, et al. Prognosis and life expectancy in chronic liver disease. Dig Dis Sci 1995; 40:1905. 4. Schemel WH. Unexpected hepatic dysfunction found by multiple laboratory screening. Anesth Analg (Cleve) 1976; 55:810.
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5. Watanecyawech M, Kelly KA Jr. Hepatic diseases unsuspected before surgery. NY State J Med 1975; 75:1278. 6. Ngai SH. Effects of anesthetics on various organs. N Engl J Med 1980; 302:564. 7. Cooperman LH. Effects of anesthesia on the splanchnic circulation. Br J Anaesth 1972; 44:967. 8. Handle DD, Summerskill WHJ. Surgery in acute hepatitis. JAMA 1963; 184:257. 9. Powell-Jackson P, Greenway B, Williams R. Adverse effects of exploratory laparotomy in patients with unsuspected liver disease. Br J Surg 1982; 69:419. 10. Shaldon S, Sherlock S. Virus hepatitis with features of prolonged bile retention. Br Med J 1957; 2:734. 11. Hardy KJ, Hughes ESR. Laparotomy in viral hepatitis. Med J Aust 1968; 1:710. 12. Strauss AA, Strauss SF, Schwartz AH, et al. Decompression by drainage of the common bile duct in subacute and chronic jaundice: a report of 73 cases with hepatitis or concomitant biliary duct infection as cause. Am J Surg 1959; 97:137. 13. Bourke JB, Cannon P, Ritchie HD. Laparotomy for jaundice. Lancet 1967; ii:521. 14. Farrell GC. Postoperative hepatic dysfunction. In Zakim D, Boyer TD, eds. Hepatology: a textbook of liver disease, 2nd edn. Philadelphia: WB Saunders, 1990:869. 15. Johnson RD, O’Connor ML, Kerr RM. Extreme serum elevations of aspartate aminotransferase. Am J Gastro 1995; 90:1244. 16. Friedman LS, Maddrey WC. Surgery in the patient with liver disease. Med Clin North Am 1987; 71:453. 17. Hargrove MD. Chronic active hepatitis: possible adverse effect of exploratory laparotomy. Surgery 1971; 68:771. 18. Brolin RE, Bradley LJ, Taliwal RV. Unsuspected cirrhosis discovered during elective obesity operations. Arch Surg 1998; 133:84. 19. Greenwood SM, Leffler CT, Minkowitz S. The increased mortality rate of open liver biopsy in alcoholic hepatitis. Surg Gynecol Obstet 1972; 134:600. 20. Eckhauser F, Appelman H, O’Leary T, et al. Hepatic pathology as a determinant of prognosis after portal decompression. Am J Surg 1980; 139:105. 21. Kanel G, Kaplan M, Zawacki I, et al. Survival in patients with post necrotic cirrhosis and Laennec’s cirrhosis undergoing therapeutic portacaval shunt. Gastroenterology 1977; 73:679. 22. Bell RH, Miyai K, Orloff MJ. Outcomes in cirrhotic patients with acute alcoholic hepatitis after emergency portacaval shunt for bleeding esophageal varices. Am J Surg 1984; 147:78. 23. Reichle R, Fahmy W, Golsorkhi M. Prospective comparative clinical trial with distal splenorenal and mesocaval shunts. Am J Surg 1979; 137:13. 24. Mikkelsen W. Therapeutic portacaval shunt. Preliminary data on controlled trial. Arch Surg 1974; 108:302. 25. Pande N, Resnick R, Yee W, et al. Cirrhotic portal hypertension. Morbidity of continued alcoholism. Gastroenterology 1978; 74:64. 26. Garrison RN, Cryer HM, Howard DA, et al. Clarification of risk factors for abdominal operations in patients with hepatic cirrhosis. Ann Surg 1984; 199:648. 27. Klemperer JD, Ko W, Connolly M, et al. Cardiac operations in patients with cirrhosis. Ann Thorac Surg 1998; 65:85. 28. Morris JJ, Hellman CL, Gawey BJ, et al. Three patients requiring both coronary artery bypass surgery and orthotopic liver transplantation. J Cardiothorac Vasc Anesth 1995; 9:311. 29. Pollard RJ, Sidi A, Gibby GL. Aortic stenosis with end stage liver disease: prioritizing surgical and anesthetic therapies. J Clin Anesth 1998; 10:253. 30. Brown MW, Burk RE. Development of intractable ascites following upper abdominal surgery in patients with cirrhosis. Am J Med 1986; 80:879. 31. Chapman CB, Snell AM, Rowntree LG. Decompensated portal cirrhosis. Report on one hundred and twelve cases. Clinical
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45. 46. 47.
48.
49.
50.
51.
52.
53.
54. 55. 56.
features of the ascitic stage of cirrhosis of the liver. JAMA 1981; 97:237. Leonetti JP, Aranha GV, Wilkinson WA, et al. Umbilical herniorrhaphy cirrhotic patients. Arch Surg 1894; 119:441. Yonemoto RH, Davidson CS. Herniorrhaphy in cirrhosis of the liver with ascites. N Engl J Med 1956; 255:733. Maniatis AG, Hunt CM. Therapy for spontaneous umbilical hernia rupture. Am J Gastro 1995; 90:310. Hurst RD. Management of groin hernias in patients with ascites. Ann Surg 1992; 216:696. Bloch RS, Allaben RD, Wait AJ. Cholecystectomy in patients with cirrhosis. Arch Surg 1985; 120:669. Bornman PC, Terblanche I. Subtotal cholecystectomy: for the difficult gallbladder in portal hypertension and cholecystitis. Surgery 1985; 98:1. Yerdel MA, Tsuge H, MiMura H, et al. Laparoscopic cholecystectomy cirrhotic patients: expanding indications. Surg Laparosc Endosc 1993; 3:180. Yerdel MA, Koksoy C, Aras N, et al. Laparoscopic versus open cholecytectomy in cirrhotic patients: a prospective study. Surg Laparosc Endosc 1997; 7:483. Sleeman D, Namias N, Levi D, et al. Laparoscopic cholecystectomy in cirrhotic patients. J Am Coll Sum 1998; 187:400. Gopalswamy N. Risks of intra-abdominal nonshunt surgery in cirrhotics. Dig Dis 1998; 16:225. D’Alburquerque LA, de Miranda MP, Genzini T, et al. Laparoscopic cholecystectomy in cirrhotic patients. Surg Laparosc Endosc 1995; 5:272. Friel CM. Laparoscopic cholecystectomy in patients with hepatic cirrhosis: a five-year experience. J Gastrointest Surg 1999; 3:286. Mansour A, Watson W, Shayani V, et al. Abdominal operations in patients with cirrhosis: still a major surgical challenge. Surgery 1997; 122:730. Doberneck RC, Sterlin WA, Allison DC. Morbidity and mortality after operation in nonbleeding cirrhotics. Am J Surg 1983; 146:306. Aranha GV, Greenlee HB. Intra-abdominal surgery in patients with advanced cirrhosis. Arch Surg 1986; 121:275. Bernstein DE. Recombinant factor VIIa corrects prothrombin time in cirrhotic patients: a preliminary study. Gastroenterology 1997; 113:1930. Berstein DE. Effectiveness of the recombinant factor VIIa in patients with the coagulopathy of advanced Child’s B and C cirrhosis. Semin Thromb Hemost 2000; 26:437. Jeffers L. Safety and efficacy of recombinant factor VIIa in patients with liver disease undergoing laparoscopic liver biopsy. Gastroenterology 2002; 123:118. Slappendel R, Huvers FC, Benraad B, et al. Use of recombinant factor VIIa to reduce postoperative bleeding after total hip arthroplasty in a patient with cirrhosis and thrombocytopenia. Anesthesiology 2002; 96:1525. Wehbi MA, Obideen K, Martinez EJ, et al. Recombinant VVI (RFVIIA) as a safe and effective therapeutic option for the correction of moderate to severe hepatic coagulopathy. Hepatology 2003; 34:667A. Ziser A, Plevak DJ, Weisner RH, et al. Morbidity and mortality in cirrhotic patients undergoing anesthesia and surgery. Anesthesiology 1999; 90:42. Farrell GC. Liver disease due to anaesthetic agents. In Farrell GC, ed: Drug-induced liver disease. Edinburgh: Churchill Livingstone, 1994:389. Touloukian J, Koplowitz N. Halothane-induced hepatic disease. Semin Liver Dis 1981; 1:134. Cousins MJ, Plummer JL, Hau PM. Risk factors for halothane hepatitis. Aust NZ J Surg 1989; 59:5. Friedman LS. The risk of surgery in patients with liver disease. Hepatology 1999; 29:1617.
Chapter 58 PREOPERATIVE AND POSTOPERATIVE HEPATIC DYSFUNCTIONS
57. Strunin L. Anesthetic management of patients with liver disease. In: Millward-Sadler GH, Wright R, Arthur MJP, eds. Wright’s liver and biliary disease. London: Saunders; 1992:1381. 58. Cowan RE, Jackson BT, Grainger SL, et al. Effects of anesthetics and abdominal surgery on the liver blood flow. Hepatology 1991; 14:1161. 59. Gibson PR, Dudley FJ. Ischemic hepatitis: clinical features, diagnosis and prognosis. Aust NZ I Med 1984; 14:822. 60. Johnson RD, O’Connor ML, Kerr RM. Extreme serum elevations of aspartate aminotransferase. Am J Gastro 1995; 90:1244. 61. Brittain RS, Marchioro TL, Hermann G, et al. Accidental hepatic artery ligation in humans. Am J Surg 1964; 107:822. 62. Farrell GC. Paracetamol-induced hepatotoxicity. In: Farrell GC, ed: Drug-induced liver disease. Edinburgh: Churchill Livingstone;1994:205. 63. Kumar S, Rex DK. Failure of physicians to recognize acetaminophen hepatotoxicity in chronic alcoholics. Ann Intern Med 1991; 151:1189. 64. Gottlieb JE, Menashe PI, Cruz E. Gastrointestinal complications in critically ill patients: the intensivists’ overview. Am J Gastroenterol 1986; 81:227. 65. LaMont JT, Isselhacher KJ. Postoperative jaundice. N Engl J Med 1974; 288:305. 66. Schmid M, Hefti ML, Gattiker R, et al. Benign postoperative intrahepatic cholestasis. N Engl J Med 1965; 272:545.
67. Kantrowitz PA, Jones WA, Greenberger NJ, et al. Severe postoperative hyperbilirubinemia simulating obstructive jaundice. N Engl J Med 1967; 276:591. 68. Boekhorst T, Urlus M, Doesburg W, et al. Etiologic factors of jaundice severely ill patients. A retrospective study in patients admitted to intensive care unit with severe trauma or with septic intraabdominal complications following surgery and without evidence of bile duct obstruction. J Hepatol 1988; 7:111. 69. Waxman K. Postoperative multiple organ failure. Crit Care Clin 1987; 3:429. 70. Moossa AR, Easter DW, Van Sonnenberg E, et al. Laparoscopic injuries to the bile duct. A cause for concern. Ann Surg 1992; 215:203. 71. Davidoff AM, Pappas TN, Murran EA, et al. Mechanisms of major biliary injury during laparoscopic cholecystectomy. Ann Surg 1992; 215:196. 72. Thompson JS, Bragg LE, Hodgson PE, et al. Postoperative pancreatitis. Surg Gynecol Obstet 1988; 167:377. 73. Frazee RC, Nagorney UM, Mucha P. Acute acalculous cholecystitis. Mayo Clin Proc 1989; 64:163. 74. Becker CD, Burckhardt B, Terrier F. Ultrasound in postoperative acalculous cholecystitis. Gastrointest Radiol 1986; 11:47. 75. Baker AL, Rosenberg IH. Hepatic complications of total parenteral nutrition. Am J Med 1987; 82:489.
