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ADVANCES IN CLINICAL CHEMISTRY VOLUME 37 BOARD OF EDITORS Kaiser I. Aziz Galal Ghourab Walter G. Guder E. D. Janus S...

Author: Herbert E. Spiegel

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ADVANCES IN CLINICAL CHEMISTRY VOLUME 37

BOARD OF EDITORS

Kaiser I. Aziz Galal Ghourab Walter G. Guder E. D. Janus Sheshadri Narayanan Francesco Salvatore It-Koon Tan

Milos Tichy Masayuki Totani Casper H. van Aswegen Abraham van den Ende Istvan Vermes Henning von Schenk Zhen Hua Yang

Advances in CLINICAL CHEMISTRY Edited by HERBERT E. SPIEGEL Applied Science & Technology Associates, Inc. Cedar Grove, New Jersey

Associate Regional Editors Gerard Nowacki Fundacja Rozwoju Diagnostyki Laboratoryjnej Krakow, Poland

Kwang-Jen Hsiao Veterans General Hospital Taipei, Taiwan

VOLUME 37

Amsterdam Boston London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo

This book is printed on acid-free paper.

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C 2003, Elsevier Science (USA). Copyright

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CONTENTS

CONTRIBUTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

PUBLISHER’S NOTE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi

PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xiii

Clinical Applications of Cytokine Assays

C. K. WONG AND C. W. K. LAM 1. 2. 3. 4. 5.

Cytokines: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytokines and Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytokine Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Application of Cytokine Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 14 20 28 32 32

Current Concepts on Diagnosis and Treatment of Acute Pancreatitis

B. KUSNIERZ-CABALA, B. KEDRA, AND M. SIERZEGA 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathophysiology of Acute Pancreatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnosis of Acute Pancreatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

47 49 51 67 70 70

Mitochondrial DNA Mutations and Oxidative Stress in Mitochondrial Diseases

YAU-HUEI WEI AND HSIN-CHEN LEE 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Brief Review of Studies on Mitochondrial Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Genetic Map and Structural Characteristics of Human Mitochondrial DNA . . . Transmission by Maternal Inheritance and Random Segregation of mtDNA during Mitosis and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

84 85 86 87

vi 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

CONTENTS

Susceptibility of mtDNA to Oxidative Damage and Mutation . . . . . . . . . . . . . . . . . . . . . . . Classification of Disease-Associated mtDNA Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Consequences of mtDNA Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemical Basis for How mtDNA Mutations Cause Mitochondrial Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress and Oxidative Damage Elicited by mtDNA Mutations . . . . . . . . . . . . Mitochondrial Diseases Caused by Mutations in Nuclear DNA . . . . . . . . . . . . . . . . . . . . . Cybrids for Studies of Mitochondrial Diseases: Applications and Limitations . . . . . Effect of mtDNA Mutations on the Apoptosis of Human Cells . . . . . . . . . . . . . . . . . . . . . . Therapy for Mitochondrial Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

90 90 97 100 102 104 107 111 113 115 116

Autoantibodies to dsDNA, Ro/SSA, and La/SSB in Systemic Lupus Erythematosus

JIEN-WEN CHIEN AND CHING-YUANG LIN 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Basic Studies of Autoantibodies to dsDNA, Ro/SSA, and La/SSB . . . . . . . . . . . . . . . . . . 3. Clinical Aspects of Anti-dsDNA, Anti-Ro/SSA, and Anti-La/SSB Autoantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Discussion and Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

129 133 145 153 154

Pathobiochemistry of Nephrotic Syndrome

VLADIMÍR TESARˇ , TOMA´ Sˇ ZIMA, AND MARTA KALOUSOVA´ 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and Function of the Glomerular Capillary Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis of Proteinuria in Acquired Forms of Nephrotic Syndrome . . . . . . . . . . . . Biochemical Signs and Clinical Symptoms of Nephrotic Syndrome . . . . . . . . . . . . . . . . Diagnosis of Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment of Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

174 174 185 196 205 206 207 208

Total Antioxidant Capacity

GRZEGORZ BARTOSZ 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Determines Total Antioxidant Capacity? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Total Antioxidant Capacity of Body Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

220 221 236 241

CONTENTS

5. 6. 7. 8. 9. 10. 11.

Total Antioxidant Capacity of Tissue Homogenates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Total Antioxidant Capacity of Food and Beverages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Total Antioxidant Capacity of Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antioxidant Activity of Individual Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of Nutrition on Total Antioxidant Activity of Body Fluids . . . . . . . . . . . . . . . . . . . Effect of Physical Exercise on Blood Plasma Total Antioxidant Capacity . . . . . . . . . . Alterations in Total Antioxidant Capacity of Body Fluids and Tissue Homogenates in Diseases and Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Points of Concern. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 246 248 251 252 254 259 260 269 272 272

Lymphoid Malignancies: Immunophenotypic Analysis

AMY CHADBURN AND SHESHADRI NARAYANAN 1. 2. 3. 4. 5. 6.

Introduction: Immunophenotypic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Background: Immunophenotypic Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Normal Lymphoid Cell Development and Antigen Expression . . . . . . . . . . . . . . . . . . . . . . Neoplastic Lymphoid Cell Antigen Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Selected Biochemical Consequences of Neoplastic Transformation . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

293 295 303 308 325 329 329

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

355

This Page Intentionally Left Blank

CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

GRZEGORZ BARTOSZ (219), Department of Molecular Biophysics, University of Lód´z, Lód´z, Poland, and Department of Cell Biochemistry and Biology, University of Rzeszów, Rzeszów, Poland ,

,

AMY CHADBURN (293), Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, New York B. KEDRA (47), First Department of General and Gastrointestinal Surgery, Collegium Medicum Jagiellonian University, Krakow, Poland B. KUSNIERZ-CABALA (47), Department of Clinical Biochemistry, Collegium Medicum Jagiellonian University, Krakow, Poland JIEN-WEN CHIEN (129), Department of Pediatrics, Taipei Veterans General Hospital, Taipei, Taiwan, Republic of China MARTA KALOUSOVA´ (173), Institute of Medical Biochemistry and Institute of Clinical Biochemistry, First Faculty of Medicine and University Hospital, Prague, Czech Republic C. W. K. LAM (1), Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong HSIN-CHEN LEE (83), Institute of Biochemistry, Chung Shan Medical University, Taichung, Taiwan, Republic of China CHING-YUANG LIN (129), Changhua Christian Hospital, and National Yang-Ming University, School of Medicine, Taipei, Taiwan, Republic of China SHESHADRI NARAYANAN (293), Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, New York

ix

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CONTRIBUTORS

M. SIERZEGA (47), Department of Clinical Biochemistry, Collegium Medicum Jagiellonian University, Krakow, Poland VLADIMÍR TESARˇ (173), First Department of Medicine, Division of Nephrology, First Faculty of Medicine and University Hospital, Prague, Czech Republic YAU-HUEI WEI (83), Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, Republic of China C. K. WONG (1), Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong TOMA´ Sˇ ZIMA (173), Institute of Clinical Chemistry, First Faculty of Medicine and University Hospital, Prague, Czech Republic

PUBLISHER’S NOTE This volume marks the last in the series to be edited by Dr. Herbert Spiegel, in whose able hands it has been since he took on the position in 1983 from Dr. Morton K. Schwartz and Dr. A. L. Latner. In the period since then, beginning with Volume 24 until now with Volume 37, Dr. Spiegel has successfully commissioned top quality reviews from world-renowned experts. This quality is reflected in the ongoing acclaim there is for the Advances in Clinical Chemistry series and the large number of citations that the series receives annually. We would like to acknowledge the significant contribution of Dr. Spiegel to the success of this series and thank him for all of the time and effort he has put in to establish its position as one of the leading reference works in the field. We wish him all the best in his retirement. At the same time we would like to welcome Dr. Gregory Makowski, from the University of Connecticut Health Center in Farmington, who has taken up the Editor’s baton. With his research interests in genetic diseases, toxicology, and point-of-care testing, Dr. Makowski is the ideal person to develop this series further.

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PREFACE After 20 years of service as the executive editor of this series, I will now become emeritus. It has been an honor to work with the many distinguished and talented scientists who have significantly contributed to the scholarship and quality of each volume. This international cadre of highly professional authors and editors were a delight to work with. They were a repository of an awesome fund and diversity of knowledge, with which I was privileged to be associated. I am equally appreciative of the unflagging support of the publisher and its staff without which these volumes could not be produced. During my tenure, I have also enjoyed the support and encouragement of my wife, Joanne. She is and has always been a good counselor as well as best friend, and I wish to express my thanks. This volume continues our objective of expanding the intellectual horizon of clinical chemistry. Included are chapters on: Clinical Applications of Cytokine Assays; Diagnosis and Treatment of Acute Pancreatitis; Mitochondrial Mutations and Mitochondrial Diseases; Pathobiochemistry of Nephrotic Syndrome; Total Antioxidant Capacity; Autoantibodies to dsDNA, Ro/SSA, and LaSSB in Systemic Lupus Erythematosis; and Lymphoid Malignancies and Immunosuppressive Analysis. The meld of analytical, anatomical, subcellular, and molecular sciences represented by these subjects will continue to evolve and expand. Clinical chemistry is a vibrant and vital profession. Future volumes, their editors, and their contributors will undoubtedly be an important part of the practice and science of clinical chemistry. As is my custom, I would like to dedicate this volume. It is dedicated to the readership, which has been attentive and responsive over the years. It is dedicated to the future, which looks bright and challenging. Finally, it is dedicated to my son Girard, whose courage is an inspiration to me. HERBERT E. SPIEGEL

xiii


ADVANCES IN CLINICAL CHEMISTRY, VOL. 37

CLINICAL APPLICATIONS OF CYTOKINE ASSAYS C. K. Wong and C. W. K. Lam Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong

1. Cytokines: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Basic Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Recently Discovered Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Intracellular Signal Transduction of Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Cytokines and Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Interleukins and Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Chemokines and Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cytokine Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Enzyme-Linked Immunospot (ELISpot) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Flow-Cytometric Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Molecular Biology Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Clinical Application of Cytokine Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Therapeutic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Diagnostic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 1 2 11 14 14 18 20 21 22 22 23 26 28 28 31 32 32

1. Cytokines: An Overview 1.1. BASIC CONCEPTS Cytokines are a family of extracellular polypeptides capable of mediating intercellular communication through an array of diverse cellular responses upon their binding to cognate receptors on the surfaces of target cells. The term cytokine was originally used to designate molecules produced by cells of the immune system, before it became apparent that molecules outside the immune system have similar modes of production and action (E7). Currently, lymphokines and monokines, 1 C 2003, Elsevier Science (USA). All rights reserved. Copyright 0065-2423/03 $35.00

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WONG AND LAM

which are secreted by lymphocytes and monocytes, and other secreted products of neutrophils, mast cells, endothelial cells, fbroblasts, astrocytes, and other cell types are included among the cytokines. Since 1980, when interferon-α (IFN-α) became the first cytokine isolated for its inhibition of viral replication in cells, cytokines have come to comprise at least 23 distinct interleukins (IL), tumor necrosis factor (TNF), IFNs, colony-stimulating factors (CSFs), chemokines, a variety of growth factors, including transforming growth factor-β (TGF-β), and the family of fibroblast growth factors. Cytokines possess several common properties. First, they all act locally to modulate the behavior of adjacent cells in a manner described as paracrine functioning [e.g., activation of eosinophils by T helper cell type 2 (Th2) cytokines including the release of IL-3 and IL-5 from Th2 lymphocytes]. Some act as autokines on cells that secrete them (e.g., eotaxin release from eosinophils) or as endocrines on cells at distant sites (e.g., the effect of IL-1 on liver cells). Second, cytokines form a network in exerting their functions. In some cases, interrupting the function of a single cytokine such as IL-1 or TNF-α may affect many downstream cytokines and their actions (e.g., the release of IL-6 and IFN-γ from lymphocytes). Third, cytokine function is frequently redundant, with more than one cytokine exhibiting the same biological activity; for example, those of IL-1α, IL-1β, and TNF-α are virtually indistinguishable. Consequently, the inhibition of a single cytokine may not be effective because other cytokines with the same function can fulfill the role of the inhibited cytokine. Fourth, cytokine function may be pleiotropic; for example, IL-1 influences the hypothalamus, liver, and macrophages in the control of thermoregulation, acute-phase response, and inflammation, respectively. Fifth, similar to other polypeptide regulatory factors, cytokines act on cells via specific surface receptors (E7, R5). These receptors are membrane-bound proteins, many of which appear to be related. For example, both p55 and p75 subunits are required to compose the IL-2-binding sites and generate a high-affinity receptor and for IL-2 to deliver a signal to the cell (B8, L8). This review will describe the biological functions and regulation of the recently identified cytokines—interleukins, chemokines, and peptide growth factors—their intracellular signal transduction and role in certain diseases, and different cytokine assay methods and their clinical applications. 1.2. RECENTLY DISCOVERED CYTOKINES 1.2.1. Interleukins Interleukins comprise a group of cytokines secreted by leukocytes involved in signaling between cells of the immune system. At least 23 distinct ILs have been identified. Because IL-1 to IL-10 have been described in a previous review article (E7), only the recently discovered ILs continuing from IL-11 are described in what follows.

CLINICAL APPLICATIONS OF CYTOKINE ASSAYS

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1.2.1.1. IL-11. IL-11 is a multifunctional hematopoietic cytokine, which was originally identified as a factor produced by an IL-1α-stimulated primate stromal cell line. It was also independently discovered as an adipogenesis inhibitory factor. IL-11 has been reported to have diverse effects on the lymphopoietic and myeloid/erythroid cells in vitro. These include expansion of T cell-dependent Ig-secreting B cells, proliferation and differentiation of megakaryocytic progenitors and a variety of myeloid and erythroid precursor cells, and multiplication of pluripotential hematopoietic progenitors. In vivo administration of IL-11 elevated the number of circulating neutrophils and platelets and increased the number of megakaryocytes in the spleen of normal mice. In several models of severe myelosuppression induced by chemotherapy and/or irradiation and in bone marrow transplant models, there was multilineage hematopoietic stimulation following treatment with recombinant IL-11. In these models, accelerated recovery of platelets was a consistent observation. These results from preclinical studies suggested that this cytokine may be an effective agent in the treatment of myelosuppression and thrombocytopenia associated with cancer chemotherapy and bone marrow transplantation (G6). 1.2.1.2. IL-12. IL-12, also known as natural killer (NK) cell stimulatory factor (NKSF) or cytotoxic lymphocyte maturation factor (CLMF), is a pleiotropic proinflammatory cytokine, which is primarily produced by antigen-presenting cells (monocytes, macrophages, dendritic cells, and B cells) and neutrophils (L2). It is a heterodimeric cytokine of 70 kDa comprising a p35 subunit for signal transduction and a p40 subunit for receptor binding. IL-12 can induce IFN-γ and T helper cell type 1 (Th1) cytokine production, but can suppress Th2 cell development (K2, M4). It has multiple effects on T lymphocytes and NK cells, including the ability to stimulate cytotoxicity, proliferation, cytokine production, and development of Th1 subset (W14, H9). IL-12 has been shown to play an important role in inflammatory reactions in systemic lupus erythematosus (SLE) (W17, W19) and allergic asthma (W16). 1.2.1.3. IL-13. IL-13, a Th2 cytokine, has multiple effects on the differentiation and functions of monocytes/macrophages. IL-13 can suppress the cytotoxic functions of monocytes/macrophages. It can also suppress the production of proinflammatory cytokines and upregulate the production of IL-1 by monocytes/macrophages. Recently, IL-12 and IL-13 have been proposed to play pivotal roles in the Th2-polarized immune response to inhaled allergens (W11). IL-12 is a critical determinant of Th1-mediated immune responses, and it has been shown that deficiency in this cytokine can lead to Th2-polarized immune responses. IL-13, on the other hand, has recently been shown to be a critical mediator of the effector arm of the allergic response. Overproduction of this cytokine has been shown to induce many common features of the allergic diseases, such as airway hyperresponsiveness, eosinophilic inflammation, immunoglobulin E (IgE) production, mucus hypersecretion, and subepithelial fibrosis. Substantial evidence suggests

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that an imbalance in the production of these two critical immunoregulatory cytokines occurs in the lungs of atopic and asthmatic individuals, such that IL-13 is overproduced and IL-12 production is impaired (W11). 1.2.1.4. IL-14. IL-14 can be produced by aggressive intermediate (diffuse large cell) lymphomas of B cell-type non-Hodgkin’s lymphoma (NHL-B) in patients with lymphomatous effusions. The autocrine or paracrine production of IL-14 may play a significant role in the rapid proliferation of aggressive NHL-B (F4). 1.2.1.5. IL-15. IL-15 is a pleiotropic cytokine, which functions in NK cell development, lymphocyte homeostasis, and peripheral immune functions. It is produced by several cell types (including fibroblasts, keratinocytes, endothelial cells, and macrophages) in response to endotoxin or microbial infection. In turn, IL-15 has been shown to act on various cells of the immune system, including T and B lymphocytes, NK cells, monocytes, eosinophils, and circulating neutrophils. Moreover, IL-15 was initially observed to induce cytoskeletal rearrangements, enhance phagocytosis, increase the synthesis of several cellular proteins, produce chemokines, and delay apoptosis of neutrophils (F2). IL-5 is important in NK cell, NK-T cell, and intestinal intraepithelial lymphocyte development and function. A role for IL-15 during rheumatoid arthritis and malignancies such as adult T cell leukemia suggests that dysregulation of IL-15 may result in deleterious effects for the host (F2). 1.2.1.6. IL-16. IL-16 was originally described as a lymphocyte chemoattractant factor (C4). It is produced by CD8+ T cells upon induction of lectins, histamine, or serotonin (L1). It is also produced by CD4+ T cells stimulated with lectins, antigen, or anti-CD3 antibodies (C5) and from eosinophils (L13), epithelial cells (C5), and mast cells (R8). Recently, IL-16 has been found to induce eosinophil chemotaxis (C10), and plasma IL-16 is elevated in allergic rhinitis (P11). 1.2.1.7. IL-17. IL-17 is another novel proinflammatory Th1 cytokine produced by activated T helper cells (A1). It is capable of inducing the production of proinflammatory cytokine IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF), inflammatory mediator prostaglandin E2, leukemia inhibitory factor (LIF), and intercellular adhesion molecule-1 (ICAM-1), the proliferation of T cells, as well as the growth and differentiation of CD34+ human progenitors into neutrophils (F5, F6). IL-17 also appears to play an upstream role in T cell-triggered inflammation and hematopoiesis by stimulating stromal cells to secrete other proinflammatory and hematopoietic cytokines (F6). Moreover, it enhances the release of NO in cartilage from patients with osteoarthritis (A12). Elevated plasma IL-17 concentration has been reported in patients with rheumatoid arthritis (RA) (C7), systemic sclerosis (K8), Helicobacter pylori-infected gastric mucosa (L21), and SLE (W17). 1.2.1.8. IL-18. IL-18, formerly called IFN-γ -inducing factor, is a novel proinflammatory cytokine related to the IL-1 family; it is produced by Kupffer cells, and


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activates macrophages, keratinocytes, intestinal epithelial cells, osteoblasts, and adrenal cortical cells (D14). It plays an important role in the Th1 response to toxic shock and shares functional similarities with IL-12 (D14). Human IL-1 receptor protein is a functional component of the IL-18 receptor (T8). IL-18 receptors are selectively expressed on murine Th1 cells, but not Th2 cells (X2). The primary functions of IL-18 include the induction of IFN-γ and TNF-α in T cells and NK cells (P12, D14), and IL-8 in eosinophils (W5), the upregulation of Th1 cytokines including IL-2, GM-CSF, and IFN-γ (D14), the stimulation of the proliferation of activated T cells (D14), and the enhancement of Fas ligand (FasL) expression in NK and cytotoxic T lymphocytes (CTLs) (D2). A murine model of allergic asthma has indicated that IL-18 can increase allergic sensitization, serum IgE, Th2 cytokines, and airway eosinophilia (K6, W10). Clinical studies have also shown that mRNA levels of IL-18, IL-10, IL-13, and regulated-upon-activation normal T cell-expressed and secreted (RANTES) protein increase in nasal mucosa after nasal allergen provocation in patients with allergic rhinitis (K5). Elevated IL-18 levels have also been shown in the urine of nephrotic patients (M8) and the serum of patients with multiple sclerosis (N6), as well as in adult-onset Still’s disease (K1), type I diabetes mellitus (N5), viral infection (H6, P8), sepsis (E5), SLE (W17, W19), allergic asthma (W16), and inflammatory rheumatic disease (M11). 1.2.1.9. IL-19. Novel cytokine IL-19 shares 21% amino acid homology with IL-10. It is a pleiotropic cytokine with important immunoregulatory functions whose actions influence the activities of many of the cell types in the immune system. The expression of IL-19 mRNA can be induced in monocytes by lipopolysaccharide (LPS) with a slight delay compared to the expression of IL-10 mRNA (2 hr poststimulation for IL-10 and 4 hr for IL-19) (G1). Although treatment of monocytes with IL-4 or IL-13 did not induce de novo expression of IL-19, these cytokines did potentiate IL-19 gene expression in LPS-stimulated monocytes (G1). In addition, GM-CSF was capable of directly inducing IL-19 gene expression in monocytes. However, IL-19 does not bind or signal through the canonical IL-10 receptor complex, suggesting the existence of an IL-19-specific receptor complex (G1). 1.2.1.10. IL-20. IL-20 is a novel member of the expanding family of IL-10-like cytokines, which includes the other novel cytokines IL-19 and IL-22 (B11, X1). It is presumed that the bioactive form of IL-20 is a noncovalently linked homodimer, similar to IL-10. Although the physiological function of IL-20 has not been identified, three lines of evidence support a role for IL-20 and its receptor in inflammatory skin diseases such as psoriasis. For example, overexpression of IL-20 in transgenic mice results in neonatal lethality with skin abnormalities similar to those observed in human psoriatic skin (B11). These include several hallmark characteristics of this multigenic diseases such as increased proliferation of keratinocytes in the basal and the suprabasal layers of the epidermis, aberrant epidermal differentiation, and infiltration of immune cells into the skin (R3). Recombinant IL-20 protein

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stimulates a signal transduction pathway through signal transducers and activators of transcription (STAT) by IL-20 receptor complexes of two types (D15). Furthermore, the expression of both IL-20 receptor subunits is markedly upregulated in human psoriatic skin compared to normal skin. IL-20 can enhance the expression of several proinflammatory genes, such as those for TNF-α, macrophage inhibitory factor-related protein-14, and monocyte chemotactic protein (MCP)-1 in keratinocyte cells stimulated with IL-1α (B11). 1.2.1.11. IL-21. IL-21, a Th1 cytokine, is most closely related to IL-2 and IL-15, with the receptor designated IL-21 receptor (P3). The common γ -chain (γc ) is an indispensable subunit of the functional receptor complexes for IL-4, IL-7, IL-9, and IL-15 as well as IL-2. The γc is also shared with the IL-21 receptor (A11, V4). IL-21 binds to the IL-21 receptor and can activate Janus kinase 1 (JAK1), JAK3, STAT1, and STAT3 (A11). In vitro assays suggest that IL-21 has a role in the proliferation and maturation of NK cell populations from bone marrow, the proliferation of mature B-cell populations costimulated with anti-CD40, and the proliferation of T cells costimulated with anti-CD3 (P3). 1.2.1.12. IL-22. IL-10-related T cell-derived inducible factor (IL-TIF; provisionally designated IL-22) is a cytokine distantly related to IL-10 and is produced by activated T cells (D16). IL-22 receptor, a new member of the interferon receptor family, and CRF2-4, a member of the class II cytokine receptor family, join together to enable IL-22 signaling. Cell lines that respond to IL-22 by activation of STATs 1, 3, and 5, but unresponsive to IL-10, have been identified (X1). In contrast to IL-10, IL-22 does not inhibit the LPS-induced production of proinflammatory cytokines by monocytes, but it has a modest inhibitory effect on IL-4 production from Th2 cells (X1). 1.2.1.13. IL-23. The novel cytokine IL-23 is composed of two subunits, p19 and the p40 subunit of IL-12. The factor p19 shows no biological activity by itself. Instead it combines with the p40 subunit of IL-12 to form a biologically active, composite cytokine, IL-23. Activated dendritic cells secrete detectable levels of IL-23. IL-23 binds to IL-12 receptor β1, but fails to engage IL-12 receptor β2. Nonetheless, IL-23 activates STAT4 in phytohemagglutinin (PHA) blast T cells. It induces strong proliferation of mouse memory [CD4(+)CD45Rb(low)] T cells, a unique activity of IL-23; IL-12 has no effect on this cell population. Similar to IL-12, human IL-23 stimulates IFN-γ production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells (O1). A transgenic mouse model demonstrated that the expression of p19 in multiple tissues could induce a striking phenotype characterized by abnormal growth, systemic inflammation, infertility, and death before 3 month of age. Founder animals had infiltrates of lymphocytes and macrophages in skin, lung, liver, pancreas, and digestive tract and were anemic. The serum concentrations of the proinflammatory cytokines TNF-α and IL-1 were elevated, and the number of circulating neutrophils was increased. Moreover, ubiquitous expression of p19 also resulted in constitutive expression of acute-phase proteins in the liver. Bone marrow transfer experiments


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showed that hematopoietic cells are the source of biologically active p19. These findings indicate that p19 shares biological properties with IL-6, IL-12, and G-CSF and that cell-specific expression is required for its biological activity (W9). 1.2.2. Chemokines 1.2.2.1. Introduction. The chemokines are a large family of chemotactic cytokines, with more than 40 members; they can facilitate leukocyte migration and positioning as well as other processes such as angiogenesis, leukocyte degranulation, inflammation, and hematopoietic development. They are divided into four groups, designated CXC(α), CC(β), C(γ ), and CX3C, depending on the configuration of cysteine residues near the N-terminal (L17, N4) (Table 1). The CXC(α) and CC(β) groups, in contrast to the C(γ ) and CX3C chemokines, contain many members and have been extensively studied. The CXC chemokines predominantly TABLE 1 CHEMOKINE FAMILIES Family

Members

CXC

B cell-attracting chemokine 1 (BCA-1) β-Thromboglobulin (β-TG) Chemokines-connective tissue-activating peptide-III (CTAP-III) Epithelial neutrophil-activating peptide-78 (ENA-78) Granulocyte chemotactic protein-2 (GCP-2) Growth-related oncogene (GRO)-α, β, γ IFN-inducible protein-10 (IP-10) IL-8 Monokine induced by interferon-γ (Mig) Neutrophil-activating peptide-2 (NAP-2) Platelet basic protein (PBP) Platelet factor 4 (PF4) Stromal cell-derived factor-1 (SDF-1) T cell-attracting chemokine CXC receptor ligand 11 (I-TAC)

CC

Activation-regulated chemokine (PARC) Alternative macrophage activation-associated CC chemokine (AMAC)-1 Eotaxin-1 Eotaxin-2 Hemofiltrate CC chemokine (HCC)-1 I-309 Macrophage inflammatory protein (MIP) Macrophage-derived chemokine (MDC) Monocyte chemotactic protein (MCP) RANTES Secondary lymphoid tissue chemokine (SLC) Thymus- and activation-regulated chemokine (TARC) Thymus-expressed CC chemokine (TECK)

C

Single C motif-1 (SCM-1)/lymphotactin

CX3C

Fractalkine

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WONG AND LAM TABLE 2 CHARACTERIZED FUNCTIONS OF SOME MAJOR CHEMOKINES Chemokine CXC IL-8, ENA, GRO

IP-10, MIG

CC MCP-1

MIP-1α

Eotaxin RANTES

Cell Specificity

Known Function

Neutrophils Endothelial cells Smooth muscle Hepatocytes T lymphocytes Monocytes Endothelial cells

Chemotaxis, activation Angiogenic Proliferation Proliferation Chemotaxis, IFN production Chemotaxis Angiostatic

Monocytes T lymphocytes Fibroblasts Smooth muscle Basophil/mast cells Monocytes T lymphocytes Basophils Eosinophils Monocytes T lymphocytes Eosinophils Basophils

Chemotaxis, activation Chemotaxis, IL-4 production TGF-β and collagen production Proliferation Activation Chemotaxis, activation Chemotaxis, IFN production Activation Activation, chemotaxis Chemotaxis Chemotaxis, activation Chemotaxis, activation Activation

target neutrophils, whereas the CC chemokines target a variety of cell types including macrophages, basophils, T lymphocytes, eosinophils, and NK cells, but not neutrophils (Table 2). The CC chemokines are divided into several structural subgroups including a group composed of the MCPs and eotaxin (L19), which has been strongly implicated in asthma and other allergic diseases. Lymphotactin and fractalkine are the only proteins so far described with a C and a CX3C motif, respectively (M5). They both act on lymphoid cells including T lymphocytes and NK cells, and fractalkine is also active on monocytes and polymorphonuclear cells (PMNs) (M5). Chemokines interact with seven-transmembrane-domain, G protein-coupled receptors including 11 CC (CCR1–11), six CXC (CXCR1–6), and one CX3C (CX3CR1) receptors. For example, recent studies have indicated that polarized Th1 and Th2 populations show differential receptor expression and responsiveness to chemokines (B14). The regulation of receptor expression during activation or deactivation of monocytes is as important as regulation of chemokine production for tuning the chemokine system (S5). Chemokines are redundant in their action on target cells. Furthermore, a given leukocyte population usually has receptors for and responds to different molecules (Table 3). For example, macrophages respond to the widest range of chemokines

CLINICAL APPLICATIONS OF CYTOKINE ASSAYS TABLE 3 CHEMOKINES AND THEIR RECEPTORS ON RESPONDING CELLS Cell Type

Receptor

Chemokine

Activated T cell

CCR5 (Th1) CXCR3(Th1) CCR1 CCR2 CCR7 CX3CR1 CCR3 (Th2) CCR4 (Th2)

MIP-1α, β; RANTES IP-10, MIG, I-TAC MCP-3, 4; MIP-1α, RANTES MCP-1, 2, 3, 4, 5 MIP-3β, SLC Fractalkine MCP-3, 4; Eotaxin-1, 2; RANTES TARC, MDC

Basophil

CCR2 CCR3

MCP-1, 2, 3, 4, 5 MCP-3, 4; Eotaxin-1, 2; RANTES

B cell

CXCR5 CXCR4

BCA-1 SDF-1

Dendritic cell

CCR1 (Immature) CCR3 (Immature) CCR4 (Immature) CCR5 (Immature) CCR6 (Immature) CCR2 CXCR4(Mature) CCR7 (Mature)

MCP-3, 4; MIP-1α; RANTES MCP-3, MCP-4; Eotaxin-1, 2; RANTES TARC, MDC MIP-1 α, β; RANTES MIP-3 α MCP-1, 2, 3, 4, 5 SDF-1 MIP-3β, SLC

Eosinophil

CCR3 CCR1 CCR2

MCP-3, 4; Eotaxin-1, 2; RANTES MCP-3, 4; MIP-1α, RANTES, HCC-1 MCP-1, 2, 3, 4, 5

Monocyte

CCR1 CCR2 CCR4 CCR5 CCR8 CX3CR1 CXCR4

MCP-3, 4; MIP-1α; RANTES; HCC-1 MCP-1, 2, 3, 4, 5 TARC, MDC MIP-1α, β; RANTES I-309 Fractalkine SDF-1

Natural killer cell

CCR2 CCR5 CX3CR1 CXCR3

MCP-1, 2, 3, 4, 5 MIP-1α, β; RANTES Fractalkine IP-10, MIG, I-TAC

Neutrophil

CXCR1 CXCR2

IL-8, GCP-2 IL-8, GCP-2, GRO, ENA-78, NAP-2

Resting T cell

CCR7 XCR1 CXCR4

MIP-3β, SLC ymphotactin SDF-1

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including most CC chemokines, fractalkine (CX3C), and certain CXC molecules (e.g., stromal cell-derived factor-1) (S5). Probably all cell types can produce chemokines under appropriate conditions. Inducible and constitutive productions are two general modes of chemokine production. Inflammatory cytokines (e.g., IL-8, IP-10, MCP-1–4, and microphage inflammatory proteins, MIPs) are induced by immune and inflammatory stimuli and mediate recruitment on demand. Other chemokines are produced constitutively and play a role in regulating the basal trafficking of leukocytes (e.g., TARC, ELC, PARC, EBI-1-ligand chemokine, SDF-1). Some chemokines, such as MDC, are produced both in a tonic (e.g., thymus) and in an inducible way (M5). 1.2.2.2. CC Chemokines. The CC chemokine eotaxin, synthesized by airway epithelium, alveolar macrophages, bronchial smooth muscle, and eosinophils (R2, N1), is a potent and specific stimulator of eosinophil chemotaxis (G2). Eotaxin binds exclusively to CC chemokine receptor-3 (CCR3) on eosinophils to mediate proinflammatory activities such as chemotaxis, stimulation of respiratory burst and adhesion of eosinophils to endothelial cells, degranulation (F7), and upregulation of CD11b (R2). CCR3 is expressed in a high number of receptors per eosinophil, 4 × 104 to 4 × 105 (R2). CCR3 is also expressed on basophils, eosinophils, lymphocytes, and dendritic cells and in response to eotaxin, RANTES, MCP-2, MCP-3, and MCP-4. Besides eotaxin, several CC chemokines have been shown to induce eosinophil migration, including MCP-2, MCP-3, MCP-4, RANTES, and MIP-1α; many of these same chemokines act on basophils as well (N4). However, the most potent in vitro eosinophil chemoattractants are eotaxin-1 and -2, MCP-3 and -4, and RANTES. Although many of these chemokines have been shown to bind to several chemokine receptors, CCR3 is the receptor that mediates the majority of the eosinophil chemotactic effect (P9). Although eosinophil migration in response to eotaxin-1, eotaxin-2, MCP-4, and RANTES is mediated by CCR3, eosinophil migration in response to macrophage-derived chemokine (MDC) appears to involve a receptor other than CCR3 or CCR4 (B12). The selectivity of the CC chemokine subfamily for T cells, monocytes, eosinophils, and basophils has led to numerous studies of their roles in allergic inflammation. 1.2.3. Peptide Growth Factors Peptide growth factors are a group of cytokines with relevance to liver, and include transforming growth factor-β (TGF-β), hepatocyte growth factor (HGF), heparin-binding growth factors I and II (HBGF I and II), insulin-like growth factors I and II (IGF I and II), and platelet-derived growth factor (PDGF) (M3). Most of these peptides are mitogens for hepatocytes; IGF-1, HBGF-II, and PDGF are growth factors for hepatic stellate cells. Many peptide growth factors are produced by both parenchymal and nonparenchymal liver cells. The TNF-α and IL-6 that influence hepatic regeneration are belived to derive from Kupffer cells. TGFβ1, which is primarily a product of Kupffer cells and stellate cells, inhibits liver cell growth (M2).


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1.3. INTRACELLULAR SIGNAL TRANSDUCTION OF CYTOKINES Cytokines are known to signal by interactions with cell-surface receptors coupled to cytoplasmic components that transmit the signal. Over the past decade, intracellular signal transduction mechanisms mediated by cytokine have been studied extensively. Binding of cytokines to their respective receptors rapidly induces activation of multiple intracellular signaling pathways, including JAK– STAT, phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), Ras–Raf– extracellular signal-regulated kinases (ERKs), c-Jun NH2-terminal protein kinases (JNKs)/stress-activated protein kinases (SAPKs), and p38 mitogen-activated protein kinase (MAPK) signaling pathways (D8). Each pathway is further regulated by various inhibitory mechanisms to modulate the response and turn off the pathway in the absence of inducer. 1.3.1. Ras–Raf–MAPK Signaling Pathway MAPKs are serine and threonine kinases that can be activated by phosphorylation in kinase cascades. Three distinct MAPK pathways and more than 12 MAPKs have been identified in mammalian cells. MEK/MAPK kinase is the family of protein kinases upstream of the MAPK. MEK-1 specifically phosphorylates and activates the MAPK regulatory threonine and tyrosine residues present in the Thr–Glu–Tyr amino-acid motif. The p42/p44 MAPK (ERK) is activated by growth factors like IL-5 for the regulation of cell proliferation, transformation, and differentiation (D8). The p38 MAPK is activated by osmotic stress, ultraviolet (UV) irradiation, and proinflammatory cytokines including TNF-α (R1); and JNK/SAPK is potently activated by irradiation and other environmental stresses such as hyperosmolarity (D9). Both p38 MAPK and JNK are responsible for the regulation of apoptosis, stress response, and inflammation (H10). c-RAF-1 is one of many kinds of MAPK kinase kinase (MAPKKK), and RAS binding with GTP is upstream of RAF. The switch between RAS–GTP and RAS–GDP can activate downstream kinases such as MAPKKK (D6). A common intermediate pathway initiating from cytokine receptors is the Ras/Raf/MEK/ERK (MAPK) cascade, which can result in the phosphorylation and activation of additional downstream kinases and transcription factors such as p90Rsk, CREB, Elk, and Egr-1 (M10). We and other groups have found that IL-3, IL-5, and GM-CSF can activate the MAPK pathway in eosinophils and an eosinophilic leukemic cell line, the EoL-1 cells (M7, A3, B16, W20). 1.3.2. JAK–STAT Signaling Pathway JAK is a family of cytoplasmic tyrosine kinases that are associated with cytokine receptors and play a major role in the initial steps of cytokine signaling. Upon ligand binding, JAKs are activated by trans-phosphorylation of two receptor-bound JAK molecules and subsequently phosphorylate a number of substrates including the cytokine receptor (D3, S3, W8). The phosphorylated receptor

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Chemokine receptor Cytokine receptor α

Lyn

Gαβγ

JAK2

PI3K Gβγ

Gα

βc

PKB RAS

PLC PIP2

STAT1α

RAF-1

DAG

IP3

PKC

[Ca2+]i

MAPKK MAPK Survival

Regulation of gene transcription growth differentiation apoptosis

nucleus FIG. 1. Major chemokine and IL-3/IL-5/GM-CSF-mediated intracellular signal transduction pathways in granulocytes.

then provides docking sites for a variety of Src homology 2 (SH-2)-domaincontaining proteins, including a novel family of cytoplasmic transcription factors, STATs. STATs are then phosphorylated on a single tyrosine residue by the JAKs, after which the STATs dimerize, migrate into the nucleus, and regulate gene transcription. This pathway is triggered by both cytokines and interferons, and it rapidly allows the transduction of an extracellular signal into the nucleus (L9). There is a general consensus that the activation of IL-3/IL-5/GM-CSF receptors results in the rapid activation of JAK2 (Fig. 1). When JAK2 is activated, it phosphorylates a number of tyrosine residues in the β chain of these receptors including Y577, Y612, Y695, and Y750 (D17, I1, V1, P4). These sites then form docking sites for STAT proteins (C11, W21). STAT5 seems to be the most predominant STAT activated by these receptors. After receptor β-chain phosphorylation, STAT5 binds to one or more tyrosine residues through its SH-2 domain and is phosphorylated by JAK2, after which it can dimerize, translocate to the nucleus, and regulate gene expression (C11, W21).


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The RAS–RAF–MAPK signaling pathway can cross-react with the JAK–STAT pathway in eosinophils (Fig. 1). It has been shown that MAPK interacts with the α subunit of IFN-α/β receptor and the activation of early-response genes by IFNs requires tyrosine phosphorylation of STAT. Therefore, MAPK can regulate IFN-α and IFN-β activation of early-response genes by modifying the JAK–STAT signaling cascade (D5). 1.3.3. PI3K and Akt/Protein Kinase B (PKB) The lipid kinase PI3K, which generates the signaling molecule phosphatidylinositol 3,4,5-triphosphate, is involved in the regulation of multiple cellular processes induced by cytokines (T7). IL-3/IL-5/GM-CSF rapidly activates PI3K in multiple cell types, an event which is dependent on tyrosine phosphorylation (C13) (Fig. 1). PKB is the downstream target of PI3K activation, for example, IL-3/ IL-5/GM-CSF activation in human granulocytes (C13). GM-CSF and IL-5 activate MAP kinases through the signaling pathway of JAK2–tyrosine-phosphorylated beta-chain-PI3K–Ras in eosinophils (H11). Th2 cytokines differentially activate PI3K and MAPK signal transduction pathways with distinct functional consequences, thereby showing complex regulation of eosinophil effector functions (C13). For example, IL-5-mediated chemotaxis in eosinophils is related to the activation of MAPK, PI3K, and a tyrosine kinase (P1). 1.3.4. The Nuclear Factor-κB (NF-κB) Pathway NF-κB is a protein transcription factor that functions to enhance the transcription of a variety of genes, including at least 27 different cytokines and growth factors, adhesion molecules, immunoreceptors, and acute-phase proteins (B10, Y2). Therefore, NF-κB plays key roles in regulating cytokine-mediated inflammation. NF-κB attaches to DNA in the promoter regions of target genes as a dimer composed of two Rel family proteins: p50 and RelA (p65). In the NF-κB heterodimer, both subunits contact DNA, but only RelA contains a transactivation domain in the C-terminal end of the protein, which activates transcription by direct interaction with the basal transcription apparatus (S4). Although NF-κB is classically defined as a p50/RelA heterodimer, other combinations of Rel proteins can function identically to NF-κB. In quiescent cells, NF-κB is sequestered in the cytoplasm through its interaction with the inhibitors IκB-α, IκB-β, or IκB-ε. TNF-α and GM-CSF can activate NF-κB in eosinophils for cytokine release (e.g., IL-8) (Y3). Binding of TNF-α, IL-1 β, or LPS at the cell surface signals phosphorylation and addition of ubiquitin to the inhibitory unit (IκB-α or p105). These events lead to recognition and proteolysis of the inhibitor, exposing the nuclear localization signal (NLS) on p50 and RelA. It allows NF-κB to be transported to the cell nucleus, where its dimers are free to bind to specific motifs in the promoter regions of cytokine genes and initiate transcription. Cytokine mRNA is then translated into proteins, which are secreted by cells. Nuclear binding of NF-κB also stimulates production

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of IκB-α and p105, which inhibits further activation of NF-κB by trapping NF-κB in the cytoplasmic compartment. As a result, it can lead to the termination of new cytokine transcription and hence suppression of the inflammatory response (G5). A panel of inflammatory factors such as IL-8 can be regulated at the transcriptional level by NF-κB (B3). Inhibition of NF-κB degradation can suppress the TNF-α-induced IL-8 release from eosinophils (Y3, F3). In animal studies, NF-κB-deficient p50 (−/−) mice were found to be defective in the production of IL-5 and eotaxin, and hence the eosinophilia in the asthmatic airway (Y4). However, the T cell priming and proliferation and the expression of the cell adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were not affected. These results demonstrated a crucial role for NF-κB in the pathogenesis of asthma (Y4). Therefore, activation and translocalization of NF-κB are the basis for increased expression of many inflammatory genes and for the maintenance of chronic airway inflammation (H7). Our unpublished data also demonstrated the important role of the activation of NF-κB for the induction of eotaxin synthesis in eosinophils. Figure 1 summarizes the major chemokine and IL-3/IL-5/GM-CSF-mediated intracellular signal transduction pathways in granulocytes.