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59
HEPATOCELLULAR CARCINOMA Jordi Bruix, Concepció Bru and Josep M. Llovet Abbreviations AAIR age adjusted incidence rate AFP a1-fetoprotein AgNOR nucleolar organizer region protein ALT alanine aminotransferase BCLC barcelona-clínic-Liver-cancer CT computed tomography HBV hepatitis B virus
HCC HCV HIV MELD MRI NASH PCNA
hepatocellular carcinoma hepatitis C virus human immunodeficiency virus model for end-stage liver disease magnetic resonance non-alcoholic steatohepatitis proliferating cell nuclear antigen
INTRODUCTION Some years ago it was common to consider hepatocellular carcinoma (HCC) as an infrequent cancer in the western world, as it was seen as a neoplasm mostly restricted to sub-Saharan Africa and Asia. At the same time, it was assumed that the diagnosis would always be made when the tumor was already advanced and thus, treatment would be unfeasible and the prognosis very grim. These concepts induced a nihilistic approach to the clinical management of patients diagnosed with HCC. However, in recent decades the situation has changed dramatically. It has been acknowledged that the incidence of HCC has increased in the western world1–3 and that this neoplasm is now the leading cause of death in patients with cirrhosis.4,5 At the same time, it has been shown that diagnosis can be achieved at an early stage when effective therapy is feasible and long-term survival is not anecdotal.6 The present chapter will summarize the most relevant aspects regarding the epidemiology, pathogenesis, diagnosis, and treatment of this neoplasm.
EPIDEMIOLOGY Recent estimations indicate that primary liver cancer is now the fifth most common cancer in the world and the third cause of cancerrelated mortality.1,2 More than half a million cases are diagnosed every year on a worldwide basis, but there are relevant geographic differences in incidence (Table 59-1).3 Eastern Asia and sub-Saharan Africa account for most cases and the annual incidence rates there largely exceed 15/100 000 habitants. Incidence rates are intermediate (between 5 and 15/100 000) in Mediterranean countries and southern Europe, while the rate is low (below 5/100 000) in northern Europe and the USA.3 However, slight changes have been detected in some specific areas. Incidence rates have decreased in Hong Kong, Shanghai, and Singapore,3 while effective vaccination against the hepatitis B virus (HBV) has significantly reduced the incidence in Taiwan.7 The same effect can be expected in other areas where this virus is the main oncogenic agent for HCC development and if vaccination is implemented. In contrast, the incidence has increased in Australia, the UK,8 Canada,9 and the USA;10,11 this probably reflects the spread of hepatitis C virus (HCV) infection. This has occurred later than in countries such as Japan12 where the
PEI PHT PS RCT TNM US TAE
percutaneous ethanol injection portal hypertension performance status randomized controlled trial tumor node metastasis ultrasonography transarterial embolization
incidence may have reached a plateau,13 or even initiated a decrease, as described in some registries in Italy. In addition to the heterogeneous geographical incidence, there are some differences of race and ethnicity. It is likely that these racerelated differences reflect different exposure to risk factors and time of acquisition, rather than genetic predisposition. Accordingly, the difference in HCC incidence according to race may vanish in populations of mixed ethnicity but with homogeneous risk profile. In all areas and cancer registries males have a higher prevalence than females. The magnitude of the ratio varies greatly, with most countries showing values between 2:1 and 4:1. The ratio exceeds 4:1 in some particular regions of France, Italy, and Switzerland and is lower than 2:1 in South America.1,3 The male predominance may be due to specific genetic and hormonal profiles together with a higher prevalence of risk factors such as viral infection, alcoholism, and smoking. The age at which HCC appears also varies according to gender, geographic area, and risk factor associated with cancer development. In most areas female age is higher than male.1,3 In high-incidence areas where HBV is the main etiologic agent, the peak age appears after 40 years, while in low-incidence areas such as the USA, the peak of age appears beyond 75 years.1,3 As previously noted, the prevalence of risk factors has a marked geographic heterogeneity and this undoubtedly accounts for a major part of the described epidemiological pattern. HBV is widespread in Asia (with the exception of Japan) and sub-Saharan Africa. HBV is mostly acquired at birth or during early childhood and, in most areas, viral oncogenicity overlaps with that of aflatoxin B-1, a powerful oncogenic agent that contaminates food stored in humid conditions in several countries of Asia. The main risk factors in Japan, Europe, and Asia are HCV infection and alcohol intake. The spread of HCV has occurred at different decades in these areas and this explains why the peak incidence of HCC in Japan and southern Europe has been registered earlier than in the USA.
RISK FACTORS FOR HCC One of the relevant characteristics of HCC is that cirrhosis underlies HCC in almost 80% of affected individuals.4,14 Thus, any agent leading to chronic liver damage and ultimately cirrhosis should be
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Section X. Tumors of the Liver
Table 59-1. Incidence of Hepatocellular Carcinoma According to Geographical Area and Distribution of Risk Factors Geographic area
Europe Western Europe Southern Northern North America Northern Southern Asia and Africa (Japan) Eastern Asia South-Eastern Asia Middle Africa World
AAIR Male/female
Risk factors HCV
HBV
Alcohol
Other
60–70%
10–15%
20%
10%
50–60%
20%
20%
10%
20% 70%
70% 10–20%
10% 10%
Aflatoxin 10%
5.8/1.6 9.8/3.4 2.6/1.3 4.1/1.6 4.8/3.6
35.4/12.6 18.3/5.7 24.2/12.9 14.9/5.5
AAIR, age adjusted incidence rate; HCV, hepatitis C virus; HBV, hepatitis B virus. Reproduced from Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet 2003; 362:1907–1917, with permission.
seen as a risk factor for HCC. Due to their high prevalence rate the major causes of cirrhosis and HCC are HBV, HCV, and alcohol, but less prevalent conditions such as hemocromatosis, primary biliary cirrhosis, non-alcoholic steatohepatitis (NASH), and Wilson’s disease have also been associated with HCC development.
HEPATITIS B VIRUS This infective agent affects 300 million people worldwide and it constitutes the most important oncogenic factor for HCC development. The evidence linking HBV with HCC is unquestionable.15 Large cohort studies have shown that the incidence in HBV carriers is significantly increased. Seminal studies in Taiwan showed a 100-fold increase in HBV carriers,16 but several aspects should temper this value. Active viral replication implies a higher risk and longstanding active infection resulting in cirrhosis is the major event that gives rise to highly increased risk.17–19 The incidence of HCC in inactive HBV carriers without liver cirrhosis is less than 0.3%.20,21 This has been clearly shown in studies in Europe20–22 and the USA,23,24 but Asian studies in HBV carriers who acquired the infection early in life indicate that in this specific population the incidence of HCC is higher even in the absence of cirrhosis.18,19 The role of specific genotypes or mutations is not well established.22,25,26 In addition to this epidemiologic evidence, it has been shown that HBV can be integrated into the host cellular genome and induce genetic damage (instability, deletion, and rearrangements).15 In addition, some of the HBV proteins disrupt cellular functions27 and may favor neoplastic transformation, induce proliferation, and impede apoptosis.28 Interestingly, occult HBV infection may become apparent if properly investigated by molecular techniques even in the absence of serological markers of HBV itself.29,30 Accordingly, the relevance of HBV may be even higher than suggested by studies based on serologic assessment of HBV infection. Finally, the implementation of vaccination against HBV has resulted in a significant decrease of HCC incidence7 and this is the final proof of the importance of this virus in the genesis of this cancer.
1110
HEPATITIS C VIRUS The discovery of this virus has enabled the proper classification of most patients with so-called non-A, non-B hepatitis. Immediately after a serological test became available it was clear that chronic HCV infection was a major risk factor for HCC.31 The prevalence of HCV in HCC cohorts varies according to the penetration of the agent in the population of each geographic area.3 A study including 12 000 men described a 20-fold increased risk of HCC in infected individuals;32 this figure is very close to the estimated risk obtained in a meta-analysis of 21 case control studies.11 The risk is clearly related to the degree of liver damage induced by the virus.33,34 There are some case reports of so-called healthy HCV carriers with HCC,35 but several cohort studies indicate that the incidence in patients with chronic hepatitis is low (below 1%) and that the risk increases sharply when cirrhosis is established.33,34 At that time, the annual incidence ranges between 2 and 8%.4 The process from acute infection to cirrhosis may take 20–30 years.36 Hence, those studies recruiting patients with chronic hepatitis will detect HCC development if follow-up has been long enough, while those studies including patients with established cirrhosis will register the risk early during follow-up. Patients infected with the human immunodeficiency virus (HIV) are now effectively treated with combined regimes and if coinfected with the HCV they present a faster evolution to cirrhosis and thus, are at risk for HCC development. In fact, liver disease and/or HCC are the major causes of death in these patients.37
ALCOHOL The estimation of risk of HCC in alcoholics was surely overestimated until the availability of a test for HCV infection, as a noteworthy proportion of alcoholic cirrhotics are infected by HCV.38 The risk for HCC appears when alcohol intake exceeds 60 g/day and beyond this cut-off it increases linearly.39 Thereby, alcohol consumption above 80 g/day for more than 10 years is associated with a fivefold increased risk. The risk of HCC development is further increased on development of cirrhosis and the annual incidence
Chapter 59 HEPATOCELLULAR CARCINOMA
increases beyond 2% in decompensated cirrhosis.40 Coinfection with HCV or HBV exerts a synergistic affect and increases HCC risk.
AFLATOXIN This is a powerful oncogenic agent that induces a specific damage in the p53 gene (G to T transversions at codon 249).41 It is produced by fungi that contaminate food stored in humid conditions, as frequently happens in some undeveloped areas of the world. Epidemiological studies have established a correlation between the magnitude of aflatoxin intake (that can be recognized by the presence of aflatoxin-albumin adducts in sera or by the detection of aflatoxin metabolites in urine) and the incidence of HCC. However, as previously mentioned, the areas of heavy aflatoxin contamination overlap with areas of high prevalence of HBV infection. A synergistic effect induces a 60-fold increase in risk, while aflatoxin alone increases the risk by four.42
TOBACCO The role of smoking in promoting HCC has been difficult to ascertain due to the frequent confounding role of other risk factors. However, recent data suggest that smoking increases the risk for HCC.55–57 Contrarily, coffee consumption reduces the risk.58
HORMONAL COMPOUNDS Oral contraceptives have been linked to the development of adenoma and in some series of HCC. While the relationship with the first entity is well established, the causality with HCC is less robust.59 Viral infection, especially HCV, may not be ruled out in old studies and the oncogenic risk in long-term users may be due to the combination of estrogens and androgens.59 Androgenic treatment for hematologic conditions or athletic improvement also increases the risk of adenomas and has been associated with HCC development,60 but strong data are not available.
IRON AND COPPER DEPOSITION Patients with hereditary hemochromatosis have a highly increased risk of HCC upon cirrhosis development.14,43,44 If cirrhosis is not present the risk is less but there are cases of HCC in patients in whom treatment prevented the progression of the disease.45 Iron deposition is responsible for oxidative damage and cancer development and the same should happen in patients with Wilson’s disease. However, a high incidence in patients with Wilson’s disease has not been documented. Interestingly, an experimental model of copper accumulation results in cancer development46 that is effectively prevented by avoiding oxidative stress.47
NON-ALCOHOLIC STEATOHEPATITIS, OBESITY, AND DIABETES Several epidemiological studies conducted in Europe and the USA have linked obesity and diabetes to a higher HCC risk.48–50 The risk is higher in males50 and the current epidemic of overweight and obesity may prompt an increase of HCC incidence linked to this entity. Data on the prevalence and incidence of HCC in NASH patients are still limited and the available information suggests that some patients with HCC in cryptogenic cirrhosis may have indeed suffered NASH,51–53 but the magnitude of HCC risk in NASH patients who have reached cirrhosis is currently unknown.
AUTOIMMUNE HEPATITIS The risk in this entity is low. Some of the reported patients with HCC had coincidental viral infection and the annual incidence is 0.1%.
a1-ANTITRYPSIN DEFICIENCY This condition may be associated with cirrhosis and HCC in the PIZZ phenotype associated with intrahepatocellular protein deposition. Again, the confounding role of viral infection is relevant, as most patients with HCC present evidence of either HBV or HCV.
PRIMARY BILIARY CIRRHOSIS The risk of HCC appears at advanced stages when cirrhosis is established and affects mostly males.54
CIRRHOSIS As previously described, cirrhosis development through any damaging agent constitutes a major event regarding the magnitude of risk for the development of HCC.4,14 However, the risk varies according to etiology61,62 and, more importantly, according to the degree of liver function impairment. There is a steady increase in incidence from early cirrhosis without portal hypertension (PHT) to decompensated cirrhosis fitting into Child–Pugh C class.63–65 Proper evaluation of unscreened compensated cirrhotics may show a 5% prevalence of HCC and the percentage increases to 15–20% when considering patients with variceal bleeding or bacterial peritonitis.4 Older age appears in most studies as a relevant predictor but it is possibly a surrogate marker of the duration of the disease. Male sex is also a marker of increased risk, reflecting either the simultaneous oncogenic effect of coincidental agents (smoking, alcohol intake) or a higher oncogenicity related to androgens. The activity of the liver disease is another relevant factor in HCC promotion. Sustained HBV replication and higher HCV-related inflammation as reflected by increased alanine aminotransferase (ALT) levels have been shown to correlate with a higher HCC incidence. More intense disease activity can also be depicted by proliferating cell nuclear antigen (PCNA)66 or nucleolar organizer region protein (AgNOR)67 staining and recent data indicate that subjects with increased proliferation bear an increased HCC risk. Other histology findings have been linked to HCC risk. The value of large-cell dysplasia is controversial as there are studies with discrepant results.63,68 Japanese authors have described irregular regeneration of hepatocytes as a powerful predictor,69 but this awaits confirmation. The parameter that has been consistently linked to increased risk is abnormal a1-fetoprotein (AFP) concentration.4,70 It is known that chronic hepatic inflammation and regeneration may induce transient increases of this tumor marker. This is well described in both chronic HBV and HCV infection even in the absence of cirrhosis.71,72 It could be argued that increased AFP levels reflect the presence of an HCC that cannot be recognized by current imaging techniques, but this would not modify the clinical relevance of the finding.
1111
Section X. Tumors of the Liver
PATHOGENESIS The relationship between any agent inducing chronic liver injury and HCC development indicates that active inflammation with oxidative damage should be a key step in the origin of the genetic damage leading to cancer. This would be present both in viral infection and in the metabolic derangement that occurs in metal accumulation, alcohol consumption, or NASH. The molecular events responsible for the development and progression of HCC are not elucidated.73,74 Malignant hepatocytes are the result of cumulative genetic changes occurring in mature hepatocytes or in stem cells. The step-by-step process is initiated through generation of foci of dysplastic hepatocytes without overt malignant phenotype. While low-grade dysplastic nodules harbor a low malignant potential, the likelihood of evolving to HCC is significant in high-grade dysplastic foci. Thirty percent of these nodules turn into HCC after a follow-up of 5 years.75,76 Dysplastic nodules and very early HCC retain their hepa-
tocytic differentiation and do not show an extensive net of newly formed vessels.77,78 At this stage the nodules usually measure 2cm
FNAB
AFP ≥ 400 ng/mL CT-MRI-Angiography
No nodule
< 1cm
Increased AFP**
US/3m
Spiral CT
Figure 59-5. Screening strategy for early hepatocellular carcinoma detection in cirrhotic patients. (Reproduced from Bruix J, Sherman M, Llovet JM, et al. Clinical management of hepatocellular carcinoma: conclusions of the Barcelona-2000 EASL conference. J Hepatol 2001; 35:421–430, with permission.)
Normal AFP
No HCC
HCC***
Surveillance US+AFP/6m
* Available for curative treatments if diagnosed with HCC ** AFP levels to be defined *** Pathological confirmation or non-invasive criteria
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Section X. Tumors of the Liver
already indicates transition into overt malignancy that will be properly diagnosed if adequately evaluated. If the nodule is > 2 cm and the underlying liver is cirrhotic, the diagnosis of HCC can be established by non-invasive criteria: two coincident imaging techniques showing a focal lesion > 2 cm with characteristic arterial hypervascularization and washout of contrast during the venous phase (Figure 59-6), or one imaging technique with specific patterns associated with AFP > 400 ng/ml. With state of the art equipment and assessment by expert radiologists the non-invasive diagnostic criteria could be further refined. Thereby, two coincidental imaging techniques showing intense arterial uptake of contrast following by contrast washout in the venous/delayed phase would be able to set confident HCC diagnosis in nodules between 1 and 2 cm within a cirrhotic liver.98 One single technique with a specific vascular profile would suffice for nodules larger than 2 cm within cirrhosis. Finally, nodules smaller than 1 cm in diameter would still be almost impossible to be confidently diagnosed by biopsy or imaging techniques.98 The policy to follow on detection of an increased concentration of AFP is less conclusive. A minor increase in AFP may be seen in patients with chronic liver disease as a result of inflammation flares and thus, in some cases it will not be due to development of cancer.71,72 However, most investigations have shown that persistent elevation of AFP during follow-up is a strong marker of increased risk of HCC development.4 It is important to note that increased risk does not mean faster tumor progression and thus, in high-risk patients there is no basis for carrying out screening at shorter time periods. Furthermore, it is well known that up to 40–60% of patients with HCC present normal or minimally increased AFP concentrations that do not reach the diagnostic cut-off.71,72 This limitation in sensitivity and the higher accuracy of imaging techniques and guided biopsy for the diagnosis of HCC have reduced the clinical efficacy of this tumor marker that will be useful in only a minority of cases. In addition to the low diagnostic sensitivity, AFP determination also has problems in specificity. AFP increases may be observed during
inflammation flares in patients with chronic HBV and HCV infection. Accordingly, AFP should not be used as a tool for screening and diagnosis since imaging techniques have better sensitivity and specificity.70 Hence, even in the presence of increased AFP concentration, the detection of an atypical radiological pattern should raise doubts about HCC diagnosis and biopsy should be indicated.98 The use of AFP fractions such as lectin-bound AFP, desgammacarboxyprothrombin,99 or glypican100 has been proposed to surpass the efficacy of AFP but their use has not fully entered clinical practice. Proteomic techniques may help to identify new markers.101 After HCC detection and diagnosis, the key issue is to stage tumor extent properly. This should be based on state-of-the-art CT or MRI.6,96,102 Both CT and MRI are equally effective for the detection of tumor sites > 2 cm.102 However, MRI angiography is more effective for nodules below this cut-off, but there are still problems with the characterization of minute nodules < 1 cm that do not exhibit the specific profile in terms of mass recognition and vascular enhancement. Angiography has almost no role in diagnosis and staging, while lipiodol CT is not reliable.4 This oily contrast is mostly retained within HCC foci after its injection in the hepatic artery, while it is cleared from non-tumoral liver. However, it has been shown that it has both false-positive and false-negative results and hence, lacks adequate clinical usefulness. Critical aspects of HCC staging are the detection of additional tumor sites and of vascular invasion. Both events reflect advanced tumor stage and higher likelihood of dissemination prior to therapy leading to recurrence after initially effective therapies such as resection, transplantation, or percutaneous ablation. Extrahepatic spread is infrequent at early stages but should be ruled out by chest CT. Bone metastases are usually symptomatic and should be ruled out if needed by bone scintigraphy. Brain metastases are exceptional. In addition to the evaluation of tumor burden according to size, number of nodules, vascular invasion, and dissemination, it is relevant to evaluate patients in terms of liver function, general health, and the presence of associated morbidity. The degree of liver function impairment because of tumor stage and underlying cirrhosis may reduce life expectancy and at the same time limit the application of therapy. This is not the case for liver transplantation, but it is very relevant to establish the feasibility of surgical resection and of invasive procedures such as transarterial chemoembolization. Finally, assessment of cancer-related symptoms provides a rough estimation of cancer stage and, if present, may indicate poor shortterm survival.
PROGNOSTIC PREDICTION
Figure 59-6. Magnetic resonance imaging scan showing a hepatocellular carcinoma located in the right lobe. Note the additional tumor site located near to the surface of the liver. Both nodules show arterial contrast uptake and washout in the venous phase.
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Outcome prediction is relevant to offer adequate information to patients and relatives at the time of diagnosis and when treatment has been initiated. In most neoplasms the prognosis is defined by tumor stage at the time of diagnosis. However, in patients with HCC this is more complex. Cirrhosis underlies HCC in most patients and thus prognosis depends on the evolutionary stage in which the neoplasm is diagnosed, the degree of liver function impairment of the underlying cirrhosis, and the treatment received.6 Simultaneously, the impairment of liver function determines the feasibility of treatment as this may further impair liver function and even induce death.
Chapter 59 HEPATOCELLULAR CARCINOMA
According to these comments, any proposal aimed at stratifying patients into different prognostic groups or at linking staging with treatment indication has to consider this complex interaction. Systems that only consider one dimension will be unable to predict outcome accurately. This is indeed the case for the tumor node metastasis (TNM) classification,103 that just takes into account tumor burden and disregards liver function. The same applies to the Child–Pugh104 or the model for end-stage liver disease (MELD)105 systems that disregard tumor extent, or to scores that merely evaluate general health status and physical capacity such as the performance status (PS)106 or the Karnofsky index. The sole usefulness of all unidimensional systems is to identify patients with very advanced disease stage and reduced life expectancy. The classification proposed by Okuda et al.107 has been used for many years but has currently been replaced by more accurate proposals. Several multidimensional systems have been proposed in recent years in France,108 the USA,109 Spain,110,111 Italy,112,113 Austria,114 Germany,115 Hong Kong,116 and Japan,117,118 to grade patients according to life expectancy (Table 59-3). All consist of a combination of tumor parameters and liver function variables, and usually provide a stratification of patients into separate groups. However, only the Italian119,120 and the Barcelona-Clínic-LiverCancer (BCLC)121–123 proposals have been validated. In addition, the BCLC system is the only one that links staging with treatment indication (Figure 59-7). This system was developed some years ago as a result of several cohort investigations and RCTs assessing the relevant outcome predictors within different tumor stages and treatment options. It conducts a first division of the patients into the relevant evolutionary stages at which patients may be diagnosed.6 Stage 0 comprises small tumors usually < 2 cm that have not reached an invasive phenotype with increased vascularization and appearance of microscopic vascular invasion and satellites. Currently, confident diagnosis of these very early HCCs corresponding to so-called carcinoma in situ is not feasible prior to resection, but in the next few
years, these tumors should be a common result in well-conducted surveillance programs. Stage A comprises tumors diagnosed at an apparently early stage when curative treatment (resection, transplant, ablation) is feasible. Patients have a preserved liver function (Child–Pugh A or B) and present with solitary tumors or up to three nodules, each < 3 cm in size. As noted, these patients are candidates for surgery or percutaneous ablation. Survival at 5 years may range between 50 and 75%. Patients with large or multifocal disease that are asymptomatic belong to an intermediate stage (stage B). These patients are candidates for transarterial chemoembolization and, depending on baseline characteristics and treatment success, will achieve a 3-year survival around 50%. Patients who report cancerrelated symptoms (pain, constitutional syndrome) or present with vascular invasion or extrahepatic spread correspond to an advanced stage (stage C). There is no standard treatment for them and their survival at 3 years is less than 10%. Finally, patients with severe impairment of liver function (Child–Pugh C) or major physical deterioration (PS = 3) or corresponding to class 3 of the Okuda staging system correspond to stage D. Their median survival is less than 6 months. The stratification of patients into separate stages according to tumor stage, liver function, and presence of symptoms, with the development of specific prognostic tools for each, is practical from a clinical point of view and also when assessing the impact of treatment. While at intermediate and advanced stages the natural history of untreated patients is better known, as there are several studies defining this issue, this is not the case for patients diagnosed at an early stage. Some groups have reported small series of untreated patients with early-stage disease. However, in normal conditions these patients are treated with any of the available options and, if this is not the case, the same reason prompting no treatment (late diagnostic confirmation, delay in staging, refusal of treatment, associated conditions preventing therapy) may impair survival and make any analysis flawed. Hence, any attempt to define prognosis in this
Table 59-3. Prognostic and Staging Systems to Predict Outcome in Hepatocellular Carcinoma Patients Author (year)
108
n
Stuart (1996)
314
CLIP (1998)111
435
Chevret (1999)107
761
Llovet (1999)109
102
BCLC (1999)110
—
Villa (2000)112 CUPI (2002)115
96 926
JIS (2003)116 SliDe (2004)117
722 177
Prognostic variables Tumor stage
Liver function
Health status
Portal vein invasion AFP Tumor morphology AFP, portal vein invasion Portal vein invasion AFP
Albumin
—
Child–Pugh
—
Bilirubin Alkaline phosphatase —
Karnofsky Performance status
Child–Pugh
Performance status
Bilirubin Bilirubin, ascites alkaline phosphatase Child–Pugh Liver damage by LCSGJ PIVKA
— Symptoms
Portal vein invasion Metastases Tumor size, number Vascular invasion Extraheptic spread Estrogen receptor status TNM AFP TNM by LCSGJ TNM by LCSGJ
— —
AFP, a-fetoprotein; TNM, tumor node metastasis; LCSGJ, Liver Cancer Study Group of Japan; PIVKA, protein induced by vitamin K absence.