2. Cytokines and Diseases 2.1. INTERLEUKINS AND DISEASES 2.1.1. Allergic Asthma Allergic asthma is a complex and heterogeneous disease characterized by intermittent reversible obstruction and chronic inflammation of the airways, bronchial hyperreactivity, and an infiltration of lymphocytes and eosinophils into the airway submucosa (Y8). A complex network of cytokines has been shown to regulate T and B cell growth, differentiation, and effector functions. The Th cytokines can be grouped into Th1 cytokines (e.g., IL-2, IL-12, IFN-γ , and TNF-α), which induce cell-mediated immunity, and Th2 cytokines (e.g., IL-4, IL-10), which stimulate IgE production (M6). It has been shown that the integration of Th cells, mast cells, and basophils plays an important role in bronchial asthma (M6). Allergeninduced IgE synthesis can trigger eosinophils, basophils, and mast cells to release cytokines for the differentiation of Th cells into Th2 cells to secrete IL-4, IL-5, IL-10, and IL-13. Moreover, basophils, mast cells, and eosinophils act as effectors of allergic inflammation through the release of proinflammatory, vasoactive, and fibrogenic factors (histamine, peptide leukotrienes, platelet-activating factor, tryptase, chymase, etc.) that are responsible for symptoms of bronchial asthma (M6). Th2 cytokines, including IL-4 (important for IgE synthesis) and IL-5 (important for eosinophil proliferation), are crucially involved in the local infiltration


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and activation of eosinophils (Y8, B18), whereas other Th2 cytokines, IL-10 and IL-13, are important in inducing airway hyperreactivity and allergic inflammation (K5). Th2 cytokine IL-10 is an antiinflammatory cytokine that suppresses the secretion of proinflammatory cytokines (D11), allergen-induced airway inflammation, and nonspecific airway responsiveness (T9). IL-13 shares a receptor component, signaling pathways, and many biological activities with IL-4. In fact, IL-13 is also an antiinflammatory cytokine and plays a unique role in the optimal induction and maintenance of IgE production and IgE-mediated allergic responses when IL-4 production is low or absent (D10, W12). Moreover, IL-13 or IL-4 shows a synergistic effect with TNF-α or IL-5 on eosinophil activation (L20). Recently, IL-11 was found to be involved in the chronic remodeling seen in asthmatic airways and is associated with increasing severity of the disease (M16). Our recent studies indicated that allergic asthmatic patients showed higher plasma IL-18, IL-12, IL-10, and IL-13 concentrations than normal control subjects (W16). In contrast, the percentage of IFN-γ -producing Th cells assessed by the FastImmune Cytokine System (Becton Dickinson, San Jose, CA) was significantly higher in normal control subjects than asthmatic patients, but the percentage of IL-4-producing Th cells did not differ (W16). Consequently, the Th1/Th2 cell ratio was significantly higher in normal subjects than in asthmatic patients. Based on these findings, allergic asthma is characterized by an elevation of both proinflammatory and Th2 cytokines. The significantly lower ratio of Th1/Th2 cells confirms a predominance of Th2 cell response in allergic asthma (W16). 2.1.2. Systemic Lupus Erythematosus (SLE) SLE is a systemic autoimmune disorder characterized by the activation of T and polyclonal B lymphocytes, production of autoantibodies, and formation of immune complexes causing tissue and organ damage (A9). Abnormal Th cytokines are involved in the pathogenesis of autoimmune diseases (H15). Recent reports indicated that peripheral blood mononuclear cells of SLE patients show decreased in vitro production of Th1 cytokines IL-2, IFN-γ (F8), TNF-α (H14), and IL-12 (H13, L14, L15) with upregulation of Th2 cytokine IL-4 (F8) and IL-10 (L14). Such an imbalance of Th cytokines may account for the polyclonal B cell activation observed in SLE. An earlier report suggested that there was no relationship between in vitro production of Th1 and Th2 cytokines and disease activity (B4), whereas other studies demonstrated that serum concentrations of Th1 cytokines IL-12 (T6), TNF-α (D4), and IFN-γ (A6) are significantly elevated in SLE patients. Our recent findings demonstrated that plasma concentration of proinflammatory IL-18 was significantly elevated in SLE patients compared to controls (W17, W19). IL-18 can enhance the Fas ligand expression in NK and CTL, causing Fas-mediated apoptosis in epithelial cells and tissue damage in SLE. In combination with other

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proinflammatory cytokines including IL-1 and TNF-α, IL-18 must be an important cytokine for initiating and exacerbating the catabolic and inflammatory responses in SLE (D12, D13). The elevation of IL-18 might therefore be crucial for organ damage (e.g., central nervous system, vascular and renal damage, etc.) through Fas:Fas ligand-mediated apoptosis in the exacerbation of SLE. Both the studies of Tokano et al. (T6) and ours have showed that plasma IL-12 concentrations in SLE patients were significantly higher than those of normal subjects. IL-12 is similar to IL-18 because both are synthesized by macrophages in response to infection for promoting the activation of cell-mediated immunity (D12). IL-12 has been shown to induce the production of IL-18 in primates (L4). The elevation of IL-12 might therefore induce the release of IL-18 so that both plasma IL-12 and IL-18 concentrations are simultaneously elevated in SLE patients (W17). The combination of IL-12 and IL-18 is very critical for the induction of innate immune response and inflammatory reaction (D14). Another cytokine Th1 cytokine, IL-17, was previously found to increase in RA and serve as a key activator of T celldriven inflammation for the pathogenesis of RA (C6). Our results also indicated the elevation of IL-17 in SLE patients (W17). Taken together, the increased production of IL-18, IL-12, and IL-17 should exert a synergistic effect for the increased production of a panel of proinflammatory cytokines including IFN-γ , IL-2, and GM-CSF in SLE. The significant elevation of the Th2 cytokine IL-4 in SLE patients confirmed its putative role in the activation of autoreactive B lymphocytes. Ratios of Th1 and Th2 cytokines can reflect cytokine homeostasis and indicate Th1 or Th2 predominance during the development of disease. Recent studies reported different results for the correlation of Th1/Th2 ratios and SLE disease activity. One study showed positive and significant correlation of ratios and SLE disease activity index (SLEDAI) using in vitro-stimulated peripheral blood mononuclear cells (PBMCs) (V3), whereas another showed a negative correlation of the ratio of IFN-γ /IL-10-secreting cells and disease activity by enzyme-linked immunospot analysis of freshly isolated PBMCs (H2). Animal experiments using autoimmune mice showed that the Th1/Th2 cytokine ratios of IL-2, IFN-γ and IL-4, IL-10 mRNA expression in polymorphonuclear neutrophils and PBMCs were inversely related to disease severity (Y9). We found a positive correlation between IL-18/IL-4 ratio and SLEDAI, suggesting an imbalanced cytokine profile, with Th1 predominance (W17). Akahoshi et al. (A4) recently reported that the ratio of IFN-γ /IL-4 determined by Th1 and Th2 cells counted by flow cytometry using intracellular cytokine staining also did not exhibit a predominance of Th2 in SLE. In contrast, they found that there was a significant predominance of Th1 among SLE patients with WHO class IV lupus nephritis, diffuse proliferative glomerulonephritis. This finding further supported that the IL-18/IL-4 ratio significantly correlated with the development of SLE and could play an important role in inflammation and tissue injury in the exacerbation of SLE.


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2.1.3. Heart Diseases A systemic inflammatory response following cardiopulmonary bypass (CPB) can contribute to the development of postoperative complications such as cardiac dysfunction (W2). Recent studies suggested that the release of several proinflammatory cytokines including TNF-α (G10), IL-6 (S2), and IL-8 (B15) may play a crucial role in inducing myocardial ischemia–reperfusion injury and whole-body inflammatory response following CPB (W2, W3). Several groups of investigators demonstrated that the myocardium is a major source of these three cytokines following ischemia and reperfusion (L11, M12, W1). The heart has been found to be a major source of these cytokines during reperfusion after longer duration of ischemia (O3, W4) or after acute myocardial infarction (W1). Our unpublished data indicate the induction of mRNAs such as those for IL-6 and IL-8 in the right atrial myocardium after clinical CPB. Moreover, the expression of “four and a half LIM-only protein 2” (FHL2), a heart-specific protein that is exclusively expressed in myofibers, was associated with the increased production of IL-6 and IL-8 mRNA in human myocardium. 2.1.4. Viral Infection Respiratory syncytial virus (RSV) infection is a major cause of bronchiolitis in infants, whereas influenza A infection usually manifests as an upper respiratory tract infection. The immunological responses of infants to RSV infection and influenza A infection are different. In our studies of the cytokine responses during these infections, we found that the serum concentrations of IL-4, IL-5, RANTES, and soluble intercellular adhesion molecule-1 (sICAM-1) in infants with RSV infection were significantly higher than those with influenza A infection (S8). The concentration of TNF-α in nasopharyngeal aspirates was significantly lower in infants with RSV infection. Therefore, a predominant T helper cell type 2 (Th2) cytokine and related immunological response was observed in infants with RSV infection, whereas a predominantly proinflammatory cytokine response was observed in infants with influenza A infection. This may explain the different clinical manifestations of the two viral infections in infants (S8). 2.1.5. Meningococcal Disease Meningococcal disease (MCD) is the commonest infective cause of death in children in Western societies. In MCD and all forms of sepsis, there is a complex interaction among cytokines and a wide variety of immune-system and non-immunesystem cells. Cytokines that have been shown to be involved in the pathogenesis of MCD include proinflammatory cytokines, TNF-α, IL-1β, IL-2, IL-6, IL-12, LIF, and IL-18; interferons, IFN-γ , IFN-α; chemokines, IL-8, RANTES, MIP1α,β, MCP-1, GRO-α; and colony-stimulating factors, G-CSF, GM-CSF, and M-CSF (H1).

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2.1.6. Type I Diabetes Mellitus Th1 proinflammatory cytokines such as IFN-γ , IL-1β, IL-12, and TNF-α released by macrophage and T lymphocytes in the vicinity of pancreatic beta cells have been implicated in the pathogenesis of type I (insulin-dependent) diabetes mellitus. Moreover, IL-18 serum levels are increased selectively during the early, subclinical stage of type I diabetes mellitus (N5). 2.2. CHEMOKINES AND DISEASES Chemokines have been shown to play an important role in the following types of diseases: allergic reaction, cardiovascular disease, neurological disorder, transplantation reactions, and viral infection. 2.2.1. Allergic Disease Chemokines have been implicated in diverse pathophysiological functions in allergic inflammation including chemoattraction, cellular activation, hematopoiesis, homeostatic role, and modulation of T cell immune response (R6). 2.2.1.1. Chemoattraction. The interaction between leukocytes in the circulation and endothelial cells lining blood vessels is a complex network of signaling events regulating trafficking of particular leukocyte subsets (B19). The interaction is mediated by a multistep process that involves (1) leukocyte rolling along the endothelial surface, (2) rapid activation of leukocyte integrins, (3) firm adhesion to endothelial ligands through activated integrins, and (4) transmigration of leukocytes through the endothelial layer. Chemokines are thought to have a central role in the modulation of this multistep process by (1) activating both the leukocyte and the endothelium and (2) increasing leukocyte integrin and adhesion molecule interaction affinity. 2.2.1.2. Cellular Activation. Chemokines are potent cell activators; after binding to the appropriate G protein-linked, seven-transmembrane spanning receptors, chemokines elicit transient intracellular calcium flux, actin polymerization, oxidative burst with release of superoxide free radicals, exocytosis of secondary granule constituents, and increased avidity of integrins for their adhesion molecules (D1, E2). 2.2.1.3. Hematopoiesis and Angiogenesis. Chemokines regulate hematopoiesis, angiogenesis, tumor immunity, fibrosis, cell survival/apoptosis, and embryonic development (N4). For example, eotaxin has been shown to stimulate directly the release of eosinophilic progenitor cells and mature eosinophils from the bone marrow (P2). It also functions as a GM-CSF following allergic challenge in the lungs (P5). Chemokines with angiogenic properties include IL-8, ENA-78, GRO, GCP-2, and NAP-2, but the detailed role of their angiogenic properties in allergic disease is unclear (N4).


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2.2.1.4. Homeostasis. Chemokines have an important role in baseline leukocyte trafficking during development, differentiation, and immune surveillance (L18), for example, B cell trafficking into follicles, the formation of germinal centers, and the structural organization of lymphoid tissues. This has important implications in allergic reactions because the responsible leukocytes (e.g., mast cells and eosinophils) reside primarily in affected tissue rather than hematopoietic organs. The eosinophil-selective chemokine eotaxin is constitutively expressed in a variety of tissues, especially mucosal tissues, where eosinophils normally reside (C15). This indicates the critical role of chemokines in establishing the location of allergic inflammation cells. 2.2.1.5. Modulation of T Cell Immune Responses. Characterization of chemokine receptors has shown that T lymphocytes display a dynamic expression pattern. This differential expression of receptors during maturation and differentiation of T-lymphocytes is thought to allow for individual chemokine specific functionality on T lymphocytes (R4). Upon activation, T cells may express an array of chemokine receptors including CCR1, CCR2, CCR5, CXCR1, and CXCR4. They thereby become sensitive to inflammatory chemokines including MIP-1α, MIP-1β, MCP-3, and RANTES, which are thought to mediate T cell trafficking to secondary lymph nodes and sites of inflammation (W7). Elevated plasma eotaxin has also been found in patients with acute asthma (L12, T1, Y1). Moreover, several CC chemokines contribute to bronchial eosinophilia in both atopic and nonatopic asthma (Y6). Chronic inflammatory diseases of the nose and sinuses, including nasal polyposis, allergic rhinitis, and both allergic and nonallergic sinusitis, are all characterized by an eosinophil-rich inflammatory infiltrate. Upregulation of eotaxin has been detected in all these disease states (B7, M17, P9). Eotaxin mRNA is markedly upregulated in the lesions of patients with inflammatory bowel disease and can explain the mechanism of eosinophil recruitment in diseases such as ulcerative colitis and Crohn’s disease (G2). In Hodgkin’s disease, the levels of eotaxin protein have been shown to correlate directly with the extent of tissue eosinophilia (T3). Thus, the evidence suggests that there is a potential role for the chemokine eotaxin in a variety of diseases that are characterized by a tissue eosinophilia. 2.2.2. Neurological Disorders Besides the well-established role of cytokines in the immune system, several recent reports demonstrated that chemokines also play a role in the central nervous system (CNS) (B2, M13). In the CNS, chemokines are constitutively expressed by microglial cells, astrocytes, and neurons, and their expression can be increased after induction with inflammatory mediators (M13). Chemokines are involved in brain development and in the maintenance of normal brain homeostasis, and play a role in the migration, differentiation, and proliferation of glial and neuronal

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cells (M14), as well as in neuroinflammation as mediators of leukocyte infiltration (B2, M13). Chemokines and their G protein-coupled receptors are constitutively expressed at low to negligible levels in various cell types in brain. Their expression is rapidly induced by various neuroinflammatory stimuli, thereby implicating them in different neurological disorders, such as trauma (S6), stroke (W6), Alzheimer’s disease (M13), and tumor progression (Y10), and in neuroimmune diseases such as multiple sclerosis (M19) and acquired immunodeficiency syndrome-associated dementia (L5). 2.2.3. Atherosclerotic Cardiovascular Disease The chemotaxis of mononuclear leukocytes and the migration, growth, and activation of the multiple cell types within atherosclerotic lesions are critical for the chronic inflammatory and fibroproliferative response in atherosclerosis (M1). Chemokine-mediated attraction of leukocytes to tissues has been shown in atherosclerotic lesions (G8). Studies using knockout and transgenic murine models indicated that chemokine receptor/ligand interactions are crucial in the development of atherosclerosis (P6). Moreover, chemokines may also interfere with smooth muscle cell migration and growth, and platelet activation and other well-defined features of the atherosclerotic process (A2). 2.2.4. Transplantation Chemokines are important mediators in allograft rejection by virtue of their activity as regulators of leukocyte movement, adhesion, and effector function (N2). Ischemic injury enhanced by reperfusion of the allograft following transplantation is associated with microvascular stress and chemokine-mediated trafficking of leukocytes into microvascular spaces (A8). Chemokines have additional biological activities that are also relevant to diverse processes that contribute to allograft rejection. The chemokine IL-8, ENA-78, and growth-regulated oncogene-alpha (GRO-α) act as angiogenic agents, whereas other chemokines, platelet factor 4 (PF4), IFN-inducible protein-10 (IP-10), monokine induced by IFN-gamma (Mig), and stromal-derived factor-1 (SDF-1), are angiostatic factors (H4). Thus, during chronic rejection, the expression of chemokines could influence the microvasculature within the allograft.

3. Cytokine Assays As with the measurement of other analytes, cytokine assay methods changed from the original bioassays to immunoassays, flow cytometric analysis, and microarray technology.


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3.1. BIOASSAYS The first types of bioassays evaluated the effects of cytokines on whole living animals by assessing endpoints including fever and altered host resistance (H16). At present, most cytokine bioassays are performed using long-term (i.e., immortalizd) monoclonal cell lines for the evaluation of the effects of cytokines (M18). In recent years, molecular biology techniques have facilitated the insertion of cytokine receptors or reporter genes into cells (C1), allowing the custom design of target cell lines with enhanced specificity or greater ease of performance. The advantages of bioassays include (1) detecting only functional molecules, (2) often offering higher sensitivity than immunoassays, and (3) enabling the description of the function of novel molecules. On the other hand, there are many drawbacks of bioassays. First, there is poor specificity due to the pleiotropic and redundant actions of cytokines. Second, there is inherent variability with the use of living organisms, ex vivo tissue sections, or isolated cells or cell lines. Third, cytokine bioassays are time-consuming and labor-intensive. Four, most data from cell line assays form sigmoid curves, making interpretation difficult. Finally, there may be a loss of cell line sensitivity and reactivity to cytokines during cell culture (H16). There are four major types of in vitro cytokine bioassay, according to the reaction elicited by the cytokine on the reporter cell. 3.1.1. Cell Proliferation Proliferative assays may be grouped into four basic categories: survival [e.g., IL-2 assay (G4)], mitogenesis [e.g., GM-CSF assay using TF-1 cell lines (K4)], comitogenesis [e.g., thymocyte assay for IL-1 (G3)], and colony formation [e.g., assay of hematopoietic cytokines (M20)]. 3.1.2. Inhibition of Cell Growth This can be characterized as direct cytotoxicity to the reporter cells [e.g., TNF assay (W15)]. Certain cytokines have also been shown to exert an antiproliferative effect on other cells in a dose-dependent manner [e.g., TGF-β assay (Y5)]. 3.1.3. Modulation of Direct Function or Activity The most relevant bioassays measure specific activities on immune function, either as effector or regulatory, for example, assay of cytokine IL-12 by the augmentation of the cytolytic function of natural killer cells (B9), IL-10 by the inhibition of the production of TNF-α from stimulated monocytes (B13), and IL-8 and eotaxin by the chemotaxis of neutrophils and eosinophils, respectively (C2). 3.1.4. Modulation of Indirect Function Other bioassays have been developed for measurement of indirect function (T5). For example, the IL-4 bioassay evaluates changes in the expression of cell surface

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molecules and the IL-5 bioassay quantifies enhancement of antibody secretion by B cells. 3.2. IMMUNOASSAYS Immunoassays utilize the specific reaction between an antigen and an antibody and is applicable to any molecule that can elicit an antibody response. 3.2.1. Sandwich Assay (ELISA) Recently, many kinds of solid-phase enzyme-linked immunosorbent assay (ELISA) have been developed for measuring cytokine levels in cell culture supernate, serum, or plasma. These assays employ the quantitative sandwich enzyme immunoassay technique. Usually, a monoclonal antibody specific for the cytokine is precoated onto a microplate. Standards and samples are pipetted into the wells and the cytokine present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for cytokine is added to the wells. Following a washing step to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color or fluorescence develops in proportion to the cytokine bound in the initial step. The color or fluorescence development is stopped and its intensity measured by a microplate reader (C8). Many manufacturers (e.g., Amersham Pharmacia Biotech Inc., Piscataway, NJ; Biosource Europe, Nivelles, Belgium; R & D Systems, Minneapolis, MN; MBL Co., Nagoya, Japan; Chemicon Int., Temecula, CA; Endogen Inc., Woburn, MA; BD Pharmingen, San Diego, CA) market ELISA kits for a wide range of different cytokines with sensitivity at the picogram per milliliter level. 3.2.2. Competitive Enzyme Immunoassay (EIA) The enzyme immunoassay (EIA) applies a single antibody to measure small molecules. The assay works on the principle that two antigens, enzyme-labeled and unlabeled analytes, compete for binding to the limited number of binding sites on the primary antibody, which is subsequently bound to the immobilized antiIgG. The amount of labeled antigen bound is inversely proportional to the amount of unlabeled antigen (e.g., a cytokine) present in the sample (S7). 3.3. ENZYME-LINKED IMMUNOSPOT (ELISPOT) The ELISpot assay has been adapted for the detection of individual cells secreting specific cytokines or other antigens. ELISpot assays employ the quantitative sandwich ELISA technique. A monoclonal antibody specific for the cytokine is precoated onto a microplate. Cells are pipetted into the wells of the microplates. During the incubation period, the immobilized antibody (in the immediate vicinity


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of the secreting cells) binds secreted cytokine. After washing away any cells and unbound substances, a biotinylated polyclonal antibody specific for the cytokine is added the wells. Following a wash to remove any unbound biotinylated antibody, alkaline phosphatase conjugated to streptavidin is added. Unbound enzyme is removed by washing and substrate solution is added. A blue-black color spot at the site of cytokine localization can be formed and counted by the ELISpot reader system (G9). The ELISpot kit for cytokine assay is commercially available from R & D Systems. 3.4. FLOW-CYTOMETRIC ANALYSIS 3.4.1. Analysis of Intracellular Cytokines Measurement of cytokines (e.g., IFN-γ and IL-4) in the cytoplasm of cells (e.g., peripheral CD4+ Th cells) can be performed using flow cytometry (Becton Dickinson) (W16). Briefly, aliquots of heparinized whole blood are stimulated with phorbal myristate acetate (PMA) and ionomycin in the presence of brefeldin A. Brefeldin A is used to increase the sensitivity of cytokine detection through its inhibitory effect on protein secretion by interference with the function of the Golgi apparatus. Activated T cells are confirmed with the expression of activation marker CD 69 using PE-conjugated CD69 specific monoclonal antibody. Activated cells are then stained with peridinin chlorophyll protein (PerCP)-conjugated, CD4specific monoclonal antibody and treated with a lysing solution. Flourescenceactivated cell sorter (FACS) permeabilizing solution is then added and cells are stained with fluorescein isothiocyanate (FITC) and PE-conjugated, anti-cytokinespecific monoclonal antibodies (mAbs) (e.g., anti-IFN-γ and IL-4 antibodies). After washing, the percentages of various cytokine-producing T cells (e.g., % IFN-γ and % IL-4) can be analyzed using flow cytometry (Fig. 2). 3.4.2. Cytometric Bead Array (CBA) The CBA (BD Pharmingen) is a series of spectrally discrete, uniform-size microparticles that can be used simultaneously to detect different soluble analytes including cytokines in a single sample on the basis of different color using flow cytometry. This series of particles with discrete fluorescence intensities can simultaneously detect a panel of six human cytokines (IFN-γ , TNF-α, IL-2, IL-4, IL-5, and IL-10) (C9, C16) (Fig. 3; see color insert). This newly developed method can save more time, consume less sample, and achieve similar sensitivity (pg/ml) and reproducibility compared with ELISA. Moreover, the ability of the CBA to measure the concentrations of six cytokines from the same sample permits the calculation of cytokine ratios. Cytokine ratios are considered markers of various disease states such as asthma, atopy, and SLE (K3, A4). The relative balances

FIG. 2. Representative dot plots (PE anti-IL-4 vs. FITC anti-IFN-γ ) for the analysis of intracellular Th cytokines of CD4+ lymphocytes using the FastImmune Cytokine System (BD Pharmingen) by flow cytometry: (a) a control subject and (b) an allergic asthmatic patient. The numbers in the quadrants denote the percentages of Th1 and Th2 cells (W16). Reproduced with permission from C. K. Wong, C. Y. Ho, F. W. S. Ko, C. H. S. Chan, A. S. S. Ho, D. S. C. Hui, and C. W. K. Lam. Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-γ , IL-4, IL-10 and IL-13) in patients with allergic asthma. Clin. Exp. Immunol. 125: 177–183, Copyright Blackwell Science Ltd., 2001.


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FIG. 3. Standard curves of different Th cytokines determined by CBA (BD Pharmingen, CA, USA) with on a three-color Becton Dickinson FACSCaliburTM flow cytometer.

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between various cytokines is often considered more important than the absolute concentrations of cytokines in an individual. 3.5. MOLECULAR BIOLOGY METHODS 3.5.1. Quantitative Analysis of Cytokine Gene Expression Reverse transcription–polymerase chain reaction (RT–PCR) has been widely used for the detection of cytokine gene expression in clinical samples (W18, K5). However, conventional RT–PCR only offers a semiquantitative analysis. Recently, the Perkin-Elmer Corporation (Wellesley, MA) developed the TaqMan cytokine gene expression plate for real-time, in vitro quantitative evaluation of a panel of human cytokine gene expression using fluorescence detection. 3.5.2. Microarrays The Human Genome Project’s large-scale sequencing efforts have generated complete sequence data for thousands of genes (V2). An important step toward the elucidation of the roles of cytokines in various biological processes is defining cytokine gene expression profiles (I2), that is, comparing patterns of multiple gene expression in different tissues and developmental stages, in normal and disease states, or in specific in vitro conditions. The hybridization of entire cDNA populations to nucleic acid arrays, including glass microarrays or membrane arrays, has been adopted for high-throughput analysis of multiple gene expression. This technology has a wide range of applications, including (1) investigating normal biological and disease processes, for example, allergy and autoimmune diseases, (2) profiling differential gene expression, (3) discovering potential therapeutic and diagnostic drug targets for cytokine-related diseases, and (4) screening for very subtle changes in response to a particular therapeutic treatment of immunological disorders. For the membrane array of cytokine expression, the general procedures (Amersham Pharmacia Biotech; R & D Systems; SuperArray Inc., Bethesda, MD; Clontech Laboratories, Inc., Palo Alto, CA) include RNA extraction, reverse transcription into biotin- or radioisotope-labeled cDNA, hybridization with about 20 to several hundred different cDNA prespotted membranes, and signal detection using fluorescence or radioactive methods (L3). As an example, Fig. 4 and Table 4 show different chemokine genes upregulated in allergic asthmatic patients compared with normal controls, based on membrane array technology (SuperArray). cDNA microarrays are glass slides or other matrices upon which thousands or tens of thousands of genes are immobilized (NEN Life Science Products, Inc., Boston, MA; Affymetrix, Inc., Santa Clara, CA). After genes are hybridized with fluorophore-labeled cDNA samples of interest, high-resolution fluorescence techniques can be applied to perform rapid analysis of thousands of genes for gene


a

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b

FIG. 4. Expression profile of cytokine genes in human lymphocytes detected by cDNA expression array system. RNA was extracted from human peripheral blood lymphocytes from (a) normal control subjects and (b) allergic asthmatic patients. Total RNA was reverse-transcribed and labeled with biotin, and gene expressions were detected using the Human Chemokine Nonrad-GEArray Kit (SuperArray Inc., Bethesda MD, USA). Negative control genes: pUC18 DNA (1A, 2A); positive control genes: β-actin (3A, 4A) and GADPH (8B, 8C, 5A, 6A, 7A, 8A).

sequencing, mapping, expression, and polymorphism detection. Glass microarrays are rapidly becoming the technology of choice for cytokine research because of their density and flexibility (B17). Recently, differential expressions of cytokines, receptors, and other signaling molecules have been reported in polarized Th1- and Th2-cell subsets (H3). However, microarray methods may only offer a semiquantitative method compared with ELISA or CBA. TABLE 4 CHEMOKINE GENES UPREGULATED IN ALLERGIC ASTHMATIC PATIENTS

Location

Gene

Ratio of Gene Expression Levels (patient to control)

(1D) (1E) (3F) (3G) (5B) (5C) (6F) (6G)

Fractalkine IL-8 MIP-1α MIP-1β

1.11 2.06 1.23 1.86

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4. Clinical Application of Cytokine Assays 4.1. THERAPEUTIC APPLICATIONS Many cytokines have been implicated in the pathophysiology of inflammations in allergy, autoimmune diseases, and cardiopulmonary bypass surgery (W3, W16, W17). Therefore, the manipulation of cytokine synthesis or action can be a therapeutic strategy for treating inflammation and other cytokine-associated diseases. The antiinflammatory action of glucocorticoids inhibits the transcription of cytokine and chemokine mRNA, but it can also interfere with protective immune responses. Therefore, research and development for pharmaceutical agents that can interfere with the selective function of critical cytokines, their receptors, and receptor-mediated intracellular mechanisms are important (B5, B6). Table 5 shows many pharmaceutical approaches for interfering with cytokines and their receptors. They include the neutralization of cytokines (e.g., anti-IL-5 antibody) (K7) and the development of chemokine or cytokine receptor antagonists (e.g., Met-RANTES) (E3) and intracellular signal antagonists (U2). 4.1.1. Cytokine Antagonists/Inhibitors Recently, a variety of approaches including antibody neutralization experiments and gene targeting have shown nonredundant specific inhibition for selected cytokines (L6, T2, G7). For example, a double-blind, randomized, placebo-controlled trial using humanized monoclonal antibody to cytokine IL-5 (SB-240563) in patients with mild asthma showed that monoclonal antibody against IL-5 decreased blood eosinophils and sputum eosinophils, thereby offering considerable therapeutic potential for treating asthma and allergy (L6). In transplantation, an antagonist of IL-17 was found to inhibit acute but not chronic vascular rejection (T2). In allergic asthma, neutralization of chemokine MCP-1 has been shown to block the development of airway hyperresponsiveness (G7). TABLE 5 PHARMACEUTICAL APPROACHES TO INTERFERING WITH CYTOKINES AND THEIR RECEPTORS Target Molecule

Antagonist/Inhibitor

Cytokines

Corticosteroid MAPK inhibitors Humanized antibodies Antisense oligonucleotides Soluble receptors

Cytokine receptors

Receptor antagonist Humanized antibodies Antisense oligonucleotides


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4.1.2. Cytokine Receptor Antagonists/Inhibitors Receptors CCR3 and IL-5α are both expressed only on eosinophils, basophils, and certain Th2 cells (H8, S1, U1), but not on neutrophils (H12). A monoclonal antibody, 7B11, to human CCR3 blocks the chemotactic response of human eosinophils to all chemokines (H8). The CC chemokine antagonist Met-RANTES has been shown to inhibit effectively eosinophil effector functions following stimulation with RANTES, MCP-3, and eotaxin through the chemokine receptors CCR1 and CCR3 (E3). Moreover, Met-RANTES antagonizes eosinophil but not neutrophil effector functions; therefore, it is a promising agent for the treatment of eosinophil infiltration such as allergic asthma (E3, E4). Recently, two series of peptides that specifically bind to the extracellular domain of the IL-5α were identified (E6). These peptide-receptor antagonists could also provide a new modality for the inhibition of IL-5 activation on eosinophils for treatment of allergic inflammation. Chemokine blockade is also a potentially novel approach to therapy of inflammatory renal disease (R7). In laboratory and animal studies, the inhibition of proinflammatory IL-1 action by inhibitors, antibodies, or receptor antagonists has proven beneficial to the treatment of various diseases such as RA (D7), cancer, inflammation, and Alzheimer’s disease (C12). A recombinant IL-13 variant, IL-13E13K, was found to be a useful antagonist for the treatment of allergic, inflammatory, and parasitic diseases or malignancies in which IL-13 plays a central role (O2).

4.1.3. Intracellular Signal Transduction Molecules as Potential Therapeutic Targets As in the foregoing description, the action or production of cytokines is mediated through a number of signal transduction pathways, which have been elucidated recently (Y7). These include pathways integrating the activation of extracellular receptors and subsequent intracellular events leading to cytokine action and gene expression. Many inhibitors have been found to interfere with signal transduction and cytokine expression (Table 6). Most of the signal transduction inhibitors in development are aimed at inhibiting or suppressing components of the abnormal cytokine response, but in the future, there are possibilities for development of preventative and curative treatments for various cytokine-related immune diseases. Transcription factor NF-κB and activator protein-1 (AP-1) (L10) are also important in the orchestration of asthmatic inflammation. This has prompted the search for specific blockers of transcription factors such as NF-κB. The proteasome inhibitor N-cbz-Leu-Leu-leucinal, MG-132, a specific inhibitor for NF-κB activation (F3), has been found to suppress chemokine IL-8 release from epithelial cells. However, its use for treating allergic inflammation requires further investigation (Table 6).

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WONG AND LAM TABLE 6 COMPOUNDS THAT AFFECT SIGNAL TRANSDUCTION OF CYTOKINE ACTION OR PRODUCTION

Molecular Target Kinases p38 MAPK MEK

Compound

SB 203580, SB 202190 PD 98059, U0126

Mode of Action

Inhibit kinase Noncompetitive inhibitor of kinase

Cytokine Affected

IL-1, TNFα, GM-CSF, IL-6, IL-8 IL-2, TNFα, GM-CSF, IFNγ , IL-6, IL-4, IL-5, IL-13, IL-8, TGFα, FGF, EGF Multiple growth factors and cytokines

Possible Therapeutic Use

Antiinflammatory, e.g. RA (B1, C17) Cancer metasis, ischemia, antiinflammatory (F1, A5)

PI3 kinase

Wortmannin, LY 294002

Inhibit kinase

JAK2

Tryphostin, AG-490 CP-118556

Inhibits kinase Inhibits kinase

Ac-YVAD-CHO

Inhibits activity

IL-1β

Antiinflammatory (N3)

MG-132, gliotoxin

Inhibits the degradation of IκBα

TNFα, IL-1β, IL-2, IL-6, IL-12, IL-8, MIP-1, MCP-1, RANTES, C-CSF, GM-CSF, IFN-β

Antiinflammatory (B10), anticancer (Y2)

Src Proteases Caspases Transcription factor NF-κB

Multiple growth factors and cytokines Multiple growth factors and cytokines

Inhibit neutrophil (T4) and eosinophil function (B16) Anticancer (M15, A10) Anticancer (H5)

4.1.4. Cytokine Gene Therapy Gene therapy as a drug delivery system offers many advantages over current protein delivery systems. These include its ability to (1) target therapies to individual tissues or cell types, (2) locally produce proteins that can act intracellularly or in an autocrine, juxtacrine, or paracrine fashion, and (3) sustain new protein synthesis for periods up to several weeks after a single administration. Cytokine gene therapy has been evaluated for treating cancer in many clinical trials (P7). Recently, gene therapy has also offered a novel approach for the treatment of recurrent acute inflammation (M21). For example, the administration of TGF-β1 in several autoimmune diseases by somatic gene therapy approach can result in depressed inflammatory cytokine production and increased endogenous regulatory cytokine production (P10). The application of transgene-based modalities, including antisense oligonucleotide technology and gene therapy, has also been developed as a novel therapeutic strategy in the treatment of allergic asthma (A7).


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4.2. DIAGNOSTIC APPLICATIONS The expression of many cytokines has been found to correlate with disease exacerbation and severity. Therefore, the measurement of cytokine concentration may be used as a predictive parameter or disease marker for monitoring disease activity and therapeutic efficacies. 4.2.1. Allergy In food allergy, serum IL-5 was found to be elevated in infants with anaphylaxis to cow’s milk at 1 week, but became undetectable after 2 weeks on a milk-free diet (M9). It seems likely that the allergic inflammation due to cow’s milk or other food allergens can induce marked eosinophilia with an associated increase in IL-5 production. Therefore, IL-5 may serve as a marker for food allergy. Other studies have also shown that IL-5 expression correlates significantly with eosinophilia and allergic symptoms of allergic rhinitis in patients with hay fever (W13). 4.2.2. Autoimmune Diseases In the study of SLE patients, we and other authors have found that there is a correlation between SLE disease activity index (SLEDAI) and the production of IL-16 (L7) and IL-18 (W19). It was suggested that IL-16 and IL-18 may be a useful indicator of disease activity of SLE. Plasma MCP-1 concentration has also been proposed as a marker for monitoring joint inflammation in rheumatoid arthritis (E1). 4.2.3. Transplantation A recent report showed that the expression of IL-17 is closely related to the degree of the acute rejection of a renal allograft (L16). Therefore, IL-17 may be involved in the alloimmune response and serve as an early marker of acute rejection (L16). 4.2.4. Viral Infection Active infection with several viruses has been shown to reduce the ability of T cells to be activated (C3). Activated T cells (1 × 106 ) normally produced 4–8 pg/ml per hr of GM-CSF after 20 hr of activation; impaired T function resulted in a decrease to the 0.2 to 2.0 pg/ml per hour range of GM-CSF. Therefore, GM-CSF produced from T cells may be used as a marker for the suppression of T cells activation in viral infection (C14).

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5. Conclusions Cytokine research is a rapidly expanding field of immunology, cell biology, and allergy research. The explosion of knowledge in the immunobiology of cytokines can help us to identify novel cytokines, evaluate the detailed roles of cytokines in the pathogenesis of many diseases, and elucidate the detailed intracellular signal transduction mechanisms of different cytokines. The results of these studies will provide a solid biochemical basis for designing new therapeutic approaches for many immunological disorders and for developing new predictive parameters and diagnostic markers for monitoring disease severity and therapeutic efficacies. For instance, research on intracellular signaling molecules will provide interesting targets for the development of putative specific drugs for interfering with the regulatory pathways of inflammation. Achieving therapeutic specificity of inactivating certain cytokines could permit the effective resolution of disease including inflammation without causing any side effect. Strategies for the development of promising therapeutic agents require more fundamental research on the signal transduction pathways to decipher the precise underlying regulatory mechanisms of different cytokines on different target cells. In view of the development of more sensitive, accurate, and specific as well as high-throughput cytokine assay methods such as microarray and CBA methods, and the recent expanding knowledge of pathophysiological mechanisms of different cytokines in various diseases, we can expect a breakthrough in the treatment of cytokine-related diseases in the near future. REFERENCES A1. Aarvak, T., Chabaud, M., Miossec, P., and Natvig, J. B., IL-17 is produced by some proinflammatory Th1/Th0 cells but not by Th2 cells. J. Immunol. 162, 1246–1251 (1999). A2. Abi-Younes, S., Sauty, A., Mach, F., Sukhova, G. K., Libby, P., and Luster, A. D., The stromal cell-derived factor-1 chemokine is a potent platelet agonist highly expressed in atherosclerotic plaques. Circ. Res. 86, 131–138 (2000). A3. Adachi, T., and Alam, R., The mechanism of IL-5 signal transduction. Am. J. Physiol. 275, C623–C633 (1998). A4. Akahoshi, M., Nakashima, H., Tanaka, Y., Kohsaka, T., Nagano, S., Ohgami, E., Arinobu, Y., Yamaoka, K., Niiro, H., Shinozaki, M., Hirakata, H., Horiuchi, T., Otsuka, T., and Niho, Y., Th1/Th2 balance of peripheral T helper cells in systemic lupus erythematosus. Arthritis Rheum. 42, 1644–1648 (1999). A5. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R., PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270, 27489–27494 (1995). A6. Al-Janadi, M., Al-Balla, S., Al-Dalaan, A., and Raziuddin, S., Cytokine profile in systemic lupus erythematosus, rheumatoid arthritis and other rheumatic disease. J. Clin. Immunol. 13, 58–67 (1993). A7. Alvarez, D., Wiley, R. E., and Jordana, M., Cytokine therapeutics for asthma: An appraisal of current evidence and future prospects. Curr. Pharmaceut. Des. 7, 1059–1081 (2001). A8. Ambrosio, G., and Tritto, I., Reperfusion injury: Experimental evidence and clinical implications. Am. Heart J. 138, S69–S75 (1999).


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C15.

C16.

C17.

D1.

D2.

D3. D4.

D5.

D6. D7. D8.

D9.

D10. D11.

D12. D13. D14. D15.

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CURRENT CONCEPTS ON DIAGNOSIS AND TREATMENT OF ACUTE PANCREATITIS B. Kusnierz-Cabala,∗ B. Kedra,† and M. Sierzega† ∗ Department of Clinical Biochemistry, Collegium, Medicum Jagiellonian University, Krakow, Poland † First Department of General and Gastrointestinal Surgery, Collegium Medicum Jagiellonian University, Krakow, Poland

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Pathophysiology of Acute Pancreatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Diagnosis of Acute Pancreatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Biochemical Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Clinical Findings and Imaging Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Prognostic Scores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

47 49 51 51 55 56 57 67 70 70

1. Introduction The first descriptions of acute pancreatitis (AP) date to the 16th and 17th centuries (Aubert in 1579, Bonetus in 1664, and Morgagni in 1691) (A9). Portal in 1811, Clässen in 1842, Klebs in 1876, Friedreich in 1878, and Senn in 1886 reported on morphological changes in hemorrhagic necrosis and sequestration of pancreatic parenchyma (F4, S11). In 1874 Zenker identified pancreatic hemorrhage as a cause of sudden death. In 1882 Balser described pancreatic steatonecrosis and considered it to be fatal. In 1889 Reginald Fitz provided the first systematic analysis of acute pancreatitis. He presented 53 cases with complete pathological and clinical data and based on these findings distinguished hemorrhagic from suppurative and gangrenous forms of the disease. Additionally, he identified some etiologic factors, such as gallstones, alcohol, perforating gastric ulcer, and trauma. Fitz pointed out significant 47 Copyright 2003, Elsevier Science (USA). All rights reserved. 0065-2423/03 $35.00

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factors in differential diagnosis, and described pancreatic abscess, splenic vein thrombosis, and pancreatic pseudocyst. He suggested a causal relationship between pancreatitis and disseminated fat necrosis and made an important observation that “an operation in the early stages of this disease is extremely hazardous.” Fitz laid the foundations of the systematic knowledge of the pathology, symptomatology, and treatment of acute pancreatitis and contributed greatly to the progress of investigations in this field at the turn of the century (L8). Fitz’s contributions to the subject of pancreatitis fed the research performed by other investigators. Opie found a gallstone impacted in the papilla of Vater on autopsies of patients who died of “pancreatic phlegmon.” Further study based on 100 autopsies revealed the presence of the common biliopancreatic channel in 89 cases. He published his results in 1901 showing the close causal relation between the gallstones impacted in Vater’s papilla and acute pancreatitis. Based on these findings, he postulated that bile reflux into the pancreatic duct, when the gallstone obstructed the opening of the common channel, induced acute pancreatitis (O2). Moreover, he claimed that patent accessory pancreatic duct (Santorini) modified the natural course of the disease, as has been confirmed in recent clinical trials (N9). In 1912 Archibald published his results of autopsies and questioned the hypothesis of Opie. He found bile duct stones in less than 50% of patients, a small number of whom were impacted in Vater’s papilla. Based on his surgical experience and pathological reports, he suggested sphincter of Oddi spasm as the etiologic factor in acute pancreatitis. This thesis was revised after the introduction of sphincter of Oddi manometry, which initiated studies on sphincter dysfunction in acute pancreatitis (G9, T8). Alcohol was firmly established as an important etiologic factor in 1917. The 19th century witnessed the beginning of studies on the pathophysiology of acute pancreatitis. In 1867 Küne isolated an enzyme catalyzing the cleavage of peptide bonds, which was later termed trypsin (1877), and a few years later Heidenhain found trypsin in pancreatic cells in the form of inactive proenzyme. Chiari, in 1883, was the first to introduce the concept of self-digestion of the pancreas in acute pancreatitis (AP). In 1895 Hildebrand indicated the role of pancreatic trypsin and lipase in developing necrosis. Elman et al. (E1), in 1929, demonstrated elevated serum amylase levels in AP. Further progress in the pathophysiology of AP came from experimental studies. Hallenbeck injected bile to the pancreatic duct, inducing edema and necrosis of the pancreas (1953). Beck demonstrated the significant role of other pancreatic enzymes (lipase, elastase, collagenase) in the development of pancreatic necrosis (1961). In the following years other factors predisposing to acute pancreatitis were identified including transient ischemia (Day in 1960 and Eisnhard in 1961), pancreatic secretion stimuli, and autonomic nervous system. The surgical treatment of AP was delineated by Mayo-Robson (M5) and Moynihan (M18). Moynihan worked out the principles of lesser sac debridement and drainage, which are still valid. In the 1960s Wall and Cray introduced peritoneal lavage, but the results were ambiguous. In the 1980s Hollender

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(1981) published his series of patients after pancreatic resection, and Bradley (1984), Knol (1984), Warshaw (1985), Pemberton (1986), and Wertheimer (1986) provided the first results of “open-abdomen” procedures, including necrosectomy, laparostomy, or temporary abdominal closure with planned reexplorations in the treatment of severe acute pancreatitis with extensive pancreatic and peripancreatic necrosis. Recent advances in molecular biology and clinical biochemistry allowed for better understanding of the processes involved in the pathogenesis of acute pancreatitis. Many attempts have been made to identify new diagnostic tests that could help in more accurate diagnosis and early prediction of severity. Moreover, several clinical trials applied novel treatment regimens to influence a specific pathway of the inflammatory process. The aim of this chapter is to review current concepts on diagnosis and treatment of acute pancreatitis.

2. Pathophysiology of Acute Pancreatitis The two most common causes of acute pancreatitis include alcohol intake and gallstone passage through the papilla of Vater. These two conditions are responsible for the majority (ca. 80%) of AP cases. About 10% of patients developed AP after exposure to toxins (scorpion venom, methyl alcohol, organophosphates) or drugs (azathioprine, mercaptopurine, valproic acid), as a result of trauma (with disruption of the ductal system) or endoscopic interventions (endoscopic retrograde cholangiopancreatography, direct sphincter of Oddi manometry), and after abdominal and thoracic surgery (especially with the use of cardiopulmonary bypass). Acute pancreatitis may be also induced by metabolic disorders (hypertriglyceridemia, hypercalcemia), infection (mumps, coxsackievirus, human immunodeficiency virus), and infestation (ascariasis). Recently, after the introduction of sphincter of Oddi direct endoscopic manometry, the concept of a hypertensive sphincter was proposed, at least in cases of acute recurrent pancreatitis (G9, T8). Although the relationship between the major etiologic factors and AP is established, the triggering mechanisms of the disease are unclear. There have been many theories regarding the inducing mechanism in the pathogenesis of AP. Opie suggested the common channel theory, which was apparently confirmed in his autopsy series. In most cases, however, the common channel is too short to harbor a gallstone, while leaving the junction of the common bile duct and pancreatic duct behind, with subsequent bile reflux to the pancreatic duct. The so-called regurgitation theory with a duodenal reflux to the pancreatic duct was questioned by the experimental data (S12, B15) and clinical practice (endoscopic and surgical sphincterotomy). A considerable body of evidence supports the thesis introduced by Lium et al. over 50 years ago that “acute pancreatitis is the result of ductal obstruction in an actively secreting pancreas” and that pancreatic ductal hypertension concomitant with excessive stimulation of the pancreas plays the pivotal

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role (C4). The prevailing etiologic concepts could not account for the mechanism of pancreatic injury and injury to extrapancreatic tissues and distant organs. In the 1960s and 1970s a new approach to the pathogenesis of acute pancreatitis was introduced. The concept of mediators released in the course of the disease entering the systemic circulation and causing dysfunction of distant organs resulted in new treatment strategies and led to the development of new operative techniques. Since that time many potent biological mediators have been discovered; however, their role has not been completely understood. The severity of the disease is largely determined by inflammatory mediators such as cytokines, oxygen radicals, proteolytic enzymes, lipids, and gaseous mediators. Recent development in cellular and molecular biology have increased our understanding of the triggering mechanisms and those responsible for the development of the severe forms of the disease. Substantial evidence from experimental studies has confirmed the role of microcirculatory derangements in the pathogenesis of AP. The intracellular events are still poorly understood. The colocalization of digestive zymogens containing proenzymes along the normal secretory pathway with lysosomal hydrolases capable of activating zymogens is considered to be a final common pathway regardless of the inciting factor. This leads to intrapancreatic, intraacinar trypsinogen activation, which overwhelms the local and systemic defense mechanisms (α1-antitrypsin and α2-macroglobulin) with subsequent autodigestion of the pancreas and peripancreatic tissues. The activation of elastase, complement, and kinins with release of bradykinin and kallidin and liberation of lipase and phospholipase A2 leads to digestion of elastic components of pancreatic blood vessels, vascular instability, fat necrosis, and remote-organ injury. Regardless of the inciting event, a local and systemic overproduction of cytokines is characteristic of the disease. Acinar cells of the pancreas are the primary source of cytokines after an initiating event. High levels of circulating cytokines cause macrophages to migrate into various distant tissues and to produce regulatory cytokines attracting immunocytes such as monocytes, neutrophils, and lymphocytes. They induce the release of large amounts of damaging cytokines along with free radicals and vasoactive substances (e.g., nitric oxide). The net cumulative effect is hypovolemia, vascular leakage, adult respiratory distress syndrome (ARDS), and, finally, multiple-organ failure (MOF). Cytokines represent a diverse group of low-molecular-weight proteins. They are the means of cell-to-cell communication and share important characteristics: they act on many different target cells, exhibit redundancy (many cytokines may induce the same biological effect), and have a tendency for positive feedback with amplification and cascades of inflammatory agents. They act in extremely small concentrations and through highly specific cell surface receptors (N6). Interleukin 1 β (IL-1) and tumor necrosis factor α (TNF-α) hold the central position in most local and systemic manifestations of the disease; they are responsible

ACUTE PANCREATITIS

51

for almost all detrimental consequences of sepsis (fever, hypoperfusion, shock, metabolic acidosis, cardiac dysfunction, disseminated intravascular coagulation). They are the primary inducers of (IL-6) and (IL-8), which are in turn reliable measures of disease severity. Numerous interactions exist among (IL-1), TNF and oxygen radicals, elastase, interferons α and γ , as well as (IL-2) and (IL-10). Another important mediator in acute pancreatitis is platelet-activating factor (PAF), which is not a cytokine, but a potent vasodilator and leukocyte activator implicated in the development of systemic inflammatory response syndrome (SIRS). The production of PAF is closely tied to the production of IL-1 and TNF. Inhibition of PAF production attenuates IL-1 and TNF production (N6).

3. Diagnosis of Acute Pancreatitis 3.1. BIOCHEMICAL TESTS 3.1.1. Laboratory Tests Used in the Diagnosis and Differentiation of Acute Pancreatitis (AP) From Other Acute Abdominal Diseases 3.1.1.1. α-Amylase (EC 3.2.1.1) 3.1.1.1.1. Amylase in serum. An at least fivefold increase in this enzyme activity is accepted for the diagnosis of AP (some authors use an increase by 10–15 times). This assay can be considered a sensitive diagnostic method only when the patient is hospitalized within a few hours from the first sensations of pain. This is linked to a clear increase in the enzyme activity as early as 2 or 3 hr from the first sensations of pain, reaching a excretory maximum in the first 24 hr and a quick normalization in the following 3 or 4 days. An increase in the enzyme activity lasting for nearly 10 days usually indicates complications in the course of AP (pseudocysts, pancreas necrosis) (B5, B10, R4). When assaying, it must be remembered that amylase activity can be within reference ranges despite increasing symptoms of AP. This is usually the case when the enzyme activity measurement was performed a few days after the actual onset of the disease, the pancreas has been nearly completely damaged, or the test was performed in serum with a significantly increased level of triglycerides. During amylase determination it should be borne in mind that an extrapancreatic increase in the enzyme activity is possible, which is connected with stomach or duodenum ulcer perforation, salivary gland infection, acute renal insufficiency, abdominal injuries, or intestinal obstruction (B10). A similar diagnostic sensitivity is typical of an increased activity of amylase in urine, which is usually diagnosed earlier and lasts for a longer period of time than in serum (S4). An increase in urinary amylase in AP is mainly attributed to the inhibition of tubular resorption by numerous low-molecular-weight proteins and peptides released from the pancreas altered during infection. Due to the low test specificity, the diagnostic value of determining the ratio of amylase clearance to

52


creatinine clearance (Cam /Ccr ), which usually amounts to 1–4% and can increase in AP up to 15%, is disputable (B10, J2). 3.1.1.1.2. Pancreatic isozymes. In AP there is a significant increase in molecular forms P2 (approximately 91% of patients) and P3 (approximately 98% of patients) (P2, W5) as well as the presence of the isozyme P4, which does not appear in healthy controls (P2, N2). In the presence of a cyst, the P4 and P5 isozyme fractions are increased to >15% of total amylase activity. In the absence of a cyst, the P4 and P5 fractions make up 2 for alcoholic AP and L/A < 2 for the biliary etiology of AP (sensitivity 84%, specificity 89%), whereas Tenner and Steinberg (T5, S20) and Kazmierczak et al. (K4) suggested significant L/A ratios of 4.0 and 4.2, respectively (Table 2). 3.1.1.3. Additional Tests Used in AP Diagnosis. Carboxylesterase may become a useful test for pancreatitis, although a recent study has shown the test to have a questionable value (A2). The applicability of carboxypeptidase A (EC 3.4.17.1) (B19), DNase (deoxyribonuclease I, EC 3.1.21.1) (M14), and lactoferrin is still being investigated; for the time being there are insufficient data to judge their worth for the diagnosis of pancreatitis (L15). Another useful enzyme in AP diagnosis is elastase 1 (pancreatic elastase, EC 3.4.21.36), which increases in acute pancreatitis and relapsing chronic pancreatitis. Increases exceed those observed for serum amylase, they persist longer, and values correlate better with the clinical symptoms (T6). TABLE 2 BIOCHEMICAL PARAMETERS IN THE EARLY DIFFERENTIATION BETWEEN NONALCOHOLIC a AND ALCOHOLIC PANCREATITIS

Marker Amylase Lipase L/A ratio ALT AST ALP MCV Urine amylase IL-6 CRP

Cutoff

Sensitivity (%)

Specificity (%)

PPV

NPV

Diagnostic Accuracy

Ref.

450 U/L 1150 U/L 4000 U/L 2 4.2 70 U/L 84 U/L 60 U/L 110 U/L 100 U/L 96 fl 3000 U/L 2.7 pg/ml 11 mg/L

81 52 23 84 96 84 91 81 91 84 66 62.5 87.5 6.3

75 87 97 89 57 69 83 59 80 50 81 74 83 91.7

0.43 — — 0.66 — 0.39 — 0.32 — 0.28 0.46 0.36 — —

0.94 — — 0.96 — 0.95 — 0.93 — 0.93 0.92 0.89 — —

0.76 0.72 0.65 0.88 0.74 0.72 0.87 0.63 0.85 0.56 0.78 0.72 0.85 0.57

S22 K4 K4 S22 K4 S22 K4 S22 K4 S22 S22 S22 P7 P7

a ALT, . . .; AST, aspartate aminotransferase; ALP, alkaline phosphatase; MCV, . . .; (IL-6), interleukin-6; CRP, C-reactive protein. PPV, NPV, as in Table 1.

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Much interest has been given to the determination of both trypsin (EC 3.4.21.4) and its zymogen—trypsinogen—which has two forms that differ in their electrophoretic mobility and isoelectric points; the anionic form is trypsinogen-2 and the cationic form is tripsinogen-1 (G1, K8, L15). After an attack of pancreatitis, serum immunoreactive trypsin increases in parallel with serum amylase activity to values up to 400 times the upper reference limit (T3). Kimland et al. (K8) recognized that in pancreatitis there was a proportionally higher increase in trypsinogen-2 than of trypsinogen-1, and that trypsinogen-2 concentrations remained elevated longer than those of either trypsinogen-1 or amylase (K8). Shortly thereafter Itkonen et al. (16) confirmed that in pancreatitis, trypsinogen-2 concentrations were elevated up to 50-fold compared with healthy controls, whereas trypsinogen-1 concentrations were elevated only 10- to 15-fold (16). As a test sample, urine has the advantage of being easily available and suitable for rapid determinations (H5). Hedström et al. (H5) showed that trypsinogen-2 in urine is a marker with high accuracy for diagnosis (sensitivity of 86–93%, specificity of 90– 95%), but lower accuracy for the evaluation of AP severity (sensitivity of 26–68%, specificity of 80–90%). The sensitivity and specificity of urinary trypsinogen-2 are superior to those of urinary amylase, serum amylase, and pancreatic amylase (G1) (Table 1). 3.1.2. Tests Necessary for the Assessment of Patient’s Clinical Condition 3.1.2.1. White Cell Count. AP is most often accompanied by an increase in leukocytosis up to (10–20) × 103/mm3 with a shift to the left in the white cell image (>80% cells are granulocytes). 3.1.2.2. Hematocrit. In the beginning stage an increase in hematocrit value even to over 55% is observed (flow of fluid out of the placenta). A decrease in the hematocrit value by approximately 3% is linked to a decrease in hemoglobin concentration by 1 g/dl. 3.1.2.3. Glucose. In the course of the disease, disturbances in the carbohydrate system with sudden states of hypoglycemia and hyperglycemia occur. 3.1.2.4. Bilirubin. An increase in the bilirubin level and an accompanying increase in aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities most often result from obstructive jaundice developed as a consequence of pancreas head edema and pressure exerted on the papilla of Vater (cholestasis). 3.1.2.5. Electrolytes. The development of hypocalcemia and hypomagnesemia among approximately 25% of patients is observed. 3.1.2.6. Albumin. A significant decrease in the albumin level is characteristic of a septic shock, which is a frequent AP complication. A decrease in the oncotic blood pressure, a pancreatic circulation deficiency, as well as water overaccumulation in lungs and respiratory insufficiency (ARDS) are observed.

ACUTE PANCREATITIS

55

3.1.2.7. Coagulation. In the beginning stage of the disease, a minor time elongation (prothrombin time, PT; thrombin time; TT; activate partial thromboplastin time, APTT) and a decrease in platelet count are observed. In the later stage of the disease, disseminated intravascular coagulation (DIC) can develop with disseminated thrombotic and hemorrhagic foci, endothelium damage, coagulation, and fibrinolysis factor consumption. Also observed are a decrease in fibrinogen, an increase in its degradation products, reptilase time elongation, an increase in D-dimer levels, and a decrease in antithrombin III (AT III) activity (B10, J2). 3.2. CLINICAL FINDINGS AND IMAGING STUDIES 3.2.1. Clinical Findings Abdominal pain is the predominant symptom in patients with AP. Typically, the pain is located in the upper abdomen and often radiates through to the back or both flanks. The onset of pain may be associated with a heavy meal or alcohol abuse. The intensity increases rapidly, but its onset is less sudden than in the case of a perforated peptic ulcer. Mild pain may be partially relieved by sitting up or by lying down, but usually body position has little influence on the intensity of pain. The second most prominent symptoms are nausea and vomiting, which are almost invariably present (B7, R3). Physical examination of the abdomen reveals tenderness most marked in the epigastrium but sometimes present throughout. Bowel sounds are decreased or absent. Usually there are no masses palpable; their presence most often indicates complications of AP, such as a pseudocyst or an abscess. In necrotizing pancreatitis the abdomen may be distended due to the intraperitoneal collection of fluid. The temperature is usually slightly elevated (100–101◦ F) in uncomplicated cases. Physical examination may reveal pleural effusion, especially on the left side. In severe acute pancreatitis, the patient may present with symptoms of dehydration including tachycardia and hypotension. In about 1% of patients, a bluish color is present around the umbilicus (Cullen’s sign) or in the flanks of the abdomen (Grey Turner’s sign) as a result of necrotizing pancreatitis and collection of blood from the retroperitoneal space. 3.2.2. Imaging Studies The sensitivity and specificity of plain film and contrast study in acute pancreatitis are low. Therefore, they are mostly used to demonstrate complications of AP. Percutaneous sonography is usually the imaging method of choice in patients with acute abdominal distress due to its wide availability, but in the case of AP the distended intestine often impairs adequate visualization of the pancreas. Still, sonography may be used as an excellent imaging modality for short-term follow-up studies, particularly in extremely ill patients who are unable to undergo computed tomography (CT).

56


Contrast-enhanced computed tomography is the most important imaging tool for the diagnosis of severe acute pancreatitis due to its excellent capacity to demonstrate early inflammatory changes as well as complications, in particular pancreatic necrosis (R3, B3, C1). The morphological changes found in CT include pancreatic enlargement, reduced attenuation or a mixed pattern, dilatation and irregularity of the pancreatic duct, soft tissue edema, and pancreatic or peripancreatic fluid collections. Because the viable pancreas “enhances” after intravenous injection of the contrast material, lack of enhancement suggests necrosis of that part of the pancreas. The overall sensitivity and detection rates for pancreatic necrosis are about 85% and 90%, respectively (B12, D1, M17). Magnetic resonance imaging is comparable to CT in its capacity to diagnose acute pancreatitis and provide precise information regarding the severity of the disease (A4, L9, P10, R12). However, due to the limited availability for emergency diagnosis it has not been widely used for routine clinical practice. 3.3. CLASSIFICATION The first classification system of acute pancreatitis was introduced by Fitz (F3) in 1889. Although this system was based on histological changes found during postmortem examination and was of limited value for clinical application, it initiated further studies on more useful classifications. In subsequent years, the number of definitions used to describe a particular course of acute pancreatitis gradually increased, which led to a number of ambiguities and made it impossible to make reliable interinstitutional comparisons (J4). Even the first generally accepted classification system, approved in 1963 during the Marseille Symposium (S2), with revisions at Cambridge (S3) and Marseille (S16), did not resolve existing differences. Development of modern diagnostic methods including ultrasound (US), computed tomography (CT), and magnetic resonance imaging (MRI) as well as recognition of the natural course of acute pancreatitis resulted in a consensus agreement in 1992 in Atlanta (B15). This clinically useful classification system based upon current imaging methods and natural history information precisely defines various courses of acute pancreatitis and associated complications as follows: Acute pancreatitis: An acute inflammatory process of the pancreas, with variable involvement of other regional tissues or remote organ systems. Severe acute pancreatitis: Acute pancreatitis associated with organ failure and/or local complications, such as necrosis, abscess, or pseudocyst. Mild acute pancreatitis: Acute pancreatitis associated with minimal organ dysfunction and an uneventful recovery; it lacks the described features of severe acute pancreatitis.

ACUTE PANCREATITIS

57

Acute fluid collection: Located in or near the pancreas, occurs early in the course of acute pancreatitis, and always lacks a wall of granulation or fibrous tissue. Pancreatic necrosis: A diffuse or focal area(s) of nonviable pancreatic parenchyma, typically associated with peripancreatic fat necrosis. Acute pseudocyst: A collection of pancreatic juice enclosed by a wall of fibrous or granulation tissue, which arises as a consequence of acute pancreatitis, pancreatic trauma, or chronic pancreatitis. Pancreatic abscess: A circumscribed intraabdominal collection of pus, usually in proximity to the pancreas, containing little or no pancreatic necrosis, which arises as a consequence of acute pancreatitis or pancreatic trauma. 3.4. PROGNOSTIC SCORES Acute pancreatitis is usually a self-limiting disease, which regresses spontaneously without further complications. However, in about 20% of cases it leads to organ failure and/or local complications and is associated with high morbidity and mortality rates (B15). Therefore, numerous attempts have been made to predict early the severe course of acute pancreatitis and to assess the possibility of complications. Objective identification of the risk of complications or death is essential for selection of those patients who should be hospitalized in the intensive care unit (ICU) and be subjected to more expensive and aggressive investigations. Moreover, it also permits interinstitutional comparison of data stratified for severity at admission and at the time of therapy. 3.4.1. Biochemical Markers Many attempts have been made to introduce into clinical practice a biochemical marker that could predict severity more accurately and more quickly than multifactorial scoring systems. Recent studies have paid increasing attention to the role of circulation disorders and hypoxia, which can constitute one of the mechanisms leading to the development of AP; these offer a significant starting point for further discussion of accompanying serious complications, both local (pancreatic necrosis, acute pseudocyst, pancreatic abscess) and systemic (SIRS, ARDS, MOF) (A1, B13, B23, R5, S9, S19, S24). An attempt to explain the phenomenon was made by Toyama et al. (T10), who claimed that even 4 hr of complete pancreas ischemia cannot be a direct cause of AP development. Although ischemia itself did not initiate AP, in most cases it led to cell necrosis. These observations suggest that necrotic lesions occur in the early stage of the inflammatory process, and not only as a complication of ongoing AP as was claimed before (T10). Ischemia is a cause of organ damage as well as of the development of a complex local interaction among markers released from endothelial cells, infiltrating leukocytes, and macrophages (K1, M6). The ischemic lesions are accompanied among others by

58


the release of oxygen free radicals, proteolytic enzymes, arachidonic acid metabolites, and other inflammation metabolites, including an increased level of cytokines (D4, R10). 3.4.1.1. Cytokines. A significant role in inflammatory reaction initiation in the course of AP is attributed to tumor necrosis factor-α (TNF-α), interleukin 1 (IL-1), and interleukin-6 (IL-6), the concentrations of which increase proportionally to the severity of acute pancreatitis (C2, G10, H8, N7). It is only after a significant simplification that early lesions connected with the activation of proinflammatory cytokines can be categorized in a simple relationship in which the release of TNF-α, a central cytokine in the cascade of proinflammatory markers, initiates a response, that is, an increase in the production of IL-6 (A6, S18), and after 24 hr also IL-8. The released cytokines lead to an increase in expression of endothelial adhesion molecules, which significantly increases the cytotoxic activity dependent upon neutrophils (B10). Apart from plasma TNF-α increase in AP, a decrease in the expression of receptors for TNF-α (TNFRI and TNFRII) is reported, together with a simultaneous increase in the level of circulating soluble receptors, sTNFRI and sTNFRII (D4), the role of which is to inhibit TNF-α by capturing, slowly releasing, or eliminating this cytokine (N6). TNF-α develops apoptosis is through the TNFRI receptor, whereas the binding of TNF-α with the TNFRII receptor activates NF-κB induction (C5). Determining the levels of soluble receptors sTNFRI (sTNFR55) and sTNFRII (sTNFR75) can be particularly helpful in the early determination of the lesion course and can be an alternative for those patients diagnosed with complicated AP who are problematic cases in determining the growing concentration of TNF-α even in a severe clinical course of the disease due to methodological difficulties related to TNF-α assaying, its short half-life of from 5 to 25 min, its phase release character, and possible bindings with proteins (P1). A considerably high level of sTNFRI was observed in patients who died of AP (D3, V2). This is indicated in studies by Schölmerich et al. (S8), who claim that in AP the levels of proinflammatory cytokines increase simultaneously with an increase in the concentration of anticytokines, to which soluble receptors belong. Similarly, the studies by McKay et al. (M8) showed a statistically significant increase in TNF-α concentration in patients with AP, in complicated pancreatic necrosis compared to the mild course of the disease (classification based on APACHE II score). The highest concentrations are released in the first and the third days from the diagnosis (M8). The research by Exley et al. (E2), however, suggests that an increase in the TNF-α concentration within 24 hr (cutoff >35 ng/ml) indicates a severe and complicated course of AP (N8, S6). The role of TNF-α in the development of AP and its complications is also confirmed in numerous studies including those by DeBeaux et al. (D3), Paajanen et al. (P1), Larvin (L5), Uomo et al. (U4), Grewal et al. (G4, G5), Gukovskaya et al. (G10), Heresbach et al. (H6), and Baxter et al. (B4).

ACUTE PANCREATITIS

59

The role played by IL-6 in AP pathogenesis is no different from the role observed in other inflammation processes, and the relatively high stability of IL-6 makes it possible to assay its concentration in every stage of disease development (D3, D4, M6, M8, N6, S8, X1). In spite of this, there are insufficient data to clearly determine a time interval in which the observed increase in concentration of IL-6 would indicate the uncontrolled development of inflammation (M8). Experimental studies showed that the peak concentration of IL-6 occurs after the TNF-α and IL-1β concentrations have reached their maximum approximately 4 hr from the first effect of the activity of the marker initiating an inflammatory response (B1). Clinical studies evaluating the IL-6 concentration in relation to the severity of infection and its process indicate its high diagnostic usefulness, significantly higher than that of the remaining cytokines (I5, P8, W10) (Table 3). The studies by Viedema et al. (V2) and Leser et al. (L11) showed considerably higher levels of IL-6 concentrations in serum of patients with a severe clinical course of AP and simultaneously minor oscillations difficult to determine in patients with mild, edematous AP. Heath et al. (H3) claimed that the peak of IL-6 secretion occurs 24–48 hr from the onset of the first symptoms of the disease. Pezzilli et al. (P8) showed that simultaneously assaying the lipase activity and the IL-6 level in serum makes it possible both to accurately distinguish AP from other acute abdominal diseases (sensitivity 94%) and make an early prediction of complications (P8) [Table 3]. What has recently enjoyed considerable interest among clinicians is the determination of IL-8 levels in patients diagnosed with AP. IL-8 is a specific chemokine, which strongly activates neutrophils, contributing to their accumulation in inflammatory foci. It results in organ damage and development of SIRS, septic shock, and multiple organ failure (MOF) (A6, G8, H1). Studies of the role of IL-8 in the prediction of the severe clinical outcome of AP have focused mainly on comparing levels of IL-8 with plasma levels of neutrophil elastase (G8, H1). This has helped determine that the level of IL-8, unmeasurable in healthy humans, demonstrates a characteristic increase in concentration in patients with AP. This finding makes it particularly possible to single out patients with severe forms of the disease from those diagnosed with mild AP (B9, D3, K14, P5, R6). Particularly high IL-8, sTNFRI, and sTNFRII concentrations have been observed in patients with SIRS, septic shock, or MOF (M6, M8). The role of IL-8 as a prognostic marker is confirmed by studies by Rau et al. (R6), which indicate that it is possible to predict pancreatic necrosis with a sensitivity of 72% and a specificity of 75% within 48 hr from the beginning of AP development (cutoff 112 pg/ml) (Table 3). IL-10 is considered one of the strongest inhibitors of neutrophil functions as well as the factor inhibiting the synthesis of IL-8 and IL-1α initiated by lipopoly saccharide (LPS) (S6, S8). As in the case of IL-8, IL-10 is not detectable in the serum of healthy humans, and its concentration clearly increases in patients with AP (especially in the first 72 hr of the disease course). Significantly higher concentrations

60


TABLE 3 CLINICAL EFFICIENCY OF TESTS IN THE EARLY ASSESSMENT OF THE PROGNOSIS OF ACUTE PANCREATITISa Marker

Specificity (%)

Sensitivity (%)

PPV

NPV

Ref.

Urine trypsinogen-2

80 90

68 26

0.62 0.55

0.82 0.72

H5 H5

Serum trypsinogen-2

80 90

47 42

0.53 0.67

0.76 0.77

H5 H5

TAP

100 58 83

85 73 72

0.60 0.39 0.44

1.0 0.86 0.94

T3 N3 N3

CRP

71 91 — 79 0.0 86 84 82 80 72–78 70–76 95

84 37 — 90 90 61 83 82 71 64–83 64–86 8.0

0.48 0.58 0.66 — 0.0 0.37 — — — — — —

0.93 0.82 0.90 — 0.75 0.94 — — — — — —

P1 P1 L5 H3 N3 N3 R8 S23 R8 R6 R11 P5

74

85

0.58

0.92

N3

TAP + CRP CAPAP

85

59

—

—

P9

59–74

45–68

—

—

K7

Pancreatic elastase-1

85

66

—

—

M12

PMN-Elastase

— —

93 71

0.80 0.60

— —

D10 G7

Neopterin

76–92

46–92

1.0

0.98

U4

IL-6

100 — — — 71 91

83 — — 70 100 82

— 0.71 0.91 0.45 — —

— 1.0 0.82 — — —

P8 L5 L11 H3 H3 S23

IL-8

75 81 75 93

82 100 72 16

— — — 0.43

— — — 0.77

S23 P5 R6 P1

92 88–91 —

94 88–95 —

— — 0.85

— — 0.96

R6 R11 L5

70

76

—

—

K5

PAP

TNFα PCT Ribonuclease

61

ACUTE PANCREATITIS TABLE 3 (Continued) Marker PLA2

Specificity (%)

Sensitivity (%)

PPV

NPV

Ref.

—

—

0.79

0.88

L5

hPASP/PCPB

64

91

—

—

R8

hPASP

60

86

—

—

R8

hPSTI LDH α2-Macroglobulin

— 100 —

79

—

—

P3

88

—

—

R8

0.82

0.67

L5

—

a TAP, Trypsinogen-activation peptide; CRP, C-reactive protein; CAPAP, carboxypeptidaseactivation peptide; PAP, pancreatis-associated protein; PMN, polymorphonuclear neutrophil; IL, interleukin; TNF-α, tumor necrosis factor-α; PCT, procalcitonin; PLA2, phospholipase A2; h, human; PASP, pancreatic-specific protein; PCPB, procarboxypeptidase B; PSTI, pancreatic secretory trypsin inhibitor; LDH, lactate dehydrogenase; PPV, NPV as in Table 1.

are observed mainly in patients diagnosed with a severe and complicated form of AP compared to AP forms with a mild course at the early stage of the disease (B9, C3, D7, S6, S15). The finding of low values may be due to altered downregulation of the immune system response in those patients (P6). 3.4.1.2. Phospholipase A2 (EC 3.1.1.4). Phospholipase A2 plays an important role in the development of complications of acute pancreatitis. It is responsible for the conversion of the phospholipids lecitin and cephalin, the main components of the cell membrane, to lysolecitin and lysocephalin, which show a strong toxic activity (N4, S5). These compounds cause damage to blood capillaries and an increase in their permeability, as well as act on the surfactant of air vesicles (N4, R1). Phospholipase A2 is responsible for pulmonary deficiency in the form of ADRS, which is one of the most serious systemic complications in the course of AP (G6, R13). Lysolecitin activity also results in pancreatic necrosis. Phospholipase A2 (PLA2) increases the activation of proinflammatory mediators such as plateletactivating mediator (PAF) (R1). Increasing serum concentration of PLA2 seems to reflect ongoing systemic inflammation in severe AP associated with SIRS (H7). Aufenanger et al. (A10) demonstrated that the activity of the pancreatic isozyme of PLA2 (pa PLA2) in serum is strongly elevated in severe acute pancreatitis and that it allows a discrimination between mild and severe forms of this disease in the early phase (Table 3). 3.4.1.3. Platelet-Activating Factor (PAF). The role of PAF in the pathogenesis of AP is linked to a very strong effect on microcirculation, which increases the permeability of vessels both through a direct action and an indirect action by activating other endothelial cells and leading to the release of prostaglandins, kinins, leukotrienes, tromboksans, and free oxygen radicals (K9, K10, S8). This aspect gains particular significance in relation to developing AP, in which the

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polymorphonuclear neutrophil (PMN) degranulation process leads to a significant increase in proteolytic enzymes (elastases, cathepsins, collagenases), increasing tissue damage (K10). Clinical studies on the application of PAF antagonists in the treatment of AP have been undertaken (B10, K10). Research by Kingsnorth et al. (K10) indicates that these substances (e.g., Lexipafant) have a positive effect, manifested in fewer complications, lack of multiorgan failure, and a lower mortality rate in these patients compared to patients treated with conventional procedures (K9, K10). 3.4.1.4. Acute-Phase Proteins. In the search for a reliable indicator of the severity of the AP course, new methodological approaches have contributed to the introduction of inflammatory mediators such as acute-phase proteins. In studies on individual acute-phase proteins in the diagnosis of AP, much attention has been focused on C-reactive protein (CRP), which particularly responds to the presence of necrotic foci (K13). Studies by Imrie (I3) drew attention to the advantages of introducing a CPR concentration measurement as a parameter complementing the Glasgow or APACHE prognostic scores, which significantly increases sensitivity and specificity in early assessment of AP complications. Lankisch et al. (L2) showed that an increase in CRP concentration by over 10 times and remaining at the same level for approximately 72 hr particularly singles out those patients who have experienced a multiple-organ failure in the course of alcoholic acute pancreatitis (K14). In Büchler’s (B22, K13) studies, a CRP concentration above 100 mg/L indicates that pancreatic necrosis is likely to occur in 95% of cases with a severe form of AP. These data were only partly confirmed in the studies by Wilson (W7), who noted that it is necessary to accept far higher cutoff values, amounting to 210 mg/L, at which sensitivity for predicting pancreatic necrosis amounted to 79%. In order to increase sensitivity for predicting pancreatic necrosis, it is recommended that CRP (cutoff >120 mg/L at 48 hr) and PMN-elastase (cutoff >400 µg/L at 24 hr) be measured simultaneously; their joint sensitivity is estimated to be 100% and their specificity to be over 95% (B10). An increase in concentration on the second day of the disease was confirmed in studies by Heath et al. (H3), who also showed there is a positive correlation between an increase in IL-6 level and CRP (an increase in CRP occurs approximately 24 hr after an increase in the IL-6 level) (H3, L11). PMN-elastase is not an acute-phase protein, but as an enzyme from activated neutrophils, it is a sensitive and specific marker of the inflammatory state with a diagnostic value similar to or even higher than that of CRP. Its increase occurs earlier and is characterized by a higher dynamism of changes, which correlates with the clinical course of the disease (D9, D10, I2, M1, U1). An attempt to use acute-phase proteins as early markers of the differentiation of edematous from necrotizing AP was made by Rau et al. (R8), who assayed CRP concentration and human pancreatic-specific protein (hPASP) successively

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for 14 days from admission to the hospital. This research showed that the levels of both parameters are significantly higher in necrotizing acute pancreatitis for CRP (>140 mg/L between days 2 and 7), whereas the highest hPASP values are observed between days 2 and 14 of the disease (>200 ng/ml) (R8). Within the first 4 days from the onset of inflammation signs, it is possible to differentiate both forms of the disease with a sensitivity of 86% and a specificity of 60% for hPASP, and a sensitivity of 71% and a specificity of 80% for CRP (Table 3). The relevancy of assaying the CRP concentration in the course of AP is confirmed by Kemppainen et al. (K7), who showed that the sensitivity for assaying hPASP in early diagnosis of AP was only between 38% and 53% and specificity was between 59% and 74%, far lower than values that can be achieved through the application of CRP measurements only. Although CRP and hPASP show no correlation with either the extent of intrapancreatic and extrapancreatic necrosis or disease etiology, their similar values, both prognostic and diagnostic, suggest that these markers can be a significant complement to research conducted among AP patients (K7, P4). The research by Wilson (W7) showed that among patients with a severe and complicated clinical course of AP, the α1-antiprotease (AAT) concentration remained at a high level throughout, although no such relationship was observed among patients diagnosed with mild AP. Moreover, as suggested in Goodman’s (G3) studies, in the course of AP a progressive decrease in the α2-macroglobulin (AMG) level is observed, which is visible especially within the first few days of the disease and is related mainly to the intensification of phagocytosis by macrophages in lymph glands and hepatocytes. Due to the participation of AMG in the process of the inactivation and elimination of the protease–antiprotease complexes from circulation by establishing stable compounds, low concentrations of AMG can be an important inflammation severity indicator (S6). These observations are confirmed by Browder’s (B18) studies, which showed an increase in AMG synthesis as a positive prognostic marker in the course of AP indicating the recovery of the patient. Human pancreatic secretory trypsin inhibitor (hPSTI) can be potentially assayed as an indicator of necrotic complications in AP (O1). This protein is an inhibitor of trypsinogen, which is produced in acinar cells in the quantity of approximately 2% of the potential content of trypsin in pancreas. Trypsin binds with its inhibitor hPSTI, then with AMG, and only this complex, trypsin–α2-macroglobulin, is eliminated from plasma (B10). Pezzili (P3) suggests that early attempts to determine the severity of the AP process based on the measurement of hPSTI within 24 hr from the first sensations of pain show a sensitivity of 79%, whereas an increase in CRP concentration has a sensitivity of 29% only (Table 3). 3.4.1.5. Trypsinogen-Activating Peptide (TAP). The most reliable way to assess trypsinogen activation is to measure trypsinogen-activation peptide (TAP) in serum, urine, pancreatic tissue, or ascitic fluid (H5, M4). Studies on TAP in

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human AP have most commonly focused on urinary TAP collected within 48 hr after the onset of symptoms (F5, H5, W9). The determination of TAP in the urine of patients with AP has been shown to be a good predictor of the severity of the disease (H5, M4), being the earliest marker of necrosis within the first 3 days (M4). TAP measurements on admission suggest that this assay may not act as a primary diagnostic test for AP, although it remains a very good marker for local complications of AP. In addition, TAP routine determination may help identify high-risk patients within the first days of the disease (F5, H5, M4). 3.4.1.6. Poly-C-Specific Ribonuclease (P-RNase) (EC 3.1.27.5). Warshaw and Fournier (W3) showed that an increase in plasma enzyme activity of pancreatic P-RNase in patients with AP may indicate necrotic lesions, and is one of the few direct markers of pancreatic tissue injury (N1, W4). Due to the time-consuming and cumbersome nature of the P-RNase assay procedure and the development of effective visualization techniques providing direct information on the structure of the inflamed pancreas, the diagnostic utility of the P-RNase assay has not been extensively studied (Table 3). 3.4.1.7. Neopterin. Neopterin is a low-molecular-weight substance derived from guanosine triphosphate (GTP) via the enzyme GTP-cyclohydrolase 1. Numerous investigators have demonstrated that neopterin, a product of human macrophages stimulated by γ interferon and other cytokines, is a useful in vivo marker of the activation of cellular immunity (U4, W1). Increased values of neopterin in body fluids have been reported in patients with malignancy, infections, and several inflammatory states. Kaufmann et al. (K3) found elevated plasma neopterin concentrations in patients with acute pancreatitis. The highest concentrations were found in clinically severe disease with systemic complications such as abnormalities of physiological parameters and development of MOF leading to a high mortality rate of 50%. These data confirm and extend earlier observations reported by Uomo and co-workers (U4). The studies by Uomo et al. (U4) demonstrated the clinical applicability of assaying neopterin concentrations for the prediction of a potentially severe progression of the disease as early as in the first day compared to patients diagnosed with a mild form of AP. Higher concentrations enduring in these patients for 6 subsequent days of the observation indicate it is possible to use the assaying of neopterin both as a prognostic factor and as a parameter to help monitor the course of the disease (U4) (Table3). 3.4.1.8. Ascorbic Acid (AA). Experimental (S1, S7, S24) and clinical (B13, B17) studies have provided some evidence for the concept that oxidative stress is the common pathway for the initiation of AP (B14). The most abundant endogenous antioxidant in the aqueous phase is ascorbic acid (AA), a bioactive form of vitamin C, which scavenges oxygen-derived free radicals produced by activated neutrophils and the hypoxanthine–xanthine oxidase system (D12). Scott et al.