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Section X. Tumors of the Liver
HCC
Stage A–C Okuda 1-2, PST 0-2, Child–Pugh A–B
Stage 0 PST 0, Child–Pugh A
Very early stage (0) Single < 2 cm Carcinoma in situ
Early stage (A) Single or 3 nodules < 3 cm, PS 0
Stage D Okuda, PST >2, Child–Pugh C
Intermediate stage (B) Multinodular, PS 0
Advanced stage (C) Portal invasion, N1, M1, PS 1-2
Terminal stage (D)
Single Portal invasion, N1, M1 Portal pressure/bilirubin
Increased
Normal
Resection
3 nodules < 3 cm
Associated diseases
No
Liver transplantation (CLT/LDLT) Curative treatments 50%–75% at 5 years
No
Yes
Yes
PEI/RF
Chemoembolization
New Agents
Randomized controlled trials 40%–50% at 3 years vs 10% at 3 years
Symptomatic treatment
Figure 59-7. BCLC staging and treatment strategy. Patients are divided into separate stages according to tumor burden, liver function, and physical condition. This stratification allows a rough estimation of life expectancy and guide treatment indication. (Reproduced from reference 110 with permission).
stage will have to incorporate the treatment options to be applied and use the relevant predictors within them. As previously mentioned, the natural history of the so-called nonsurgical HCC is widely studied. Optimally, the analysis of these patients should exclude those cases diagnosed at an end-stage. Otherwise, the prognostic predictors will merely identify the markers of end-stage disease. Years ago we took advantage of the two prospective RCTs comparing transarterial embolization (TAE)124 and tamoxifen versus no treatment and joined the two control groups of these investigations into a cohort of 102 patients who presented with large multifocal HCC, but who did not report a heavily impaired physical status (PS > 2) or advanced liver disease (Child–Pugh C).110 Their 1-, 2-, and 3-year survival was 54, 40, and 28%, respectively and we identified the presence of cancer-related symptoms (PS 1 or 2, constitutional syndrome) and detection of vascular invasion or extrahepatic spread as independent predictors of survival. Accordingly, asymptomatic patients without vascular invasion or extrahepatic spread showed a 1-, 2-, and 3-year survival of 80, 65, and 50%, respectively, while these figures decreased to 29, 16, and 8% in those with at least one adverse characteristic (Figure 59-8).109,110 As exposed, the current prognostic estimation in patients with HCC is derived from rough assessment of tumor stage combined
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with liver function evaluation and registration of cancer-related symptoms. However, it is expected that current effort in translational research will facilitate basing the prediction on the identification of markers associated with the activation of the mechanisms that govern cancer progression and dissemination.
TREATMENT There are several options to be considered in patients with HCC, but only a few can achieve long-term cure. These include surgical resection, liver transplantation, and percutaneous ablation.6 Unfortunately, the success of these approaches is restricted to patients diagnosed at an early stage and currently, this involves fewer than 40% of patients evaluated in referral units. Most patients are diagnosed at a more advanced stage and the only option that has been shown to have a positive impact on survival is transarterial chemoembolization.125 However, its applicability is reduced to 1000 48 62 118 26
10–18% 10% 11% 16% 13% 10 days or intervention required Septic shock or surgical intervention
Pancreatitis
Perforation Infection
Basket impaction
Spontaneous release or repeat endoscopy
Febrile or septic illness for >3 days or endoscopic or radiologic intervention Radiologic intervention
Surgical intervention
(Data from Cotton PB, Lehman G, Vennes J, et al. Endoscopic sphincterotomy complications and their management: an attempt at consensus. Gastrointest Endosc 1991; 37:383–393.56)
scopic sphincterotomy. Multiple studies suggest that it is safe;48,49 however, most of the reports are either retrospective or not prospective controlled trials. One prospective study showed a significantly higher incidence of hemorrhage with endoscopic sphincterotomy (26%) versus balloon dilation (2%) with no appreciable pancreatitis.45 However this is a much higher bleeding rate than reported in the largest prospective study on the complications of endoscopic sphincterotomy and a study looking at the complication of bleeding associated with sphincterotomy.36,55 The largest prospective study comparing these two modalities found the incidence of bleeding as a complication as defined by the consensus conference was 0%, but “self-limited” or bleeding which required some form of endoscopic intervention occurred in 27% of the ES group and 10.5% of the balloon dilation group. Unfortunately, the definition of a “complication” is not specifically delineated in many of these studies and they fail to use the definitions of complications that were developed by a consensus conference on complications of endoscopic sphincterotomy (Table 64-1).56 The largest US study, which included academic and private-practice endoscopists, and the European study
both came to the conclusion that balloon dilation should be avoided in routine practice.46,50 EBD may have a role in several clinical situations, such as in patients with Billroth II anatomy where complete sphincterotomy is technically more difficult due to the inverted position of the major papilla, and in patients with cirrhosis who have a high rate of hemorrhage at endoscopic sphincterotomy.36,55 One study performed EBD in 21 patients with cirrhosis and coagulopathy and compared them to 20 historical control patients undergoing ES at the same institution.57 The bleeding rate was 30% in the ES group and 0% in the EBD group and all but one of the bleeding cases occurred in Child–Pugh class C patients.
COMPLEX STONE EXTRACTION In any large series, there will be a small percentage of intraductal calculi which are either inaccessible due to failed cannulation or prior surgery, such as a Billroth II or Roux en Y anastomosis. Another common cause is failure of standard methods of stone extraction. These patients will either have to undergo surgical extraction, either
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Section XI. Diseases of the Biliary Tract
Figure 64-8. Endoscopic papillary balloon dilation. The stone is located above the balloon next to the wire, and the balloon waist is in the papilla.
laparoscopically or open, or use a variety of specialized techniques to access and/or fragment the stones. One of the commonest techniques is the combined percutaneous–endoscopic approach, where the interventional radiologist will access the biliary tree by a percutaneous transhepatic puncture using fluoroscopic guidance and place a guidewire through the needle and across the major papilla. The endoscopist can then grasp the guidewire, pull it through the endoscope, and use it to perform a sphincterotomy if access to the biliary tree was the only issue.58–60 More complex cases, such as Roux en Y bilioenteric anastomoses or presence of large stones with prior failed endoscopic extraction, present more challenging issues.61,62 Extracorporeal lithotripsy is a proven modality, but is not approved for use in the USA.5,62 Direct ductoscopy, either with a mother–baby scope37,41,62,63 or percutaneous cholangioscopy with a small-caliber endoscope, provides an attractive, non-surgical alternative for extraction of large or complex stones which fail standard endoscopic or percutaneous extraction.61,64,65 An interventional radiologist can use mechanical methods to fragment the stone and push the fragments into the duodenum through a prior sphincterotomy or balloon-dilate the sphincter during the same procedure.66 Stones that fail mechanical lithotripsy can be fragmented with a piezoelectric lithotripsy probe or with a holmium-YAG laser37,61,64–66 (Figure 64-9).
Figure 64-9. A Transhepatic cholangiogram in a patient with left intrahepatic duct stones and a choledochoduodenostomy due to duct trauma during laparoscopic cholecystectomy. B Ureteroscope passed through a 12 French biliary catheter. C Endoscopic view of stone and electrohydraulic lithotripsy probe. D Duct cleared of all stones.
A
B
C
D
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Chapter 64 NON-SURGICAL MANAGEMENT OF GALLSTONES
ERCP IN PREGNANCY Gallbladder crystals form “sludge” which is common in pregnancy, is usually asymptomatic, but is associated with a higher incidence of stone formation, especially if it appears during the first trimester.67 Symptomatic gallstone disease is a not uncommon occurrence in pregnancy and the physician has additional concerns, including the risk of any intervention on both the mother and the fetus, as well as the effects of the radiation exposure on the fetus if fluoroscopy is needed during ERCP. Surgical cholecystectomy is considered relatively safe for the mother with both the open and laparoscopic technique, but increased fetal wastage is reported with open procedures,68 whereas the relative safety of laparoscopy has been demonstrated in multiple studies.69,70 Management of CDS presents another level of complexity. There are data on the amount of radiation exposure that is considered safe.71 There are only a few large series on ERCP in pregnancy.70,72,73 The patients in these series who underwent ERCP and ES for presumed or proven gallstone disease had no increased incidence of complications or apparent untoward effects on the fetus. The administration of standard medication for conscious sedation was well tolerated by mother and fetus. Fetal monitoring was used in only one study.73 The patient is placed in the standard prone position or the left lateral decubitus position and a lead shield is placed between the cathode-ray tube and the patient to protect the fetus from ionizing radiation (Figure 64-10). We place a dosimeter over the sacrum and anterior abdomen to document the radiation dose to the skin surface. The recorded dose was 59 mRem, which was well within the current recommended guidelines.71,74 A phantom model was used in another study73 to estimate the fetal dose which ranged from 310 ± 160 mrad. It is best to minimize fluoroscopy time and avoid fixed exposures to reduce radiation to the minimal amount necessary to complete the procedure successfully.
ERCP IN PEDIATRICS ERCP has been an accepted modality in adults for 30 years. Its use in the pediatric population has been a little slower to evolve but guide-
lines have been developed.75 The indication for ERCP in the pediatric population is so infrequent that most pediatric gastroenterologists either have not been trained or cannot maintain proficiency for this examination, and the procedures are performed by adult endoscopists. There are several situations that are unique in this population. Infants may need to be done with a pediatric-size endoscope with 8.0-mm outside diameter and 2.0-mm operating channel.76 The only problem is that the channel size limits the range of therapeutic instruments that can be used. The procedure is technically similar to adults, with the exception that a higher percentage of cases are performed with deep sedation or general anesthesia and general anesthesia can be safely administered in the ERCP suite.77 Most reports include patients 18 years of age and under,78–80 and do not separate adolescents, who may act more like the adult population, from children. However, one study found no difference in success or complications rates between children and adolescents.81 None of these studies report a complication rate any higher than in the adult population, especially when the indication was for suspected choledocholithiasis. Another series advises caution, reporting post-ERCP pancreatitis in 28.6% of pediatric patients, and 40% when the procedure was performed for suspected biliary pathology. They defined pancreatitis as any pain and elevated amylase post-ERCP but the clinical significance of the pancreatitis was not clear.82 Endoscopic therapy in this patient population is best left to the expert endoscopists who work closely with the pediatrician in each instance.
ENDOSCOPY IN CHOLECYSTECTOMY COMPLICATIONS The incidence of complications after cholecystectomy has not changed markedly in the era of laparoscopic cholecystectomy, although the number of duct injuries may have increased slightly.83 The management is best approached through the cooperative effort of the operating surgeon, endoscopist, and interventional radiologist. The preferred approach is established and may depend on the local expertise available. Complicated injuries are best palliated as much Figure 64-10. Endoscopic retrograde cholangiopancreatography positioning of the dosimeters and lead shielding for the pregnant patient who requires fluoroscopy.