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(S10) and Braganza et al. (B17) demonstrated that plasma AA was significantly below normal on admission to hospital for AP, and that this fall was greater in AP than in other causes of acute abdominal distress (B14, B17, S10). Bonham et al. (B13) reported that during a 5-day study patients with severe AP had significantly lower plasma AA concentration than those with mild pancreatitis (B14). According to Sies (S13) and Cochrone (C6), vitamin C supplies in the organism in the course of AP can be reduced by 84%–96%. Crandon et al. (C9) suggested that patients with AP might have increased urinary AA losses, but Mason et al. (M2) found that the 24-hr urinary excretion of vitamin C was the lowest the first day after the operation and that it returned to normal after the eighth day. The data derived from the measurement of urinary AA concentration in patients with mild and severe AP (B13) suggest that the depletion of plasma AA cannot be explained by increased urinary losses (B14). Based on the studies that indicated the relationship between a decrease in vitamin C concentration and morphological changes within the pancreas, it was suggested that assaying the level of vitamin C can constitute a useful indicator of pancreatic necrosis. 3.4.1.9. Procalcitonin (PCT). Recent studies have drawn attention to PCT as an early indicator of SIRS, which can be extremely useful in the early prediction of AP severity (M19). PCT, a 116-amino-acid propeptide of calcitonin with a long half-life (25–30 hr) (M9, M10), has been found in the systemic circulation to appear in high concentrations in patients with severe bacterial or fungal infections and sepsis (A3, A7, A8, K2, M11, M15, R11). Despite studies carried out for years, the role of PCT in the pathomechanism of inflammatory reaction and the place of its production remain unknown (M16). It has been proved in experimental conditions that the release of PCT is induced by LPS and is accompanied by an increase in IL-6 and TNF-α concentrations (A7, D2, M10, N5, W6). Many studies compared the prognostic value of measuring PCT, CRP, TNF-α, and IL-6 in patients with a septic shock, which accompanies the complicated form of AP as well as peritonitis. PCT turned out to be a better prognostic parameter indicating the progression or regression of inflammation compared to IL-6, whereas CRP and TNF-α concentrations in the group of the patients who died did not differ from the concentrations among the patients who survived. PCT concentrations have also been used in attempts to specify the etiologic factor that induces the series of changes leading to the development of AP (B20). The persistence of a significant increase in the PCT concentration in the early stage of development can indicate biliary AP (B20, M11), whereas a successive increase in the concentrations observed in subsequent days indicates rather the development of infected pancreatic necrosis (M11, R6). Until now only through fine needle aspiration (FNA) has early diagnosis of infected necrosis been possible (R6, R11). Monitoring the patient’s condition based on PCT measurement performed from the very admission to the hospital can be a sensitive and specific

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parameter specifying possible recommendations for surgical intervention and, in the period after necrosis drainage, a valuable source of information on possible postsurgery complications (R6). The relevance of PCT measurements for predicting necrotic complications of AP is confirmed among others by Rau et al. (R6), who found significantly higher PCT and IL-8 levels in a group of patients where the further course of the disease was characterized by the development of infected pancreatic necrosis compared to patients with an edematous form of AP or aseptic necrosis (Table 3). One of the serious, life-threatening AP complications is ARDS, and the possibility of using PCT assay for the differentiation of infective etiology from noninfective etiology is exceptionally useful and has become an integral part of the therapeutic procedure for patients diagnosed with AP (B21). PCT level measurement can fill the existing diagnostic gap (first 2 days) between CRP measurement, which is a good marker of the assessment of AP course severity only after approximately 36–72 hr from the development of lesions, and the diagnosis of AP. An additional advantage of PCT measurement over classical acute-phase proteins is that there is no increase in PCT concentration in response to surgical trauma (M11, R6). 3.4.2. Scoring Systems Ranson et al. (R4) were the first to evaluate the usefulness of a multifactor scoring system in predicting an unfavorable course of acute pancreatitis. Due to its simplicity, it has become a standard system used worldwide, and initiated the objective assessment and description of the clinical picture as a possible basis for early definition of risk classes and standards for prospective evaluation of acute pancreatitis. Because the initial series that provided the basis for the Ranson criteria consisted mainly of alcohol-induced AP, an appropriate modification was proposed for gallstone pancreatitis (R2). Although it was generally accepted that a severe attack of acute pancreatitis is predicted by the presence of three or more positive factors, the Ranson criteria attracted much criticism as to methodological layout and practical implications. Many reports claimed that the Ranson signs have a poor predictive power and the observed differences cannot always be attributed to experimental biases or the heterogeneity of the populations studied. This observation was confirmed in a meta-analysis performed by DeBernardinis et al. (D5). A scoring system (Glasgow) similar to that of Ranson was proposed by Imrie et al. (14) with further modifications (B11, C7) and was found to be effective in predicting the severity of AP for the major two common etiologies, alcohol and biliary (M3). However, the major disadvantage of the Ranson and Glasgow systems is the necessity of 48-hr data collection following admission, which does not allow early allocation of patients to appropriate investigative groups. The Acute Physiology and Chronic Health Evaluation (APACHE II) system (K12) is another scoring system, which was validated in a number of prospective clinical studies on acute pancreatitis (D6, K10, M7, O3, W8). A score of at least

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8 points on admission to the hospital is considered as an indicator of severe pancreatitis. As opposed to the Ranson and the Glasgow criteria, the APACHE system can be used at admission and then in monitoring the clinical course of an individual patient. However, even this complex scoring system has some disadvantages. APACHE II has been shown by some authors to be of limited utility in the early prognostic evaluation of acute pancreatitis because of its low positive predictive value (D11, L4). Other multifactor scoring systems, including the Simplified Acute Physiology Score (SAPS) and the Bernard Organ Failure Scoring System (OFS) (B8), require prospective studies for adequate assessment. 3.4.3. Imaging Techniques As new imaging tools have become available, such as CT and MRI, many attempts have been made to evaluate imaging criteria for assessing the severity of acute pancreatitis. The first severity index of acute pancreatitis was developed in 1990 by Balthazar et al. (B2). The CT Scoring Index (CTSI) is a 10-point system based on the degree and the type of changes in pancreatic parenchyma and peripancreatic tissues as well as the extent of pancreatic necrosis. The majority of studies confirm its clinical utility for prediction of severity of AP (K6, L1, M20, S14, V1); however, some authors report CT to be ineffective (L13, L14). Computed tomography is undoubtedly the most accurate method for determining the extent of necrosis in acute pancreatitis; debate exists, however, regarding patient selection and the optimal timing of CT (M20, L3), especially in the patients with Ranson score below 2. Although contrast medium has been found to aggravate acute pancreatitis in animal models, some recent studies proved the safety of contrast-enhanced CT in humans (U3, H9). 3.4.4. Other Clinical assessment of severity in AP is still one of the most useful approaches. The accuracy of initial assessment on admission is about 40–68%, depending on the experience of the examining clinician (C7), and increases to 70–80% after 48 hr (H2). A study performed by Uhl et al. (U2) revealed no differences in the severity of acute pancreatitis caused by gallstones, alcohol, and other factors. The same findings were published by Lankisch et al. (L1).

4. Therapy Despite more than a century of research endeavor, there is no specific medical treatment for acute pancreatitis (B16, K11). Conservative management is a method of choice in the treatment of edematous AP. These patients do not have multiple

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organ failure and do not have to be treated in the ICU. Emergency or early (during 24 hr) endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic sphincterotomy (ES) are the preferred approaches for management of acute biliary pancreatitis (F1). ES for identifiable common bile duct stones (CBD) is associated with high success rate and low morbidity (L12). The overall morbidity was reduced especially by the reduction of biliary sepsis, but the local and systemic complications were not significantly reduced (F1). ES should be complemented by sphincter of Oddi manometry. To reduce recurrent acute biliary pancreatitis, elective laparoscopic cholecystectomy (LC) should be performed after disappearance of acute symptoms (2–12 weeks of the initial presentation) (L12). LC during the first week of admission is associated with an increase of operative complications and rate of conversion and longer postoperative stay (T1). The management of patients with severe necrotizing acute pancreatitis requires admission to the ICU. The natural course of severe AP progresses in two phases. The first 2 weeks is characterized by the systemic inflammatory response syndrome, sometimes with MOF syndrome. The second phase, beginning 2 weeks after the onset of the disease, is dominated by sepsis-related complications resulting from infection of pancreatic necrosis (B24). More than 80% of deaths in AP are due to septic complications as a consequence of bacterial infection of pancreatic necrosis (B7). Severity is evaluated by multiscoring systems, including the Ranson and the Imrie criteria. Beger and Büchler developed a definition of severity: the presence of pancreatic necrosis, which is a marker of severity. According to the Atlanta Symposium, severe acute pancreatitis should be defined by the presence of organ failure, local complications (necrosis, pseudocysts, and abscesses), or both. Patients with identified necrosis (on a contrast-enhanced CT scan) or organ failure (manifested respiratory, renal, cardiovascular insufficiency) require monitoring and supportive care (T2). In patients with sterile necrosis a policy of conservative treatment is adopted (L10). It is one of the most important aspects of treatment of these patients. They need aggressive fluid resuscitation. Occasionally, pressors such as dopamine and levoephedrine are required to maintain cardiac output. A Swan–Ganz catheter can differentiate between pancreatitis-induced respiratory distress syndrome and congestive heart failure. Careful monitoring of sodium, potassium, and calcium is required. Pulmonary complications can range from atelectesis to adult respiratory distress syndrome. These patients should be monitored with continuous-pulse oximetry. If saturation is less then 92%, the patient should be provided supplemental oxygen (T2). Hydration to maintain renal output and cardiovascular stability may cause worsening of pulmonary function, requiring intubation with mechanical ventilation. Renal insufficiency sometimes requires dialysis. To reduce the amount of proinflammatory cytokines such as interleukin-1, interleukin-6, and tumor necrosis factor-α, patients with sepsis and SIRS during severe AP require continuous

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venovenous hemodiafiltration (K15). Patients with AP are routinely placed on “no oral intake.” Gastric secretion is routinely removed by placement of gastric tubes, although they are not necessary in all patients, but are effective in patients with protracted nausea, vomiting, or paralytic ileus. To decrease gastric acid secretion, H2-blocking agents or proton pump inhibitors are used. When duodenal pH is increased to a value of more than 4.5, secretin stimulation of the pancreas does not occur (S17). There was no apparent effect on survival or difference in complication rates after administration of direct inhibitors of pancreatic secretion: glucagon, calcitonin, and somatostatin and its analogue, octreotide (B6, L17, M7). Studies with aprotinin (Trasylol) do not demonstrate any significant benefit for patients with mild or severe acute pancreatitis (L7). Double-blind, randomized studies of Lexipafant, an antagonist of platelet-activating factor, in the treatment of severe AP gave promising results (J3). Severe AP is characterized by a high catabolism and patients require adequate nutrition. Some investigators suggest using early total parenteral nutrition (TPN) to decrease the stimuli for pancreatic secretion. In patients suspected to have hypertriglyceridemia, it is prudent to limit the lipid content. Other investigators suggest enteral nutrition by enteral tube put into the small intestine using an endoscope or intraoperatively. This method seems to be more physiological due to prevention of bacterial translocation. One of the most marked symptoms of AP is acute pain, which can be treated with epidural anesthesia. It needs to be kept in mind that in acute biliary pancreatitis, before endoscopic sphincterotomy, patients should not be given opioids due to sphincter of Oddi constriction. Secondary pancreatic infection is a major cause of death in patients with acute necrotizing pancreatitis (T2). Prophylactic antibiotics may reduce the incidence of infection in necrotizing pancreatitis (A5). Imipenem, ciprofloxacin, and ofloxacin were found to have high pancreatic tissue levels and high bacterial activity against bacteria known to cause infected necrosis and sepsis in these patients (T2). Due to the high frequency of fungal infections, ketoconazole (W2) or fluconazole together with antibiotics should be given patients with severe AP. The presence or absence of infection can be established using fine-needle aspiration (A5, R9). Treatment of infected necrosis by surgical debridement is currently the standard approach (L10). Surgery remains the gold standard in the treatment of infected pancreatic necrosis (G2). Techniques of debridement and postoperative management have been the subject of debate in the literature (A5, B24, F2, R7, T9); most authors have concluded that each method has a role in specific patients. The conventional surgical approach with debridement and Penrose/sump drainage is the oldest method, adequate for patients who will not need reexploration. An aggressive operative approach involving open packing and frequent, planned reoperations is a better method for controlling pancreatic sepsis. After debridement, the septic area is packed with a ring of nonadherent petrolatum gauze. The abdomen is left

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open, and packing is changed every 24–48 hr. To allow easy reentry to the abdominal cavity, a synthetic mesh or zipper is sutured into the abdominal fascia. Necrosectomy and subsequent continuous lavage by means of double-lumen drainage tubes using 6–48 L of isotonic saline per day is an alternative method of treatment. Percutaneous drainage assisted by CT or ultrasonography is adequate therapy for pancreatic pseudocysts and abscesses (M13). Surgical drainage is required when percutaneous drainage is not effective. Treatment of necrotizing acute pancreatitis should be performed in hospitals with suitable knowledge, equipment, and experience in this area.

5. Conclusions In this chapter we have reviewed current concepts on diagnosis and treatment of acute pancreatitis. Despite numerous attempts to find an ideal diagnostic marker, combined use of physical examination, biochemical tests, and imaging studies is the gold standard for identifying patients with AP. Several biochemical tests including TAP determination are very promising, but require further studies. A considerable number of candidate markers for predicting the severity of acute pancreatitis is available. However, neither a single clinical scoring system nor a single biological marker is found to be useful in general clinical practice. Further research should provide more evidence for the utility of combined scoring systems including biochemical markers, clinical evaluation, and CT. REFERENCES A1. Agarwal, N., and Pitchumoni, C. S., Assessment of severity of acute pancreatitis. Am. J. Gastroenterol. 86, 1385–1391 (1991). A2. Aho, H. J., Sternby, B., Kallajaki, M., and Nevalainen, T. J., Carboxyl ester lipase in human tissue and in acute pancreatitis. Int. J. Pancreatol. 5, 123–134 (1989). A3. Al-Nawas, B., Krammer, L., and Shah, P. M., Procalcitonin in diagnosis of severe infections. Eur. J. Med. Res. 1, 331–333 (1995/1996). A4. Amano, Y., Oishi, T., Takahashi, M., and Kumazaki, T., Nonenhanced magnetic resonance imaging of mild acute pancreatitis. Abdom. Imaging 26, 59–63 (2001). A5. Ashley, S. W., Perez, A., Pierce, D. C., Brooks, D. C., Moore, F. D., Jr., Whang, E. E., Banks, P. A., and Zinner, M. J., Necrotizing pancreatitis: Contemporary analysis of 99 consecutive cases. Ann. Surg. 234, 579–583 (2001). A6. Asimakopoulos, G., Mechanisms of the systemic inflammatory response. Perfusion 14, 269–277 (1999). A7. Assicot, M., Induction of circulating procalcitonin following intravenous administrations of endotoxin in humans. In “International Conference on Endotoxins. Amsterdam IV” [Abstract]. 1993. A8. Assicot, M., Gendrel, D., Carsin, H., Raymond, J., Guilbaud, J., and Bohuon, C., High serum procalcitonin concentrations in patients with sepsis and infection. Lancet 341, 515–518 (1993).

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M4. M5. M6. M7. M8.

M9. M10. M11.

M12.

M13. M14. M15. M16. M17. M18. M19.

M20.

N1. N2.

N3.

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MITOCHONDRIAL DNA MUTATIONS AND OXIDATIVE STRESS IN MITOCHONDRIAL DISEASES Yau-Huei Wei∗ and Hsin-Chen Lee† ∗ Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, Republic of China † Institute of Biochemistry, Chung Shan Medical University, Taichung, Taiwan, Republic of China

1. 2. 3. 4.

5. 6.

7. 8. 9. 10.

11. 12. 13. 14.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Brief Review of Studies on Mitochondrial Diseases . . . . . . . . . . . . . . . . . . . . . . . . . The Genetic Map and Structural Characteristics of Human Mitochondrial DNA. . . . . Transmission by Maternal Inheritance and Random Segregation of mtDNA during Mitosis and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Maternal Inheritance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Heteroplasmy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Segregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Threshold Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Susceptibility of mtDNA to Oxidative Damage and Mutation . . . . . . . . . . . . . . . . . . . Classification of Disease-Associated mtDNA Mutations . . . . . . . . . . . . . . . . . . . . . . . 6.1. Large-Scale Rearrangements of mtDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Point Mutations in tRNA Genes of mtDNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Point Mutations in rRNA Genes of mtDNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Point Mutations in Protein-Coding Genes of mtDNA . . . . . . . . . . . . . . . . . . . . . Molecular Consequences of mtDNA Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemical Basis for How mtDNA Mutations Cause Mitochondrial Dysfunction. . . Oxidative Stress and Oxidative Damage Elicited by mtDNA Mutations . . . . . . . . . . . Mitochondrial Diseases Caused by Mutations in Nuclear DNA . . . . . . . . . . . . . . . . . . 10.1. Mutations in Nuclear Genes That Affect Mitochondrial Respiratory Enzymes . 10.2. Nuclear Gene Mutations Involved in the Maintenance of mtDNA Copy Number or mtDNA Integrity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3. Mutations in Nuclear Genes and Animal Models of Mitochondrial Diseases . . . Cybrids for Studies of Mitochondrial Diseases: Applications and Limitations . . . . . . Effect of mtDNA Mutations on the Apoptosis of Human Cells . . . . . . . . . . . . . . . . . . Therapy for Mitochondrial Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Biochemical defects and mutations in mitochondrial DNA (mtDNA) have been increasingly recognized as etiologic factors in a wide spectrum of mitochondrial diseases (S4, S14, W7, Z3). The central roles of mitochondria in energy production, the generation of reactive oxygen species (ROS), and the initiation of apoptosis in human cells have suggested possible mechanisms of pathogenesis for mitochondrial disease (S14). As the major intracellular supplier of energy in the form of ATP, mitochondria consume more than 90% of the oxygen uptake of the human cell. Under normal physiological conditions, about 1–5% of the oxygen consumed by mitochondria is converted to superoxide anions, hydrogen peroxide, and hydroxyl radicals (B3, C3, N6, T12). These detrimental by-products of respiration are usually disposed of by the antioxidant defense system in mitochondria, which includes manganese–superoxide dismutase (MnSOD), catalase, and glutathione peroxidase (GPx). However, in aging tissues and under some pathological states a significant fraction of ROS and free radicals may escape the antioxidant system and cause oxidative damage to lipids, proteins, nucleic acids, and other subcellular structures such as membranes (B3, F3, P7, P9, R1, R2, S2, S17, W5, W7). As a consequence of this and other damage, the bioenergetic functions of mitochondria in tissue cells decline with age and are defective in patients with mitochondrial diseases. In the past decade, a number of investigators have demonstrated that respiratory function of mitochondria gradually declines with age in various human tissues (C13, H13, L10, P4, T10, Y5). This not only causes insufficient production of ATP, but also elicits an increase of generation of ROS and free radicals by the electron transport chain of mitochondria (B3, S15, W7). The defects in the respiratory chain that are commonly found in the affected tissues of patients with mitochondrial diseases are much more severe and have more profound consequences. Abnormal mitochondria and impaired respiratory function are the most distinctive characteristics in patients with mitochondrial myopathy, mitochondrial encephalomyopathy (A3, A5, G8, P8, T3, T4, Z1), and other mitochondrial diseases. The most common and best-defined mitochondrial diseases include chronic progressive external ophthalmoplegia (CPEO) (H7, W1), Kearns–Sayre syndrome (KSS) (C4, S10), myoclonic epilepsy with ragged-red fibers (MERRF) syndrome (S9), mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes (MELAS) syndrome (D1, G4, G5, N5, S13), and Leigh disease (P5). Mitochondrial disease may be manifested either as isolated symptoms (e.g., myopathy) or together with other multisystem disorders including encephalomyopathy, peripheral neuropathy, limb muscle weakness, endocrinopathy, nephropathy, and hepatic dysfunction (A3, C4, M7, N5). Patients with any of these diseases usually suffer from defective energy metabolism of mitochondria and most of them have specific mutation(s) of mtDNA in the affected tissues (C1, C9, H1, K3, K4, M11, M15, O1, P1, S14, S22, T3, T4).

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In this chapter we review the defects of respiratory function and DNA mutations in the mitochondrial genome and nuclear DNA underlying mitochondrial diseases and discuss the roles that oxidative stress, oxidative damage, and apoptosis may play in the pathogenesis of this group of overt metabolic disorders. The cell cultures and animal models for studies of mitochondrial diseases and potential therapies are also discussed.

2. A Brief Review of Studies on Mitochondrial Diseases In 1962, Luft et al. (L14) first identified a rare hypermetabolic disorder, which was characterized by elevated body temperature, structural abnormalities in mitochondria, and uncoupling of oxidative phosphorylation from respiration. During the next few decades, a large number of patients with biochemical and morphological evidence of respiratory chain defects were described. In 1988, the discovery of the first pathogenic mtDNA mutations, including point mutations (W2) and largescale deletions (H7), established a new paradigm for clinical diagnosis, study of pathogenesis, and management of mitochondrial diseases (W1). In the past decade or so, more than 150 deletions and 80 point mutations of mtDNA have been identified in patients afflicted with diverse mitochondrial diseases (M15). The clinical manifestations of this group of disease range from relatively mild and late-onset conditions to frequently fatal syndromes, such as MELAS and Leigh disease. In addition to the primary biochemical defects of mitochondrial diseases, mitochondrial respiratory chain dysfunction has been demonstrated to play an important role in aging, neurodegenerative diseases, and infertility. On the other hand, a number of recent clinical and molecular studies have demonstrated that some of the mitochondrial diseases are caused by defects in nuclear genes related to oxidative phosphorylation. The identification of the nuclear genes responsible for oxidative phosphorylation-related diseases has proceeded at a much slower pace as compared with the discovery and characterization of disease-associated mtDNA mutations. In 1995, the mutation in a nuclear gene coding for succinate dehydrogenase was first discovered in a family with an autosomal recessive form of Leigh disease (B7). It is fair to say that the majority of mitochondrial diseases are caused by mtDNA mutation(s), although there is an increasing number of nuclear DNA mutations identified to be responsible for several familial mitochondrial disorders (W1). Although a wide spectrum of mtDNA mutations has been demonstrated to be associated with various mitochondrial diseases, the molecular mechanisms of pathogenesis of these diseases have remained elusive. Animal models for mtDNA mutation-elicited mitochondrial diseases were not available until the generation in 2000 of the first mice carrying a large-scale mtDNA deletion (I4). The discovery of pathogenic mtDNA mutations and the establishment of animal models with mtDNA mutation have provided an opportunity for researchers to

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conduct studies at the molecular and the cellular levels and gain new insights into the pathogenesis of mitochondrial diseases (W1). However, in the course of these studies many fundamental questions concerning the pathological mechanisms and the relationship between mtDNA genotype and clinical phenotype of mitochondrial diseases have remained unanswered.

3. The Genetic Map and Structural Characteristics of Human Mitochondrial DNA The mitochondrial respiratory chain is composed of four enzyme complexes: NADH–coenzyme Q (CoQ) reductase (Complex I), succinate–CoQ reductase (Complex II), ubiquinol–cytochrome c reductase (Complex III), and cytochrome c oxidase (Complex IV). NADH or FADH2, which are continually generated from various dehydrogenases in biological oxidation of simple sugars, amino acids, and fatty acids, channels electrons to Complex I or II of the respiratory chain. Coenzyme Q shuttles electrons between these two complexes and Complex III, and the molecular oxygen ultimately accepts electrons from Complex IV. Mitochondria produce ATP by the coupling of a respiration-generated proton gradient with the proton-driven phosphorylation of ADP by F0F1-ATPase (Complex V). The oxidative phosphorylation system contains over 80 polypeptides. Only 13 of them are encoded by mtDNA, which is contained within mitochondria, and all the other proteins that reside in the mitochondrion are nuclear gene products. Mitochondria depend on nuclear genes for the synthesis and assembly of the enzymes for mtDNA replication, transcription, translation, and repair (T1). The proteins involved in heme synthesis, substrate oxidation by TCA cycle, degradation of fatty acids by β-oxidation, part of the urea cycle, and regulation of apoptosis that occurs in mitochondria are all made by the genes in nuclear DNA. Human mtDNA is a circular, double-stranded DNA molecule of 16,569 base pairs (bp), whose complete nucleotide sequence was published in 1981 (A4). In contrast to nuclear DNA, mtDNA does not have introns. The displacement loop (D-loop) is the only noncoding region (ca. 1.1 kb) in human mtDNA, which contains the initiation site (OH ) for mtDNA replication and the promoters for transcription of both heavy (H) and light (L) strands of mtDNA. Transcription of both strands of mtDNA produces polycistronic transcripts, which are processed to release 13 mRNAs, 22 tRNAs, and 2 rRNAs. The 2 rRNAs, 14 tRNAs, and 12 polypeptides are encoded on the H strand, and the L strand codes for 8 tRNAs and 1 polypeptide. The 13 polypeptides encoded by mtDNA together with polypeptides encoded by the nuclear DNA are assembled in the mitochondria to form respiratory enzyme Complexes I, III, IV, and V. Only Complex II is encoded entirely by the nuclear DNA. Interspaced among the polypeptide-coding sequences are the 22 tRNA and 2 rRNA genes, whose products are essential for protein synthesis in mitochondria. All the genes encoded in mtDNA are thus important for mitochondrial respiration and


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oxidative phosphorylation, and any mtDNA mutation that leads to impaired expression of these genes would be expected to cause a deficiency in energy metabolism.

4. Transmission by Maternal Inheritance and Random Segregation of mtDNA during Mitosis and Development There are several important characteristics of the genetics of the mitochondrial genome that are different from those of nuclear genetics. They affect the clinical manifestation and transmission of mitochondrial diseases and are discussed in detail in what follows. 4.1. MATERNAL INHERITANCE Human mitochondria and mtDNA are maternally inherited, that is, mtDNA is transmitted from a mother to her children (G3, H11). In the mature mammalian oocyte the mtDNA copy number increases to approximately 100,000, whereas it is decreased to about 100 copies in the spermatozoon (J5). It has been observed that a small number of paternal mitochondria may enter the egg during fertilization (A6, G10). However, even though the paternal mtDNA may constitute a small fraction of mtDNA in the fertilized oocyte, the paternal mitochondria and mtDNA are rapidly degraded and eliminated after fertilization and in the early phase of embryogenesis (K1, S20). Thus, most (but not all) of mtDNA mutations are transmitted through the maternal lineage, and affected males with an mtDNA disease cannot transmit the genetic defect in mtDNA to the next generation (E1). Almost all the pathogenic point mutations of mtDNA are maternally inherited. However, for unknown reasons, large-scale deletions, insertions, and tandem duplications of mtDNA are mostly sporadic and are not transmitted through the maternal lineage (S8, Z3). 4.2. HETEROPLASMY A typical human cell usually contains hundreds of mitochondria and thousands copies of mtDNA (R3). Theoretically, all of the mtDNA molecules in an individual are identical. This condition is termed homoplasmy. However, two or more mtDNA genotypes may coexist within a single mitochondrion, a cell, an organ, or an individual. This is called heteroplasmy. Although the common pathogenic point mutations of mtDNA associated with Leber’s hereditary optic neuropathy (LHON) are almost invariably homoplasmic, most pathogenic mtDNA mutations in the human are heteroplasmic (S4). The degree of heteroplasmy (or the proportion) of a pathogenic mtDNA mutation may differ among mitochondria, among cells, and among tissues (mosaicism) of an affected individual. It has been demonstrated that the proportion of mtDNA with a pathogenic mutation is usually higher than 70%

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in the affected tissues of patients with mitochondrial diseases (P1, W1). However, there are few cases in which the peripheral tissues contain higher levels of mutant mtDNA than does the target tissue (P1). 4.3. SEGREGATION During cell division, different proportions of mutant mtDNA may be transmitted to the daughter cells in a random fashion. Random segregation of mtDNA molecules during mitosis and during development could lead to high levels of mutant mtDNA in some cells, but low levels in others (C8, H12, J6). Uneven partitioning of the mitochondrial genomes during cytokinesis can lead to differences in mtDNA genotype between daughter cells and result in random genetic drift (J6). Moreover, the degree of heteroplasmy within the tissue cells can change during the life span of the affected individual (W5). In a number of patients, an increase of the mutation load is associated with the progression of the mitochondrial disease. The defective mitochondria harboring high levels of mutant mtDNA may proliferate through an unknown mechanism and thus result in an increase in the proportion of mutant mtDNA within the postmitotic tissue cells. On the other hand, in rapidly dividing cells, mutant mtDNA is often present at a relatively lower proportion (S8). This may result from the possibility that the cells harboring high levels of mutant mtDNA fail to divide, but those with a low mutant load divide rapidly. However, it remains to be investigated whether a selection mechanism might be operating during the life span of the individual. Large-scale deletions, insertions, and tandem duplications of mtDNA are usually not found in blood cells, and the proportions of mtDNA with point mutations in blood cells are generally lower than those in muscle of patients with mitochondrial disease (P1, W5). Thus, the absence of mtDNA mutation in blood samples cannot be used to exclude mitochondrial disease (L7, P1). On the other hand, higher levels of mutant mtDNA are usually found in postmitotic tissues such as cardiac and skeletal muscles and skin tissue of patients. Large-scale deletions or point mutations of mtDNA are generally detectable in muscle biopsies of about 70% of patients with mitochondrial disease (L7, P1). Some of these patients are affected by mutations in nuclear DNA. Other, unknown mutations in mtDNA or nuclear DNA are present in the rest of the patients. It is well established that mitochondrial function defects are not severe until the proportion of mutant mtDNA reaches a high level, which forms the basis of the concept of the “threshold effect.” In skeletal muscle, the level of the A3243G mutation is related to the severity of strokelike episodes, epilepsy, and dementia in patients with MELAS syndrome (C6, H14, P1). Similarly, the level of the A8344G mutation is correlated with the degree of cerebellar ataxia and myoclonus in patients with MERRF syndrome (C6). Thus, molecular genetic analysis of mtDNA mutations in muscle biopsies usually provides more definitive diagnosis of


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mitochondrial disease (P1). However, the relationship between mtDNA genotype and clinical phenotype is more complex for most of the pathogenic mtDNA mutations (P1, P2, W1). On the other hand, dramatic differences in the degree of heteroplasmy have usually been seen in pedigrees carrying a pathogenic mtDNA mutation. A woman within a pedigree harboring heteroplasmic mutant mtDNA generally transmits a variable amount of mutant mtDNA to each of her children (H14, P1). This can result in a wide range of different clinical phenotypes in the next generation—from asymptomatic individuals to severely affected offspring with progressive fatal neuromuscular disease (H14). One explanation for the rapid changes in genotype is a genetic bottleneck of mtDNA (B10, H12). During oogenesis, the mtDNA is amplified to approximately 100,000 copies in each of the mature oocytes. The bottleneck hypothesis suggests that the number of mtDNAs is reduced during oogenesis to a relatively small number before massive expansion, and thus only a small proportion of the mtDNAs repopulate the oocytes from which offspring develop after fertilization (B10, H11, J6, M6, P11). The germline bottleneck would lead to the loss of some mtDNA mutations or the development of individuals harboring higher levels of mutant mtDNA (H14). Moreover, the extreme segregation of mtDNA could also arise from the selective replication of only a small number of mtDNA molecules and eventually repopulate the somatic cells of an individual in the next generation (J7, P11). 4.4. THRESHOLD EFFECT It has been demonstrated that mtDNA mutations exhibit a threshold effect in eliciting mitochondrial disorders (H14, P11). The proportion of mutated mtDNA molecules in a heteroplasmic population within cells and within mitochondria affects the severity of the biochemical defect, but the dosage effect does not necessarily follow a linear correlation (S3). Once the mutant mtDNAs reach a critical level, the phenotypes at the cellular and the tissue levels change rapidly from normal to abnormal (H14). It was found that the threshold for expression of biochemical defects is approximately 65% mutant for a large-scale deletion (H3, P1) and up to 95% for tRNA mutations of mtDNA (C11, P2). Moreover, the organs most often affected in patients with mitochondrial disorders are high-energy-demanding tissues, such as skeletal and cardiac muscles and the central nervous system (P1, W1). Therefore, the threshold is dependent not only on the type of mtDNA mutation, but also on the energy demand of individual cells and tissues (L7, P1, S3). Therefore, the variability in the heteroplasmy of mtDNA mutation in different tissues of affected individuals together with tissue-specific differences in the threshold of mutant mtDNA and the varied energy demands of different organs could lead to highly variable clinical phenotypes observed in patients with mtDNA disease (P1, P11, W1).

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5. Susceptibility of mtDNA to Oxidative Damage and Mutation The mitochondrial genome has a mutation rate 17-fold greater than that of nuclear DNA (R2). This has been ascribed to the distinctive characteristics of mtDNA. Unlike nuclear DNA, mtDNA is a naked, compact DNA molecule without protective histones, and replicates rapidly without proofreading and efficient DNA-repair systems. It is known that mtDNA is transiently attached to the mitochondrial inner membrane, in which a considerable amount of ROS is continually produced by the respiratory chain (W5, W8). Moreover, certain regions of the mtDNA (e.g., D-loop) have been demonstrated to be particularly sensitive to oxidative insult of ROS and are prone to mutation (H9, H10, M12). It has been shown that the hotspots for oxidative modification and mutation of mtDNA are located at or near unusual structures including bent, antibent, and non-B DNA sequences in human mtDNA (H9, H10). These characteristics have rendered mtDNA vulnerable to attack by ROS and free radicals, which are continually generated by electron leakage of the respiratory chain. In addition, several mtDNA mutations and oxidative damage occur more frequently and accumulate at relatively higher levels in sun-exposed skin (P3, Y1), the lung of cigarette smokers (F1, L4), and the oral tissues of individuals with a habit of chewing betel quid (L5). These observations suggest that there is a high susceptibility of mtDNA to oxidative damage and mutation elicited by endogenous as well as exogenous oxidative stress. Moreover, these somatic mtDNA mutations and oxidative damage contribute to the ever-increasing alterations of structure and function of mtDNA that generally occur in the affected tissues of patients with mitochondrial disease (K7, M12, P1).

6. Classification of Disease-Associated mtDNA Mutations Because mitochondrial diseases are a genetically heterogeneous group of disorders associated with impaired oxidative phosphorylation (P13, S4, Z3), patients may display a wide range of clinical symptoms in various combinations (N2, P1, S7). These diseases can affect any tissue in the body, but brain and skeletal muscle are most frequently involved. Disorders of the mitochondrial respiratory system often cause mitochondrial myopathy, which may be the primary clinical manifestation or an associated symptom of a mitochondrial disease (A6, B5, C4, G4, G5, G8, H7, N2, Z1, Z3). Moreover, patients with impaired oxidative phosphorylation often have associated common neurological clinical features, including paresis of extraocular muscle, retinitis pigmentosa, deafness, seizures, ataxia, strokelike episodes, dementia, and peripheral neuropathy (B5, E5, G4, G5, N2, N5, P5, P12, S9, S10, W1, W2). In addition, patients may have nonneurological symptoms, and


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they may present with cardiomyopathy, diabetes, or endocrine dysfunction (A3, H14, K4, M7, O1, P1, T5, W4). Although DNA mutations in nuclear DNA may cause mitochondrial dysfunction, the majority of genetically defined mitochondrial diseases are caused by mutations in mtDNA (M15, P1, S4). Point mutations and deletions of mtDNA have been reported to be associated with or responsible for mitochondrial myopathies and/or encephalomyopathies (M15, P1, S4). Patients with such diseases usually manifest major clinical symptoms early in life and at a later stage may develop additional multisystem disorders such as encephalopathy and/or peripheral neuropathy. Most of the mitochondrial myopathies occur sporadically and are often caused by large-scale mtDNA deletions (P1). However, there are several reports on maternally inherited mitochondrial myopathy and familial mitochondrial myopathy. These patients usually harbor a specific mtDNA mutation and often exhibit defects in NADH–CoQ reductase and/or cytochrome c oxidase. Human mtDNA mutations include rearrangements in which mtDNA genes are deleted or duplicated, point mutations in tRNA or rRNA genes, and point mutations in polypeptide-coding genes causing missense mutations that may change a critical function of the affected polypeptides constituting the oxidative phosphorylation system (M15). The molecular biology and biochemical and pathological consequences of each type of these mtDNA mutations are described in detail in what follows. 6.1. LARGE-SCALE REARRANGEMENTS OF mtDNA Large-scale deletions of mtDNA were the first mutations in mtDNA to be associated with human disease (H7). These mtDNA deletions are usually heteroplasmic, and generally remove several genes encoding subunits of respiratory enzymes and tRNA genes in the mitochondrial genome (S4). It has been established that mtDNA with a deletion is rarely transmitted from affected women to their children and mtDNA deletions are usually sporadic (S4). On the other hand, an mtDNA duplication produces an mtDNA molecule that is larger than the wild-type mtDNA and contains two tandemly arranged mtDNA molecules consisting of a full-length mtDNA and a deleted mtDNA (S14). Large-scale deletions of mtDNA are commonly associated with CPEO, KSS, and Pearson’s syndrome (S4, M15). KSS is characterized by early onset of ophthalmoplegia and retinitis pigmentosa, and may be accompanied by cerebellar ataxia or cardiac conduction block (H7, S4). However, deletions alone or a host of large-scale mtDNA rearrangements comprising deletions and duplications have been identified in other mitochondrial disorders including diabetes mellitus and deafness, Wolfram syndrome, MELAS, KSS–MELAS overlapping syndrome, and Leigh disease (S4, W1).

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WEI AND LEE TABLE 1 POINT MUTATIONS IN rRNA/tRNA GENES OF mtDNA ASSOCIATED WITH MITOCHONDRIAL DISEASESa

Gene Location

mtDNA Mutation

12S rRNA

T1095C C1310T A1438G A1555G

16S rRNA

C3093G

tRNAPhe

G583A A606G T618C G1606A G1642A A3243G A3243G A3243G A3243T G3249A T3250C A3251G A3252G C3254G C3256T A3260G T3264C T3271C T3271C T3273C C3275A A3288G T3291C A3302G C3303T A4269G

tRNAVal tRNALeu(UUR)

tRNAIle

tRNAIle

tRNAMet tRNATrp

T4274C T4285C A4295G G4298A A4300G G4309A A4317G C4320T T4409C G4450A G5521A G5549A

Diseaseb Sensorineural hearing loss Diabetes mellitus Diabetes mellitus Aminoglycoside-induced deafness or maternally inherited deafness MELAS MELAS Exercise intolerance/myoglobinuria Mitochondrial myopathy Ataxia, myoclonus, and deafness MELAS MELAS CPEO Diabetes/deafness Mitochondrial myopathy KSS Mitochondrial myopathy/CPEO Mitochondrial myopathy MELAS Mitochondrial myopathy MELAS Maternal myopathy and cardiomyopathy Diabetes mellitus MELAS Diabetes mellitus Ocular myopathy LHON Myopathy MELAS Mitochondrial myopathy Maternal myopathy and cardiomyopathy Fatal infantile cardiomyopathy plus a MELAS-associated cardiomyopathy CPEO CPEO Maternally inherited hypertrophic cardiomyopathy CPEO/multiple sclerosis Maternally inherited cardiomyopathy CPEO Fatal infantile cardiomyopathy plus a MELAS-associated cardiomyopathy Mitochondrial encephalocardiomyopathy Mitochondrial myopathy Myopathy Mitochondrial myopathy Dementia/chorea


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TABLE 1 (Continued) Gene Location tRNAAla tRNAAsn tRNACys tRNATyr tRNASer(UCN)

tRNAAsp tRNALys

tRNAGly

tRNAHis tRNASer(AGY) tRNALeu(CUN) tRNALeu(CUN)

tRNAGlu tRNAThr

mtDNA Mutation T5628C A5692G G5703A A5814G T5874G A7445G G7497A T7510C T7511C T7512C G7543A A8296G G8328A G8342A A8344G A8348G T8356C G8363A T9997C T10010C A10044G G12192A C12258A A12308G T12311C G12315A A12320G T14709C G15915A A15923G A15924G

Diseaseb CPEO CPEO CPEO, mitochondrial myopathy Mitochondrial encephalopathy Exercise intolerance Sensorineural hearing loss Mitochondrial myopathy Sensorineural hearing loss Sensorineural hearing loss MERRF/MELAS, progressive encephalopathy Myoclonic epilepsy and psychromotor trgression Diabetes/deafness/MERRF Mitochondrial encephalopathy PEO/myoclonus MERRF Cardiomyopathy MERRF MERRF Maternal inherited hypertrophic cardiomyopathy Progressive encephalopathy Gastrointestinal reflux/sudden infant death syndrome Maternally inherited cardiomyopathy Diabetes mellitus/deafness CPEO CPEO CPEO Mitochondrial myopathy Mitochondrial myopathy/diabetes mellitus Mitochondrial myopathy Lethal infantile mitochondrial myopathy Lethal infantile mitochondrial myopathy

a

This table is based upon MITOMAP [M15]. MELAS, Mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes; MERRF, myoclonic epilepsy with ragged-red fibers; CPEO, chronic progressive external ophthalmoplegia; LHON, Leber hereditary optic neuropathy; NARP, neurogenic muscle weakness, ataxia, and retinitis pigmentosa. b

6.2. POINT MUTATIONS IN tRNA GENES OF mtDNA A number of distinctive syndromes have been shown to be associated with specific point mutations of mtDNA (Table 1) (M15, S4, S14). Several point mutations have been reported to occur at tRNA genes in the mitochondrial genome. For example, the A8344G mutation is present in patients with MERRF syndrome (S9), whereas the A3243G mutation of mtDNA was first identified in a subgroup of patients with MELAS syndrome (G4). MERRF syndrome was the first

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mitochondrial disease in which a mitochondrial protein synthesis defect was identified as arising from a point mutation in a tRNA gene (S9). It has been reported that over 80% of MERRF patients harbor the A8344G mutation in the tRNALys gene of human mtDNA. A small percentage of MERRF patients harbor a T-to-C mutation at nucleotide pair (np) 8356 of mtDNA (S4). On the other hand, point mutations at np 3243, 3271, and 3291 in the tRNALeu(UUR) gene of mtDNA are associated with MELAS (G4, G5). About 80% of MELAS patients harbor the A3243G mutation of mtDNA in the affected tissues (G4, P1). The other mutations in the tRNALeu(UUR) gene, such as a T-to-C transition at np 3271 of mtDNA, are also found to associate with MELAS syndrome, but with much lower incidence (G5). It has been generally recognized that the relationship between mtDNA genotype and clinical phenotype is not straightforward. The A3243G mutation in the tRNALeu(UUR) gene of mtDNA, which is predominantly associated with MELAS syndrome, may also manifest clinically as CPEO, mitochondrial myopathy, diabetes mellitus, and deafness (P1, S4). 6.3. POINT MUTATIONS IN rRNA GENES OF mtDNA An A1555G mutation in a mitochondrial 12S ribosome RNA gene was reported to be responsible for nonsyndromic hearing impairment (Table 1) (P12). This mutation is homoplasmic and was first detected in affected patients with nonsyndromic sensorineural deafness, but not all individuals carrying such a homoplasmic mutation of mtDNA develop deafness. This A1555G mutation combined with a recessively segregating autosomal gene mutation or exposure to aminoglycosides was proposed to be involved in the pathogenesis of such a type of deafness (E5). On the other hand, we found a novel C3093G point mutation in the mitochondrial 16S rRNA gene in a patient with MELAS syndrome, diabetes mellitus, hyperthyroidism, and cardiomyopathy (H14). This mutation was found to coexist with the A3243G mutation of mtDNA at high proportions in the skeletal muscle of the proband. Interestingly, the C3093G mutation was very low in blood cells and hair follicles of her mother, but was enriched only in the skeletal muscle of the proband. The C3093G mutation was not transmitted to the three sons of the proband, who are currently asymptomatic. Based on the clinical features and the molecular genetics of the proband and her family members, we suggested that the two point mutations in mtDNA caused the disease in a synergistic manner. 6.4. POINT MUTATIONS IN PROTEIN-CODING GENES OF mtDNA On the other hand, several point mutations in the mitochondrial genome affect protein-coding genes (Table 2). The T-to-G or T-to-C mutation in the ATPase 6 gene at np 8993 of mtDNA is one of the pathogenic mutations for Leigh disease (L7, P1, S4). The G11778A, G3460A, and T14484C mutations have been associated with LHON (L7, P1, S4).