Fluoroscopy tower
Dosimeters Endoscope
Lead shielding
Radiation source
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Section XI. Diseases of the Biliary Tract
as possible locally and referred to a center with specific expertise in complex biliary problems.
BILIARY LEAKS Postoperative biliary leaks are the more common complication of cholecystectomy.83 The typical presentation is abdominal pain and tenderness, with nausea and vomiting less frequent. Mild elevation of AST, ALT, and alkaline phosphatase occur but bilirubin level is normal or minimally elevated.84 The diagnosis can be suggested by demonstrating a fluid collection on transabdominal ultrasound, but this finding is not sensitive and may be non-specific. Radionuclide scanning is the most sensitive for demonstration of leaks.84 ERCP can correctly identify the source of the leak in most instances and offers a modality for therapy.85–89 The method of endoscopic approach has been variable, with the common denominator being obliteration of the transpapillary gradient.87 High levels of success have been reported with ES alone, 7 F90,91 or 10 F stenting alone88,91 or in combination with ES89,91 and nasobiliary stents.89 The largest retrospective study found only a 66% success with sphincterotomy alone.91 None of these options has been approached in a prospective systematic fashion, and in some instances small leaks may resolve spontaneously.88,91 The drawback to stent placement is that a second procedure is required for stent removal. Nasobiliary stenting has the advantage of allowing re-opacification of the biliary tree and then removal of the stent once closure is documented. The major difficulty with nasobiliary stents is accidental dislodgement and this problem has made us reserve this technique for complicated leaks or leaks that have failed conventional management, with evidence for persistent leak despite prior stent or ES. Success is high with endoscopic therapy for simple leaks that are demonstrable at ERCP, especially for cystic duct stump or duct of Luschka leaks – 100% success was reported in four series.85,87,89,91,92 Biliary leaks with large biloma should have the biloma drained by an interventional radiologist to prevent infection within the peritoneal cavity. Complex leaks and leaks associated with biliary strictures present a more complex problem92 and will be addressed below.
DUCT INJURIES One large series92 classifies bile duct injuries as follows. Type A has a bile leak from the cystic duct stump or from a peripheral radicle and management is typically endoscopic. Type B is a leak from a major biliary radicle with or without duct stricture. Type C is a duct stricture with no leak, and type D is complete transection of a duct. It is extremely rare that endoscopy can manage a type D lesion93 and this is left to the surgeon and the interventional radiologist. The management of type B and C lesions is possible endoscopically if the lesion involves the common bile duct and/or common hepatic duct and does not extend into the bifurcation. The endoscopic approach is to place a 10 F stent through the stenosis/leak area at the first ERCP, and 2–3 months later reassess the lesion. If the leak has closed and the stenosis has resolved then no further treatment is needed (Figure 64-11). If the stricture is still evident, then two92 or three 10 F stents94 are placed for 6–12 months, usually with good long-term results.92,94,95 Strictures at the level of the bifurcation have
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a high failure rate for endoscopic management92,94,95 and are more amenable to surgical intervention.96,97 One of the rare complications of cholecystectomy is a documented leak by radionuclide scanning and a “normal” ERCP which may represent an injury to the right hepatic duct and, depending on the anatomy of the injury, can have a complex presentation (Figure 64-12). The common situation is a left-sided dominant system and the lateral aspect of the right lobe is supplied by an aberrant duct. If the cystic duct is very close to or actually inserts into the aberrant duct, then the anatomy for the injury to occur is present.98,99 The patient can present with pain and positive HIDDA scan, and the ERCP reveals no leak; however the endoscopist should be alert to this lesion if there is a paucity of ducts in the lateral aspect of the right lobe (Figure 64-12a). The lesion can be demonstrated by transhepatic cholangiography or by injecting a subhepatic drain if it is present. Management is a surgical Roux en Y hepaticojejunostomy.99 A second presentation is right upper quadrant pain, abnormal liver tests with a normal bilirubin, and again the ERCP is “normal,” and a magnetic resonance imaging scan can demon-strate a clipped segment with dilated ducts in the right lobe (Figure 64-12b). The most complex presentation would be represented by Figure 64-12d. The patient presents with pain and abnormal LFTs and ERCP demonstrates a leak (Figure 64-13a) which resolves with stenting. However, pain or fever may occur later and MRCP shows a dilated segment in the lateral aspect of the right lobe (Figure 64-13b).
COMPLICATIONS The complications of ERCP are well known, and the prevalence can depend on the definition of the investigator but the criteria shown in Table 64-1 should be used.56 Some complications are related to the diagnostic portion of the procedure and others only if therapy is performed. Major predictive factors for complications are expertise of the endoscopist, and performance of a precut to gain access to the bile duct. A complication rate of 4.6% was reported in patients undergoing ERCP within 30 days of cholecystectomy for suspected choledocholithiasis, significantly lower than the overall 9.5% rate for sphincterotomy in general.36 The incidence of “nuisance” bleeding, defined as bleeding during or immediately postprocedure, which requires endoscopic therapy has decreased with the introduction of microprocessor-controlled electrosurgical units. Significant bleeding with > 3 g fall in hemoglobin or need for transfusions still occurs and most such cases are delayed bleeds after the patient has been discharged.55 Many of these bleeds resolve with supportive measures or re-endoscopy, and the need for angiography or surgery for control is rare. Pancreatitis remains the commonest complication of sphincterotomy, but is much more common if the indication is for sphincter of Oddi dysfunction as compared to gallstones.36 Elevation of serum amylase and lipase levels is common postprocedure and is not predictive of evolving pancreatitis. The more relevant laboratory data are an elevation of the white blood cell count, and AST, which is the most predictive of prolongation of hospitalization.100 Cholangitis is primarily related to failure to extract stones. One should avoid high-pressure injection of contrast in a duct that has been obstructed as significant bacteremia occurs if biliary pressures exceed 30 cm of H2O.101 If there is a concern about the amount of contrast to be injected, one should aspirate
Chapter 64 NON-SURGICAL MANAGEMENT OF GALLSTONES Figure 64-11. A Endoscopic retrograde cholangiopancreatography demonstrating a large leak from the common bile duct beside a T-tube with stenosis at the level of the take-off of the cystic duct. B 10 French biliary stent placed with the proximal end above the T-tube. C At 3-month follow-up ERCP there is resolution of the narrowing and leak.
B
A
C
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Section XI. Diseases of the Biliary Tract
Figure 64-12. Most isolated right hepatic duct (RHD) injuries occur if the cystic duct inserts in close proximity or from the right hepatic duct. A common association with this anomaly is the dominant left hepatic duct system where the first bifurcation of the left system supplies the middle portion of the liver. The three lower figures demonstrate the various presentation of this form of duct injury.
Left hepatic duct Aberrant right duct Common bile duct
A
Leak from RHD– Cystic stump clipped
B
Both ducts clipped
C
No clips Both ducts leaking
D
RHD clippedCystic stump Leaking
Figure 64-13. A Endoscopic retrograde cholangiopancreatography shows a cholangiogram with contrast leaking from the cystic duct to a subhepatic drain. Note the lack of ducts in the lateral aspect of the right lobe. B Magnetic resonance imaging cholangiogram showing a dilated duct system in the same region.
A
B
some of the bile prior to contrast injection. Perforation is the least common complication, but is more common when CDS is the indication for the procedure. Perforations can be secondary to endoscope trauma and are usually large and require immediate surgical closure.102 Instrumentation perforation at the papilla related to the sphincterotomy or guidewire perforations in the bile duct can be managed conservatively initially as most are microperforations, and are treated with nothing by mouth, intravenous fluids, nasogastric suction and broad-spectrum antibiotics.102,103 A surgical consultation should be obtained in all instances, but surgical exploration is only needed for failure to respond to conservative treatment.102,103 The patients should be followed with frequent CT scans to document the course of the disease.104 Indications for surgery include large
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intraperitoneal or retroperitoneal fluid collections and deterioration in clinical status.102
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Chapter 64 NON-SURGICAL MANAGEMENT OF GALLSTONES
4. Bennion LJ, Grundy SM. Risk factors for the development of cholelithiasis in man. N Engl J Med. 5. Adamek HE, Kudis V, Jakobs R, et al. Impact of gallbladder on the outcomes in patients with retained bile duct stones treated with extracorporeal shockwave lithotropsy. Endoscopy 2002; 34:624–627. 6. Lai KH, Lin LF, Lo GH, et al. Does cholecystectomy after endoscopic sphincterotomy prevent the recurrence of biliary complications? Gastrointest Endosc 1999; 49:483–487. 7. Saito M, Tsuyuguchi T, Yamaguchi T, et al. Long-term outcome of endoscopic papillotomy for choledocholithiasis with cholecystolithiasis. Gastrointest Endosc 2000; 51:540–545. 8. Conway JD, Russo MW, Shrestha R. Endoscopic stent insertion into the gallbladder for symptomatic gallbladder disease in patients with end-stage liver disease. Gastrointest Endosc 2005; 61:32–36. 9. Houghton PW, Jenkinson LR, Donaldson LA. Cholecystectomy in the elderly: a prospective study. Br J Surg 1985; 72:220–222. 10. Firilas A, Duke BE, Max MH. Laparoscopic cholecystectomy in the elderly. Surg Endosc 1996; 10:33–35. 11. Spira RM, Nissan A, Zamir O, et al. Percutaneous transhepatic cholecystectomy and delayed laparoscopic cholecystectomy in critically ill patients with acute calculus cholecystitis. Am J Surg 2002; 183:62–66. 12. Pigott JP, Williams GB. Cholecystectomy in the elderly: a prospective study. Am J Surg 1988; 155:408–411. 13. Wong SKH, Yu SCH, Lam YH, Chung SSC. Percutaneous cholecystostomy and endoscopic cholecystolithotripsy in the management of acute cholecystitis. Surg Endosc 1999; 13:48–52. 14. Sosna J, Kruskal JB, Copel I, et al. US-guided percutaneous cholecystostomy: features predicting culture-positive bile and clinical outcome. Radiology 2004; 230:785–791. 15. Boland GW, Lee MJ, Mueller PR, et al. Gallstones in critically ill patients with acute calculous cholecystitis treated by percutaneous cholecystostomy: nonsurgical therapeutic options. Am J Roentgenol 1994; 162:1101–1103. 16. Hatjidakis AA, Karampekios S, Prassopoulos P, et al. Maturation of the tract after percutaneous cholecystostomy with regard to the access route. Cardiovasc Interv Radiol 1998; 21:36–40. 17. Fan ST, Lai ECS, Mok FPT, et al. Early treatment of acute biliary pancreatitis by endoscopic papillotomy. N Engl J Med 1993; 328:228–232. 18. Pezzilli RP, Billi P, Barakat B, et al. Effects of early ductal decompression in human biliary acute pancreatitis. Pancreas 1998; 16:165–168. 19. Neoptolemos JP, Carr-Locke DL, London NJ, et al. Controlled trial of urgent endoscopic retrograde cholangiopancreatography and endoscopic sphincterotomy versus conservative treatment for acute pancreatitis due to gallstones. Lancet 1988; 2:979–983. 20. Wang CH, Mo LR, Lin RC, et al. Rapid diagnosis of choledocholithiasis using biochemical tests in patients undergoing laparoscopic cholecystectomy. HepatoGastroenterology 2001; 48:619–621. 21. Prat F, Meduri B, Ducot B, et al. Prediction of common bile duct stones by noninvasive tests. Ann Surg 1999; 229:362–368. 22. Barr LL, Frame BC, Coulanjon A. Proposed criteria for preoperative endoscopic retrograde cholangiography in candidates for laparoscopic cholecystectomy. Surg Endosc 1999; 13:778–781. 23. Santucci L, Natalini G, Sarpi L, et al. Selective endoscopic retrograde cholangiography and preoperative bile duct stone removal in patients scheduled for laparoscopic cholecystectomy: a prospective study. Am J Gastro 1996; 91:1326–1330. 24. Majeed AW, Ross B, Johnson AG, Reed MW. Common duct diameter as an independent predictor of choledocholithiasis: is it useful? Clin Radiol 1999; 54:170–172.
25. Lichtenbaum RA, McMullen HF, Newman RM. Preoperative abdominal ultrasound may be misleading in the risk stratification for presence of common bile duct abnormalities. Surg Endosc 2000; 14:254–257. 26. Pezzilli R, Billi P, Barakat B, et al. Ultrasonographic evaluation of the common bile duct in biliary acute pancreatitis patients: comparison with endoscopic retrograde cholangiopancreatography. J Ultrasound Med 1999; 18:391–394. 27. Prat F, Edery J, Meduri B, et al. Early EUS of the bile duct before endoscopic sphincterotomy for acute biliary pancreatitis. Gastrointest Endosc 2001; 54:724–729. 28. Liu CL, Lo CM, Chan JK, et al. Detection of choledocholithiasis by EUS in acute pancreatitis: a prospective evaluation in 100 consecutive patients. Gastrointest Endosc 2001; 54:325–330. 29. Prat F, Amouyal G, Amouyal P, et al. Prospective controlled study of endoscopic ultrasonography and endoscopic retrograde cholangiography in patients with suspected common bile duct lithiasis. Lancet 1996; 347:75–79. 30. Amouyal P, Amouyal G, Levy P, et al. Diagnosis of choledocholithiasis by endoscopic ultrasonography. Gastroenterol 1994; 106:1062–1067. 31. Kohut M, Nowak A, Nowakowska-Dulawa E, et al. Endosonography with linear array instead of endoscopic retrograde cholangiography as the diagnostic tool in patients with moderate suspicion of common bile duct stones. World J Gastroenterol 2003; 9:612–614. 32. Kohut M, Nowakowsky-Dulawa E, Marek T, et al. Accuracy of linear endoscopic ultrasonography in the evaluation of patients with suspected common bile duct stones. Endoscopy 2002; 34:299–303. 33. Tseng LJ, Jao YT, Mo LR, Lin RC. Over-the-wire US catheter probe as an adjunct to ERCP in the detection of choledocholithiasis. Gastrointest Endosc 2001; 54:720–723. 34. Catanzaro A, Pfau P, Isenberg GA, et al. Clinical utility of intraductal US for evaluation of choledocholithiasis. Gastrointest Endosc 2003; 57:648–652. 35. Buscarini E, Tansini P, Vallisa D, et al. EUS for suspected choledocholithiasis: do benefits outweigh costs? A prospective, controlled study. Gastrointest Endosc 2003; 54:510–518. 36. Freeman ML, Nelson DB, Sherman S, et al. Complications of endoscopic biliary sphincterotomy. N Engl J Med 1996; 335:909–918. 37. Borgaonkar MR. Passage of a bile duct stone. Gastrointest Endosc 2003; 57:721. 38. Cunningham JT, Cotton PB, Hawes RH, et al. Mechanical lithotripsy for biliary stones: a 5 year university experience. Am J Gastroenterol 2000; 95:2474–2475. 39. Cipolletta L, Costamagna G, Bianco MAA, et al. Endoscopic mechanical lithotripsy of difficult common bile duct stones. Br J Surg 1997; 84:1407–1409. 40. Sorbi D, Van OS E, Aberger FJ, et al. Clinical application of a new disposable lithotripter: a prospective multicenter study. Gastrointest Endosc 1999; 49:210–213. 41. Katsinelos P, Galanis I, Pilpilidis I, et al. The effect of indwelling endoprosthesis on stone size for fragmentation after long-term treatment with biliary stenting for large stones. Surg Endosc 2003; 17:1552–1555. 42. Cotton PB, Forbes A, Leung FW, Dineen L. Endoscopic stenting for long term treatment of large bile duct stones. Gastrointest Endosc 1987; 33:411–412. 43. Bergman JJ, Rauws EA, Tijssen JG, et al. Biliary endoprosthesis in elderly patients with endoscopically irretrievable common bile duct stones: report on 117 patients. Gastrointest Endosc 1995; 42:195–201. 44. Hui CK, Lai KC, Wong WM, et al. Retained common bile duct stones: a comparison between biliary stenting and complete clearance of stone by electrohydraulic lithotripsy. Aliment Pharmacol Ther 2003; 17:289–296.
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45. Lin CK, Lai KH, Chan HH, et al. Endoscopic balloon dilation is a safe method in the management of common bile duct stones. Digest Liver Dis 2004; 36:68–72. 46. Arnold JC, Benz C, Martin WR, et al. Endoscopic papillary balloon dilation vs. sphincterotomy for removal of common bile duct stones: a prospective randomized pilot study. Endoscopy 2001; 33:563–567. 47. Bergman JJ, Rauws EA, Fockens P, et al. Randomised trial of endoscopic balloon dilation versus endoscopic sphincterotomy for removal of bile duct stones. Lancet 1997; 349: 1124–1129. 48. Mathuna PM, White P, Clarke E, et al. Endoscopic balloon sphincteroplasty (papillary dilation) for bile duct stones: efficacy, safety, and follow-up in 100 patients. Gastrointest Endosc 1995; 42:468–474. 49. Tanaka S, Sawayama T, Yoshioka T. Endoscopic papillary balloon dilation and endoscopic sphincterotomy for bile duct stones: long-term outcomes in a prospective randomized controlled trial. Gastrointest Endosc 2004; 59:614–618. 50. DiSario JA, Freeman ML, Bjorkman DJ, et al. Endoscopic balloon dilation compared with sphincterotomy for extraction of bile duct stones. Gastroenterology 2004; 127:1291–1299. 51. Sugiyama M, Atomi Y. Endoscopic papillary balloon dilation causes transient pancreatobiliary and duodenobiliary reflux. Gastrointest Endosc 2004; 60:186–190. 52. Sugiyama M, Atomi Y. Does endoscopic sphincterotomy cause prolonged pancreatobiliary reflux? Am J Gastroenterol 1999; 94:795–798. 53. Isayama H, Komatsu Y, Inoue Y, et al. Preserved function of the Oddi sphincter after endoscopic papillary balloon dilation. Hepato-Gastroenterology 2003; 50:1787–1791. 54. Kawabe T, Komatsu Y, Isayama H, et al. Histological analysis of the papilla after endoscopic papillary balloon dilation. Hepato-Gastroenterology 2003; 50:919–923. 55. Perini RF, Sadurski R, Cotton BP, et al. Post-sphincterotomy bleeding after the introduction of microprocessor-controlled electrosurgery: does the new technology make the difference? Gastrointest Endosc 2005; 61:53–57. 56. Cotton PB, Lehman G, Vennes J, et al. Endoscopic sphincterotomy complications and their management: an attempt at consensus. Gastrointest Endosc 1991; 37:383–393. 57. Park DH, Kim MH, Lee SK, et al. Endoscopic sphincterotomy vs. endoscopic papillary dilation for choledocholithiasis in patients with liver cirrhosis and coagulopathy. Gastrointest Endosc 2004; 60:180–185. 58. Ponchon T, Valette PJ, Bory R, et al. Evaluation of a combined percutaneous-endoscopic procedure for the treatment of choledocholithiasis and benign papillary stenosis. Endoscopy 1987; 19:164–166. 59. Chespak LW, Ring EJ, Shapiro HA, et al. Multidisciplinary approach to complex endoscopic biliary intervention. Radiology 1989; 173:995–997. 60. Calvo MM, Bujanda L, Heras I, et al. The rendezvous technique for the treatment of choledocholithiasis. Gastrointest Endosc 2001; 54:511–513. 61. van der Velden JJ, Berger MY, Bonjer JH, et al. Percutaneous treatment of bile duct stones in patients treated unsuccessfully with endoscopic retrograde procedures. Gastrointest Endosc 2000; 51:418–422. 62. Adamek HE, Maier M, Jakobs R, et al. Management of retained bile duct stones: a prospective open trial comparing extracorporeal and intracorporeal lithotripsy. Gastrointest Endosc 1996; 44:40–47. 63. Weikert U, Muhlen E, Janssen J, et al. The holmium-YAG laser: a suitable instrument for stone fragmentation in choledocholithiasis. The assessment of the results of its use under babyscopic control. Dtsche Med Wochensch 1999; 124:514–518.
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64. Yamakawa T. Percutaneous cholangioscopy for the management of retained biliary tract stones and intrahepatic stones. Endoscopy 1989; 21:333–337. 65. Kusano T, Masato F, Isa T, et al. Percutaneous transhepatic cholangioscopic lithotripsy and change of biliary manometry patterns. Hepato-Gastroendterology 1999; 46:2153–2158. 66. Chikamori F, Nishio S, LeMaster JC. Percutaneous papillary balloon dilation as a therapeutic option for cholecystocholedocholithiasis in the era of laparoscopic cholecystectomy. Surg Today 1999; 29:856–861. 67. Maringhini A, Ciambra M, Baccelliere R, et al. Biliary sludge and gallstones in pregnancy: incidence, risk factors, and natural history. Ann Intern Med 1993; 119:116–120. 68. Hiatt JR, Gordon-Hiatt JC, Williams RA, Klein SR. Biliary disease in pregnancy: strategy for surgical management. Am J Surg 1986; 151:263–265. 69. Glasgow RE, Visser BC, Harris HW, et al. Changing management of gallstones disease during pregnancy. Surg Endosc 1998; 12:241–246. 70. Sungler P, Heinerman PM, Steiner H, et al. Laparoscopic cholecystectomy and interventional endoscopy for gallstone complications during pregnancy. Surg Endoscopy 2000; 14:267–271. 71. Medical radiation exposure of pregnant and potentially pregnant women. NCRP report no. 54. Washington, DC: National Council on Radiation Protecton and Measurements; 1977. 72. Jamidar PA, Beck GJ, Hoffman BJ, et al. Endoscopic retrograde cholangiopancreatography in pregnancy. Am J Gastroenterol 1995; 90:1263–1267. 73. Tham TCK, Vandervoort J, Wong RCK, et al. Safety of ERCP during pregnancy. Am J Gastroenterol 2003; 98:308–311. 74. Hoffman BJ, Cunningham JT. Radiation exposure to the pregnant patient during ERCP. Gastrointest Endosc 1992; 38:253. 75. Fox VL, Werlin SL, Heyman MB. Subcommittee on endoscopy procedures of the patient care committee of the North American Society for Pediatric Gastroenterology Nutrition. Endoscopic retrograde cholangiopancreatography in children. J Pediatr Gastroenterol Nutr 2000; 30:335–342. 76. Kato S, Kamagata S, Asakura T, et al. A newly developed smallcaliber videoduodenoscope for endoscopic retrograde cholangiopancreatography in children. J Clin Gastroenterol 2003; 37:102–104. 77. Wengrower D, Gozal D, Gozal Y, et al. Complicated endoscopic pediatric procedures using deep sedation and general anesthesia are safe in the endoscopy suite. Scand J Gastroenterol 2004; 39:283–286. 78. Guelrud M, Mendoza S, Jaen D, et al. ERCP and endoscopic sphincterotomy in infants and children with jaundice due to common bile duct stones. Gastrointest Endosc 1992; 38:450–453. 79. Newman KD, Powell DM, Holcomb III GW. The management of choledocholithiasis in children in the era of laparoscopic cholecystectomy. J Pediatr Surg 1997; 32:1116–1119. 80. Tarnasky PR, Tagge EP, Hebra A, et al. Minimally invasive therapy for choledocholithaisis in children. Gastrointest Endosc 1998; 47:189–192. 81. Pfau PR, Chelimsky GG, Kinnard MF, et al. Endoscopic retrograde cholangiopancreatography in children and adolescents. J Pediatr Gastroenterol Nutr 2002; 35:619–623. 82. Prasil P, Laberge JM, Barkun A, Flageole H. Endoscopic retrograde cholangiopancreatography in children: a surgeon’s perspective. J Pediatr Surg 2001; 36:733–735. 83. The Southern Surgeons Club. A prospective analysis of 1518 laparoscopic cholecystectomies. N Engl J Med 1991; 324:1075–1078. 84. Brugge WR, Rosenberg DJ, Alavi A. Diagnosis of postoperative bile leaks. Am J Gastroenterol 1994; 89:2178–2183.