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TABLE 2 POINT MUTATIONS IN PROTEIN-CODING GENES OF mtDNA ASSOCIATED WITH MITOCHONDRIAL DISEASESa

Gene Location ND1

ND2

COI

COII

ATP6

COIII

ND3 ND4L ND4 ND4 ND5

ND6

mtDNA Mutation

Amino Acid Change

T3308C G3316A T3394C T3394C G3460A G3496T C3497T G3635A A4136G T4160C T4216C C4640A A4917G G5244A G5920A G6930A G7444A T7587C T7671A G7896A T8993G T8993C T9101C T9176G G9438A G9738T G9804A G9952A T9957C T10191C

Met–Thr Ala–Thr Tyr–His Tyr–His Ala–Thr Ala–Ser Ala–Val Ser–Asn Tyr–Cys eu–Pro Tyr–His Ile–Met Asp–Asn Gly–Ser Trp–TER Gly–TER TER–Lys Met–Thr Met–Lys W–TER Leu–Arg Leu–Pro Ile–Thr Leu–Arg Gly–Ser Ala–Ser Ala–Thr Trp–TER Phe–Leu Ser–Pro

T10663C A11084G G11778A G11832A A12026G G13513A A13514G A13528G G13708A G13730A G14453A G14459A T14484C

Val–Ala Thr–Ala Arg–His Trp–TER Ile–Val Asp–Asn Asp–Gly Thr–Ala Ala–Thr Gly–Glu Ala–Val Ala–Val Met–Val

Diseaseb MELAS NIDDM; LHON; PEO LHON NIDDM LHON LHON LHON LHON LHON LHON LHON LHON LHON LHON Myoglobinuria, exercise intolerance Multisystem disorder LHON Mitochondrial encephalomyopathy Mitochondrial myopathy Mutisystem disorder NARP NARP/Leigh syndrome LHON Leigh syndrome LHON LHON LHON Mitochondrial encephalopathy Progressive encephalopathy; MELAS Epilepsy, strokes, optic atrophy, and cognitive decline LHON MELAS LHON Exercise intolerance Diabetes mellitus MELAS MELAS LHON-like LHON LHON MELAS LHON + dystonia LHON (continues)

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WEI AND LEE TABLE 2 (Continued)

Gene Location

Cyt b

mtDNA Mutation

Amino Acid Change

C14568T G15059A G15150A T15197C G15242A G15257A G15615A G15762A G15812A

Gly–Ser Gly–TER Trp–TER Ser–Pro Gly–TER Asp–Asn Gly–Asp Gly–Glu Val–Met

Diseaseb LHON Mitochondrial myopathy Exercise intolerance Exercise intolerance Mitochondrial encephalomyopathy LHON Exercise intolerance Mitochondrial myopathy LHON

a

This table is based upon MITOMAP [M15]. MELAS, Mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes; CPEO, chronic progressive external ophthalmoplegia; LHON, Leber hereditary optic neuropathy; NARP, neurogenic muscle weakness, ataxia, and retinitis pigmentosa; NIDDM, non-insulin-dependent diabetes mellitus; TER, termination. b

Besides Leigh disease, NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) is another disease caused by the T8993G mutation in the ATPase 6 gene of mtDNA (L7, S4). NARP is characterized by neurogenic muscle weakness, sensory neuropathy, ataxia, and retinitis pigmentosa, and in some cases also may be associated with dementia. Leigh disease is characterized by symmetrical areas of necrosis involving midbrain, basal ganglia, thalamus, pons, and optic nerves (S4). Individuals with the proportion of T8993G mutant mtDNA less than 70% are usually asymptomatic. Leigh disease is associated with a proportion of the mutant mtDNA above 90%. Patients with the proportion of the mutant mtDNA in muscle or blood between 70% and 90% generally develop NARP with various degrees of development delay (S4). LHON has been reported to be associated with mtDNA mutations causing Complex I defect (W2). The primary mutation associated with LHON is a G-to-A mutation in the ND4 gene at np 11778 of mtDNA (S4, W2). The G11778A transition is the most common mtDNA mutation for LHON and accounts for over 50% of all cases. A T3460C mutation in the ND1 gene and a T14484C mutation in the ND6 gene have been reported to be the primary mutations in about 10% of LHON patients (S4). The three primary LHON mutations are each associated with acute or subacute onset of bilateral visual loss, which commonly occurs in young males. The predominance of affected males and the observation that the majority of females with homoplasmic LHON mutation never develop visual impairment suggest that the mtDNA mutation itself is not the only determinant of the disease phenotype (S4).


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Although the number of disease-related mtDNA mutations is increasing, the molecular consequences and the underlying molecular mechanisms in these pathogenic mtDNA mutations remain partially understood. The most intriguing observation is that not only can the same mutation in the same gene (e.g., tRNALeu(UUR) gene) cause different diseases (e.g., MELAS syndrome vs. diabetes mellitus), but also different mtDNA mutations result in the same disease (P1). In fact, little is known about the relationship between genotype and phenotype of mitochondrial DNA disease (P1).

7. Molecular Consequences of mtDNA Mutations In the past decade, a variety of mtDNA mutations have been reported to have pathogenic roles underlying a range of mitochondrial diseases (M15). However, aside from biochemical defects in respiration, the molecular consequences of these mtDNA mutations remain poorly understood. Recently, the consequences of several mtDNA mutations have been extensively studied at the molecular and the cellular levels. The A3243G mutation occurs in the termination-associated sequence (TAS), which is the binding site of mtDNA for the mitochondrial transcription termination factor (mTERF) (C12, K9). The binding of mTERF at this site normally serves to selectively increase the rate of transcription of the upstream 12S and 16S rRNA genes, but not the downstream heavy-strand genes. This thus ensures that sufficient 12S and 16S rRNAs are synthesized for the translation of mRNAs encoding all of the polypeptides in mitochondria. In vitro experiments have shown that the mutation does indeed reduce the affinity of mTEFR for the DNA (C12) and cause a decrease in the rate of termination of the rRNA gene transcription (H5). However, an analysis of the A3243G cybrids did not reveal any significant difference from the controls in the relative steady state levels of the two rRNA species that are encoded upstream of the termination site and of the mRNAs encoded downstream (C12, K3, K5). Moreover, it was suggested that alterations in the processing of RNA 19, an incompletely processed transcript corresponding to the 16S rRNA + tRNALeu(UUR) + ND1 reported to accumulate in A3243G mutant cells (S5), contribute to the reduced rate of synthesis and steady-state levels of mtDNA-encoded polypeptides. Defects in RNA processing (K3) or in the termination of RNA synthesis at the TAS site near the tRNALeu(UUR) gene (H5) have thus been proposed to be responsible for the decrease in respiratory function in affected tissues of patients with MELAS syndrome. However, solid data from in vivo studies are needed to support these notions. Several studies revealed that the A3243G mutation is associated with the decrease in protein synthesis of affected cells (C11, S5). When the ratio of mutant

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mtDNA to total mtDNA exceeded 95%, the protein synthesis activity suddenly decreased (C11, K3). The protein synthesis defect may be due to the reduced level of aminoacyl-tRNA. Mutations in the tRNA gene may affect the stability of the tRNA molecule, resulting in a low steady-state level of aminoacyl-tRNA (B6, C11, Y4). The A3243G mutation occurs at position 14 in the consensus structure of tRNA, and the base at this position is typically involved in the tertiary folding of tRNA. This mutation has thus been proposed to destabilize the tertiary structure of the tRNA molecule, causing it to be more susceptible to nuclease attack. Yasukawa et al. (Y4) reported that both the mutant tRNALeu(UUR) at np 3243 (A-to-G) and that at np 3271 (T-to-C) were markedly unstable in the respective cybrid cells, resulting in a significant decrease of steady-state levels of the tRNA. The total amounts of leucyl-tRNALeu(UUR) with A3243G or T3271C mutations were estimated to be less than 30% that of the wild-type counterpart (Y4). In the study of A8344G mutant tRNALys in MERRF syndrome, it was found that an approximately 50% reduction of aminoacyl-tRNALys leads to severe impairment of protein synthesis and the production of premature polypeptides (E2). Moreover, the half-life of tRNAIle transcribed by the mitochondrial tRNAIle gene with an A4296G point mutation has also been shown to be reduced both in vitro and in vivo (Y2). Furthermore, Chomyn et al. (C11) demonstrated that the A3243G mutation affects both the steady-state level and the aminoacylation efficiency of tRNALeu(UUR). They further suggested that these defects possibly reduce the rate of assembly of mRNA with ribosomes, which leads to a decrease in the rate of mitochondrial translation (C11). However, a number of reports suggest that a decrease in protein synthesis cannot explain the decline in respiratory function or oxygen consumption of the affected cells (D6, F2, H4). In cybrids with homoplasmic T3271C mutant mtDNA, Complex I activity was severely reduced, but protein synthesis was only slightly lower than that of the normal control (H4). On the other hand, posttranscriptional modification of mitochondrial tRNA molecules is essential for the maintenance of structural and functional properties including correct folding, aminoacylation, and codon recognition. Several lines of evidence revealed that mutant tRNALeu(UUR) with A3243G or T3271C mutation (Y4) and tRNALys with A8344G point mutation (Y3) are deficient in a posttranscriptional modification at the anticodon wobble position. In one of the studies of A8344G mutation, it was found that the mutant cybrid cells produce abortive polypeptides and exhibit reduced synthesis of mitochondrial proteins (Y3). Interestingly, despite the fact that mutant tRNALeu(UUR) molecules with A3243G or T3271C mutation carry mutations at different positions, both of them were found to cause deficiency in the modification at the wobble position (Y4). Although the translation rates are maintained at near-normal levels or are only slightly decreased, both of the MELAS-associated mutations lead to the production of abnormal proteins in mitochondria. The deficiency in uridine modification at the wobble position in the mutant tRNALeu(UUR) gene strongly suggests mistranslation


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by these mutant tRNAs according to the mitochondrial wobble rule. Because the wobble modification is presumably essential for the tRNA to decode their cognate codons, the modification defects lead to mistranslation, which has been suggested as a common mechanism in the pathogenesis of mitochondrial diseases caused by a mutation in the tRNA genes of mtDNA. However, the mutant tRNAIle with A4296G mutation was shown to have normal posttranslational modification (Y2). Thus, the posttranscriptional modification deficiency seems not to be a universal phenomenon in all mutations of mitochondrial tRNA genes. The T8993G mutation changes a highly conserved leucine for arginine in the ATPase 6 subunit, which is a component of the proton conduction channel (F0) of Complex V (S3, S4). Although the T8993G mutation was suggested to cause an impairment of the F0F1-ATPase complex possibly through this proton channel defect, the effect of this mutation has not been fully elucidated. The mitochondria isolated from the cybrids carrying T8993G mutant mtDNA were found to exhibit a reduced rate of state 3 respiration. Baracca et al. (B2) demonstrated that the substitution of Leu156 with Arg of F0F1-ATPase subunit a, resulting from the T8993G mutation, causes a reduction in the rate of ATP synthesis in platelet submitochondrial particles. However, both ATPase and ATP-driven proton translocation through F0 were not significantly affected (B2). Recently, Nijtmans et al. (N3) showed that mutant subunit ATPase 6 is associated with impaired assembly of Complex V in human cells. In cybrids harboring mtDNA with a 4,977-bp deletion, it was shown that the respiratory function decreases with an increase in proportion of deleted mtDNA (W6). Moreover, studies of cybrids harboring over 60% of deleted mtDNAs showed a sharp decline in the activities of respiratory enzymes and in the synthesis of mtDNA-encoded proteins (H3). In mouse cybrids with a predominant amount of mtDNA with a 4,696-bp deletion, the deleted mtDNA was shown to be associated with a simultaneous decrease in the activity of cytochrome c oxidase (COX) and in mitochondrial translation (I3). It was suggested that large-scale mtDNA deletions result in impaired mitochondrial translation due to loss of tRNA genes encoded by deleted sequences. However, the synthesis of the fusion proteins in the cybrids harboring a deletion, which eliminates tRNA genes, indicates that the missing tRNAs were transcribed from the wild-type genomes (H3). One model was thus proposed that even though mitochondria with deleted mtDNA miss the deleted tRNA genes, all mtDNA products required for keeping normal structure and function could be provided by mitochondria harboring wild-type mtDNA through mitochondrial fusion and subsequent exchange of their genetic contents (N1). Once the proportion of the deleted mtDNA molecules reaches a critical level, mitochondrial translation may become limiting due to the lack of sufficient tRNAs transcribed solely from remaining wild-type mtDNA. Under such conditions, the translation may be shifted from complementation to competition of the tRNAs in the cells, resulting in the progressive inhibition of overall translation of mtDNA-encoded polypeptides.

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8. Biochemical Basis for How mtDNA Mutations Cause Mitochondrial Dysfunction Deficient respiratory chain function caused by mtDNA mutations could affect various biochemical functions of mitochondria, such as mitochondrial membrane potential, ATP synthesis, ATP/ADP ratio, ROS production, and calcium homeostasis (S14). In cybrids harboring mtDNA with a 4,977-bp deletion, it was shown that the respiratory function decreases with an increase in the proportion of deleted mtDNA (W6). In cybrids containing less than 55% of deleted mtDNA, the ATP synthesis rate and ATP/ADP ratio were found to be maintained at levels similar to those of cybrids with intact mtDNA (P10). Once the proportion of the deleted mtDNA exceeds this threshold, the mitochondrial membrane potential, the rate of ATP synthesis, and the ATP/ADP ratio in the cybrids are dramatically decreased (P10). These findings provided solid evidence to suggest that the bioenergetic deficiencies caused by the accumulation of mtDNA deletions contribute to cellular function defects in the target tissues of patients with mitochondrial disease. In the study of cultured skin fibroblasts harboring mtDNA with A3243G mutation or A8344G mutation, James et al. (J4) demonstrated that mitochondrial membrane potential and respiratory rate are significantly decreased in mutant cells. They showed that the two mutations in mitochondrial tRNA genes lead to the assembly of bioenergetically incompetent mitochondria, resulting in functional defects of Complexes I and IV (J4). According to the observation of a significant increase in the cell volume occupied by secondary lysosomes and residual bodies, they suggested that mitochondrial degradation is increased in the tissue cells of patients with either MELAS or MERRF syndrome (J4). Moreover, the capacity for mitochondrial ATP synthesis and the ability to maintain ATP/ADP ratio were further demonstrated to decline in skin fibroblasts of patients with MELAS or MERRF syndrome as compared to skin fibroblasts from healthy subjects (J3). In addition, it was demonstrated that cells containing mtDNA with A3243G mutation or A8344G mutation are particularly sensitive to increased ATP demand (J3). Interestingly, these findings were further confirmed in cybrids containing different proportions of mtDNA with the A3243G mutation (P2). In a recent study, we demonstrated that cybrids harboring over 90% of the A3243G mutant mtDNA have significantly lower respiratory rate and decreased electron transfer activities, and thereby lower ATP/ADP ratio and reduced energy charge (P2). Importantly, we further demonstrated that the defects in respiratory function elicited by the A3243G mutation of mtDNA cause an increase in oxidative stress as indicated by the decrease in the GSH/GSSG ratio and enhanced oxidative damage to lipids in cybrids harboring high levels of the mutant mtDNA (P2). Moreover, cybrids harboring high proportions of the A3243G mutant mtDNA were found to be much more susceptible to an exogenous oxidant, tert-butylhydroperoxide (P2). Therefore, these


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observations provided important biochemical evidence to suggest that these mitochondrial tRNA mutations disrupt mitochondrial protein synthesis and decrease the electron transfer activities and the energy metabolism of mitochondria. These biochemical defects occur concurrently with a decrease in cellular respiration and decreases in the membrane potential and proton gradient across the mitochondrial inner membrane. Because the proton gradient is the driving force for ATP synthesis, its decrease results in a decline of the maximal rate of ATP synthesis. In addition, the mitochondrial membrane potential is also involved in driving Ca2+ uptake through the Ca2+ uniporter, buffering the transient Ca2+ influxes, and modulating cytoplasmic Ca2+ (G9). Decreasing the mitochondrial membrane potential will certainly affect cellular calcium homeostasis. It was shown that the decrease in mitochondrial membrane potential in MELAS fibroblasts led to elevated intracellular calcium and impaired the ability of the cells to handle excess calcium influx (M16). Moreover, Brini et al. (B9) showed that there is a derangement of mitochondrial Ca2+ homeostasis in cultured cells from MERRF patients, but not in those from NARP patients. Treatment of the MERRF cells with drugs affecting organellar Ca2+ transport restored both the agonist-dependent mitochondrial Ca2+ uptake and the ensuing stimulation of ATP production (B9). These results thus suggested an important role for the perturbation of Ca2+ homeostasis in the pathogenesis of various mitochondrial diseases caused by mtDNA mutations. On the other hand, defective respiratory function elicited by the mtDNA mutation contributes to an increase in the production of ROS and free radicals, thereby causing higher oxidative stress and severe oxidative damage in affected cells (P2, W6). Because either enhanced oxidative stress or disruption of calcium homeostasis is an important factor in the triggering of cell death, mitochondrial dysfunction in tissue cells from MELAS and MERRF patients may contribute significantly to the pathogenesis of these diseases. Histochemical analysis of muscle biopsies from patients with mitochondrial myopathies typically reveals ragged-red fibers on Gomori trichrome staining and some of these fibers stain strongly for succinate dehydrogenase. The ragged-red fibers reflect mitochondrial proliferation, but the trigger for this is not known. These fibers often stain negatively for COX in affected tissues from patients with CPEO, KSS, MERRF, or Leigh disease but stain positively in affected tissues of MELAS patients. Müller-Höcker and co-workers detected even higher densities of cytochrome c oxidase-deficient fibers in the heart muscle of patients with KSS or CPEO syndrome (M18, M19, M20, M21). In some patients with CPEO syndrome, the mitochondria were loosely coupled and the F0F1-ATPase activity was deficient in the skeletal muscle (M21). Recently, Zeviani et al. (Z2) found that the activities of Complexes I and IV are significantly decreased in the endocardial biopsies and cultured cybrid cells of children with idiopathic hypertrophic cardiomyopathy and mitochondrial myopathy. Pitkänen and co-workers (P8) reported severe defects in Complex I and Complexes I + III of the respiratory chain in four patients with

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familial mitochondrial myopathy. Silvestri et al. (S13) revealed a direct correlation between the proportion of the A3243G mutation and defective COX function in single-muscle fibers. Recently, Inoue et al. (I4) described the generation of mice with mitochondrial dysfunction by introduction of mitochondria with mouse mutant mtDNA with a 4,696-bp deletion into mouse zygotes. The results showed that accumulation of the deleted mtDNA induced mitochondrial dysfunction in various tissues. In cross-section studies of skeletal muscles, they found a good correlation between COX deficiency and predominance of the deleted mtDNA (I4). All muscle fibers containing more than 85% of the deleted mtDNA were negative for COX, whereas those with less than 85% were positive for COX. Moreover, heart tissue of the mice with predominantly deleted mtDNA also showed a mosaic distribution of COXnegative and COX-positive cardiomyocytes (I4). This group of investigators further examined the cardiac mitochondria in mouse heart carrying the deleted mtDNA and the results showed that accumulation of deleted mtDNA induced progressive reduction of COX activity and abnormal morphology of mitochondrial cristae (N1). They thus suggested that accumulation of high levels (>91.6%) of deleted mtDNA induces complete reduction of mitochondrial translation due to the lack of six tRNA genes in the 4,696-bp-deleted mtDNA. Moreover, the reduction of all translation products encoded by mtDNA was found to result in abnormal assembly of respiratory enzyme complexes in mitochondrial inner membranes, leading to abnormal structure and function of mitochondria in affected cells (N1).

9. Oxidative Stress and Oxidative Damage Elicited by mtDNA Mutations Under normal physiological conditions, ROS and free radicals (e.g., ubisemiquinone and flavosemiquinone) are generated and maintained at a relatively high steady-state level in mitochondria of tissue cells (B8, F3, N6, T12, W8). It has been shown that the respiratory enzyme Complex I and the protonmotive Q cycle operating in Complex III are the major sites that generate ROS in the respiratory chain (B8, F3, K10, T12, W8). Under nornal physiological conditions, one normal rat liver mitochondrion could produce about 3 × 107 superoxide anions in a day (R2). To cope with the free radicals and ROS by-products generated in aerobic metabolism, human cells have developed a defense system consisting of an array of antioxidant enzymes including MnSOD, copper/zinc superoxide dismutase (Cu/ZnSOD), glutathione peroxidase, and catalase (C3, F3, Y6). MnSOD and Cu/ZnSOD convert superoxide anions to hydrogen peroxide, which is then transformed to water by glutathione peroxidase or by catalase (C3, F3, Y6). Although these enzymes together with other antioxidants can dispose of ROS and free radicals, the fraction of these that may escape these cellular defense mechanisms


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is increased with age or in some diseases (B1, B3, C3, P4, S15, W5, W7). A defect in any of these antioxidant enzymes will lead to an increase of oxidative stress as a result of increased intracellular levels of ROS and free radicals, which may cause oxidative damage to cellular constituents including DNA, RNA, proteins, and membrane lipids (B3, S2, S17). It has been observed that when the respiratory chain is blocked by respiratory inhibitors, the levels of ROS and free radicals generated by mitochondria are elevated (G1, S21). These results are consistent with the observation of an increase in the production of ROS in ρ 0 cells completely lacking mtDNA (M14). Interestingly, it was shown that Complex I deficiency leads to increased production of superoxide anions and induction of MnSOD (L15, P7, P9). The induction of MnSOD is most likely a response of the affected cells to the change in the redox status rather than a result of the Complex I defect per se. Moreover, Luo et al. (L15) provided direct evidence to prove that Complex I deficiency is associated with an excessive production of hydroxyl radicals and lipid peroxides in cultured cells. The resultant damage may contribute to the early onset of cardiomyopathy, cataracts, and death in early infancy in affected patients. Recently, Geromel et al. (G2) further demonstrated that the T8993G point mutation of mtDNA induces overproduction of superoxide anions in cultured skin fibroblasts from two patients with cytochrome c oxidase deficiency. These observations are consistent with earlier work demonstrating that dysfunction in the electron transport chain results in an increase of oxidative stress in the human cell (B1, C3, W5). Recently, we demonstrated that the 8-OHdG content of human cells containing the A8344G mutation of mtDNA is significantly higher than that of control cells under oxidative stress (M1). Moreover, it was observed that skin fibroblasts harboring the A3243G mutation or the A8344G mutation of mtDNA contained more secondary lysosomes and residual bodies, which suggests that the degradative pathways for disposal of waste biological molecules are defective in these cells (J4). The improper accumulation or incomplete degradation of dysfunctional biomolecules is another source of stress imposed on affected tissues. Bandy and Davison (B1) reported that DNA mutations in mitochondria cause an increase in oxidative stress of tissue cells. Using cytoplasmic transfer techniques, we constructed a series of cybrids and demonstrated that both the glutathione disulfide/reduced glutathione (GSSG/GSH) ratio and the lipid peroxide content in cybrids harboring >95% mutant mtDNA were significantly higher than those of control cybrids that contained undetectable mutant mtDNA (P2). Moreover, the specific content of 8-OHdG and lipid peroxides was also demonstrated to be significantly increased in cybrids harboring over 68% of the 4,977-bp-deleted mtDNA as compared with that of the cybrids containing undetectable mutant mtDNA (W6). Therefore, these observations provided strong support to the notion that oxidative damage to vital biomolecules increased as a result of the elevation of the intracellular oxidative stress elicited by mitochondrial disease-associated mtDNA mutations.

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By the use of mice lacking the heart/muscle adenine nucleotide translocator (ANT1), Esposito et al. (E4) showed that mitochondria isolated from skeletal muscle, heart, and brain of mutant mice produced markedly increased levels of H2O2. The absence of ANT1 blocks the exchange of ADP and ATP across the mitochondrial inner membrane, thus inhibiting oxidative phosphorylation. These findings indicate that inhibition of oxidative phosphorylation does increase mitochondrial production of ROS. On the other hand, Melov et al. (M9) reported that mice lacking mitochondrial MnSOD exhibit a tissue-specific inhibition of the respiratory enzymes such as NADH dehydrogenase and succinate dehydrogenase. It was found that accumulation of oxidative DNA damage was enhanced in the skeletal muscle of these mice. MnSOD is an intramitochondrial free-radical-scavenging enzyme, which is the first line of defense against superoxide anions produced as a by-product of respiration. These findings suggest that biochemical defects in the respiratory chain can contribute to the increase of mitochondrial production of ROS, which may play a role in the onset and/or progression of mitochondrial diseases. Once the antioxidant defenses are insufficient to dispose of the ROS, an increase in the incidence and abundance of mtDNA mutations in the affected tissues is expected. Therefore, besides ATP deficiency, alterations in cellular redox status or increased production of ROS also could contribute to the pathogenesis of mitochondrial disease (W6, W7).

10. Mitochondrial Diseases Caused by Mutations in Nuclear DNA A number of recent clinical and molecular biological studies suggested that mitochondrial diseases may be caused by defects in nuclear genes coding for proteins related to or involved in respiration and oxidative phosphorylation (L8, S3, S14, S19, V2). The nuclear genes encode hundreds of proteins, which include structural components of respiratory enzyme complexes, proteins required for the assembly of the respiratory enzyme complexes, factors involved in the biogenesis of mitochondria, and factors and proteins essential for the maintenance of mtDNA copy number or mtDNA integrity (S14, S19, V2). Any mutations in nuclear DNA-encoded respiratory enzyme subunits or defects of nuclear genes coding for proteins involved in the maintenance of mtDNA copy number or mtDNA integrity can result in mitochondrial dysfunction. 10.1. MUTATIONS IN NUCLEAR GENES THAT AFFECT MITOCHONDRIAL RESPIRATORY ENZYMES In 1995, Bourgeron et al. (B7) first reported that mutation in a nuclear gene coding for succinate dehydrogenase may result in mitochondrial disease. In 1998,


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the first mutation in nuclear DNA whose product is a component of Complex I was identified in a patient with Leigh disease (V1). Several disease-associated mutations were also found in nuclear genes encoding subunits of Complexes I and II (V2). Moreover, mutations in the SURF-1 gene, an assembly factor involved in the early phase of COX formation, have been reported in several patients with Leigh disease or COX deficiency (V2). 10.2. NUCLEAR GENE MUTATIONS INVOLVED IN THE MAINTENANCE OF mtDNA COPY NUMBER OR mtDNA INTEGRITY Several mutations of the nuclear genes coding for proteins involved in the maintenance of mtDNA copy number or mtDNA integrity were recently identified in patients with autosomal recessive disorders such as mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and autosomal-dominant progressive external ophthalmoplegia (adPEO) (S19). MNGIE patients may have multiple mtDNA deletions and/or mtDNA depletion. Multiple deletions of mtDNA have also been detected and characterized in adPEO. In these diseases, defects in nuclear genes cause secondary mtDNA loss or large-scale deletions, which in turn lead to mitochondrial dysfunction. Moreover, the features of these diseases resemble those caused by mtDNA mutations, but they are all transmitted in a Mendelian fashion. Mutations of the heart/skeletal muscle isoform of the adenine nucleotide translocator (ANT) type 1 gene on chromosome 4q34 were identified in several patients with adPEO (K2). ANT1 is involved in the control of the mitochondrial nucleoside/nucleotide pool and may play a critical role in the maintenance of mtDNA. Recently, Spelbrink et al. (S16) reported that mutations in the gene coding for Twinkle, a phage T7 gene 4-like protein localized in mitochondria, are associated with mtDNA deletions in some patients with adPEO. This gene codes for a helicase-like protein, and has thus been suggested to play a critical role in the maintenance of mtDNA integrity. As a result of this finding, a small portion of mitochondrial diseases (e.g., adPEO) may be considered as “helicase diseases.” Moreover, mutations in DNA polymerase γ were also reported to associate with PEO (V3). Because DNA polymerase γ is responsible for mtDNA synthesis and repair, mutations in the gene have been identified in a number of patients with MNGIE and adPEO (N4). On the other hand, mutations in the gene coding for thymidine phosphorylase have been identified in some patients with MNGIE and adPEO (N4). Saada et al. (S1) further reported that mutations in the thymidine kinase-2 gene, expressed in mitochondria, represent a new etiology for mtDNA-depletion syndrome. These studies have given new insights into the mechanisms of mtDNA maintenance in mammals. These findings suggest that defect of these nuclear genes that disturb mitochondrial nucleoside/nucleotide pools and/or the replication of mtDNA can affect mtDNA copy number and mtDNA integrity, thereby resulting in mitochondrial dysfunction.

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WEI AND LEE TABLE 3 ANIMAL MODELS OF MITOCHONDRIAL DISORDERS Model

Mitochondrial Defect

MnSOD-deficient mice

Increase in ROS level

Gpx1-deficient mice

Increase in ROS level and in lipid peroxidation level in liver; decrease in liver mitochondrial respiratory control ratio and power output index Impaired mitochondrial transport of ATP/ADP, defect in coupled respiration MtDNA depletion, respiratory chain deficiency MtDNA depletion, respiratory chain deficiency

Ant1-deficient mice

Tfam-deficient mice (germline Tfam knockout) Tfam-deficient mice (Heart and skeletal muscle-specific Tfam knockout) Tfam-deficient mice (β cell-specific Tfam knockout)

Heteroplasmic mtDNA-deletion mice

MtDNA depletion, respiratory chain deficiency

Respiratory chain deficiency

Phenotype

Ref.

Dilated cardiomyopathy; neuronal degeneration of the basal ganglia and brain stem Growth retardation

L11, M9

Mitochondrial myopathy and hypertrophic cardiomyopathy

G6

Homozygous knockouts die at embryonic day 9.5

L3

Dilated cardiomyopathy, atrioventricular heart conduction blocks

W4

Mitochondrial diabetes, impaired stimulus-secretion coupling in β cells in young mice, loss of β cells in older mice Mitochondrial myopathy, kidney failure

S12

E3

I4

10.3. MUTATIONS IN NUCLEAR GENES AND ANIMAL MODELS OF MITOCHONDRIAL DISEASES Using gene-knockout technology, a mouse line lacking the nuclear gene encoding MnSOD was first generated in 1995 (Table 3) (L11). MnSOD is an intramitochondrial free-radical-scavenging enzyme, which is encoded by sod 2 gene in nuclear DNA and is regulated by the redox status of the cell. Li et al. (L11) reported that mice lacking MnSOD died within the first 10 days of life due to dilated cardiomyopathy, accumulation of lipids in liver and skeletal muscle, and metabolic acidosis. A severe reduction in succinate dehydrogenase and aconitase activities of the heart was observed. These results indicate that MnSOD is required for the maintenance of the integrity of mitochondrial enzymes that contain iron or iron–sulfur centers.


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In 1997, Graham et al. (G6) reported that mice lacking the nuclear gene encoding the heart/muscle isoform of ANT1 exhibit a severe defect in mitochondrial respiration, a dramatic proliferation of mitochondria in skeletal muscle, and cardiac hypertrophy. The mutant mice developed mitochondrial myopathy and cardiomyopathy associated with ragged-red muscle fibers, lactic acidosis, and severe exercise intolerance, which are similar to observations in patients with mtDNA diseases (G6, W1). In 1998, Larsson et al. (L3) reported that mice with heterozygous disruption of the nuclear gene encoding the mitochondrial transcription factor A (Tfam) exhibit reduced mtDNA copy number and respiratory chain deficiency in the heart. Tfam is directly involved in the regulation of transcription and replication of mtDNA. It was found that homologous knockout embryos exhibited severe mtDNA depletion with defective respiration and oxidative phosphorylation, and died prior to embryonic stage E10.5 (L3). These findings suggest that Tfam regulates mtDNA copy number and is essential for mitochondrial biogenesis and embryonic development. Moreover, by tissue-specific disruption of the gene encoding Tfam in heart and muscle, Wang et al. (W4) showed that mutant mice developed a mosaic cardiac-specific progressive respiratory chain deficiency, dilated cardiomyopathy, and atrioventricular heart condition block and died at 2–4 weeks of age. This mouse model reproduces biochemical and physiological features of the dilated cardiomyopathy of Kearns–Sayre syndrome. In addition, Silva et al. (S12) demonstrated that mutant mice with tissue-specific disruption of the Tfam gene in pancreatic beta cells developed diabetes from the age of approximately 5 weeks and displayed severe mtDNA depletion and deficiency in respiration and oxidative phosphorylation in islets at the ages of 7–9 weeks. They further noted that the islets isolated from 7- to 9-week-old mutant mice exhibited reduction of the mitochondrial membrane potential, impaired Ca2+ signaling, and decreased insulin release in response to glucose stimulation (S12). This animal model reproduces the beta-cell pathology of mitochondrial diabetes. Because Tfam has a direct role in the regulation of mtDNA gene expression, these animal models have provided evidence that the respiratory chain is critical for normal heart function and insulin secretion. Based on the observation that phenotypes of the mutant mice are similar to those observed in patients with mitochondrial diseases (Table 3), it was suggested that these mice will provide good animal models for future study of pathogenesis and development of potential therapy for mitochondrial diseases (L2).

11. Cybrids for Studies of Mitochondrial Diseases: Applications and Limitations Although point mutations and large-scale deletions of mtDNA have been established to be closely associated with mitochondrial diseases (C9, L1, L7, M15, S4),

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there has been limited evidence that accumulation of these pathogenic mutant mtDNAs in tissues is responsible for the expressions of various clinical phenotypes. There are several limitations in using primary cell lines from patients with mitochondrial disease (e.g., fibroblasts and myoblasts) (J1). They are not immortal, and usually grow very slowly in regular culture media. Some of the cell lines do not retain a fully physiological, differentiated phenotype in culture. In addition, they tend to lose mutant mtDNA as a result of mitotic segregation (J1). Most importantly, one cannot rule out the effect of the nuclear background on the phenotypic expression of mitochondrial dysfunction under examination. Cybrids are trans-mitochondrial cytoplast hybrid cell lines in which patientderived mtDNA is transferred by cytoplast fusion to a tester recipient line (ρ 0 cells) lacking mtDNA. Considering that all these cybrid lines share the same nuclear background, the alteration observed in cybrids should provide a clear-cut answer to the question as to whether accumulation of the mutant mtDNA and resultant expression of mitochondrial dysfunction are responsible for the expression of disease phenotype. Cybrids have been used to confirm the pathogenicity of mtDNA mutations, elucidate the molecular mechanisms by which they impair mitochondrial function, and demonstrate the threshold effect of mtDNA mutation at the cell level (Table 4) (C11, D5, D6, E2, H2, H3, K3, P3, P10, W6). By constructing cybrids in which mutant mtDNAs derived from patients are intercellularly transferred into ρ 0 cells, it was demonstrated that mtDNA mutations are themselves directly responsible for decreased respiratory chain activity and/or oxygen consumption without nuclear gene involvement (D5). On the other hand, cybrids have also been used to demonstrate the important contributions of the nuclear background in mitochondrial diseases and aging (D5, I5). Unfortunately, despite the fact that cybrids have provided useful models for studies of mtDNA diseases, many fundamental questions remain unanswered (J1). They do not retain a fully physiological and differentiated phenotype in culture. It has been shown that glycolysis generates enough ATP for cultured human cells with oxidative phosphorylation deficiency to grow and divide if glucose is available. Any phenomenon observed under this condition cannot be ascribed to a decrease of ATP availability. This makes cultured cells different from human tissue cells, where ATP synthesis by mitochondria is the major source of cellular ATP (J1). Moreover, because cybrids are tumor-derived, they have a risk of exhibiting genetic instability in long-term culture and cannot reproduce the characteristics of differentiated cells in the body (J1). In addition, because of strong selection for efficient growth, genetic or epigenetic changes that mask or modify the mutant phenotype in cybrids can be unwittingly selected (J1). Therefore, to resolve these issues, establishment of animals with pathogenic mutant mtDNA is required. These animals could provide a model for studying


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TABLE 4 CYBRID MODELS OF MITOCHONDRIAL DISORDERS Cybrid Harboring mtDNA Mutation A3243G

T3271C

G5703A

A8344G

tRNALeu(UUR)

tRNALeu(UUR)

tRNAAsn

tRNALys


Ref.

Decrease in mitochondrial protein synthesis rates Increase in amounts of RNA19 Decrease in the steady-state levels of mtDNA-encoded polypeptides Aminoacylation deficiency Reduced association of mRNA with ribosomes Decrease in total amounts of leucyl-tRNALeu(UUR) Deficient in posttranscriptional modification at the anticodon wobble position Lower oxygen consumption rate Decrease in respiratory enzyme activities Lower ATP/ADP ratio Decline in energy charge Decrease in GSH/GSSG ratio Increase in lipid peroxides More susceptible to oxidant stress Decrease in mitochondrial protein synthesis rates Decrease in respiratory function Low Complex I activity Increase in amounts of RNA19 Decrease in total amounts of leucyl-tRNALeu(UUR) Deficient in posttranscriptional modification at the anticodon wobble position Severe defects in oxidative phosphorylation function Decrease in mitochondrial protein synthesis rates Decrease in the steady-state levels of tRNAAsn Decrease in the stability of the tRNAAsn secondary or tertiary structure Severe defects in respiratory chain activity Decrease in mitochondrial protein synthesis rates Decrease in the steady-state levels of mitochondrial translation products Decrease in total amount of aminoacyl-tRNALys

C11, K4, K6 K4, K6 K4 C11 C11 Y4 Y4 D5, P2 D5, K4, K6, P2 P2 P2 P2 P2 P2, W9 H4, K6 K6 H4 K6 Y4 Y4 H1 H1 H1 H1 M8 M8, Y3 M8 E2

(continues)

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WEI AND LEE TABLE 4 (Continued)

Cybrid Harboring mtDNA Mutation

T8356C


tRNALys

T8993G

ATPase 6

G3460A

ND1

G11778A

ND4

4,977 bp-deletion

Deficient in posttranscriptional modification at the anticodon wobble position Increase in 8-OHdG content More susceptible to oxidant stress Severe defects in respiratory chain activity Decrease in mitochondrial protein synthesis rates Decrease in the steady-state levels of mitochondrial translation products Decrease in ATP synthesis capacity Impaired ATP synthase assembly Decrease in ATP synthase stability Reduced state 3 respiration Reduced ADP/O ratio Decrease in the maximal respiration rate Decrease in Complex I activity More sensitive to Fas-induced apoptosis Decrease in oxygen consumption rate More susceptible to oxidant stress More sensitive to Fas-induced apoptosis Decrease in mitochondrial membrane potential Decrease in ATP synthesis rate Decrease in ATP/ADP ratio Decrease in respiratory enzyme activities Increase in 8-OHdG content Increase in lipid peroxides

Ref. Y3 M1 W9 M8 M8 M8 N3 N3 N3 T11 T11 B11 B11 D2 B11, V4 W9 D2 P10 P10 P10 W6 W6 W6

exactly how pathogenic mtDNA mutations cause mitochondrial dysfunction in various tissues. Recently, cybrid technology has also provided a system for introducing or selecting for specific mtDNA mutations in the whole organism (I4). Mouse mtDNAs with mutations accumulated in somatic tissues or cells were trapped into ρ 0 cells for isolation of mtDNA-repopulated cybrids (I3). Inoue et al. (I4) obtained mice carrying mouse mtDNA with a 4,696-bp deletion by electrofusion of fertilized mouse eggs with enucleated cybrids. They found that the introduced deleted mtDNA was transmitted maternally, and its accumulation induced mitochondrial dysfunction in various tissues. This is the first animal model for studies of mitochondrial diseases caused by large-scale deletions of mtDNA. Although the first mouse model of mtDNA disease has been established (I4), it is noteworthy to point out that some observations from the animal model of mtDNA disease are different from the features of the disease phenotype. The


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transmission of deleted mtDNA through the female germline to the following generations is rarely seen in patients with pathogenic deleted mtDNA. Moreover, a large amount of deleted mtDNA was found in mitotic tissues of mutant mice, but human mitotic tissues usually do not accumulate deleted mtDNA. Furthermore, most of these mice died because of kidney failure (I4). Kidney failure is not a typical symptom of mitochondrial disease, though it has been reported in several patients with mtDNA deletions. This raises interesting questions about the genetics of pathogenic mtDNA mutations, which should be addressed in future investigations of the animal model (L2). In theory, this type of mouse model should offer an opportunity for researchers to better understand the pathogenesis of specific mutations in mtDNA and defect in the mitochondrial respiration and oxidative phosphorylation system and to test therapeutic strategies for the development of treatments for mitochondrial disease (L2).

12. Effect of mtDNA Mutations on the Apoptosis of Human Cells Apoptosis, or programmed cell death, is an evolutionarily conserved mechanism essential for morphogenesis, development, and tissue homeostasis (H6). Recently, it was proposed that apoptosis plays an important role in some pathological conditions, including neurological disorders (S18). Moreover, dysregulation of apoptosis has also been implicated in the pathogenesis of various human diseases such as cancer and autoimmune and neurodegenerative disorders (T9). Mitochondria have been established as playing a critical role in the triggering and mediation of apoptosis (G7, K8, L6). Translocation of proapoptotic proteins to mitochondrial outer membranes, opening of permeability transition pores, and release of cytochrome c and other proapoptotic proteins from mitochondria have been demonstrated to precede nuclear DNA fragmentation and apoptosis (K8, L6). Thus, it is highly plausible that apoptosis is a contributory factor to the pathogenesis of mitochondrial diseases (R1). Recently, Mirabella et al. (M13) reported the presence of DNA fragmentation and the expression of apoptosis-associated proteins (Fas, p75, and caspase-3) in muscle biopsies of patients with point mutations and deletions of mtDNA. They found that in patients carrying single mtDNA deletions or point mutations in tRNA genes (tRNALys, tRNALeu(UUR), tRNAIle , and tRNATrp), the degree of apoptosis in muscle biopsies matched the number of mutated genomes and the severity of both mitochondrial myopathy and the neurological phenotype of the patients. However, only modest or no signs of apoptosis were observed in muscle of patients with mitochondrial diseases associated with point mutations in structural genes, such as NARP and LHON, in spite of the presence of high proportions of the mutated genomes (97–100%) (M13). Moreover, COX-negative muscle fibers were found to harbor more apoptotic nuclei than COX-positive muscle fibers (M13).

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COX-negative fibers together with ragged-red fiber (RRF) in muscle are considered the hallmarks of mitochondrial myopathy (S4). COX deficiency is one of the most common enzymatic defects in patients with mitochondrial diseases irrespective of the presence or absence of a known mutation in the mitochondrial or nuclear genome (S4). Recently, Di Giovanni et al. (D4) further showed that in patients with cytochrome c oxidase deficiency a variable number of muscle fibers exhibited apoptotic nuclei that matched with the level of respiratory enzyme reduction and roughly correlated with muscle weakness. However, no signs of apoptosis were observed in patients with deficiencies of Complexes I and II and without muscle weakness (D4). These observations suggest that apoptosis has a potential pathological role in the mitochondrial diseases associated with COX deficiency. On the other hand, Sciacco et al. (S6) recently reported that there was no apoptosis in muscle fibers and no increase in the expression of both Fas and Bcl-2 apoptosis-related proteins in 33 muscle biopsies from patients with genetically different mitochondrial disorders. However, all their patients presented only mild to moderate muscle weakness. It is possible that the impairment in energy metabolism in muscle of these patients was not severe enough to reach the threshold of mitochondrial defect needed to trigger apoptosis. Massive apoptosis was observed in cultured skin fibroblasts harboring over 90% of the T8993G mutant mtDNA (G2). Moreover, oxidative stress induced by superoxide anions was found in fibroblasts harboring the T8993G mtDNA mutation in the ATPase 6 gene. Interestingly, apoptosis can be abolished by treatment with perfluoro-tris-phenyl nitrone (TAPBN), an antioxidant spin-trap molecule (G2). These observations suggest that overproduction of superoxide anions associated with the T8993G mutation of mtDNA is sufficient to trigger apoptosis. This superoxide anion-induced apoptosis may be involved in the pathogenesis of the T8993G mutation of mtDNA in brain of patients with NARP or Leigh disease characterized by symmetrical bilateral necrotic lesions in the brain stem, basal ganglia, thalamus, and spinal cord. It has been demonstrated that cells lacking mtDNA (ρ 0 cells) can undergo apoptosis (J2, J8, M5). Interestingly, it was demonstrated that cybrids with a high proportion of mtDNA carrying a single deletion or a point mutation are very sensitive to cell death triggered by Fas (D2) or oxidative stress (H8, W9). Recently, Wang et al. (W3) provided in vivo evidence that respiratory chain deficiency predisposes cells to apoptosis. They found massive apoptosis in Tfam-knockout embryos at embryonic stage 9.5, and apoptosis was increased in the heart of the tissue-specific Tfam-knockout mice (W3). Moreover, the human osteosarcoma (143B)-derived, mtDNA-less ρ 0 cells were found to be more susceptible to apoptosis induced by different stimuli in vitro, such as anti-Fas antibody, tumor necrosis factor-α, and staurosporine (H8, W3). These results suggest that an increase in cell apoptosis is a pathogenic event involved in the pathogenesis of mitochondrial diseases associated with mtDNA mutations. However, an osteosarcoma-derived ρ 0 cell line


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was recently reported to be less susceptible to apoptosis under some conditions (D3). More systematic studies are warranted to evaluate the roles that mtDNA and mtDNA mutations play in apoptosis associated with mitochondrial diseases.

13. Therapy for Mitochondrial Diseases Despite major advances in our understanding of mitochondrial diseases, no effective therapy is available. Several pharmacological agents have been used to treat mitochondrial diseases (C10, T6). Although some improvements have been reported in isolated cases, a lack of response has also been reported in some patients for each of the agents. Due to the critical role of the respiratory chain in energy metabolism, it is difficult to develop effective biochemical and pharmacological therapies to bypass the defect in the respiratory chain caused by a pathogenic mtDNA mutation. Treatment for the majority of patients with mitochondrial disease is primarily supportive in nature. Compounds such as riboflavin, α-tocopherol (vitamin E), ascorbate (vitamin C), menadione (vitamin K3), succinate, and nicotinamide have been used to bypass defects in the respiratory chain (A7, B4, L13, M2, M17, O2, P6, S10). Menadione and ascorbate accept electrons from reduced coenzyme Q10 (CoQ10H2) and transfer them to cytochrome c. Although there is no hard evidence of a convincing clinical benefit, these agents have been used for some time and might be theoretically beneficial to patients and rarely cause significant side effects (C9). These artificial electron acceptors could improve symptoms in patients with respiratory chain disease. On the other hand, coenzyme Q10 (CoQ10) has also been used for treating patients with mitochondrial disorders (A1, A2, C2, C5, I1, I2, L9, L12). CoQ10 is an essential component of the respiratory chain, and functions as a mobile electron transfer component between membranous flavoprotein dehydrogenases and Complex III in the inner mitochondrial membrane. In addition to serving as an electron transporter in the respiratory chain, CoQ10 has another function as a free radical scavenger. Treatment with CoQ10 has been reported to improve the symptoms, cerebrospinal fluid (CSF) lactate levels, and cardiac conduction in some patients with mitochondrial myopathies (A1, A2, C2, C5, I1, I2, L9). Potential benefits from CoQ10 were suggested for nonneurological features of mitochondrial disease (L12). Moreover, CoQ10, menadione, ascorbate, and superoxide dismutase-mimetic manganese-5,10,15,20-tetrakis (4-benzoic acid) porphyrin (MnTBAP) have been shown to mediate their antioxidant effects on the ROS and the oxidative stress involved in neuronal death in mitochondrial diseases (S4, T6). There is increasing evidence to support the role of ROS in cell death (R1), and antioxidants appear to have benefits in animal models of mitochondrial disease (W1). For example, Melov et al. (M9) reported that mice lacking mitochondrial MnSOD exhibited a

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tissue-specific inhibition of the respiratory-chain enzyme NADH dehydrogenase and succinate dehydrogenase and increased accumulation of oxidative DNA damage. Defective MnSOD in these mice resulted in dilated cardiomyopathy, hepatic lipid accumulation, and early neonatal death (M9, M10). Interestingly, the investigators further reported that treatment with the antioxidant MnTBAP was able to prevent the mice lacking mitochondrial MnSOD from developing cardiomyopathy and dramatically prolonged their survival (M10). However, these mutant mice then developed neurological phenotypes similar to those observed in patients with mitochondrial abnormalities such as Leigh disease. They thus proposed that progressive neuropathology is caused by excessive mitochondrial production of ROS due to the failure of MnTBAP to cross the blood–brain barrier (M10). This MnSOD–knockout mouse model may serve as a useful system for screening new drugs for treating mitochondrial disorders. Some therapeutic strategies that selectively inhibit the replication of mutant mtDNA are being investigated to give the wild-type genome a distinct replicative advantage. It was recently reported that oligomycin induces a decrease in the cellular content of a pathogenic mutation in the ATPase 6 gene of human mtDNA (M4). Moreover, synthetic polyamide nucleic acid or peptide nucleic acids (PNAs), which are complementary to a short mtDNA sequence with a point mutation or a deletion breakpoint, have been used as antisense probes to selectively inhibit replication of mutant mtDNA (T7, T8). Selective inhibition of mutant mtDNA replication has been demonstrated in vitro by sequence-specific antigenomic PNAs (T7, T8). In addition, it has been reported that PNAs can be taken up by cultured human cells and successfully imported into mitochondria in intact cells by the addition of the presequence peptide of the nuclear-encoded subunit VIII of human cytochrome c oxidase (C7). This approach may reduce the level of mutant mtDNA below the critical threshold and thereby correct the biochemical defects in the affected tissues of patients harboring the mutant mtDNA. A number of alternative strategies have also been investigated with the aim of correcting for mtDNA defects. A method is being developed in which modified genes or gene products are introduced into mitochondria via the protein import machinery to complement a defect in mtDNA gene. Kolesnikova et al. (K6) recently reported that modified cytoplasmic tRNAs can be specifically targeted to mitochondria and participate in mitochondrial translation in yeast. They suppressed the mutations in mtDNA by tRNAs imported from the cytoplasm. This will provide a possible strategy for treatment of mitochondrial tRNA gene disorders. An alternative strategy is to express the mtDNA protein-encoding gene within the nucleus. Recently, Manfredi et al. (M3) successfully expressed wild-type ATPase 6 protein allotopically from nucleus-transfected constructs encoding an amino-terminal mitochondrial targeting signal appended to a recoded ATPase 6 gene (made compatible with the universal genetic code). The recoded and allotopically expressed mtATPase 6 was found to be able to rescue a deficiency in


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ATP synthesis in cybrids harboring homoplasmic mtDNA with the T8993G point mutation. In addition, it was revealed that mutant mtDNAs are rare or undetectable in satellite cells cultured from the muscle biopsy specimens of patients with largescale deletions or those with tRNA point mutations of mtDNA (S11). Because satellite cells are responsible for muscle fiber regeneration, promoting muscle regeneration might be useful for restoring wild-type mitochondrial genomes into disease muscle fibers and for correcting biochemical defects. Based on the use of a biopsy procedure (S11) as well as a short period of concentric exercise training (T2), the enhanced incorporation of satellite cells led to a remarkable increase in the ratio of wild-type to mutant mtDNA and in the proportion of muscle fibers with normal respiratory-chain function. This has provided a novel strategy for improving the bioenergetic and the physiological functioning of muscle in patients with mtDNA mutations.

14. Concluding Remarks In the past decade, point mutations and large-scale deletions of the mitochondrial genome have been shown to be associated with or responsible for many respiratory-chain disorders. A portion of patients with so-called “mitochondrial diseases” manifest mitochondrial myopathy, either alone or in combination with other multisystem disorders. The clinical spectrum of these diseases has expanded from the classical mitochondrial myopathies to disorders as diverse as mitochondrial encephalomyopathy, cardiomyopathy, endocrinopathy, hearing impairment, and diabetes mellitus. The involvement of mtDNA mutation in the pathogenesis of some of these diseases can explain many of the characteristic features of these mitochondrial disorders, for instance, maternal transmission, varying degrees of multisystem involvement, and wide range of clinical manifestations with different degrees of severity. However, in practice it is very difficult to differentiate mitochondrial myopathies caused by point mutation of mtDNA from those caused by mtDNA deletion or nuclear DNA mutation. Even more intriguing is that either idiopathic dilated or hypertrophic mitochondrial cardiomyopathy can be caused by or associated with different point mutations in tRNA or rRNA genes of the mitochondrial genome. Moreover, cardiomyopathies are developed at a later stage in some of patients with mitochondrial diseases and are manifested as one of the secondary symptoms or multisystem disorders. This agedependent progression (deterioration) of the diseases may be caused or aggravated, at least partly, by the aging-associated mtDNA mutation and oxidative damage to vital biomolecules in the target tissues of patients. The primary pathogenic mtDNA mutation(s) in the affected tissues not only causes respiratory-chain defects and insufficient ATP production, but also elicits the elevation of oxidative

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stress as a result of enhanced electron leakage and ROS production of the defective electron transport chain. Therefore, patients with mitochondrial myopathy usually undergo a rapid clinical course and exhibit premature aging, often dying before early childhood. Because mitochondria play a key role in the initiation and execution of apoptosis of human cells, several investigators have examined the involvement of apoptosis in the pathogenesis of mitochondrial diseases. Most studies showed that human cells harboring mtDNA mutations are more susceptible to apoptosis compared with controls. It is noteworthy that in patients carrying single mtDNA deletions or point mutations in tRNA genes (tRNALys, tRNALeu(UUR), tRNAIle , and tRNATrp) the degree of apoptosis in muscle biopsies was found to match the number of mutated mtDNAs and the severity of both mitochondrial myopathy and the neurological phenotype of the patients (M13). We also demonstrated that skin fibroblasts and cybrids harboring a 4,977-bp deletion of mtDNA are more susceptible to apoptosis under ultraviolet irradiation and treatment with proapoptotic agents such as staurosporine and hydrogen peroxide (H8). Based on recent advances in molecular and cellular biological studies, we suggest that mtDNA mutation and a small number of nuclear DNA mutations cause respiratory-chain dysfunction and generate more ROS and free radicals in inner mitochondrial membranes. This not only results in lower efficiency of oxidative phosphorylation and increase in oxidative stress, but also leads to cell apoptosis of affected tissues, which may also play an important role in the pathophysiology and the progression of some, if not most, mitochondrial diseases. With this integrated view of the pathogenesis of mitochondrial diseases in mind, treatment of patients may include redox therapy, paced exercise training, and genetic manipulations at the early embryonic stage or on the affected tissues of adult patients. ACKNOWLEDGMENTS Part of the work reported in this chapter was supported by research grants from the National Science Council (NSC90-2320-B010-078 and NSC90-2320-B010-079) and the National Health Research Institutes (NHRI-EX91-9120BN) of the Republic of China.

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H3.

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AUTOANTIBODIES TO dsDNA, Ro/SSA, AND La/SSB IN SYSTEMIC LUPUS ERYTHEMATOSUS Jien-Wen Chien∗ and Ching-Yuang Lin† ,‡ † Department of Pediatrics, Changhua Christian Hospital, Changhua, 500, Taiwan ‡ National Yang-Ming University, School of Medicine, Taipei, Taiwan

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. History of Systemic Lupus Erythematosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Treatment and Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Basic Studies of Autoantibodies to dsDNA, Ro/SSA, and La/SSB. . . . . . . . . . . . . . . . . 2.1. Mechanism of Self-Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. History of Autoantibodies to dsDNA, Ro/SSA, and La/SSB . . . . . . . . . . . . . . . . . 2.3. Molecular Structures of Anti-dsDNA, Anti-Ro/SSA, and Anti-La/SSB Autoantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Mechanism of Anti-dsDNA, Anti-Ro/SSA, and Anti-La/SSB Autoantibody Production in Patients with SLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Direct Cytotoxic Effects of Anti-dsDNA, Anti-Ro/SSA, and Anti-La/SSB Autoantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Clinical Aspects of Anti-dsDNA, Anti-Ro/SSA, and Anti-La/SSB Autoantibodies. . . . 3.1. Assays for Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Anti-dsDNA Autoantibodies and Lupus Nephritis . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Clinical and Serological Correlations of Anti-Ro/SSA and Anti-La/SSB Autoantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Discussion and Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Systemic lupus erythematosus (SLE) is a multisystemic disease characterized by a wide variety of autoantibodies leading to highly heterogeneous clinical manifestations. The target antigens of these autoantibodies include autoantigens in 129 Copyright 2003, Elsevier Science (USA). All rights reserved. 0065-2423/03 $35.00

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extracellular space, in intracellular cytoplasm, or inside the nucleus. These various autoantibodies, produced via several putative mechanisms, lead to various kinds of organ damage. In this chapter we review recent progress on three of the best-studied autoantibodies of SLE: anti-double-stranded DNA (anti-dsDNA), anti-Ro/SSA, and anti-La/SSB. 1.1. HISTORY OF SYSTEMIC LUPUS ERYTHEMATOSUS How an ancient Roman family name, Lupus (“wolf”), came to have a disease association is obscure (D8, pp. 515–516). The earliest known medical use of “lupus” appeared in a 10th-century biography of St. Martin (S24). Hundreds of years passed before several authors first recognized the varied components of SLE (B10). The first major event of the modern study of the disease was the development of the lupus erythematosus cell test by Hargraves in 1949 (H4); it was followed by the introduction of the immunofluorescent antinuclear antibody test in 1958 (F13). The preliminary criteria for the classification of SLE were first published in 1971 (C21) and were revised in 1982 (T1). The third revision with two changes was completed in 1997 (H12). 1.2. EPIDEMIOLOGY Cases of the disease show a female preponderance of 8:1, which is thought to be due to a synergistic effect of female hormones (L16). The overall prevalence of SLE ranges from 14.6 to 50.8 cases per 100,000 people (H10), although a study in the United States has put the prevalence as high as 1 in 1,177 people (L8). Prevalence data from worldwide studies range from 12.5 to 39 cases per 100,000 people in White populations. Surveys from China show a prevalence of 40–70 cases per 100,000 people (H10). There are also racial differences in the incidence of SLE, ranging from 31/100,000 among Asian females to 4.4/100,000 in White females, with Black females falling in between at 19.8/100,000 (L16). Published studies from Sweden, England, Japan, and Curacao Caribbean island of showed average incidence rates of 5.8, 6.8, 5.3, and 7.9 cases per 100,000, respectively, for women (H13). The reasons for these geographic differences in prevalence and incidence are unclear. The differences may be due to genetic, environmental, or other host factors. 1.3. PATHOGENESIS 1.3.1. Immunology SLE is the prototype systemic autoimmune disease, with a hallmark of autoantibody production. The mechanism leading to loss of self-tolerance and production of autoantibodies is discussed in Section 2.

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1.3.2. Genetics The importance of genetics in the pathogenesis of SLE in humans has been explored in two ways: association and linkage. The first association study was done before 1971 and revealed an association between the HLA region of HLA-B8 and lupus in humans (G15). Later data from northern Europe indicated that the association was closer with DR3 than it is with B8 (J3). The effects of other genes apart from the B8–DR3 haplotype are described in more than 100 publications (S9). The association between HLA alleles and the level of various autoantibodies has also been studied (S9). Anti-Ro and anti-La are powerfully associated with DR3/DR2 heterozygotes at the DQ alleles. Anti-Sm and anti-nRNP tend to be associated with alleles on haplotypes containing a DR4 or a DQ3 allele. The HLA association of antibodies against DNA appears to be less consistent through many studies. The strong association between human lupus and a deficiency of the early components of the classic complement system provides valuable guidance in the study of the pathogenesis of SLE. Complete deficiency of C1q is almost always associated with lupus (over 90%) (S19). Defects in each of the three C1q peptide chains encoded by three genes in tandem on chromosome 1 have been described in human lupus. Seventy-five percent of patients with complement deficiency of C4 have lupus. The deficiency of any of four alleles of the two C4 genes, C4A and C4B, is associated with lupus. The deficiency of C4A appears to be the closest association (F4). About 33% of European patients with C2 deficiency also have lupus (W2). These patients also tend to have a higher frequency of anti-Ro autoantibody (P6). Beside complement genes, many members of the family of Fcγ receptors (Fcγ Rs) also have been associated with lupus. These members include Fcγ RIIIB (C19, E7), Fcγ RIIA (S25), and Fcγ RIIIA (W15). In the case of Fcγ RIIA, an arginine substitution for histidine in amino-acid position 131 results in reduced affinity for immunoglobulin G2 (IgG2) and is associated with lupus in both African Americans and Koreans (S25). In Fcγ RIIIA receptors, alanine at amino-acid position 176 instead of phenylalanine results in higher binding affinity of natural killer cell receptors for IgG1 and IgG3 (W15). Examples of other genes found to be associated with lupus include that for interleukin-10 (IL-10) (E9, T8) [which also has been associated with anti-Ro production (L8)] and the fas gene [which is responsible for the MRL lpr/lpr mouse model of lupus, but rarely occurrs in humans (W16)]. The first linkage study in human lupus was done by Bias et al. (B15) using poorly informative markers. More recent studies revealed a possible linkage on chromosome 1 (H5, T10). Genome scan studies from Minnesota (G1) and Oklahoma (M23) revealed 16 and 12 possible linkages, respectively. The two studies have been reviewed by Harley et al. (J3).

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Young-Sun and Edward reviewed the genetics of a murine lupus model and presented a three-step pathogenic pathway model of lupus pathogenesis (Y6). By using their three congenic strains—B6.Sle1, B6.Sle2, and B6.Sle3—they found that the pathogenesis of lupus nephritis is a complicated, multistep process resulting from multiple abnormalities within the various checkpoints of a three-step pathogenic pathway. These abnormalities include genes with effects on apoptosis, cytokine production, signal transduction, inflammatory mechanisms, and costimulation within the immune system. Just as with murine models, knowledge of the genetics of human lupus also provides hints concerning the mechanism of autoantibody production in SLE. This will be discussed in Section 2. 1.3.3. Environmental Factors Two of the best-recognized environmental factors are ultraviolet radiation (UVR) and viral infection. UVR exposure from sunlight is the best-established environmental factor in both induction and exacerbation of SLE. Cutaneous lesions tend to occur in sun-exposed areas of lupus patients. Experimental reproduction of cutaneous lupus lesions by exposure to UVR has been reported in both animals and humans (L17, N2). UVR can stimulate the cell surface expression of nuclear antigens, including Ro, on the surface of keratinocytes, as well as induce E-selectin and intercellular adhesion molecule-1 (ICAM-1) expression on dermal endothelial cells, which leads to the margination and local migration of lymphocytes (M17). Circulating anti-Ro binds keratinocytes that express this surface Ro antigen, leading to antibody-dependent cellular cytotoxicity by infiltrating cytotoxic lymphocytes and the subsequent destruction of basal keratinocytes (N3). Viral infection is a possible triggering factor. Various viruses are known to be associated with SLE, including myxoviruses, reoviruses, measles, and rubella (A22). The pathogenesis of UVR and viruses in generating autoantibodies will be discussed in a later section. 1.4. TREATMENT AND PROGNOSIS There is no cure for SLE. Guillermo et al. (G16) found only 10 randomized controlled trials during the past 5 years, 5 for lupus nephritis and 5 for all SLE patients. Compared to conventional therapies for lupus nephritis, a monthly bolus with intravenous cyclophosphamide is more effective than a monthly bolus with methyl prednisolone, but has significant side effects (including amenorrhea, cervical dysplasia, avascular necrosis, and herpes zoster) in both groups (G14). In order to avoid these side effects, more recent therapies have been developed. However, neither plasmapheresis (W1), intravenous immunoglobulin (B21), recombinant human DNase (rhDNase) (D3), nor mycophenolate mofetil (C14) was shown to be more effective than conventional therapy. However, some of these


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studies included only a small number of patients and limited follow-up duration, and further evaluation is necessary. There are also therapies based on knowledge of the pathogenesis of SLE, mainly for the removal of anti-dsDNA autoantibodies. We will discuss these therapies in a later section. The prognosis of SLE has much improved during recent decades. The overall 10-year survival rate in retrospective series is 75–85% (U1). The major cause of early death is usually active disease, whereas the leading cause of late death is atherosclerosis (A2). Infection is a major cause of mortality in all stages of SLE (G16). In our experience with SLE in children, the 5-year renal and patient survival rates were 93.1% and 91.08%, respectively (Y4). Several features of SLE have been associated with mortality in a multivariate model (A3). These features include renal damage, thrombocytopenia, very active disease at presentation, and lung involvement. Despite the large decrease in the mortality of SLE patients, there are still many issues to be resolved (G16).

2. Basic Studies of Autoantibodies to dsDNA, Ro/SSA, and La/SSB 2.1. MECHANISM OF SELF-TOLERANCE The key to the highly efficient ability of vertebral lymphocytes to detect numerous foreign antigens is the possibility of generating autoantibodies. In normal individuals, the immune system has evolved tolerance mechanisms to discriminate between self and non-self molecules to avoid self-reactivity. These tolerance mechanisms (Table 1) can be classified into two broad categories according to the location where they act. Central tolerance acts at the early stage of the development of both T lymphocytes (in thymus) and B lymphocytes (in bone marrow), whereas peripheral tolerance includes a panel of different strategies of the immune system to prevent the generation of an active immune response against self or usually harmless environmental proteins. The major mechanism of central tolerance is TABLE 1 MECHANISMS OF SELF-TOLERANCE Central

Peripheral

Deletion Receptor editing

Deletion Anergy Indifference Interclonal competition Follicular exclusion

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clonal deletion. Interaction between potential self-active lymphocytes with a high concentration of multivalent self-antigen can induce a strong signal to them and result in physical elimination of these lymphocytes by means of apoptosis (N3). Compared with clonal deletion, receptor editing plays a minor role. Tiegs et al. (T5) found that transgenic bone marrow B cells encountering membrane-bound neoself-antigen modify their receptors by expressing the V(D)J recombinase activator genes and assembling endogenously encoded immunoglobulin variable genes. This autoantigen-directed change in the specificity of newly generated lymphocytes is termed receptor editing. Because the entire repertoire of T cell-receptor (TCR) specificities is generated within the thymus and the TCR sequence on a given T cell does not change once that cell has emigrated from the thymus (D2, C17), the central mechanism of self-tolerance is the major mechanism of T cell tolerance. Notwithstanding the central mechanism of clonal deletion just mentioned, there is abundant evidence that self-reactive T and B lymphocytes persist in normal hosts (B29). These lymphocytes escape from central clonal deletion probably due to their low affinity to self-antigen or lack of the expression of the self-antigen in thymus or bone marrow. After “leaking” from their original site of formation, these selfreactive lymphocytes must be either deleted or tolerated by the host via a peripheral tolerance mechanism. Clonal deletion also plays a role in peripheral tolerance. Administration of large amounts of soluble antigen to normal or transgenic mice was shown to lead to rapid apoptotic death in germinal-center B cells with high affinity for the antigen (H2, P9, S12). Another mechanism of peripheral tolerance, clonal anergy for B cells, was demonstrated by experiments with a doubletransgenic mouse encoding both hen egg lysozyme (HEL) and anti-HEL (G11). There were detectable peripheral functionally inactivated lymphocytes (producing anti-HEL antibodies) that failed to proliferate and respond to antigenic stimuli (HEL, the neo-self-antigen). IgM on B cells was markedly downregulated due to posttranslational changes involving selective retention and degradation of IgM in a pre-Golgi department (B8). A threshold receptor occupancy by self-antigen of between 25% and 50% is required to induce the phenotypic and functional changes associated with anergy (H6). The life span of the anergic B cells was markedly reduced to 3–4 days compared with 4–5 weeks for nontolerant immunoglobulin (F15). However, the anergic state is reversible, as demonstrated by upregulation of IgM and antibody production after repeated exposure to T cell helper or T-independent stimuli such as lipopolysaccharide (LPS) (A4). Clonal anergy is also involved in T cell tolerance. Mechanisms of clonal anergy of T cells include (1) absence of B7-mediated costimulation (S4, M10), (2) use by T cells of the CTLA-4 receptor rather than CD28 to recognize B7 molecules (P2), and (3) coexpression of the death receptor, Fas (CD95), and its ligand, FasL, by repeatedly encountering persistent self-antigens (V5, N1). In contrast to clonal anergy, clonal indifference (ignorance)


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TABLE 2 POSSIBLE DISRUPTION OF SELF-TOLERANCE IN PATIENTS WITH SLE Abnormal B cell tolerance Abnormal central deletion Abnormal maintenance of peripheral B cell anergy Selective production of autoreactive B cells by the stimulation of prolonged abnormal autoantigens Abnormal T cell tolerance Abnormal central deletion Abnormal T cell anergy Increased activation of T helper cells Abnormalities in regulatory T cells Other immune abnormalities Increased autoantigen translocation in skin after UV radiation Increased apoptotic waste-containing autoantigens Defect of early components of classic pathway of complement system Exposure to environmental antigens with molecular mimicry

results in functionally normal self-reactive lymphocytes. However, these lymphocytes are unaffected by the presence of self-reactive antigens (O4). The mechanism of self-ignorance may include (1) anatomic sequestration of the self-antigen from immunocompetent lymphocytes or (2) presentation of the antigen to lymphocytes in the absence of second signals (V6). Interclonal competition and follicular exclusion involve mechanisms between existing and newly formed clones of cells for space, growth factors, and nutrients in order to keep a constant total number of lymphocytes (B7, F11, F12). Competition should favor foreign-specific over anti-self-specific antigens. Antibodies with self-activity do not enter the follicle, and die within 1–3 days (F14). In summary, impairment of all the foregoing mechanisms during a multistep process may be involved in the loss of self-tolerance in patients with SLE (Table 2). Self-reactive anergic lymphocytes may be reactivated when a genetically vulnerable person faces an environmental trigger from viral infection leading to sustained inflammation. The precise mechanism of anti-Ro/SSA, anti-La/SSB, and antidsDNA antibodies will be discussed in the following sections. 2.2. HISTORY OF AUTOANTIBODIES TO dsDNA, Ro/SSA, AND La/SSB The first discovery of serum factor in human lupus with affinity for tissue nuclei was from three laboratories almost simultaneously in 1957 (H14, C7, R11). Clark et al. in 1969 first identified antibodies (Abs) that reacted with Ro antigen in patients with SLE (C20). Alspaugh et al. (A16) in 1975 identified antibodies to SSA antigen in patients with Sjögren’s syndrome (SS). Anti-Ro and anti-SSA Abs were later shown to target the same antigen (A15).

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2.3. MOLECULAR STRUCTURES OF ANTI-dsDNA, ANTI-Ro/SSA, AND ANTI-La/SSB AUTOANTIBODIES Knowledge of the structures of autoantibodies provides clues to their antigen sources and the mechanism of their production. 2.3.1. Anti-dsDNA Antibodies Pathogenic anti-dsDNA antibodies from human lupus are different from nonpathogenic anti-dsDNA antibodies from normal people. Results from structural studies reveal that the production of these pathogenic antibodies is antigen-driven and helper T cell-dependent. 2.3.1.1. Pathogenic versus Nonpathogenic anti-dsDNA Antibodies. The germline repertoire of light- and heavy-chain variable regions that may encode DNAspecific antibodies is very large in mice. Based upon sequence analyses by Marion et al., there are an estimated 50 VH genes and 36 VL genes that may be used to encode anti-DNA V regions in the mouse (M6). These germline genes can produce anti-DNA antibodies. Moreover, these antibodies are not totally deleted by the central tolerance mechanism and can “leak” to the periphery. Thereafter, these anti-DNA antibodies can be detected in the plasma of normal humans (H11, R11, R18, S5). However, there are many differences between the anti-DNA antibodies of healthy humans and those of human lupus patients. Nonpathogenic anti-DNA antibodies of normal individuals usually belong to the IgM isotype, polyreact with single-stranded DNA (ssDNA) with low affinity, and lack complement-fixation ability. On the other hand, pathogenic anti-DNA antibodies from human lupus patients usually belong to the IgG isotype, react with dsDNA with high affinity, and have complement-fixation ability (R11, R16, E2). The problem is then to determine which antigen sources by what mechanisms elicit pathogenic antidsDNA antibodies. Efforts from earlier studies trying to elicit pathogenic antidsDNA antibodies from immunized nonautoimmune mice with synthetic dsDNAs (L9, L10, M20) or Z-form dsDNA (T6) failed to produce anti-dsDNA. These elicited antibodies do not bind to mammalian B-form dsDNA with random nucleotide sequences. The resulting lack of immunogenicity of dsDNA from these studies, together with the finding that normal humans make anti-DNA antibodies as part of their normal “natural” immune repertoires, have led to the hypothesis that “pathogenic” anti-DNA antibodies from human lupus are simply derived from the by-product of generalized, polyclonal B cell activation (K5, K6). This early hypothesis faced difficulties from recent studies of structural analysis of antidsDNA antibodies. 2.3.1.2. Anti-dsDNA Antibodies Are Antigen-Driven. There is evidence that variable regions of both heavy and light chains of both murine and human monoclonal anti-dsDNA antibodies are produced after an affinity maturation process via isotype switching, clonal selection, and somatic mutation (D5, D9, W11, C16, V3). These findings suggest that anti-dsDNA antibodies are produced by an


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antigen-driven, T cell-dependent pathway. First, as mentioned previously, pathogenic anti-dsDNA antibodies are usually of IgG isotype. This suggests they are produced after isotype switching. Second, although the germline repertoire of the genes that encode anti-dsDNA antibodies is very large, recurrent preferred genes are expressed (M6). This means they are produced after clonal selection. Third, recurrent, somatically derived variable-region structures, particularly arginines in the third complementarity-determining region of the heavy chain (VH-CDR3), have also been recurrent and preferentially expressed in monoclonal anti-DNA antibodies (E4, K12). In addition, specificity for dsDNA is also strongly correlated with the relative position of arginines in VH-CDR3 (M20, R1, B3). This suggests they are produced after antigen-driven somatic mutation. Direct evidence of DNA as an antigen source to drive autoantibody production was derived from experiments using combined DNA–protein as an immunogen to immunize mice. The mice immunized with the DNA–peptide complex produce anti-dsDNA antibodies with pathogenic properties (D7). Further evidence comes from the study of antibodies produced by autoimmune mice during their life. Anti-DNA antibodies first appear around 2 months of age in female NZB/W mice, and are in significant titer by 4–5 months (M24). Following the development of anti-DNA antibodies, glomerular Ig deposits first appear around 3–4 months of age and continue to increase in severity with increasing age (L5, H16). The first histological signs of glomerulonephritis in these mice appear at around 6 months of age (L5) and coincide with a class shift in anti-DNA from IgM to IgG (S28, P1). In summary, structural analyses of anti-DNA antibodies support the hypothesis that autoimmunity to DNA is both initiated and sustained as a clonally selected, antigen-specific immune response to DNA. The initial autoimmune response to DNA is dominated by lower affinity IgM antibody primarily specific for singlestranded, denatured mammalian DNA. As the autoimmune response progresses, the antibody isotype switches to IgG, and the antibody acquires increasing affinity for native, double-stranded DNA. 2.3.2. Anti-Ro/SSA and Anti-La/SSB Antibodies Anti-Ro/SSA antibody was identified in the serum of more than 10% of patients with connective tissue disease (CTD) (B20), making these autoantibodies the most prevalent of antibodies to ribonucleoprotein (RNP) antigens. The molecular weight of the major antigenic peptide is about 60 kDa (Y1). Other peptides related to anti-Ro autoantibodies include 52-kDa Ro, 54-kDa Ro, a second 60-kDa Ro, 57kDa Ro, and calreticulin. There are four Ro-associated RNAs, hY1, hY3, hY4, and hY5, with length ranging from 84 to 112 bases. All four have been sequenced (K3, W14, O1). The La peptide contains 408 amino acids and has a predicted molecular weight of 46.7 kDa (C10, C11, C15). The La peptide appears to bind any RNA with a polyuridine 3′ end (M9, S26). At least one isolated Ro particle also contains

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the La peptide, thereby providing a structural basis for the association of anti-Ro and anti-La autoantibodies in patient serum (H3). Meilof et al. showed there was closely coordinated production of anti-Ro/SSA and anti-La/SSB in SLE during a 10-year follow-up (M15). This suggests the common antigenic source of the two autoantibodies. Ro has a role in the discard pathway for 5S RNA (O2). La plays multiple roles in cells, such as a shuttle protein to carry RNA transcripts from the nucleus to the cytoplasm (B1, B2) or a termination factor for RNA polymerase III (G12, G13). Scofield et al. reviewed the fine specificity of the autoimmune response to the Ro and the La antigens and concluded that multiple epitopes of the antigens are involved in antigen-driven immune response (S6). 2.4. MECHANISM OF ANTI-dsDNA, ANTI-Ro/SSA, AND ANTI-La/SSB AUTOANTIBODY PRODUCTION IN PATIENTS WITH SLE 2.4.1. Sources of Autoantigens 2.4.1.1. Introduction. It is well known that native mammalian DNA is poorly immunogenic and has never been found to be able to induce anti-DNA antibodies (M3, M6). However, analysis of somatic mutations revealed that the induction of anti-dsDNA autoantibodies from SLE patients is antigen-driven and thus T celldependent. What are the antigen sources that induce anti-dsDNA autoantibody production? The possible sources of autoantigens are (1) autoantigens altered by apoptotic cells, (2) autoantigens with endogenous or exogenous adjuvants, and (3) exogenous DNA (viral or bacterial) with molecular mimicry, which induces antibodies that cross-react with self-antigens. We discuss the role of apoptosis first. 2.4.1.2. Apoptosis as a Source of Antigens. There have been many studies regarding apoptosis as the major etiopathogenetic factor of SLE in the last decade. As we discuss in the following sections, accumulation of apoptotic waste serves as a source for autoantigens that induce and maintain autoimmune responses. Apoptotic cell processes which become a source of autoantigens are (1) increased apoptosis in patients with SLE, (2) decreased apoptotic cell removal, and (3) alteration and presentation of autoantigens by the apoptotic process. 2.4.1.2.1. Increased apoptosis. Evidence of increased apoptosis in SLE is demonstrated by the fact that lymphocytes of both SLE patients and lupus mice show an increased rate of apoptosis ex vivo (E6, V4, L22). In addition, elevated levels of circulating oligonucleosomes are noted in SLE (R21, A19). The reason for increased apoptosis in SLE patients was revealed by studies of two environmental factors linked with SLE: UV radiation and viral infection. UV radiation (UVB) was shown to cause apoptosis of keratinocytes and formation of “surface blebs.” The Ro antigen is then expressed in the surface of the blebs and may contribute to the induction of autoimmunity to Ro (C4). Viral infection, on the other hand, is well associated with disease relapse.


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2.4.1.2.2. Decreased apoptotic cell removal. The possible causes of decreased apoptotic cell removal are (1) decreased phagocytosis function of macrophages in patients with SLE, (2) decreased level of early components of complement of the classic pathway, (3) decreased serum amyloid P component (SAP), and (4) elevated plasma level of nucleosomes in lupus patients. Normally, cells undergoing apoptosis are quickly removed in vivo by noninflammatory engulfment phagocytosis. A reduced phagocytic activity of SLE patients’s polymorphonuclear leukocytes (PMN), monocytes, and macrophages is well established (B23, L6, S34, V7). Herrmann et al. found that the percentage of macrophages engulfing apoptotic cell material was significantly reduced in SLE compared to that in control patients (H9). They subsequently demonstrated that the phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with SLE was impaired (H8). Long-lasting presentation of apoptotic cell-derived self-antigens may continuously challenge T cell self-tolerance and render proteolytically cleaved autologous apoptotic material immunogenic. As mentioned previously, the strongest known single genetic risk factor for the development of human SLE is complete deficiency of one of the components of the classical complement pathway (C1q, C1r, C1s, C4, and C2) (W4). Individuals with complete deficiency of C1q have the highest prevalence of SLE and the most severe manifestation of the disease. The best-understood function of the complement system is the clearance of immune complexes. Complement can bind to IgG– and IgM–antigen complexes and promote their transport and clearance by erythrocytes and mononuclear phagocytes bearing complement receptors (D1). Deficiency of complement will result in deposition of immune complex and organ injury. The injured organ subsequently elicits inflammatory reaction, releases autoantigens, and thus induces autoimmunity (W3). On the other hand, C1q has a role in clearing apoptotic cells. Studies have shown that apoptotic keratinocytes can be specifically recognized by human C1q (K8). C1q deposition was localized to the surface blebs of apoptotic cells, which suggests that C1q may be critical for proper recognition, clearance, and processing of self-antigen contained within surface blebs generated by apoptotic cells. In addition to the clearance effect, complement may also play a role in self-tolerance. In the HEL double-transgenic model, autoreactive B cells become anergic by downregulated surface IgM when their B cell receptor is doublelinked with souble HEL autoantigen (G11). Decreased complement may result in decreased strength of signal in selection (C1). Serum amyloid P component (SAP) is the single normal circulating protein that shows specific calcium-dependent binding to DNA and chromatin in physiological conditions. It can bind and solubilize the chromatin exposed by cell death and binds in vivo to apoptotic cells. Mice with targeted deletion of SAP spontaneously develop antinuclear autoimmunity and severe glomerulonephritis (B16). Thus SAP has an important role in inhibiting the formation of pathogenic autoantibodies against chromatin and DNA by binding to chromatin and regulating its production.

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Nucleosomes also play a role in decreased apoptotic cell removal. Nucleosomes can induce a strong, dose-dependent inhibition of phagocytosis of apoptotic thymocytes by young, preautoimmune macrophages of MRL+/+ mice (L1). 2.4.1.2.3. Role of apoptotic enzymes. When cells undergo apoptosis, two distinct populations of blebs are clustered with autoantigens that present at the surface of apoptotic cells (C3). The smaller blebs contain fragmented endoplasmic reticulum and ribosomes as well as the ribonucleoprotein Ro. The larger blebs (apoptotic bodies) contain nucleosomal DNA, Ro, La, and the small nuclear ribonucleoproteins. How do these autoantigens induce autoimmunity? Mere increase in apoptosis is not sufficient to trigger autoantibody production. In other chronic inflammatory diseases, such as mixed connective tissue disorder (MCTD), Wegener’s granulomatosis, Takayasu arteritis, and polyarteritis nodosa, increased apoptosis of peripheral blood mononuclear cells was also noted (L22). Autoantigens can become immunogenic only after being processed specifically by apoptotic cells. Evidence shows that the autoantigens clustered in membrane blebs of apoptotic cells have been modified either by being selectively cleaved and phosphorylated by intracellular proteases specific to the apoptotic process (C5, U2) or by undergoing oxidative modification by reactive oxygen species (C3). In addition, the majority of autoantigens of human systemic autoimmune disease are efficiently cleaved by granzyme B in vitro and during cytotoxic lymphocyte granule-induced death, generating unique fragments not observed during other forms of apoptosis (A20). Cells undergoing apoptosis then express these unique altered autoantigens and reveal cryptic epitopes at the cell surface of apoptotic bodies and serve as the source of autoantigens. 2.4.1.3. DNA with Adjuvants and Molecular Mimicry 2.4.1.3.1. Autoantigens with adjuvants. DNA also becomes more immunogenic if it is conjugated with either endogenous self-activator (natural adjuvant) or exogenous adjuvant from viral or bacterial peptides. Experimentally, anti-dsDNA antibodies have been successfully induced by a complex of native calf thymus DNA and a highly immunogenic DNA-binding peptide Fus1 (D7). Dendritic cells can be activated by endogenous signals received from cells that are stressed, virally infected, or killed necrotically. These cells contain endogenous activating substances, which function as natural adjuvants (G2). Viruses can induce apoptosis of human keratinocytes. These apoptotic cells can express viral antigens and autoantigens, which are coclustered in specific subsets within surface blebs and then cause challenge to self-tolerance if not cleared and processed properly (R15). Andreassen et al. showed that T cell lines specific for polyomavirus T antigen recognize T antigen complexed with nucleosomes and trigger B cells to produce anti-DNA antibodies (A20). 2.4.1.3.2. Antigens with molecular mimicry. There is also evidence that antibodies induced by viruses or bacteria can cross-react with autoantigens.


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2.4.1.3.3. Viruses. Rabbits have been induced to produce anti-RNP antibodies by immunization with the p30 gag protein, a protein of several mammalian C-type viruses (Q1). Blomberg et al. found an increased frequency of antibodies that were cross-reactive with baboon endogenous retrovirus and murine leukemia virus among 72 SLE patients compared with 88 controls (B17). Plotz found that autoantibodies in patients with lupus are antiidiotype antibodies to antiviral antibodies (P4). 2.4.1.3.4. Bacteria. Sharma et al. found that the cross-reactivity of IgG1 (T-dependent) anti-DNA with phosphorylcholine (PC), a dominant epitope on pneumococcal cell wall polysaccharide, is greater than that of IgG2 (T-independent) (S10). A peptide surrogate for dsDNA, which was identified from a phage peptide library using either murine monoclonal (G6) or human lupus polyclonal (S29) anti-dsDNA antibody, induced the production of anti-dsDNA antibodies in nonautoimmune BALB/c mice (G6) and in rabbits (S29). Although the motif peptide RLTSSLRYNP exhibits homology with a chromosome-associated polypeptide, it does not show any similarity with antigens of suspected infectious agents (S29). 2.4.2. Presentations of Autoantigens: Bystander Dendritic Cells or B cells Direct evidence that apoptotic cells can induce autoimmunity comes from experiments in which normal mice injected with syngeneic apoptotic thymocytes developed antinuclear, anticardiolipin, and anti-ssDNA antibodies. These mice suffered from immunoglobulin G deposition in the glomeruli several months after immunization (M16). How do apoptotic cells induce production of autoantibodies? Under physiological conditions, asynchronously dead cells are removed by phagocytosis and no inflammatory response is induced. The single dying cells are outnumbered by their living neighbors, who contribute to the clearance of these cells in combination with professional scavenger macrophages (W17, S2). Immature dendritic cells are interspersed among professional phagocytes. These phagocytes compete for the phagocytosis of apoptotic cells. They also secrete a vast array of factors when engulfing apoptotic cells (V10, F2), including IL-10, which selectively blocks dendritic cell maturation (B4). However, coculture of dendritic cells with apoptotic cells leads dendritic cells to acquire relevant antigens and stimulate major histocompatability complex (MHC) class I-restricted cytotoxic T lymphocytes by phagocytosing apoptotic cells (A12). Thus, when massive apoptosis occurs in conditions such as viral infections or ischemic insults, together with decreased apoptotic cell removal due to causes described in the previous section, a higher number of dendritic cells can internalize apoptotic cells efficiently after 60 min (R17). Therefore, massive apoptosis may facilitate dendritic cell recruitment for the clearance of apoptotic cells.