Chapter 64 NON-SURGICAL MANAGEMENT OF GALLSTONES
85. Foutch PG, Harlan JR, Hoefer M. Endoscopic therapy for patients with a post-operative biliary leak. Gastrointest Endosc 1993; 39:416–421. 86. Kozarek RA, Ball TJ, Patterson DJ, et al. Endoscopic treatment of biliary injury in the era of laparoscopic cholecystectomy. Gastrointest Endosc 1994; 40:10–16. 87. Bjorkman DJ, Carr-Locke DL, Lichtenstein DR, et al. Postsurgical bile leaks: endoscopic obliteration of the transpapillary pressure gradient is enough. Am J Gastroenterol 1995; 90:2128–2133. 88. Wootton FT, Hoffman BJ, Marsh WH, Cunningham JT. Biliary complications following laparoscopic cholecystectomy. Gastrointest Endosc 1992; 38:183–1185. 89. Christoforidis E, Goulimaris I, Tsalis K, et al. The endoscopic management of persistent bile leakage after laparoscopic cholecystectomy. Surg Endosc 2002; 16:843–846. 90. Hoffman BJ, Cunningham JT, Marsh WH. Ensdoscopic management of biliary fistulas with small caliber stents. Am J Gastroenterol 1990; 85:705–707. 91. Kaffes AJ, Hourigan L, De Luca N, et al. Impact of endoscopic intervention in 100 patients with suspected postcholecystectomy bile leaks. Gastrointest Endosc 2005; 61:269–275. 92. Bergman JJHG, van de Brink GR, Rauws EAJ, et al. Treatment of bile duct lesions after laparoscopic cholecystectomy. Gut 1996; 38:141–147. 93. Baron TH, Feitoza AB, Nagorney DM. Successful endoscopic treatment of a complete transaction of the bile duct complicating laparoscopic cholecystectomy. Gastrointest Endosc 2003; 57:765–769. 94. Borowicz MR, Adams DB, Simpson JP, Cunningham JT. Management of biliary strictures due to laparoscopic cholecystectomy. J Surg Res 1995; 58:86–89.
95. Draganov P, Hoffman BJ, Marsh WH, et al. Long-term outcome in patients with benign biliary strictures treated endoscopically with multiple stents. Gastrointest Endosc 2002; 55:680–685. 96. De Wit LH, Rauws EAJ, Gouma DJ. Surgical management of iatrogenic bile duct injury. Scand J Gastroenterol 1999; 34:89–94. 97. Adams DB, Borowicz MR, Wootton III FT, Cunningham JT. Bile duct complications after laparoscopic cholecystectomy. Surg Endoscopy 1993; 7:79–83. 98. Kalayci C, Aisen A, Canal D, et al. Magnetic resonance cholangiopancreatography documents bile leak site after cholecystectomy in patients with aberrant right hepatic duct where ERCP fails. Gastrointest Endosc 2000; 52:277–281. 99. Lillemoe KD, Petrofski JA, Choti MA, et al. Isolated right segmental hepatic duct injury: a diagnostic and therapeutic challenge. J Gastrointest Surg 2000; 4:168–177. 100. Wojtun S, Gil M, Gil J. Recognition of ERC-induced pancreatitis in patients with choledocholithiasis by an analysis of laboratory findings. Hepato-Gastroenterol 2000; 47:550–553. 101. Yoshimoto H, Ikeda S, Tanaka M, Matusmoto S. Relationship of biliary pressure to cholangiovenous reflux during endoscopic retrograde balloon catheter cholangiography. Digest Dis Sci 1989; 34:16–20. 102. Stapfer M, Selby RR, Stain SC, et al. Management of duodenal perforations after endoscopic retrograde cholangiopancreatography and sphincterotomy. Ann Surg 2000; 232:191–198. 103. Enns R, Eloubeidi MA, Mergener K, et al. ERCP-related perforations: risk factors and management. Endoscopy 2002; 34:293–298. 104. Zissen R, Shapiro-Feinberg M, Oscadchy A, et al. Retroperitoneal perforation during endoscopic sphincterotomy: imaging findings. Abdom Imaging 2000; 25:279–282.
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65
SURGERY OF THE BILIARY TRACT Daniel Tseng and John Hunter Abbreviations ERCP endoscopic retrograde cholangiopancreatography LC laparoscopic cholecystectomy
MRCP
magnetic resonance cholangiopancreatography
INTRODUCTION The extrahepatic biliary tract extends from the right and left hepatic ducts exiting the liver to the ampulla of Vater. It has always been an area troublesome to surgeons because of its tenuous blood supply, difficult anatomy, and unforgiving complications when disturbed. As surgeons enter a new age of minimally invasive surgery and endoscopic techniques, the limits are constantly tested and the old ways are redefined. Fortunately, patients have benefited from technologic advances with decreased hospital stays, diminished pain, small incisions, shorter recovery period, and a lower morbidity and mortality compared with the past. The purpose of this chapter is to provide an update on the management of problems of the biliary tract, specifically addressing the surgical options.
GALLBLADDER Cholecystectomy accounted for 442 000 of the nearly 40 million surgical procedures performed in the USA in 2001. It has surpassed appendectomy as the most frequently performed operation by the general surgeon.1 Since the first laparoscopic cholecystectomy (LC) was performed in 1985, the use of minimally invasive techniques in the treatment of gallbladder problems has expanded dramatically. The surgeon is now able not only to remove the gallbladder laparoscopically, but also to perform many other interventions that in the past required open intervention. While LC has dramatically improved the general surgeon’s ability to care for patients, it has also brought new dilemmas to surgery. Despite innovative surgical techniques and equipment, major complications still occur and may do so in a different manner than occurred previously. Therefore surgeons are required to manage challenges that did not exist prior to the development of minimally invasive surgery.
CHOLELITHIASIS In 2001, the Centers for Disease Control reported in their National Hospital Discharge Survey that cholelithiasis accounted for 367 000 of the 3.2 million hospital admissions, with an average length of stay for these patients of nearly 4 days.1 With approximately 10–15% or 20 million of adults in the USA carrying gallstones, this disease costs individuals and society a great deal.
PTD
percutaneous transhepatic biliary drainage
Gallstone composition consists primarily of cholesterol and bile pigments. An imbalance of the natural production of bile acids, phospholipids, and cholesterol predisposes individuals to the formation of lithogenic bile anywhere in the biliary system. The mnemonic of the four Fs (female, fat, forty, and fertile) characterizes patients who are classically found to have cholelithiasis associated with cholesterol stones. Other factors attributable to the formation of cholelithiasis include gallbladder dysmotility, nutrition, prior bowel surgery, hormonal changes, blood dyscrasias, medications, and infection. Genetic factors may play a role in the unusually high 80% incidence of gallstones in women of the Pima Indians in the USA, as well as the increased risk of gallstones in first-degree relatives.2,3 Fortunately, the majority of patients with cholelithiasis never develop symptoms. A review of the major published literature on the natural history of gallstones reveals that symptoms occur at approximately 1–2% per year, with complications developing in only 2–6% (Table 65-1). Most biliary symptoms result from pressure within the biliary system or result from inflammatory processes in or around the biliary system such as cholecystitis or pancreatitis (Table 65-2). Visceral sensation from the gallbladder can mimic pathologic states such as gastroesophageal reflux disease, angina, and appendicitis. Most commonly the visceral pain is referred to the right shoulder. Parietal irritation from either an acute or chronic condition results from overdistension of the gallbladder and irritation of the peritoneum causing pain located in the right upper quadrant.
INDICATIONS FOR CHOLECYSTECTOMY Once symptoms from cholidocholithiasis develop, recurrence can be expected in approximately 50% of patients over the course of 1 year.4 More recent data demonstrate a significant improvement in quality of life after cholecystectomy for biliary colic in the majority of patients and can be an indication for cholecystectomy in goodrisk patients.5 Although symptoms can be controlled medically, complications can be life-threatening. Complications occur in most series at less than 1% per year and represent an indication for surgery (Table 65-3).6,7 The diminished pain, smaller incisions, shorter hospitalizations, and faster recovery associated with LC have prompted surgeons to advise patients to have their gallbladder removed before complications occur.
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Table 65-1. Natural History of Gallstones Author (year)
Gracie (1982)8 McSherry (1985)9 Friedman (1989)10 Cucchiaro (1990)11 Wada (1993)12 Juhasz (1994)13 Attili (1995)14 Angelico (1997)15 Halldestam (2004)16
Table 65-3. Complications of Cholelithiasis
No. of patients
Length of disease (years)
Biliary pain (%)
Biliary complications (%)
123 135 123 125 680 110 118 426 120
15 4 5 5 13 6 10 10 7
18 17
2 0 6 2 3 5 3
11 20 9 26 38 7
5
Table 65-2. Symptoms of Cholelithiasis Epigastric or right upper quadrant abdominal pain Fatty food intolerance Heartburn Bloating Belching Jaundice Flatulence
Acute Cholecystitis Simply defined, acute cholecystitis is inflammation of the gallbladder as a result of obstruction of the outflow of bile from the cystic duct. Causes for cystic duct obstruction include gallstones, biliary sludge, or neoplasms that result in overgrowth of native bacteria present within the gastrointestinal tract. The most common Gram-negative organisms identified are Escherichia coli and Klebsiella spp., and the anaerobes Clostridium and Bacteroides are second most common; and Gram-positive organisms are rare. While infection of the bile is not uncommon, most acute cholecystitis occurs with sterile bile. Signs and symptoms of acute cholecystitis include abdominal pain and tenderness, fever, nausea, vomiting, positive Murphy’s sign, and a palpable fullness in the right upper quadrant. Unlike biliary colic, these symptoms can be somewhat insidious and are constant in nature, worsening over time. Whereas colic subsides over time, true acute cholecystitis does not, and therefore often requires admission to hospital for treatment. Laboratory examination often reveals an elevated white blood cell count with or without a significant shift in the differential towards polymorphonuclear cells. Ultrasonographic description of acute cholecystitis includes a thickened edematous gallbladder wall, pericholecystic fluid, and sonographic Murphy’s sign. Once the diagnosis of acute cholecystitis has been made, initial therapy is resuscitative in nature. Initiation of intravenous fluid resuscitation and broad-spectrum antibiotics for Gram-negative and anaerobic bacteria is paramount in preparation for surgery. Appropriate response to therapy allows the patient time to recover from the initial insult prior to proceeding to the operating room. Although cholecystitis is not an emergent operation in most instances, urgent removal of the gallbladder should be performed during the same hospitalization. The earlier and more edematous phases of acute cholecystitis facilitate LC.