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2.4.3. Sustained Autoantibody Inducers: Role of Cytokines The role of cytokines, especially interferon-α (IFN-α), was reviewed by Rönnblom and Alm (R12). The relationship of cytokines and SLE was first demonstrated by increasing serum levels of IFN-α in SLE patients (H15, Y7, B5). The evidence that IFN-α plays a role in the pathogenesis of SLE includes (1) the levels of IFN-α are correlated with disease activity (Y7, B5, B11) and (2) IFN-α therapy of patients with nonautoimmune disease frequently results in production of anti-dsDNA antibodies or SLE (R14, K1, R13, I1). IFN-α belongs to the type I IFNs. Type I IFNs have the ability to promote the survival and differentiation of antigen-activated T helper cells (M8, A5, S17, B18). They also promote maturation of monocyte-derived dendritic cells (S1). Thus type I IFNs might play a central role in initiating the immune response to antigens (R12). Serum from SLE patients with immune complexes containing anti-DNA antibodies and DNA induced the production of IFN-α in normal blood leukocytes in vitro (C6, V1, V2). Anti-DNA antibodies were able to convert DNA motifs, such as the CpG-rich unmethylated DNA of a plasmid (V2), into a potent IFN-α inducer in natural IFN-α-producing cells (NIPCs). In the presence of viral infection, IFN-α is produced. IFN-α, as well as IL-12 and IFN-γ , can help dendritic cells to activate naive autoimmune T cells, which subsequently stimulate B cells to produce autoantibodies (R12). Then the immune complexes form and act as endogenous IFN-α inducers. Thus a vicious cycle of sustained IFN-α secretion and generation of autoimmune T and B cells is established (R12). 2.4.4. Summary Impaired function of phagocytes and deficiency of early components of the classic pathway of the complement system result in increased apoptotic waste. This waste, including nucleosomes and other autoantigens, is formed and altered by the protease of apoptotic cells. They are expressed on the surface of apoptotic blebs and activate bystander dendritic cells. Subsequently the dendritic cells activate helper T cells, which then help B cells to generate high-affinity autoantibodies (Fig. 1). 2.5. DIRECT CYTOTOXIC EFFECTS OF ANTI-dsDNA, ANTI-Ro/SSA, AND ANTI-La/SSB AUTOANTIBODIES Autoantibodies lead to organ damage via various possible mechanisms. They can form a circulating immune complex after binding with respective antigen. The immune complex is then deposited within respective organs and causes damage. The autoantibodies can also form an immune complex via an in situ pathway. They react with trapped circulating antigens or endogenous antigens within respective organs and thus cause organ damage. In the last decade, there also has


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FIG. 1. Mechanism of autoantibody production in SLE.

been increasing evidence that autoantibodies have the ability to penetrate living cells and directly elicit cytotoxic effects on the cells. Cellular and nuclear localization of autoantibodies was initially described three decades ago (A7, A8, A9). However, some argued that they only represent a fixation artifact, with movement of immunoglobulins into cells during tissue preparation

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(C17, K11). Nevertheless, much evidence in the last decade shows that autoantibodies can penetrate living cells, bind to respective target antigens, and cause cell damage. 2.5.1. Evidence of Anti-dsDNA Antibodies Causing Cytotoxic Effects in Cells Anti-dsDNA antibodies can cause cytotoxic effects in various cultured cells in vitro. They can trigger apoptosis in cultured mesangial and endothelial cells (T9, L3). Monoclonal anti-DNA autoantibodies produced by human–human hybridoma also revealed cytotoxicity to primary cultures of lymphocytes (S11). Treatment of the various tumor cell lines with cytotoxic anti-DNA autoantibodies induced internucleosomal DNA fragmentation and annexin V binding to the cell surface characteristic of the apoptotic pathway of cell death (K10). 2.5.2. Mechanism of Anti-dsDNA Antibody Penetration into Cells There is evidence of binding (A14, K9, O6, R8, Z1, Z2) and penetration (A6, A10) of anti-dsDNA antibodies or anti-Ro antibodies (L13) to live cells, with subsequent cellular localization (K9, G9, Y2, Z1, Z2). Many putative receptors for anti-dsDNA on the membrane of various cell types have been noted. Some workers found a 30-kDa protein involved in the binding and internalization of [3H]DNA via receptor-mediated endocytosis (B12, B13). Other possible receptors include nucleosomes on human leukocytes (R8), Fc receptors on human T cells (A6), DNA on mouse and human mononuclear cells (O6), a 94-kDa protein on several cells lines (J2), DNase-resistant target on human fibroblasts and PK 15 cells (K9), membrane determinant precisely resembling DNA in murine renal tubular cells (Z1), Hp8 on human and murine tubular cells (Z2), ribosomal P protein on rat and human glomerular mesangial cells (S30, S31), brush border myosin 110 kDa on rat hepatoma cells, and a diverse set of membrane proteins on a series of human tumor cell lines (R3). After penetrating into cells, these autoantibodies may localize within the cytoplasm or the nucleus, or recycle back to the cell membrane (R6). How do the antibodies localize to the nucleus? Conformational analysis of the antibodies indicates that their antigen-binding regions share a tertiary structure resembling nuclear localization sequences (F6). There are three types of interactions of anti-dsDNA antibodies with living cells (R6): (1) prolonged residence on the membrane, with rabbit-complement prompt cytolysis results; (2) cytoplasmic localization; and (3) nuclear localization. Once internalized, the antibodies move to the location of their target ligands and can then bind to the cross-reactive targets. Various target ligands have been found, including (1) common cellular antigens [Sm/RNP, cytoskeleton, laminin, proteoglycans, heparan sulfate (H1), and elongation factor-2 (EF-2) (A11)], (2) selfantigens [naked DNA, histones, and chromatin (H1)], and (3) non-self-antigens [bacterial polysaccharides (S5), bacterial β-galactosidase (P3), and phospholipids from gram-positive bacteria (C2)].


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2.5.3. Mechanism by Which Internalized Anti-dsDNA Antibodies Cause Cellular Damage Anti-dsDNA antibodies may lead to cellular damage via DNA-hydrolyzing activity of the antibodies (S13, G10). In addition, they can arrest the cell cycle in the G0 phase (A6) or cause cell death (A7, A9, O6). Murine monoclonal anti-DNA antibodies can penetrate glomerular cells, resulting in podocyte fusion, proteinuria, and glomerular hypercellularity (V8). They also lead to cellular toxicity by the inhibition of an eukaryotic ribosomal protein (EF-2), an essential elongation factor in protein synthesis, causing the inhibition of cellular protein synthesis (R6). They also can enhance the release of proinflammatory cytokines (IL-1β, IL-8, and TNF-α) from mononuclear cells (MNCs) to augment the inflammatory reaction (S32). Anti-dsDNA antibodies also have the ability to upregulate adhesion molecules on cultured umbilical vein endothelial cells (L4). In addition, anti-dsDNA antibodies can interact with DNase 1 and inhibit endonuclease activity and therefore attenuate apoptosis (Y2). The cytoplasm-localized antibodies can be shown to recycle back to the cell surface. If they are altered during recycling, neo-antigen can be expressed to T lymphocytes with augmentation of the inflammation response (Y2). In summary, autoantibodies can be internalized into living cells of various types via various receptors on the cell membrane. Then these antibodies travel to different localizations and react with various target antigens. Finally, the functions of cells are altered by induced apoptosis or augment inflammation. 3. Clinical Aspects of Anti-dsDNA, Anti-Ro/SSA, and Anti-La/SSB Autoantibodies The highly specific properties and widespread prevalence of anti-dsDNA antibodies make them one of the most important serologic markers for SLE. Pathogenic anti-dsDNA antibodies (high-avidity, IgG isotype, complement-fixation ability) are highly specific for SLE. The prevalence of anti-dsDNA in SLE ranges from 70% to over 80% (C8, C18, K2, S23). Studies on the progression of SLE with time confirm the presence of anti-dsDNA in over 90% of all SLE patients (W9). Swaak and Smeenk followed 441 patients without any lupus symptoms but with positive anti-dsDNA, using the Farr assay (S38). They concluded that about 85% of the patients with a positive DNA-antibody finding, but without any SLE symptoms, would suffer an outbreak of the disease most likely within a few years. Anti-Ro and anti-La are less specific. They can be identified in the serum of more than 10% of patients with connective tissue disease (CTD) (B20), making these autoantibodies the most prevalent of antibodies to ribonucleoprotein (RNP) antigens. The prevalence of these antibodies in patients with lupus is 63% in Singapore (B18), 15% in Mexico (G12), and 15–57% in other Western countries

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(B6, B9, B18, M20, S38, S7). Of our patients with SLE, 53.8% had antibody to Ro antigen (C18). In the following sections we discuss the clinical relevance of these antibodies, and also detection methods, clinical significance, and clinical correlations of disease manifestations to the autoantibodies. 3.1. ASSAYS FOR DETECTION 3.1.1. Methods for the Detection of Anti-dsDNA Methods for detection of anti-dsDNA include immunofluorescence, hemagglutination, radioimmunoassay (RIA), and enzyme-linked immunoassay (ELISA). Different methods may result in discrepant results due to the heterogeneous nature of the antibodies (S20). Numerous studies that compare methods conclude that no single test is perfect. It may be necessary to combine different methods for both higher sensitivity and higher specificity. The most commonly used methods for detecting anti-dsDNA are the CLIF test, the Farr assay, and the ELISA test. 3.1.1.1. Crithidia luciliae Immunofluorescence (CLIF Test). Crithidia luciliae is a nonpathogenic flagellate. This test uses the giant mitochondrion (kinetoplast) circular dsDNA of the flagellate as the antigen in the immunofluorescence test (A1). One of the disadvantages of this test is that the circular dsDNA of the kinetoplast may contain DNA-associated histone-like proteins (T14), which are growth cycle-dependent. If the patient’s serum contains antihistone antibodies, they can result in cross-reaction, which may lead to false-positive results (D6). The fact that antihistones have been found in up to 50% of lupus patients makes the tests much less specific for the disease (R20). Another disadvantage is that the sensitivity of the assay may be low if the antigen density is low. Furthermore, if the commercial kits utilize inadequate substrate and/or coating quality, the test will result in much lower diagnostic sensitivity. 3.1.1.2. Farr Assay. This method uses the principle of liquid-phase radioimmunoassay (RIA). Under a condition of high salt concentration, the test uses ammonium sulfate to precipitate antibody–antigen complexes (W11). Both IgG and IgM isotypes (S21) of high-avidity antibodies are detected. The disadvantages of the test include the use of radioisotopes and the possibility of the cross-reaction of anti-ssDNA (W8, R19). An alternative RIA test for anti-dsDNA uses polyethylene glycol (PEG) as precipitating reagent. This PEG test can detect both highand low-avidity antibodies (R10). 3.1.1.3. Enzyme Immunoassay (ELISA). This method is generally considered to be highly sensitive. Major problems of the technique relate to the antigen used and the method of fixing the antigen. The native double-stranded form of DNA from calf thymus or salmon sperm is less suitable because it contains a high proportion of proteins (up to 25%) and RNA. It must be vigorously purified before fixing


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on the plates. On the other hand, if plasmid dsDNA is selected as the antigen, only gentle purification is necessary to get pure dsDNA without ssDNA or nuclear proteins (W10). An inappropriate coating procedure for dsDNA may reduce the specificity of the test. Only ssDNA binds to the hydrophobic surfaces coated with polystyrol, the coating material generally used in ELISAs (E8). Cationic carriers for dsDNA binding, such as poly-L-lysine and protamine sulfate, may result in false-positive antibody reactions (B24). Another binder, an amphipolar ion-pair reagent, has been shown to be fully inert to immunoglobulins. 3.1.1.4. Other Methods. Marion et al. used a competitive ELISA to measure the specificity of anti-DNA antibodies. Low-affinity, low-avidity antibodies bind to competitor DNAs in solution and thus cannot bind to solid-phase DNA subsequently (M7). A new automatic instrument platform using the immunofluorescence method (EliATM) to detect anti-dsDNA has recently been developed. We found that results from this method correlated well with results from the ELISA method (unpublished). 3.1.1.5. Comparing Methods Used for Detecting Anti-dsDNA. The CLIF assay has only moderate sensitivity (due to low antigen concentration) and moderate specificity (due to cross-reaction for nuclear proteins). The Farr assay uses high salt for precipitating high avidity of both IgG and IgM antibodies. It possesses high specificity, but is less sensitive for detecting high-avidity antibodies with low concentration. Moreover, the Farr assay cannot discriminate IgG from IgM. The ELISA test has high sensitivity for detecting anti-dsDNA with high avidity, but it has lower specificity. Other disease states may give positive results, especially when dsDNA material used as antigens is contaminated with ssDNA. Such diseases include chronic active hepatitis, rheumatoid arthritis, non-lupus connective tissue diseases (such as Sjögren’s syndrome, myasthenia gravis), drug-induced lupus, and certain infectious diseases; another confounding effect is aging (B30). Thus, only antibodies with a high titer may be correlated better with the clinical activity of lupus. Werle et al. suggested combining higher sensitivity ELISA and higher specificity Farr assays for detecting dsDNA (W10). 3.1.2. Methods for the Detection of Anti-Ro and Anti-La Methods for the detection of anti-Ro and anti-La include traditional immunodiffusion, commercial ELISA kits, and methods such as immunoblotting and immunoprecipitation. 3.1.2.1. Immunodiffusion. The traditional method using Oüchterlony double immunodiffusion is specific and sufficiently sensitive. The major drawback of the test is that it takes 2 days to complete. 3.1.2.2. ELISA. Commercial kits for detecting anti-Ro and anti-La are available. This approach has 10- to 100-fold increase in sensitivity over double immunodiffusion. The possibility of nonspecific binding is increased, so the

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specificity is less compared to immunodiffusion. In addition, a significant proportion of normal people have positive ELISA results for anti-Ro or anti-La but without precipitin formation. Thus, the results of ELISA tests should be read with caution. 3.1.2.3. Immunoblotting. One can test for the antibody against 60-kDa Ro or 52-kDa Ro with the Western blot test. The antigenicity of the Ro antigen is adversely affected by denaturation by boiling in sodium dodecyl sulfate for blotting, and so anti-Ro may became undetectable using this test. The levels of anti-Ro and anti-La may change 10- to 20-fold during the course of the disease. The relevance of this change to disease expression is not known. Scopelitis et al. found no correlation between serum anti-Ro and SLE disease activity in 88 patients (S7). However, Skinner and Maddison followed and collected serial serum of 22 SLE patients for 6 years and found that increased anti-Ro was correlated with disease activity (S18). Further studies are needed to clarify this issue. 3.2. ANTI-dsDNA AUTOANTIBODIES AND LUPUS NEPHRITIS Anti-dsDNA antibodies are both specific and pathogenic to SLE. As mentioned previously, these autoantibodies are produced by an antigen-driven mechanism. They are organ-specific, especially kidney. We will discuss the close relationship between anti-dsDNA and lupus nephritis and the proposed mechanism by which anti-dsDNA leads to lupus nephritis. 3.2.1. Anti-dsDNA Antibody as a Marker of Disease Activity The close correlation between disease activity of SLE and level of anti-dsDNA has been documented repeatedly (D4, L24). Anti-dsDNA also represents a reliable marker for acute exacerbation of disease activity (S37, T3). Some report that highavidity anti-dsDNA correlates with renal manifestations and active lupus nephritis (S37), whereas low-affinity antibodies are associated with cerebral complications (S21, S22). 3.2.2. Evidence That Anti-dsDNA Antibodies Cause Lupus Nephritis The presence of anti-dsDNA antibodies correlates with nephritis both in mice and in human patients (A21, H7). Anti-dsDNA antibodies are concentrated within human and murine glomeruli in vivo, and injection of DNA to lupus-prone mice that are positive for anti-dsDNA antibodies accelerates the progression of nephritis (E1, K7, L5). Injection of anti-dsDNA monoclonal antibodies into nonautoimmune mice also has been shown to produce nephritis (D5, O5, G7, V9). In addition, injection of bacterial DNA induces the formation of anti-DNA antibodies in nonautoimmune mice and subsequent development of nephritis (G8).


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In spite of the strong evidence just cited, lupus nephritis is not invariably correlated with serum anti-dsDNA. (NZB × NZW)F1 × NZW backcross mice may develop nephritis without anti-DNA antibodies (Y5). Patients with lupus nephritis also do not always have anti-dsDNA antibodies or have only low levels of antibodies (A23, H7, L21). Swaak et al. noted a sharp drop at the time of flareup of anti-dsDNA antibodies accompanied by low levels of C1q and C3 in patients with severe SLE flares (S36). 3.2.3. Proposed Immunopathogenic Mechanisms of Lupus Nephritis It is well understood that the pathogenesis of lupus nephritis involves immune complex formation with subsequent complement activation and induction of inflammation in the kidney (F5, S33). The pathways leading to immune complex formation can be classified into two categories. According to one, the immune complex is formed in the circulation. According to the other pathway, the immune complex is formed inside the kidney. The later can be further classified according to which antigen source is formed in the immune complex. One of the antigen sources is from the circulation, the other is the antigen of the renal tissue that is cross-reactive with anti-dsDNA antibodies. 3.2.3.1. Circulating Immune Complex. Anti-dsDNA/DNA immune complexes have long been considered responsible for the development of lupus nephritis. The level of immune complexes in SLE patients with active disease detected by monoclonal anti-DNA antibodies is higher (T7). About half of SLE patients had elevated amounts of DNA antigen in the immune complexes (N4, R2, S27). There are many other reports arguing against this mechanism as the major pathogenetic pathway of lupus nephritis. The presence of circulating DNA/antiDNA immune complexes is difficult to detect (B25, I3). Moreover, recent studies demonstrated that the levels of circulating DNA (nucleosome) immune complexes were low (B28, F8, L14). Data also suggest that DNA/anti-DNA complexes are rapidly cleared by the liver (E5) and bind poorly to glomerular basement membrane (GBM) (I2). Recent studies show that nucleosome/antinucleosome immune complexes contribute more to lupus nephritis. Serum anti-dsDNA reactivity is always associated with “antinucleosome” reactivity (A18, B26, B28, B29, C9). Even the highly purified monoclonal and polyclonal anti-dsDNA antibodies selected by affinity chromatography bind to isolated dsDNA and also to nucleosomes (C9, L23). Hybridoma-secreting anti-DNA can also form immune complexes in vitro with nucleosomes released from dying hybridomas in culture (F9). Finally, the binding of an anti-DNA antibody to a nucleosome may render the immune complex more positive and thereby make it more prone to bind to the GBM (T2). 3.2.3.2. Cross-Reactive Antibody. The ability of anti-DNA antibodies to bind to other negatively charged substances represents an attractive variant of the

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molecular mimicry theme. Many potentially relevant components of the glomerular basement membrane that can interact with anti-DNA have been described. These components include fibronectin, collagen, heparin sulfate, and laminin (F1, J1, M2, R4). Non-DNA antigens have been implicated in the mechanism of immune complex deposition (F7). Some authors believe this is the major pathway leading to immune deposit formation (M1) because it has been difficult to demonstrate initiation of nephritis by the other mechanisms. They suggest that complex deposition may modify or amplify disease activity rather than initiate the process (M1). However, others found that the binding of autoantibodies to various antigens purified from biological sources or to cultured cell lines may be mediated by nuclear antigens bound to the protein or cell of interest (L15). Thus they suggest that direct binding of autoantibodies to nonnuclear antigen epitopes on the glomerulus or GBM appears to be a less common event than the binding of autoantibodies to the glomerulus or GBM via nuclear antigens (L15). 3.2.3.3. Planted Antigen. In this hypothesis, circulating antigens lodge in the glomerulus to serve as a nidus for subsequent immune deposit formation. However, DNA, the antigen of anti-DNA, only weakly binds to intrarenal type IV collagen (B14, G5, I2) or GBM proteins such as fibronectin or laminin (B14, G17). However, the avidity of DNA to type IV collagen can be inhanced by histones predeposited on the GBM (B14, S14). Histone and other nuclear antigens have been shown to bind well to GBM in vitro and in vivo (S3, B14). Intravenous administration of DNA will deposit in GBM only after the GBM is predeposited with histone (I1, S3). The source of circulating nuclear antigens is increased apoptosis in SLE patients, as mentioned previously. In summary, each of the three hypotheses has studies to support or reject it. If we consider the recent finding about the mechanism of production of anti-dsDNA, the three hypotheses may work together in the pathogenesis of lupus nephritis. The increased apoptosis in SLE leads to an increased level of circulating nuclear antigens. These antigens are subsequently deposited in kidney before they form immune complexes in situ. This is possibly the major pathway. The antigens can also form immune complexes with anti-dsDNA in the circulation before being deposited in kidney. The final pathway is the activation of the complement system, which leads to inflammation and injury of renal tissues.

3.3. CLINICAL AND SEROLOGICAL CORRELATIONS OF ANTI-Ro/SSA AND ANTI-La/SSB AUTOANTIBODIES 3.3.1. Anti-Ro, Anti-La, and Neonatal Lupus Neonatal lupus is caused by cross-reaction between maternal antibodies and fetal tissue. It is particularly interesting because it provides a unique opportunity for studying the pathogenic role of autoantibodies.


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The diagnosis of neonatal lupus depends on the discovery of placental transfer of maternal autoantibodies to the infant, together with characteristic clinical findings. The clinical manifestations can be classified into permanent insult and transient phenomena. Congenital heart block of varied degree is a life-long insult, which accounts for 7–10% of neonatal lupus (D13). Transient manifestations (D10, E10, W6, W12) mostly involve skin (erythematosus, scaly rash over sun exposured area). As many as 50% of infants with neonatal lupus have skin involvement (L7). The other transient manifestations include involvement of the hematologic system (anemia, thrombocytopenia, leukopenia) and the liver (hepatosplenomegaly, hepatitis), and possibly pneumonitis. The mothers of infants with neonatal lupus may be asymptomatic or may have a variety of autoimmune diseases, including Sjogren’s syndrome, SLE, rheumatoid arthritis, overlap syndrome, or even leukocytoclastic vasculitis (P8, W7). Although an association between maternal connective tissue disease and cardiac involvement in neonatal lupus was suspected as early as 1901 (M22), the association between skin disease in an infant and maternal autoimmune disease was not reported until more than 50 years later. McCuistion and Schoch (M13) described an infant with a scaly erythematosus rash whose mother had systemic lupus erythematosus. The typical association of maternal antibodies to Ro/SSA was described for congenital heart block (CHB) several decades later in 1983 (R5, S8). The skin rash, as well as other transient syndromes, disappears within 6 months after birth, coinciding with the disappearance of maternal autoantibodies. The characteristic lesions of skin rash are erythematosus, annular, scaling plaques with a predilection for sun-exposed scalp and periorbital area as well as in the groin, on the soles of the feet, and in the axilla (L11, L12, M4, M11, M13, T4, W7). The lesions are often apparent in the first few postnatal months. Most affected infants have antiRo and/or anti-La. In rare cases anti-U1RNPs are causative antibodies (D17, P7). Up to 90% of cases of CHB are due to neonatal lupus. Another 10% of cases occur in infants suffering from major anatomic lesions or mesotheliomas of the atrioventricular node (D13). Most CHBs are third- and second-degree atrioventricular heart blocks (B31, S16). However, low-titer maternal anti-Ro may be associated with first-degree atrioventricular heart block as well as sinus bradycardia (B32, D15, D16). The autoantibodies mostly associated with CHB are anti-52-kDa Ro/SSA, anti-60-kDa Ro/SSA, and anti-La/SSB. Julkunen et al. (J5) examined serum from 31 mothers of children with CHD and found that incidences of anti52-kDa Ro, anti-60-kDa Ro, and anti-La were 97%, 77%, and 39%, respectively. The pathological findings at the end of gestation are calcification, fibrosis, and fatty degeneration of the cardiac conduction system, especially the artioventricular (AV) node. CHB carries a significant mortality (14–22%) (A17, B32, M14) and morbidity. Mortality is highest within the first 3 months (B32). The treatments include heart transplantation (S16) and permanent pacemaker. Two-thirds of the children required a pacemaker within 3 years after birth, and 100% by the age of 18. Pacemaker therapy can lead to a normal life expectancy (S16).

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There is much clinical and experimental evidence that anti-Ro/La antibodies are arrhythmogenic to the fetal heart: 1. There is a close correlation between CHB and fetal anti-Ro/La. 2. Lower titers of anti-Ro are associated with low risk for CHB (B22, B35, D11, D16, P5, S15, S16). 3. Significantly higher levels of anti-52-kDa Ro and anti-La were demonstrated in the mothers of infants with CHB (D13). 4. From the results of prenatal ultrasound findings, the timing of CHB is not randomly distributed throughout gestation. Bradycardia is most often identified between 18 and 24 weeks of gestation; the time coincides with a period of significantly increased passage of maternal IgG autoantibodies into the fetal circulation (D11, D16). 5. Anti-60-kDa Ro and anti-52-kDa Ro were found in acid eluate of a heart from a fetus with CHB who died at 34 weeks of gestation (R7). 6. Perfusion of newborn rabbit myocytes with anti-Ro/La results in reduction of cardiac repolarization and alteration of calcium transport (A13, A14). 7. Perfusion of young adult rabbit hearts induces AV block and inhibition of L-type calcium currents (G3). 8. Conduction defects in BALB/c mice are induced after immunization with 52-kDa Ro (M18). 9. Perfusion of human fetal heart with anti-52-kDa Ro results in AV block (B22). The pathogenic mechanisms of CHB are not fully understood. Currently proposed mechanisms include the following observations: 1. Anti-La binds to the sarcolemmal membrane of human fetal cardiocytes at 9–15 weeks of gestation, whereas binding to adult hearts was not observed (L18). 2. The unique fetal alternative splicing mRNA exon 4 products of 52-kDa Ro-encoding amino acids include a leucine zipper (C13). The leucine zipper is the major autoantigen epitope (B23, B33, D12, D14, F10, K4, M17, R9). 3. Induction of apoptosis in cultured cardiocytes results in surface accessibility of the Ro/La antigens (M17). There are many unresolved questions. 1. Only 1% of infants whose mothers have anti-Ro/La will develop neonatal lupus (P8). 2. The recurrence rate is only 25% (P8). 3. Up to 40% of mothers with infants with neonatal lupus are asymptomatic (P8, W7). Only about 10–25% of mothers fulfill the criteria for the diagnosis of SLE.


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4. Mothers of children with cutaneous neonatal lupus had a greater incidence of overt autoimmune disease (75%) than mothers of children with CHD (44%) after 7-year follow-up of 88 patients (L7). Compared with anti-dsDNA, relatively fewer studies have been performed on anti-Ro/La antibodies. However, the unique ability to demonstrate the pathogenic role of autoantibodies causing disease provides us a chance to explore the mechanism of human autoimmune disease. The possibly most important task is to identify the cofactors making the infants of mothers with anti-Ro/La vulnerable to developing neonatal lupus syndromes. 3.3.2. Anti-Ro, Anti-La, and Lupus Nephritis Is anti-Ro antibody nephritogenic? Regarding the relationship between anti-Ro antibody and lupus nephritis, conflicting results have been reported. Some researchers observed an association of anti-Ro antibody with nephritis (W5, N5). Anti-Ro antibody has been shown to be enriched in glomerular eluates from postmortem kidneys of two patients who died with lupus nephritis (M5). Skinner et al. showed that anti-Ro, anti-La, and anti-DNA antibodies all are concentrated in renal tissue (S18). In contrast, other investigators found a decreased risk of kidney involvement (B34). Zimmermann et al. showed that patients with anti-Ro antibody, determined by ELISA, but with anti-Ro60 reactivity alone (determined by immunoblotting) and nonblotting patients were apparently less prone to develop lupus nephritis (Z3). Our data revealed no association between anti-Ro or anti-La antibody titers and renal involvement (C18). However, patients with proteinuria had lower anti-Ro antibody titers. In addition, patients with class III lupus nephritis tend to have lower anti-Ro antibody titers than patients with other classes of lupus nephritis. However, possibly due to the small number of cases, this did not reach statistical significance. Possible explanations for the discrepancy include genetic differences and environmental or other, unknown factors. Further study is needed to determine anti-Ro/La and renal involvement in SLE.

4. Discussion and Conclusion Based on studies of the structure, the fine specificity, and the antigenic sources of autoantibodies, together with progress in genetics and immunology, it is possible to explore the basic pathogenetics of the prototype of autoimmune disease—SLE. Although most patients suffering from the disease have a long life, there are still fulminant cases resulting in mortality and morbidity, especially when the kidneys and/or central nervous system are involved. The highly varied clinical presentations are somewhat reflected by the various presentations of serological autoantibodies. Besides diagnosis, the main clinical application of the knowledge derived from studies of autoantibodies is for treatment. We

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have shown that if pediatric lupus nephritis is treated earlier and more aggressively, there is a better outcome, even for class IV proliferative glomerulonephritis (Y4). There are several relatively new therapeutic modalities for the treatment of SLE. Trying to eliminate pathogenic anti-dsDNAs, Ferguson et al. developed an antigenbased heteropolymer (AHP) (F3). AHP is a bispecific dsDNA × monoclonal antibody (mAb) complex (dsDNA × anti-CR1 mAb) that enables the use of the unique immune complex-binding and clearing capacity of the complement receptor (CR1) on primate erythrocytes. In vitro studies of AHP show a substantial reduction (≥90%) of anti-dsDNA titer (L20). In vivo studies in two rhesus monkeys indicate that the erythrocyte-bound antibodies are rapidly cleared from the circulation (F3). Other methods of clearing autoantibodies are extracorporeal and use the immunoadsorption principle. Hollow fibers with dextran sulfate-coated cellulose membranes can adsorb pathogenic anti-DNA subgroups of high avidity and/or cationic antibodies, anticardiolipin, and anaphylatoxins (S35). The SLE disease activity index significantly decreased after such treatment (S35). However, the indication is still controversial. We are also trying a new drug for SLE patients. Cordyceps sinensis (CS) is a traditional Chinese medicine with immunomodulatory effect. We have shown that MRL lpr/lpr mice treated daily with H1-A extract of CS for 8 weeks show progressivly reduced anti-dsDNA production (Y3). Although there was no change in immune complex formation, there were less mesangial proliferation (Y3). Can genetic-prone people be prevented from getting SLE? Ofosu-Appiah et al. fed SLE mice with kidney extract (KE) as immunomodulatory therapy (O3). The KE-fed mice had marked reduction of IgG1 and IgG3 responses, together with markedly suppressed IL-4 and IL-10 production (O3). All of these therapies are under development. The treatment of SLE is still a challenging task for the physician. The importance of the effective treatment of lupus nephritis is reflected by the results of mass urinary screening for school children in Taiwan: The most important secondary glomerulonephritis discovered by the screening is SLE with lupus nephritis (L19). In order to achieve a better prognosis, physicians need to be familiar with the effects of autoantibodies, diagnose SLE early, and treat it with appropriate methods. REFERENCES A1. Aarden, L. A., de Groot, E. R., and Feltkamp, T. E. W., Immunology of DNA. III. Crithidia luciliae, a simple substrate for the determination of anti-dsDNA with the immunofluorescence technique. Ann. N. Y. Acad. Sci. 254, 505–515 (1975). A2. Abu-Shakra, M., Urowitz, M. B., Gladman, D. D., and Gough, J., Mortality studies in systemic lupus erythematosus: Results from a single center. I. Causes of death. J. Rheumatol. 22, 1259– 1264 (1995).


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A3. Abu-Shakra, M., Urowitz, M. B., Gladman, D. D., and Gough, J., Mortality studies in systemic lupus erythematosus: Results from a single center. II. Predictor variables for mortality. J. Rheumatol. 22, 1265–1270 (1995). A4. Adams, E., Basten, A., and Goodnow, C. C., Intrinsic B cell hyporesponsiveness accounts for self-tolerance in lysozyme/anti-lysozyme double-transgenic mice. Proc. Natl. Acad. Sci. USA 87, 5687–5691 (1990). A5. Akbar, A. N., Lord, J. M., and Salmon, M., IFN-alpha and IFN-beta: A link between immune memory and chronic inflammation. Immunol. Today 21, 337–342 (2000). A6. Alarcon-Segovia, D., and Llorente, L., Antibody penetration into living cells. IV. Different effects of anti-native DNA and anti-ribonucleoprotein IgG on the cell cycle of activated T gamma cells. Clin. Exp. Immunol. 52, 365–371 (1983). A7. Alarcón-Segovia, D., Llorente, L., Fishbein, E., and D´ıaz-Jouanen, E., Abnormalities in the content of nucleic acids of peripheral blood mononuclear cells from patients with systemic lupus erythematosus. Arthritis Rheum. 25, 304–317 (1982). A8. Alarcón-Segovia, D., Ruiz-Argüelles, A., and Fishbein, E., Antibody to nuclear ribonucleoprotein penetrates live human mononuclear cells through Fc receptors. Nature 271, 67–69 (1978). A9. Alarcón-Segovia, D., Ruiz-Argüelles, A., and Llorente, L., Antibody penetration into living cells. II. Anti-ribonucleoprotein IgG penetrates into Tgamma lymphocytes causing their deletion and the abrogation of suppressor function. J. Immunol. 122, 1855–1862 (1979). A10. Alarcon-Segovia, D., Ruiz-Arguelles, A., and Llorente, L., Broken dogma: Penetration of autoantibodies into living cells. Immunol. Today 17, 163–164 (1996). A11. Alberdi, F., Dadone, J., Ryazanov, A., Isenberg, D. A., Ravirajan, C., and Reichlin, M., Crossreaction of lupus anti-dsDNA antibodies with protein translation factor EF-2. Trends Biochem. Sci. 15, 420–424 (1990). A12. Albert, M. L., Sauter, B., and Bhardwaj, N., Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392, 86–89 (1998). A13. Alexander, E. L., Buyon, J. P., Lane, J., Lafond-Walker, A., Provost, T. T., and Guarnieri, T., Anti-SS-A/Ro SS-B/La antibodies bind to neonatal rabbit cardiac cells and preferentially inhibit in vitro cardiac repolarization. J. Autoimmun. 2, 463–469 (1989). A14. Alexander, E., Buyon, J. P., Provost, T. T., and Guarnieri, T., Anti-Ro/SS-A antibodies in the pathophysiology of congenital heart block in neonatal lupus syndrome, an experimental model. In vitro evectrophysiologic and immunocytochemical studies. Arthritis Rheum. 35, 176–189 (1992). A15. Alspaugh, M. A., and Maddison, P., Resolution of the identity of certain antigen–antibody systems in systemic lupus erythematosus and Sjögren’s syndrome: An interlaboratory collaboration. Arthritis Rheum. 22, 796–798 (1979). A16. Alspaugh, M. A., and Tan, E. M., Antibodies to cellular antigens in Sjögren’s syndrome. J. Clin. Invest. 55, 1067–1073 (1975). A17. Alyward, R. D., Congenital heart block. Br. Med. J. 1, 943 (1928). A18. Amoura, Z., Chabre, H., Koutouzov, S., Lotton, C., Cabrespines, A., et al., Nucleosomerestricted antibodies are detected before anti-dsDNA and/or antihistone antibodies in serum of MRL–Mp lpr/lpr and +/+ mice, and are present in kidney eluates of lupus mice with proteinuria. Arthritis Rheum. 37, 1684–1688 (1994). A19. Amoura, Z., Piette, J.-C., Chabre, H., Cacoub, P., Papo, T., et al., Circulating plasma levels of nucleosomes in patients with systemic lupus erythematosus: Correlation with serum antinucleosome antibody titers and absence of clear association with disease activity. Arthritis Rheum. 40, 2217–2225 (1997). A20. Andreassen, K., Bredholt, G., Moens, U., Bendiksen, S., Kauric, G., and Rekvig, O. P., T cell lines specific for polyomavirus T-antigen recognize T-antigen complexed with nucleosomes: A molecular basis for anti-DNA antibody production. Eur. J. Immunol. 29, 2715–2728 (1999).

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PATHOBIOCHEMISTRY OF NEPHROTIC SYNDROME Vladim´ır Tesaˇr,∗ Tom´ aˇ s Zima,† and Marta Kalousov´ a‡ ∗ First Department of Medicine, Division of Nephrology, First Faculty of Medicine and University Hospital, Prague, Czech Republic; † Institute of Clinical Chemistry, First Faculty of Medicine and University Hospital, Prague, Czech Republic; ‡ Institute of Medical Biochemistry and Institute of Clinical Biochemistry, First Faculty of Medicine and University Hospital, Prague, Czech Republic

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Structure and Function of the Glomerular Capillary Wall . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Structure and Function of Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Structure and Function of the Glomerular Basement Membrane . . . . . . . . . . . . . . 2.3. Structure and Function of Podocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Structure and Function of Podocyte Foot Processes . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Biology of Podocyte Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Vasoactive Hormones and Podocyte Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Slit Diaphragm and Its Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Inherited Podocyte Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Pathogenesis of Proteinuria in Acquired Forms of Nephrotic Syndrome . . . . . . . . . . . . 3.1. Causes of Acquired Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Mechanisms of Proteinuria in Acquired Nephrotic Syndrome . . . . . . . . . . . . . . . . 3.3. Nephrin and Podocin in the Acquired Forms of Nephrotic Syndrome . . . . . . . . . . 3.4. Plasma Factors That May Increase Permeability of the Glomerular Capillary Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Reabsorption of Albumin in the Proximal Tubule . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Biochemical Signs and Clinical Symptoms of Nephrotic Syndrome . . . . . . . . . . . . . . . 4.1. Hypoproteinemia and Hypoalbuminemia in Nephrotic Syndrome . . . . . . . . . . . . . 4.2. Hyperlipidemia in Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Pathogenesis of Edema in Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Thromboembolic Complications of Nephrotic Syndrome. . . . . . . . . . . . . . . . . . . . 4.5. Infections in Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Protein Binding of Endogenous and Exogenous Substances. . . . . . . . . . . . . . . . . . 4.7. Changes of Renal Function in Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . 4.8. Proteinuria and Progression of Chronic Renal Insufficiency . . . . . . . . . . . . . . . . . . 5. Diagnosis of Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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6. Treatment of Nephrotic Syndrome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Nephrotic syndrome is a clinical and laboratory syndrome defined by heavy proteinuria (exceeding 3.5 g/1.73 m2 of body surface area in adults, or 40 mg/hr/m2 in children), accompanied by hypoproteinemia (mainly hypoalbuminemia), hypercholesterolemia (in severe cases also hypertriacylglycerolemia), lipiduria, and edema. Children with nephrotic syndrome may suffer mainly from life-threatening infectious complications, whereas adult nephrotics are endangered predominantly by (potentially fatal) thromboembolic complications. Persistent nephrotic syndrome may result in negative nitrogen balance and malnutrition, accelerated atherosclerosis owing to the severe hyperlipidemia (mainly hypercholesterolemia) and progression to chronic renal failure. Urinary loss of binding proteins may be accompanied by increased sensitivity to some protein-bound substances, including drugs and endogenous hormones. Remission of nephrotic syndrome may rarely evolve spontaneously; more commonly remission may be achieved either by the elimination of causative agents (e.g., drug or infection) or by immunosuppressive treatment. In some glomerular diseases (e.g., minimal change disease) remissions of nephrotic syndrome may be followed by recurrent relapses; in other glomerular diseases, nephrotic syndrome may persist for many years until the patient finally reaches end-stage renal failure (e.g., focal segmental glomerulosclerosis, diabetic nephropathy, or renal amyloidosis). Nephrotic syndrome has been the subject of many recent reviews (O7, C1). In this review we concentrate mainly on new data concerning the molecular mechanisms of glomerular permselectivity and the biology of podocytes. Inherited podocyte disorders with defined molecular deficiencies of different podocyte proteins may shed new light on the pathogenesis of more common acquired forms of nephrotic syndrome with still unknown pathogenesis (e.g., minimal change disease and focal segmental glomerulosclerosis).

2. Structure and Function of the Glomerular Capillary Wall The glomerular capillary network lined with fenestrated endothelia is surrounded by the glomerular basement membrane and visceral epithelial cells (podocytes). Podocytes cover the outer aspect of the glomerular basement membrane with their

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numerous projections called foot processes. Apical parts of foot processes near to the external surface of the glomerular basement membrane are interconnected by thin slit diaphragms (Fig. 1). Plasma is filtered through a sieve consisting of three main layers: (1) large endothelial fenestrations, (2) the dense network of the glomerular basement membrane, and (3) the slit diaphragm between podocyte processes. The glomerular capillary wall has a very high hydraulic permeability and the glomerular basement membrane and the slit diaphragm probably contribute approximately 50% each to the total hydraulic resistance of the capillary wall (D8). Foot process effacement found both in experimental models of nephrotic syndrome and in human glomerulopathies dramatically reduces the hydraulic permeability of the glomerular capillary wall (G11). The glomerular capillary wall is believed to be selective for size, charge, and shape (D2). Charge selectivity is probably based on the presence of a dense network of negatively charged proteoglycans in the glomerular basement membrane and negatively charged molecules on the surface of endothelial and epithelial cells. The size selectivity may be also in part determined by the dense network of the glomerular basement membrane, but the most restrictive part of the size-selective barrier is probably the slit diaphragm. Only small molecules with an “effective” radius of less than 1.8 nm pass freely through this diaphragm; molecules with an effective radius of more than 4.0 nm are completely restricted (the effective radius of albumin is 3.6 nm). The importance of the normal structure of both the glomerular basement membrane and the slit diaphragm for the preservation of glomerular permselectivity may be best illustrated by the fact that antibodies directed against different structural proteins of the glomerular basement membrane [proteoglycans, type IV collagen, or laminin (V2)] and antibodies directed against the main protein of the slit diaphragm, nephrin (K10), may cause massive proteinuria. 2.1. STRUCTURE AND FUNCTION OF ENDOTHELIAL CELLS Fenestrated endothelial cells usually have their bodies near the mesangial pole of the glomerular capillary; the flat, fenestrated part of the endothelial cells covers about 60% of the capillary surface (Fig. 1) and contains round pores 50–100 nm in diameter, occupying in rat about 13% of the capillary surface (E1, B11). Unlike in other fenestrated endothelia, endothelial pores in glomerular fenestrated endothelium lack a diaphragm. The luminal membrane is negatively charged due to polyanionic glycoproteins coating its surface (e.g., sialoglycoprotein and podocalyxin). Podocalyxin is believed to be the major surface polyanion of both endothelial and visceral epithelial cells (S6). Glomerular endothelial cells are able to produce endothelin and endotheliumderived relaxing factor (EDRF), synthesize and release von Willebrand factor,

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factor IXa, and factor Xa, and express HLA-DR antigens; they play an active role in the control of inflammation and coagulation. The endothelial cell barrier normally restricts the passage of formed elements (e.g., red cells) through the glomerular capillary wall, but it presents no restriction to the passage of different macromolecules. 2.2. STRUCTURE AND FUNCTION OF THE GLOMERULAR BASEMENT MEMBRANE The glomerular basement membrane (GBM) forms the backbone of the glomerular tuft. It is composed of three layers: lamina rara interna, lamina densa, and lamina rara externa. The glomerular basement membrane is composed of a network of collagen type IV molecules (H5) intertwined with nidogen to another network composed of molecules of laminin. Type IV collagen and laminin are responsible for the firmness of the glomerular basement membrane and enable adhesion of endothelial cells and podocytes as well. The network of type IV collagen restricts mainly the passage of large (globular) electroneutral molecules through the glomerular basement membrane. There are six different chains of collagen IV (α1–α6) with different locations in the basement membrane. Classic α1 and α2 chains are located mostly in the subendothelial space (lamina rara interna); whereas α3 and α4 chains are located in the lamina densa, so these different chains may form separate networks. Collagen monomers consist of a triple helix and are able to form dimers and tetramers communicating at its amino (7S domain) and carboxy (NC1 domain) termini, forming together a flexible assembly providing mechanical strength to the basement membrane and a scaffold for the alignment of other proteins. The importance of “nonclassic” collagen IV chains is best documented by their role in the pathogenesis of glomerular disease. Glomerular injury in Goodpasture syndrome is mediated by antibodies directed against the NC1 domain of α3 chain of collagen IV, the X-linked form of Alport syndrome is caused by mutation in the gene for α5 chain (T6), and familial benign hematuria (thin membrane disease) is caused by different mutations of α3 and α4 chains (B1). All these diseases are primarily characterized by hematuria, whereas nephrotic proteinuria is very uncommon as a presenting symptom. Thus, the absence or abnormal function of “nonclassic” collagen IV chains may decrease the strength of the glomerular basement membrane resulting in the formation of larger defects enabling the passage of red cells through glomerular capillary walls. Glomerular permselectivity for macromolecules does not seem to be substantially impaired. Laminin [consisting of three polypeptide chains, A, B1 (possibly replaced in GBM by S) and B2] is the most important noncollagenous protein of the glomerular basement membrane. Laminin forms a second network, which is connected to the collagen IV network probably via another protein called entactin or nidogen. Laminin is probably very important for cellular differentiation and adhesion, but its mesh clearly also contributes to the structure of the glomerular basement membrane. The postnatally common embryonic laminin-10 isoform is


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replaced by laminin-11 isoform in the glomerular basement membrane. Interestingly, mice deficient for the β2 chain of laminin (present in laminin-11 and absent in laminin-10) develop nephrotic syndrome (S14, T6). This suggests a putative role of laminin-11 isoform in maintaining normal glomerular permselectivity. Cell attachment to the glomerular basement membrane is mediated by laminin, entactin, and fibronectin, at least partly through integrin receptors on the cell surface of both endothelial and visceral epithelial cells. The electronegative charge of the glomerular basement membrane contributes to the repulsion of mostly negatively charged plasma proteins. Heparan sulfate has been shown to be the biochemical substrate of the anionic sites demonstrated in the glomerular basement membrane (K2). Heparan sulfate is a component of two main proteoglycans of the glomerular basement membrane, perlecan (H2) and aggrin (G9). Perfusion of the kidney with heparinase resulting in the removal of all glycosaminoglycans with the exception of keratan sulfate increases the permeability of the glomerular basement membrane for ferritin (K3). Loss of glomerular anionic structures was also described in experimental nephrotic syndrome induced in rats by puromycin aminonucleoside (C4). The generally accepted concept of glomerular charge selectivity has been challenged (R12). Observed differences in fractional clearances of differently charged macromolecules (namely dextrans) also could be caused by their different catabolism in proximal renal tubule (R12) (see Section 3.5). 2.3. STRUCTURE AND FUNCTION OF PODOCYTES Podocytes (glomerular epithelial cells) are highly specialized cells covering the outer layer of the glomerular basement membrane, where they form multiple projections called foot processes, which are interconnected with an interpedicellar membrane, that is, a slit diaphragm (P3) (Fig. 1). Podocytes are attached to the glomerular basement membrane, which is mainly synthesized by these cells (K24). One of the main functions of podocytes is supportive, stabilizing the structure of the glomerular capillary wall and counteracting the distention of the capillary wall and the slit diaphragm. Podocytes also represent the final barrier to the glomerular filtrate. Podocytes are polarized cells, so one can differentiate between luminal and abluminal (basal) membrane domains (the basal domain corresponds to the sole plates of the foot processes, which are embedded in the basement membrane). The slit diaphragm forms the border between the luminal and the abluminal membranes. The luminal membrane is covered with negatively charged sialoglycoproteins, including podocalyxin, podoendin, SGP115/107, and others (M7). Other proteins, including megalin/gp330, are present on the entire surface of podocytes. Antibodies in the rat model of membranous nephropathy, Heymann nephritis, are directed against megalin/gp330 (S2). Podocytes have a very well developed cytoskeleton. In the cell body microtubules and intermediated filaments (vimentin, desmin)

FIG. 1. Putative location of podocyte proteins mutated in different types of familial nephrotic syndrome.