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Cholecystitis Cholangitis Gallstone pancreatitis Gallstone ileus Pyoderma gangrenosum Mirizzi’s syndrome
In the past, it was suggested that those patients who develop complications such as acute cholecystitis have an increased morbidity with early surgical intervention, which prompted many to propose that those with cholecystitis wait for up to 6 weeks before undergoing cholecystectomy. That information was mainly based on observational studies and applied more generally to open procedures. Recent data, however, have demonstrated that outcomes are generally equivalent and may be slightly better if surgery is performed during the same hospitalization rather than waiting 4–6 weeks.17–19 Recently, a meta-analysis of three randomized prospective clinical trials revealed that patients who had undergone early LC had similar rates of complications, a trend toward fewer conversions to open procedures, and a trend toward decreased hospital stay (P < 0.06) compared with delayed LC.17 Of significance, a failure rate requiring urgent operation during the initial hospitalization in 9.3% of those who were randomized to delayed surgery was observed. Of those who were able to leave the hospital, almost 15% suffered a recurrence of their symptoms requiring readmission, and 26% of those required urgent operation. Overall, 23% of patients failed nonoperative therapy during the study period, and over half of these patients required urgent surgery. The current available literature clearly supports early surgery in the setting of acute uncomplicated cholecystitis.18,19
Cholangitis Normally bile flowing from the liver is “sterile.” Bacterial contamination of bile is normally prevented by a number of factors. The sphincter of Oddi acts as a barrier to stop the reflux of duodenal bacteria into the biliary system. Hepatic sinusoids contain Kupffer cells which remove organisms and secrete immunoglobulin A and bile salts that help sterilize the bile. The flow of bile naturally removes debris and bacterial contamination helping to create the sterile environment. Once obstruction occurs, those natural protective mechanisms are lost, and this results in backflow of organisms into the biliary tree with subsequent development of cholangitis.20 In the USA, gallstones are the major etiologic cause for the development of cholangitis. Approximately 10% of those with gallstones may go on to develop cholangitis if left untreated.21 However, when evaluating someone with cholangitis, other causes must be considered (Table 65-4). Typical organisms cultured in the blood and bile are the usual bacteria found in the gastrointestinal tract, including E. coli, Enterobacter, Enterococcus, and Klebsiella. However, instrumentation may allow Pseudomonas, skin, and oral flora to be introduced into the biliary system as well.22 Cholangitis is characterized by Charcot’s triad, which consists of fever (90%), right upper quadrant pain (70%), and jaundice (60%).23
Chapter 65 SURGERY OF THE BILIARY TRACT
Table 65-4. Causes of Cholangitis Cholelithiasis Primary biliary stones Malignant neoplasms Strictures Instrumentation Infection Primary sclerosis cholangitis
Sicker patients have associated mental status changes (10–20%), and hypotension (30%), resulting in Reynolds’ pentad.24 Laboratory abnormalities include leukocytosis, hyperbilirubinemia, and mild elevations of transaminases and alkaline phosphatase.25 Severe liver dysfunction with coagulation abnormalities can result from prolonged untreated disease, with a mortality rate of nearly 100%. Initial resuscitation with intravenous fluids, broad-spectrum antibiotics, and correction of any underlying coagulopathy is critical in the initial management of this disease. After stabilization of the patient, biliary decompression must be performed in order to allow the infection to resolve. Non-operative drainage procedures are offered first, and these include endoscopic retrograde cholangiopancreatography (ERCP) or percutaneous transhepatic biliary drainage (PTD). ERCP can be successfully performed in over 90% of patients with cholangitis and is the treatment of choice for decompression of the biliary system. In one published series of 898 patients with cholangitis treated with ERCP, the mortality rate was only 0.42% and the complication rate was 6%.26 Inability to perform endoscopic decompression usually results from difficulty in identifying and cannulating the ampulla of Vater. In patients with prior upper gastrointestinal surgery, it may prove too difficult or impossible to cannulate the ampulla. Even if cannulation is completed, debris or stones within the common bile duct may not be cleared completely. Also, individuals may have intrahepatic stones that are not amenable to removal. In these situations, PTD may be a better option. However, PTD is associated with a higher complication rate than ERCP. A number of studies have been completed demonstrating lower morbidity and mortality rates with ERCP compared to surgery in the setting of acute severe cholangitis. Lai et al.27 performed a prospective randomized trial directly comparing the use of ERCP versus surgical decompression in the setting of acute cholangitis. Eighty-two patients were randomized over a 43-month period. The results favor endoscopic drainage over surgical drainage, demonstrating a diminished need for ventilatory support (30% versus 63%), fewer complications (34% versus 66%), and fewer retained stones (7% versus 29%). Mortality was also significantly decreased (10% versus 32%) in the endoscopic drainage versus surgery groups, respectively. This study and others have demonstrated the superiority of early endoscopic decompression, reserving surgery for those who have failed the endoscopic approach.28
PERCUTANEOUS TRANSHEPATIC BILIARY DECOMPRESSION An alternative therapy for the initial treatment of obstructive cholangitis that was frequently utilized in the 1980s was PTD. This method allowed decompression of the biliary system without the
Table 65-5. Complications from Percutaneous Transhepatic Decompression Sepsis Hemobilia Intra-abdominal bleeding Cholangitis Bile leak Catheter malfunction/misplacement Hypotension Pancreatitis Pneumothorax/hemothorax
need for heavy sedation or general anesthesia. It was found to be especially helpful in the elderly and frail with multiple comorbidities. Besides being less invasive, PTD allowed access to the intrahepatic biliary system after decompression, for removal of stones through the percutaneous tract, and treatment of intrahepatic strictures via balloon dilation. Currently PTD is used most frequently when ERCP is unsuccessful. PTD-related complication rates vary between 6 and 30% (Table 65-5).22,25,29,30 However, retrospective studies have shown that PTD can be performed with much lower morbidity and mortality than early surgery for cholangitis. Chen et al.22 reported a decline in mortality of 13% with early surgery to 2% in those who had a good response to PTD followed by surgery. In this series there was no mortality related to the PTD alone. Rapid improvement in both clinical and laboratory abnormalities is expected, certainly within 72 hours if successful drainage is achieved.31
SURGICAL DECOMPRESSION IN CHOLANGITIS DUE TO CHOLELITHIASIS Open surgical drainage procedures have been associated with a high morbidity and mortality rate in the setting of acute cholangitis. The morbidity and mortality rate in the 1970s and 1980s ranged from 20 to 60% when surgical treatment was used as the first line of treatment.32 However, results are dismal if no therapeutic intervention is performed, with mortality close to 100%. Welch et al.33 in 1976 reviewed the charts of 20 patients admitted for cholangitis. Sixteen had undergone surgical exploration while the other 4 were managed non-operatively: the latter all subsequently died. Those who were explored within 24 hours had a mortality of 17%, whereas those who were explored within 24–72 hours had a mortality of 50%. Other risk factors that have been identified on univariate analysis that increase mortality following surgery include low pH, thrombocytopenia, hypoalbuminemia, multiple medical problems, and high bilirubin.34 There are several surgical options and the choice of operative approach is dependent on the location of the gallstones. The simplest approach is open common bile duct exploration with placement of a T-tube for drainage. This approach is the fastest and least morbid operation available. It allows complete external drainage of the infected bile and should be the surgical treatment of choice, especially in those who are unstable.35,36 Other additional procedures to be considered at the time of the operation that may be helpful are cholecystectomy, cholangiography, and choledochoscopy. However these procedures may add precious time to the surgery and are not necessary in the unstable patient.
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Section XI. Diseases of the Biliary Tract
If the patient is stable, clearance of the biliary system by choledochoscopy or cholangiography is helpful in identifying retained common bile duct or intrahepatic stones. Choledochoscopy is superior to conventional intraoperative cholangiography in identification of retained common bile duct stones, as demonstrated by Lau et al. in 1991.37 Their group performed a large prospective randomized trial comparing choledoscopy to routine cholangiography for the demonstration of common bile duct stones. Retained common bile duct stones at 2 weeks postoperatively were more common in the group that received cholangiogram alone (17%) versus those undergoing choledochoscopy (1%). Choledochoscopy was performed safely in the setting of the hemodynamically stable patient with acute cholangitis but was time-consuming and not recommended for unstable septic patients. Alternatively, internal drainage via choledochoenteric bypass can be considered in those who have an obstructed ampulla or very large stones that would not otherwise pass on their own. This is rather time-consuming and should only be performed in the stable patient. Options include choledochoduodenostomy or Roux en Y choledochojejunostomy. Side-to-side choledochoduodenostomy is faster and allows for a single anastomosis. Extensive kocherization of the duodenum allows tension to be removed from the anastomosis, but this may not be possible in those with severe inflammation. Roux en Y choledochojejunostomy is preferred for those who have a rather fixed and immobile duodenum.
are multiple treatment options with no clear consensus as to the best approach for the management of this problem. Currently pancreatitis is generally classified as mild, moderate, or severe forms. There are multiple scoring systems that have been developed for grading pancreatitis. Among these scoring systems are Ranson’s criteria,31 APACHE II,45 Glasgow,34 and Atlanta criteria.35 These scoring systems offer prognostic criteria of anticipated morbidity and mortality. Despite the ability to classify the degree of pancreatitis, initial therapy for pancreatitis is based on severity at presentation. All patients share a common presentation of abdominal pain, with different degrees of organ compromise detected by physical and laboratory examination. Patients admitted to the hospital are placed on strict bowel rest and require fluid and electrolyte replacement. Depending on the degree of pancreatitis, intensive care unit monitoring, antibiotics, and parenteral nutrition may be initiated. Identifying the cause of pancreatitis is paramount in preventing its recurrence. Ultrasound and computed tomography are used to identify gallstones and biliary sludge, determine biliary ductal dilation, assess the degree of pancreatitis, and detect any complications from the disease. If gallstones or biliary sludge are identified as the source of pancreatitis, multiple options currently exist as to the further treatment and are tailored to the severity of the pancreatitis.
Mild/Moderate Gallstone Pancreatitis CHOLECYSTECTOMY AFTER DRAINAGE In the past, after endoscopic drainage was completed, not all patients underwent subsequent cholecystectomy. The high-risk patients, the elderly, those with multiple comorbid conditions, or those who refused cholecystectomy were managed expectantly. Unfortunately a recurrence rate for cholangitis as high as 19% occurred if the gallbladder was left in situ.38 Multiple prospective randomized trials have demonstrated that the recurrence rate of complicated gallstone disease requiring cholecystectomy is between 14 and 40%, even after endoscopic sphincterotomy (Table 65-6). Therefore it is our policy routinely to remove the gallbladder in patients who have been cleared of common duct gallstones endoscopically once patients have been stabilized.
GALLSTONE PANCREATITIS Acute pancreatitis may be a severe disease process with a mortality rate between 5 and 15%.39,40 The presence of gallstones accounts for approximately 30–50% of patients who develop pancreatitis and remains the most common cause for acute pancreatitis. The severity of resulting gallstone pancreatitis is extremely variable, with some patients requiring prolonged intensive care unit stays. There Table 65-6. Recurrence of Gallstone Complications Requiring Cholecystectomy after Endoscopic Sphincterotomy Author (year) Tanaka (1987)41 Hammarstrom (1995)42 Targarona (1996)43 Suc (1998)44
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Patients
Follow-up
103 39 50 97
3 years 5 years 17 months 5 years
Later cholecystectomy (%) 33% 14% 37%
For those who do not have severe pancreatitis based upon available criteria, multiple therapeutic alternatives are available that depend on the local expertise (Table 65-7). Management strategy is dependent on the available resources and skills of the endoscopist, radiologist, and surgeon. Laparoscopic cholangiography is now a routine procedure; however, laparoscopic common bile duct exploration remains technically challenging, timeconsuming, and vexing for surgeons unfamiliar with the technique. Because of this, UK consensus guidelines for the treatment of biliary pancreatitis advocate the use of selective preoperative ERCP followed by interval LC (two-stage approach).46 Alternatively, for those individuals of adequate training and skill, a more technically demanding one-stage approach with LC and common bile duct exploration results in excellent outcomes.47,48 Optimal timing for LC in those with mild or moderate form of pancreatitis is still debated. Some authors advocate that cholecystectomy after ERCP and stone clearance is unnecessary because the incidence of recurrent gallstone pancreatitis is low and morbidity
Table 65-7. Treatment Alternatives for Gallstone Pancreatitis in Mild or Moderate Form ERCP with sphincterotomy alone ± interval cholecystectomy Preoperative ERCP with sphincterotomy followed by laparoscopic cholecystectomy Laparoscopic cholecystectomy alone Laparoscopic cholecystectomy with intraoperative cholangiogram and possible common bile duct exploration Laparoscopic cholecystectomy with intraoperative cholangiogram and postoperative ERCP with sphincterotomy ERCP, endoscopic retrograde cholangiopancreatography.
Chapter 65 SURGERY OF THE BILIARY TRACT
and mortality are not different.49 Still, there are significant data showing that recurrence rates of gallstone pancreatitis without cholecystectomy range between 30 and 61%.50,51 Moreover, if pancreatitis does recur, it has a much higher morbidity and mortality.52 There is a decrease in recurrent episodes of pancreatitis if the gallbladder is removed early.53 Unfortunately, the risks of complications during LC are higher in the setting of gallstone pancreatitis compared with uncomplicated gallstone disease, but studies have shown that it can still be performed safely.54 The ability to perform the operation laparoscopically successfully ranges between 90 and 100% in most series.55–63 As with cholecystitis, early surgery (