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dominate (K22); in the foot processes microfilaments containing actin, myosin, and α-actinin are primarily found (A5). Adhesion of podocytes to the basement membrane is mediated especially by β1 integrins [e.g., α3β1 binding to type IV collagen and laminin (C10)]. Other proteins, for example, talin and vinculin, also have been localized to foot processes (D7). Integrins may also, through transmembrane signaling, influence the arrangement of the cytoskeleton (C6). 2.4. STRUCTURE AND FUNCTION OF PODOCYTE FOOT PROCESSES Foot processes contain contractile structures composed of actin, myosin, α-actinin, vinkulin, and talin, which are attached to the glomerular basement membrane with the help of α3β1-integrin (B2, C10). Contractile elements of podocytes can respond to the stimulation of podocytes by vasoactive hormones and so influence the ultrafiltration coeficient Kf . Podocytes may contribute to the selectivity of the glomerular capillary wall (both size and charge selectivity). Damage to the podocytes leads to retraction (fusion) of foot processes and to proteinuria. Retracted and damaged podocytes are not able to cover all the outer surface of the glomerular basement membrane. Change of podocyte shape, retraction of foot processes, and podocyte loss can be observed in different human diseases accompanied by heavy proteinuria and nephrotic syndrome, for example, minimal change disease and focal segmental glomerulosclerosis, and also in diabetic nephropathy. Contractile elements of the podocyte foot processes, which may influence the hydraulic permeability of the glomerular capillary wall, may be regulated via vasoactive hormones. Receptors for some vasoactive hormones, for example, endothelin (R4), atrial natriuretic peptide (S9), nitric oxide (K22), and angiotensin II (Y1), have been described on the podocyte surface. Differentiation of podocytes begins in the early phase of glomerular development (the phase of the so-called S-shaped body). Foot processes are formed, while tight junctions between podocytes are opened and replaced by slit diaphragms enabling the passage of glomerular filtrate through the space between the foot processes of podocytes, with the slit diaphragm remaining as the last sieve. Electrostatic repulsion, as a result of the presence of sialylated proteins on the podocyte surface, is important for the opening of intercellular spaces (T1). In fact, sialidase administration leads to the collapse of foot processes and slit diaphragms. 2.5. BIOLOGY OF PODOCYTE PROTEINS Podocalyxin is the main sialoprotein on the surface of the podocyte body and the apex of podocytes above the level of the slit diaphragms (K16). Podocalyxin activation can be demonstrated in the later phase of glomerular development and immediately precedes the formation of urinary spaces. Podocalyxin is a type I membrane protein with four potential N-glycosylated sites and many potential sites for

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O-glycosylation (K17). The intracellular part of podocalyxin is attached to the actin cytoskeleton through ezrin (one of the actin-binding proteins of the EZM family—ezrin, moesin), is related to erythrocyte band 4.1 protein (O6). Interaction of ezrin with podocalyxin may be mediated or stabilized by another protein, Na+, H+ exchanger regulatory factor (NHERF) (O6). The significance of the cytoskeleton for the integrity of podocytes may be demonstated by the fusion of podocytes after cytochalasin administration (A4). Recently, several new podocyte-specific proteins have been identified: nephrin, megalin, podoplanin, GLEPP-1, and synaptopodin. Nephrin is a basic structural molecule of the slit diaphragm. Mutations of the nephrin gene result in congenital nephrotic syndrome of the Finnish type. The structure and function of the slit diaphragm are described in detail in Section 2.7. Megalin is a transmembrane protein of rat podocytes with a molecular weight of ca. 600 kDa belonging to the low-density lipoprotein (LDL) receptor family (F1). Megalin may play a role in the endocytosis of lipoproteins by rat podocytes (megalin is localized in clathrin-coated pits on the surface of podocytes mediating endocytosis). Antibodies against megalin are pathogenic in experimental membranous nephropathy in rats, Heymann nephritis. Megalin is probably not expressed by human podocytes. In proximal tubular epithelial cells, megalin plays an important role in the reabsorption of some protein molecules (Section 3.5). Podoplanin is an integral membrane glycoprotein of rat podocytes with a molecular weight of 43 kDa (B9). Podoplanin expression was demonstrated to be decreased in nephrotic syndrome induced in rats by the administration of puromycin aminonucleoside. Administration of antibodies directed against podoplanin induced the retraction of podocyte foot processes and temporary proteinuria in rats. Podoplanin can be of importance for maintaining the integrity of foot processes and for regulation of glomerular permeability (M3). GLEPP-1 is a membrane protein tyrosin-phosphatase with a large extracellular part containing eight fibronectin type III domains and one intracellular domain with tyrosin-phosphatase activity (T3). GLEPP-1 is located on the surface of foot processes and can contribute to maintaining their structure. In the experimental model of anti-GBM nephritis in rabbits, reduction of podocyte GLEPP-1 expression was demonstrated. Decreased expression of GLEPP-1 was demonstrated also in renal biopsies of patients with crescentic glomerulonephritis (Y2). Synaptopodin is an actin-associated protein with a molecular weight of 73.7 kDa expressed apart from other sites in foot processes of mature podocytes. Its role in podocyte biology is not exactly known, but it is believed to play a role in the motility of podocyte foot processes (M6). Synaptopodin expression is increased in human glomerulopathies with foot process fusion, but its expression cannot be demonstrated in the necrotic areas of glomerular capillary wall, in cellular crescents, and in early and late phases of focal segmental glomerulosclerosis FSGS, including collapsing FSGS and human immunodeficiency virus (HIV)-induced FSGS (K15).


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Further information about the function of these podocyte proteins and identification of further podocyte proteins and their interactions will clearly contribute to the better understanding of the mechanisms of proteinuria. 2.6. VASOACTIVE HORMONES AND PODOCYTE FUNCTION Angiotensin II is believed to regulate the glomerular filtration rate via its action on the tone of the afferent and efferent arterioles and the ultrafiltration coefficient. Its action results in the loss of selectivity of the glomerular capillary wall and increased proteinuria (I4). Moreover, angiotensin II also acts upon the mesangial cells as a growth factor and stimulates hypertrophy of podocytes. Rat podocytes express both AT1 and AT2 receptors (S10). Some hormones, for example, bradykinin, prostanoids, ATP, thrombin, endothelin, vasopresin, and norepinephrine (H4), increase the intracellular concentration of calcium in podocytes, whereas dopamine, epinephrine, and prostaglandin E2 (PGE2) increase the podocyte concentration of cAMP, and atrial natriuretic peptide (ANP) increases the podocyte concentration of cGMP. Vasoactive hormones can influence the contractility and the structure of podocyte foot processes. An increase of intracellular calcium and cAMP in podocytes leads to contraction of foot processes and a decrease of Kf , whereas an increase of cGMP can act in the opposite direction (S9). Vasoactive hormones can also change the charge on the podocyte surface and so facilitate proteinuria (P2). It has been demonstrated that podocyte damage may cause and perpetuate progressive chronic renal failure (K23, K25). Molecular pathogenesis of podocyte damage resulting in glomerulosclerosis is currently far from being fully elucidated. Vasoactive hormone (namely angiotensin II)-induced activation of podocytes may contribute to podocyte damage even in patients with chronic renal failure (A2). 2.7. SLIT DIAPHRAGM AND ITS STRUCTURE The glomerular basement membrane may restrict the filtration of large neutral and smaller negatively charged molecules. Nevertheless, the membrane between the podocyte foot processes (slit diaphragm) is the final and most important barrier preventing the passage of smaller molecules (like albumin) into the glomerular filtrate. Its width (distance between adjacent foot processes) is about 40 nm. When the intraglomerular pressure increases, the width of the slit diaphragm can probably increase (Y3). Based on electronoptic studies, a zipper-like structure with pores of 4 × 14 nm (slightly smaller than a molecule of albumin) has been proposed (R10). A high concentration of a specific α-isoform of a protein from the zonula occludens ZO-1 was found on the cytoplasmic site of the plasma membrane at the place where the slit diaphragm inserts (K27). At the same time, proteins normally present in tight junctions, like cingulin and occludin, are absent. It was hypothesized that

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the slit diaphragm represents a modified form of zonula occludens (tight junction) with increased permeability, attached to the cytoskeleton of podocytes with the help of ZO-1 protein. The molecular structure of the slit diaphragm was unknown until recently. Orikasa et al. isolated a monoclonal antibody reacting with glomeruli (designated 5-1-6) inducing massive proteinuria in rats (O5). Immunoelectronoptic studies demonstrated that nephritogenic antibody reacts with a protein with a molecular weight of about 51 kDa, which is exclusively present in the slit diaphragm (K9). Identification of the main protein of the slit diaphragm, called nephrin, was accomplished through studies of the mutated gene NPHS1 in patients with congenital nephrotic syndrome of the Finnish type (T6). The nephritogenic antibody 5-1-6 has been recently demonstrated to react with nephrin (T4). Administration of the nephritogenic antibody 5-1-6 results in the loss of nephrin and ZO-1 protein from the podocyte membrane, but the foot process structure remains well preserved despite nephrotic proteinuria (K10). This is dissimilar to congenital nephrotic syndrome, where a defect of nephrin is accompanied by foot process fusion. No explanation for this apparent paradox is available. Nephrin may be important for the normal development of slit diaphragms, but the structure of the slit diaphragm may be preserved by some other protein(s). Alternatively, foot process fusion may be the result of long-lasting (at least several weeks) proteinuria. Further studies of the structure of the slit diaphragm and its changes after the administration of the 5-1-6 antibody are therefore warranted. Nephrin belongs to the immunoglobulin superfamily; it has eight extracellular immunoglobulin-like domains and one extracellular fibronectin-like domain. The intracellular part of nephrin has no homology with any other known protein; it contains nine tyrosyl residues, which under some conditions possibly may be phosphorylated. The immunoglobulin domains are of the so-called C2 type, which are common in proteins active in intercellular interactions (adhesion). Immunoelectronoptically, nephrin has been localized exclusively to the slit diaphragm. Two molecules of nephrin are believed to extend from contralateral foot processes in parallel with the glomerular basement membrane and form, by means of homophilic interaction (similar to other adhesion molecules containing immunoglobulin-like domains, e.g., cadherins N-CAM or C-CAM) the formerly described (R10) zipper-like structure (R11), with a central filament formed by six amino-terminally localized immunoglobulin-like domains interacting with similar immunoglobulin-like domains of the opposite molecule of nephrin. The suggested structure of two nephrin molecules could approximately extend the length of the slit diaphragm (35–45 nm). Suggested pores of size 14 × 4 nm in the slit diaphragm could correspond to the space between two neighboring molecules of nephrin. The fibronectin-like domain and the area between the first six and the remaining two immunoglobulin-like domains (the so-called spacer) could enable the expansion of the slit diaphragm during an increase of the intraglomerular pressure.


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The current model of the slit diaphragm does not exclude the possibility that other (yet unidentified) proteins may contribute to their structure. CD2-associated protein (CD2AP) with a molecular weight of 80 kDa interacts with the cytoplasmic part of nephrin. Knockout mice with deficient CD2-associated protein also develop nephrotic syndrome with the fusion of podocyte foot processes (S14). CD2-associated protein is also expressed by T-lymphocytes and takes part in the interaction of T-lymphocytes with antigen-presenting cells. Putative dysfunction of T-lymphocytes in minimal change disease could be hypothetically explained by the disordered structure or function of CD2-associated protein (J2). Decreased mitogen-stimulated T-lymphocyte proliferation has been demonstrated in CD2AP knockout mice, similar to patients with minimal change disease (S14). T-lymphocyte dysfunction, however, is not specific for minimal change disease, but may be more probably the consequence than the cause of nephrotic syndrome (W1, S7) (see Section 3.4). P-cadherin is another protein whose presence has been recently demonstrated in the slit diaphragm (R5). Further studies of podocyte biology may be enhanced by the development of conditionally immortalized human podocyte cell lines, which may enter growth arrest and express different podocyte proteins (nephrin, podocin, CD2AP, synaptopodin, ZO-1, P-cadherin, and catenins) and also cyclin-dependent kinase inhibitors, for example, p27 and p57 (S3). De novo expression of cyclin-dependent kinase inhibitor p57 during glomerulogenesis coincides with the cessation of podocyte proliferation. During glomerular injury decreased expression of p57 occurs predominantly in proliferating podocytes, expressing proliferating cell nuclear antigen (PCNA) (H3). Proliferation may contribute to healing, but it may be also followed by the loss (apoptosis) of affected podocytes. The cytoplasmic part of nephrin interacts also with ZO-1 protein and actin (K10). Interaction of the antibody or toxin with the extracellular part of nephrin could thus also result in intracellular signaling (phophorylation of tyrosine residues in the cytoplasmic part of nephrin), change of the actin cytoskeleton, and foot process fusion. Indeed, increased levels of phosphotyrosine were demonstrated in renal biopsies of patients with minimal change disease and membranous nephropathy (B2). 2.8. INHERITED PODOCYTE DISORDERS Congenital nephrotic syndrome of the Finnish type is clinically characterized by heavy proteinuria present already in utero, which leads without nephrectomy and renal replacement therapy to the death of the affected children usually before the second year of life. Electronoptically, the glomerular basement membrane seems to be intact with the fusion of the podocyte foot processes. The chemical composition of the glomerular basement membrane is normal in patients with congenital nephrotic syndrome and all genes of the main proteins of the glomerular

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TABLE 1 INHERITED PODOCYTE DISORDERSa Disease

Gene

Locus

Inheritance

Protein

Congenital nephrotic syndrome (Finnish type) Familial FSGS (steroid-resistant nephrotic syndrome) Familial FSGS Familial FSGS Neonatal nephrotic syndrome (in mice)

NPHS1

19q13

AR

Nephrin

NPHS2

1q25-31

AR

Podocin

ACTN4 FSGS2? CD2AP

19q13 11q21-22 —

AD AD AR

α-Actinin ? CD2AP

a FSGS, Focal segmental glomerulosclerosis; AR, autosomal recessive; AD, autosomal dominant; the gene responsible for FSGS2 has not yet been identified; the human counterpart of the CD2AP mutation has not been identified.

basement membrane are intact. The gene for congenital nephrotic syndrome of the Finnish type (NPHS1) has been localized to the 13.1. region of the long arm of chromosome 19 and it has been demonstrated that that this gene is specifically expressed in the kidney. Subsequently, more than 40 different mutations in affected families have been identified (L3). The product of the gene NPHS1 is a protein with a molecular weight of 135 kDa specifically expressed by podocytes, called nephrin (K18) (see Section 2.7). The hypothesis that the recurrence of nephrotic syndrome in about 20% of children with congenital nephrotic syndrome after renal transplantation may be caused by the immune reaction of the recipient against normal nephrin (J2), and, similarly, that patients with Alport syndrome may develop antibodies directed against glomerular basement membrane, was not substantiated. Deposition of antinephrin antibodies along the glomerular basement membrane was not demonstrated (L1). Besides congenital nephrotic syndrome of the Finnish type (T6), nephrotic syndrome (with histologic appearance of focal segmental glomerulosclerosis) may result from mutations of other podocyte proteins (T7, K4) (Table 1). Autosomal recessive familial focal segmental glomerulosclerosis (FSGS), resulting in steroid-resistant nephrotic syndrome, is caused by mutation of the gene NPHS2, encoding another podocyte protein, podocin. Podocin is a 42-kDa podocyte integral membrane protein with moderate homology to human stomatin and MEG-2 protein of Caenorhabditis elegans (B8). Its function is currently unknown, but based on the analogy to MEG-2, it may link ion channels to the cytoskeleton. Clinically, nephrotic syndrome develops usually between 3 months and 5 years of age (C3). Patients with later onset of the disease have been described as well (T8). Recently the mutation of the NPHS2 gene has been found in nearly 30% of patients with sporadic steroid-resistant nephrotic syndrome (K6). The relation between steroid resistance and sporadic mutations of podocin (and possibly other)


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gene(s) coding the structural podocyte proteins remains to be elucidated. Familial steroid-responsive nephrotic sysndrome in childhood is not linked to the NPHS2 gene. It is probably caused by mutation in another distinct gene locus (F4). Autosomal dominant familial focal segmental glomerulosclerosis (K5) may be caused by mutation in ACTN4, encoding α-actinin-4, a widely expressed protein, one of the four α-actinin isoforms significantly expressed in podocytes. α-Actinin forms head-to-tail homodimers, thus crosslinking actin filaments and reacting with other cytoskeletal and cell-surface molecules. Thus, the molecule may be important for the maintenance of podocyte shape. Autosomal dominant FSGS typically presents with adult onset, nonnephrotic proteinuria, and slowly progressive chronic renal insufficiency. A second autosomal dominant FSGS gene (FSGS2) is linked to chromosome 11q21–22, but the mutation has not yet been identified (W8). Other mutations further increasing the genetic heterogeneity of familial FSGS probably will be identified (W7). Mice knocked out for CD2-associated protein (CD2AP) also develop nephrotic syndrome and renal failure with histologic appearance of FSGS (S14). CD2AP is an 80-kDa protein interacting with the cytoplasmic domain of nephrin (Section 2.5). Human mutation of the gene for CD2AP has not been described yet. Familial FSGS may also be only one of the components of more complex familial syndromes. In unilateral renal agenesis, FSGS is probably caused by the reduction of the renal mass (A6). FSGS may also result from mutations in the Wilms tumor suppressor gene WT1 (exon 9) as part of the Denys–Drash syndrome, which includes FSGS, Wilms tumor, and male pseudohermaphroditism (P5). Homozygous mutation in the adhesion molecule β4-integrin has been recently described as a cause of FSGS with nephrotic syndrome and epidermolysis bullosa (K1).

3. Pathogenesis of Proteinuria in Acquired Forms of Nephrotic Syndrome 3.1. CAUSES OF ACQUIRED NEPHROTIC SYNDROME Familial forms of nephrotic syndrome are very rare, but they are very important for the elucidation of the molecular pathogenesis of proteinuria in the far more common acquired glomerular diseases accompanied by nephrotic syndrome. Nephrotic syndrome may complicate the course of many primary and secondary glomerulopathies. Diabetic nephropathy is the most common cause of nephrotic proteinuria (not always accompanied by full-blown nephrotic syndrome). Lupus nephritis and renal amyloidosis are much rarer secondary glomerulopathies resulting in nephrotic syndrome. The prevalence of primary glomerulopathies differs between Blacks and Whites (focal segmental glomerulosclerosis is more common

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ˇ ET AL. TESAR TABLE 2 RELATIVE FREQUENCY OF PRIMARY GLOMERULOPATHIES LEADING TO NEPHROTIC SYNDROME IN CHILDREN AND ADULTS (O7)

Minimal change disease Focal segmental glomerulosclerosis Membranous nephropathy Membranoproliferative glomerulonephritis Other diseases

Children (%)

Adults 60 yr (%) 20 2 39 0 39

in Blacks) and varies with age (Table 2). Minimal change disease and focal segmental glomerulosclerosis are more common in children and young adults; membranous nephropathy becomes the commonest primary glomerulopathy leading to nephrotic syndrome in adults above 40 years. The etiology of primary glomerulonephritis remains to be elucidated; an etiologic factor has been identified only in a minority of patients (e.g., HIV may cause a rapidly progressive form of focal segmental glomerulosclerosis, and hepatitis B, neoplasia, or penicillamine and gold salts may induce membranous nephropathy). The mechanisms leading to increased permeability of glomerular capillary walls for proteins in patients with acquired forms of nephrotic syndrome are also relatively poorly defined. In the following we concentrate mainly on the pathogenesis of minimal change disease, focal and segmental glomerulosclerosis, and idiopathic membranous nephropathy. These three diseases are responsible for about 60–95% of nephrotic syndromes and their prevalence depends on age. Minimal change disease is the most common cause of nephrotic syndrome in children, presenting typically with rapid onset of mostly steroid-sensitive nephrotic syndrome, usually with selective proteinuria (albuminuria). Light-microscopic morphology of the kidney is normal and immunofluorescence is negative. Foot process effacement on electron microscopy is the only observed pathology. Focal and segmental glomerulosclerosis (FSGS) is characterized by the sclerosis of some segments of some glomeruli. Segmental sclerosis usually progresses into global sclerosis, affecting more and more glomeruli, finally resulting in chronic renal failure. In some patients transition from minimal change disease to FSGS could be demonstrated and according to some authors, FSGS is believed to be a variant of minimal change disease with poor prognosis. Nephrotic syndrome with the histologic appearance of minimal change disease can also recur in some patients with FSGS after renal transplantation. Idiopathic membranous nephropathy is the commonest form of nephrotic syndrome in middle-aged and elderly patients. The glomerular capillary wall is thickened due to immune deposits (containing mosty immunoglobulin G, IgG)


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located between the outer aspect of the glomerular basement membrane and podocytes, usually also with the effacement of their foot processes. During the course of the disease, immune deposits are encircled by the abundantly formed and thickened glomerular basement membrane. Some patients may also progress to end-stage renal failure. 3.2. MECHANISMS OF PROTEINURIA IN ACQUIRED NEPHROTIC SYNDROME Heavy proteinuria is caused by increased permeability of the glomerular capillary wall for macromolecules. Mainly the size, the charge, and the shape influence the passage of macromolecules through the glomerular capillary wall. The glomerular basement membrane and the slit diaphragm represent the main barriers for the filtration of macromolecules. The glomerular capillary is believed to restrict (mainly due to the electrical charge) the filtration of molecules with molecular weights higher than 10 kDa, and molecules with of more than 40–50 kDa (i.e., approximately like albumin) almost do not pass through the glomerular capillary wall. Charge selectivity is probably mediated mainly by the polyanionic glycosaminoglycans present in the glomerular basement membrane (C5, G6). This barrier restricts mostly the movement of relatively small polyanionic proteins (molecular weight 70–150 kDa), mainly albumin. Loss of charge selectivity is believed to be the main cause of albuminuria (selective proteinuria) in minimal change disease (B10). Size selectivity is probably caused by the mesh of glomerular basement proteins, which effectively restricts the passage of larger proteins with molecular weight of more than 150 kDa. Loss of size selectivity is probably the main cause of nonselective proteinuria in membranous nephropathy (S13). Proteinuric states are commonly accompanied by the fusion of podocyte foot processes. The assumption that this foot process fusion is only a nonspecific secondary response of podocytes to proteinuria has been recently challenged by the observation that foot process fusion may be caused by toxic or autoimmune damage to podocytes. Podocyte damage has an important influence on both charge and size selectivity of the glomerular capillary wall. Ultrafiltration is decreased and macromolecules massively penetrate to Bowman’s space in areas of foot process fusion and especially in areas where podocytes are completely detached from underlying the glomerular basement membrane (areas between neighboring podocytes, where “naked” glomerular basement membrane is not covered by podocytes on its outer aspect). These sites are probably the anatomical correlate of functionally demonstrated “large” pores in the glomerular capillary wall. Functional studies with fractional excretion of dextrans of different charge and molecular weight showed that fractional clearance of smaller molecules of dextran (2.6–4.8 nm) may be

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decreased in nephrotic subjects compared to controls, but fractional excretion of larger molecules of dextran (5.2–6.0 nm) is, in contrast, substantially increased in nephrotic subjects (probably caused by the opening of “large” pores) (M8). Another cause of proteinuria may be damage to the glomerular basement membrane, which can be induced in rats, for example, by the infusion of elastase into the renal artery. In this model, massive proteinuria is not accompanied by any morphological sign of podocyte damage. Glomerular permeability is increased, for example, by C5b–C9 components of complement, superoxide, hydroxyl ions, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) (via superoxide), prolonged incubation with PAF, neutralization of the electronegative charge of the glomerular capillary wall with protamine, or enzymatic degradation of the glomerular basement membrane with metalloproteinase-3 (S11). The mechanism of massive proteinuria is obviously different in each primary glomerulonephritis (C7). In minimal change disease and focal segmental glomerulosclerosis, the presence of a circulating permeability factor(s) produced by T-lymphocytes has been proposed repeatedly (G3, S5) (see Section 3.4). T-lymphocyte hybridomas from patients with minimal change disease have been demonstrated to produce a permeability factor inducing severe proteinuria in rats (K20). This factor does not seem to be toxic for podocytes, but toxins leading to podocyte damage and antibodies directed against podocytes are also able to induce nephrotic proteinuria (as discussed subsequently). In some patients with idiopathic nephrotic syndrome, diffuse podocyte damage (with foot process fusion) is also accompanied by segmental capillary collapse with hyalinosis and adhesion of glomerular basement membrane to Bowman’s capsule. The relation between diffuse podocyte damage and the development of segmental lesions, if any, has not been sufficiently clarified. In some experimental models of nephrotic syndrome (e.g., in neprotic syndrome induced by puromycin aminonucleoside or adriamycin), single administration of podocyte toxin results in reversible nephrotic syndrome histologically similar to human minimal change diseases (foot process fusion), and repeated administration of this toxin leads to persistent nephrotic syndrome histologically characterized by focal and segmental glomerulosclerosis. Proteinuria is selective (concerning mostly albumin and small proteins with molecular weight lower than that of albumin) in minimal change disease and some patients with focal segmental glomerulosclerosis. It has been proposed that selective proteinuria is caused predominantly by the loss of the charge selectivity of the glomerular capillary wall. Nonselective proteinuria (with the urinary excretion also of large molecules with molecular weight of about 200 kDa, e.g., IgG) may be caused by the concomitant loss of size selectivity.


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FSGS is probably a very heterogeneous disease (D5). Viral infections (HIV-1, parvovirus B19, and simian virus 40) have been also implicated in the pathogenesis of sporadic focal and segmental glomerulosclerosis (T2). Genetic factors, for example, angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism, are supposed to play a very important role also in the progression of renal failure in affected subjects. The DD genotype is more frequent in FSGS patients compared to those with minimal change nephrotic syndrome (L2). Children with FSGS and the II genotype are less likely to progress to chronic renal failure (F3). In membranous nephropathy, subepithelial immunodeposits containing IgG and the C3 component of complement located between podocytes and the glomerular basement membrane were implicated in the podocyte damage, which is probably mediated by the local activation of complement with subsequent formation of a membranolytic complex C5b–C9 (C10). Experimental studies demonstrated that immune deposits are formed in situ by the reaction of circulating antibodies with the antigens of the podocyte membrane or antigens previously bound to the surface of podocytes. Podocyte antigen reacting with nephritogenic antibodies was characterized in an experimental model of membranous nephropathy, socalled Heymann nephritis. This protein, originally called gp330, was then renamed megalin (F1). Unfortunately, megalin is not expressed by human podocytes and cannot be the putative pathogenic antigen in human membranous nephropathy. Podocyte damage in membranous nephropathy is probably caused by the local activation of complement with the formation of the membranolytic complex C5b–C9. Locally formed chemotactic fragments of complement (e.g., C5a) do not penetrate through the glomerular basement membrane, and that is why in membranous nephropathy glomeruli are not infiltrated with leukocytes. Proteinuria in membranous nephropathy is nonselective and it is believed to be caused by the loss of size selectivity (C4). Reactive oxygen species play a significant role also in the glomerular capillary damage and podocyte damage in Heymann nephritis. Extracellular ATP (and probably also some other vasoactive hormones) may increase podocyte superoxide production by the activation of NAD(P)H-dependent oxidases, including the main component of the complex NADPH oxidoreductase cytochrome b558 at the transcriptional and the translational levels (G8). Stimulation of podocyte superoxide production may also play an important role in the pathogenesis of vasoactive hormone-induced podocyte damage (Section 2.6). Pathogenesis of foot process fusion in various human glomerulopathies may be different. On one hand, in membranous nephropathy, foot process fusion may be the consequence of complement-induced podocyte damage (C11); on the other hand, in minimal change disease, proteinuria may be caused by direct damage to the slit diaphragm with consequent foot process fusion. In any case, foot process fusion results in the formation of large pores and proteinuria.

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IgA nephropathy is a common primary glomerulopathy with a histologic picture of mesangioproliferative glomerulonephritis with IgA deposits in the glomerular mesangium. The glomerular capillary wall is damaged only in the most severe forms of IgA nephropathy and nephrotic syndrome is not common in this type of glomerulopathy. In patients with IgA nephropathy, urinary podocyte excretion may serve as a marker of disease activity. Interestingly, although ACE inhibitor (trandolapril), angiotensin II antagonist (candesartan), and calcium antagonist (verapamil) reduced proteinuria in a similar way, trandolapril and candesartan reduced urinary podocyte excretion more significantly (N1). In membranoproliferative glomerulonephritis, circulating immune complexes may deposit not only in mesangium, but also between endothelial cells and the glomerular basement membrane with subsequent local activation of the complement system. Glomerular damage may be caused both by the membranolytic complex of complement (C5b–C9) and by infiltrating neutrophils and monocytes attracted by C5a, chemokines, PAF, and leukotrienes and activated locally by proinflammatory cytokines, e.g., TNF. Formation of reactive oxygen species (particulary hypochlorous acid formed by myeloperoxidase) and proteases (elastase, cathepsin G, and metaloproteinases) then results in serious damage to the glomerular basement membrane with nonselective proteinuria. In the patients with lupus nephritis, the histologic picture of both membranous nephropathy (type V) and proliferative glomerulonephritis (types III and IV) may be encountered. In proliferative lupus nephritis with subendothelial deposits, an important causative role of binding of cationic nucleosomes to predominantly negatively charged structures of the glomerular basement membrane with subsequent binding of antinucleosomal antibodies has been postulated. 3.3. NEPHRIN AND PODOCIN IN THE ACQUIRED FORMS OF NEPHROTIC SYNDROME Nephrin expression possibly may be changed also in the acquired forms of nephrotic syndrome (T4). Decreased transcription of the nephrin gene (decreased levels of nephrin mRNA by 40% on the 3rd day, even before the appearance of proteinuria, and by more than 80% on the 10th day) were demonstrated in rats with puromycin aminonucleoside-induced nephrotic syndrome (L6), an experimental model of minimal change disease. Podocyte damage induced by puromycin aminonucleoside is probably mediated by the metabolisation of puromycin to hypoxantin and the formation of reactive oxygen species (R9). Probucol-stimulated increased transcription of the nephrin gene suggests that in puromycin-nucleoside induced nephrotic syndrome a protein complex containing nephrin may be damaged by lipoperoxidation (L6), or the reactive oxygen species may interfere with the intracellular signaling in podocytes. In experimental Heymann nephritis, nephrin transcription was decreased by only 20%, but the measurement was possibly performed too late, not until 12 weeks


191

after induction of the disease (L6). In another study, significant decrease of nephrin mRNA, but no ZO-1 mRNA, was observed in the fourth and eighth months of passive Heymann nephritis (B5). Administration of an angiotensin-converting enzyme inhibitor (lisinopril) and an angiotensin II antagonist (L-158,809) substantially ameliorated renal damage and normalized nephrin transcription in this experimental model of human membranous nephropathy (B5). The effect of ACE inhibitors and angiotensin II antagonists on nephrin transcription may contribute to the positive influence of ACE inhibitors on glomerular size selectivity and glomerular permeability for water (R8). ACE inhibitors may increase podocyte nephrin transcription directly, through angiotensin receptors on podocytes (N2), or indirectly, through, for example, the influence of ACE inhibitors on the size and distribution of immune complexes (R6), or the activation of C5b-9 and the formation of reactive oxygen species (Y4). In the early phase of experimental membranous nephropathy (passive Heymann nephritis in rats), most slit diaphragms were still visible, but some were displaced by deposits or absent. Later (on day 7), when the rats were severely proteinuric, most slit diaphragms were absent or replaced by occluding-type junctions. Progressive decrease of nephrin staining and displacement of nephrin from podocyte foot processes could be demonstrated; on the other hand, staining for ZO-1 and CD2AP was unchanged (Y5). Human data are very sparse and so far inconclusive. Significant decrease of glomerular mRNA for nephrin was demonstrated using reverse transcriptase– polymerase chain reaction (RT-PCR) in renal biopsies of three nephrotic patients with minimal change disease, and mild decrease was present also in the biopsy of one patient with early membranous nephropathy (F5). It has been hypothesized that genetically decreased nephrin expression may predispose patients to the development of nephrotic syndrome, but decreased nephrin transcription only during the course of glomerular disease seems to be more probable. Decreased expression of nephrin was not demonstrated using in situ hybridization in renal biopsies of 56 proteinuric children with minimal change disease, focal segmental glomerulosclerosis, and membranous nephropathy (P1). Expression of nephrin and ZO-1 protein was absent only in crescents and sclerotic lesions, probably as a result of the deranged structure of the podocytes (P1). Although neither immunochemistry nor in situ hybridization may be sensitive enough to disclose small abnormalities of the nephrin distribution on the surface of podocytes, acquired glomerular diseases with nephrotic syndrome are probably not accompanied by a severe reduction of podocyte nephrin expression. Production of antibodies reacting with different segments of nephrin and preparation of nephrin knockout mice can help further elucidate the role of nephrin and the pathology of the slit diaphragm in the pathogenesis of various forms of nephrotic syndrome. Restriction of podocyte expression to a small segment of the podocyte membrane should stimulate further studies for identifying the podocyte-specific regulatory elements, which could be directed in the future by

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specific interventions enabling the development of gene therapy for glomerular diseases. Recently, mutation of the NPHS2 gene (encoding the protein podocin) has been found in nearly 30% of patients with sporadic steroid-resistant nephrotic syndrome (K6). It is possible that a substantial portion of steroid-resistant patients with FSGS may have sporadic mutations of several different genes for (yet-unrecognized) podocyte proteins. 3.4. PLASMA FACTORS THAT MAY INCREASE PERMEABILITY OF THE GLOMERULAR CAPILLARY WALL The role of circulating factors increasing glomerular permeability in the pathogenesis of nephrotic syndrome in minimal change disease and focal segmental glomerulosclerosis was suggested over 15 years ago (S6, G3). Minimal change diseases and focal segmental glomerulosclerosis may be two extremes of one continuum with similar pathogenesis. In an experimental model of nephrotic syndrome in rats, a single administration of puromycin aminonucleoside or adriamycin results in reversible podocyte damage with foot process fusion, whereas repeated administration of the same substances leads to focal segmental glomerulosclerosis progressing to end-stage renal failure. Focal segmental glomerulosclerosis (FSGS) is characterized by the focal accumulation of extracellular matrix and lipids in segments of some glomeruli. Although idiopathic FSGS occurs mostly sporadically, it may be hereditary, either with autosomal dominant or autosomal recessive inheritance (Section 2.8). In these hereditary forms of FSGS, mutations of the podocyte cytoskeletal protein podocin (B8) and α-actinin have been demonstrated (K5). Mutations of podocin have been recently described also in some children and adolescents with sporadic, mostly steroid resistant FSGS (C3, K6). Possibly, even in some steroid-resistant adults, FSGS may be caused by the mutation of podocin; in others, the circulating permeability factors may play a role (G5, K6). The role of circulating permeability factor(s) in the pathogenesis of FSGS is supported by the common recurrence of FSGS after renal transplantation (I5) and the influence of plasma exchange on posttransplant proteinuria (A7). A smaller and only temporary effect of plasma exchange on proteinuria has been demonstrated also in patients with steroid-resistant FSGS in native kidneys (F3) and studies in vitro showing the increase of glomerular permeability by the sera of patients with FSGS (S5). In two patients with kidneys transplanted from patients suffering from FSGS, proteinuria disappeared during 1 year after renal transplantation (R3). Recurrence of nephrotic syndrome after renal transplantation can be predicted using bioassay detecting changes in the size of isolated glomeruli exposed to various oncotic pressures and to sera containing (or not containing) factors increasing


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glomerular permeability. Unfortunately, this bioassay cannot reliably differentiate between patients with FSGS and those with other glomerular diseases accompanied by nephrotic syndrome (S5, D1, P4). Molecular identification of circulating permeability factors has not been successful. It cannot be excluded that increased glomerular permeability may be caused by several different factors. Immunoadsorption with protein A suggested that immunoglobulin or immunoglobulinbound substance could be the circulating permeability factor (D3), whereas other studies suggested the role of a protein with a molecular weight of 30–50 kDa (S12) or the factor inhibiting inducible NO synthase in mesangial cells (T5). The cellular source of the circulating permeability factor also remains elusive. T-lymphocytes were suggested as a putative source of this factor already in the early 1970s (S8). In the 1990s hybridomas from T-lymphocytes of patients with nephrotic syndrome with minimal change disease were constructed (K20). These hybridomas produced the factor inducing proteinuria in rats. Unfortunately, it was not possible to reproduce these studies. Using a subtractive DNA library (“relapse” vs. “remission”), 84 differently transcribed transcripts were identified in T-cellenriched peripheral blood mononuclear cells in the relapse of minimal change disease (S1, C9). At least 18 identified transcripts were closely involved in the T cell receptor-mediated complex signaling cascade, including components of the T cell receptor and cytoskeletal scaffold. On one hand, the authors demonstrated increased expression levels of Fyb/Slap, L-plastin, and grancalcin; on the other hand, decreased levels of interleukin-12 (IL-12) receptor β2 mRNA suggest the development of T helper 2 phenotype in minimal change disease (S1). These new methods should help to identify yet-unknown genes supposedly playing important roles in the pathogenesis of increased glomerular permeability not only in minimal change disease, but also in other forms of acquired nephrotic syndrome (C9). Normal serum was recently demonstrated to contain substances inhibiting permeability factor(s). These inhibitors are not present in sera of patients with FSGS (S11). Some of these inhibitors have been identified as apolipoproteins of the high-density lipoprotein (HDL) complex, for example, apo J, apo E2, and apo E4 (C2). Inhibitors of the permeability factors may be lost in urine in patients with nephrotic syndrome and their presence in urine has been documented (G5). FSGS may thus be caused not only by the (increased) production of permeability factors, but also by the urinary loss of their inhibitors. Bioassay (S5) is not able to differentiate between increased production of permeability factors and the loss of their inhibitors. Increased permeability was confirmed by this bioassay even in patients with FSGS and the documented mutation of the podocin gene, apparently without increased production of the permeability factors (G5). Sporadic forms of FSGS can be subdivided into three types: FSGS with mutations of the genes for podocyte proteins (podocin, α-actinin, or others, yet unidentified), FSGS with increased production of the permeability factor(s), and FSGS

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caused by deficiency of inhibitors of circulating permeability factors. This classification may have putative therapeutic consequences. Immunosuppression seems to be indicated only in patients with increased production of permeability factor(s); deficient inhibitors of permeability factors could be replaced by exogenous administration, and patients with mutations of genes for podocyte proteins are apparently resistant to steroid and cytotoxic treatment. 3.5. REABSORPTION OF ALBUMIN IN THE PROXIMAL TUBULE The proximal tubule is generally believed to be physiologically exposed to very small concentrations of filtered albumin. In proteinuric states, the amount of filtered and reabsorbed albumin is markedly higher and increased reabsorption of albumin and other proteins may be toxic to tubuli and may lead together with other factors to tubulointerstitial fibrosis and progressive renal insufficiency. Reabsorbed proteins can stimulate tubular synthesis of chemokines (RANTES and MCP-1) (W3, Z2) and endothelin (Z3). Elucidation of the relations between increased glomerular protein trafficking and tubulointerstitial damage depends on a detailed understanding of the mechanisms of tubular reabsorption of albumin and other proteins in the proximal tubule. Histochemical and ultrastructural characteristics of protein endocytosis in the proximal tubule have been studied since the beginning of the 1960s. Endocytic vacuoles fuse with lysosomes, where degradation of protein occurs. Physiological amounts of filtered albumin are reabsorbed via a high-affinity, low-capacity mechanism, whereas under conditions of increased glomerular filtration of albumin, an additional high-capacity mechanism is activated. As proved on isolated tubular cells and in rat kidneys, binding of albumin to a multifunction receptor (e.g., megalin) occurs prior to its endocytosis (C8). Recently, it has been suggested that albumin (and other proteins) degraded in lysosomes are mostly regurgitated to the tubular lumen and appear in the urine as small peptides with a molecular weight of about 10 kDa. Because common methods measure only intact albumin, overall albumin (intact + degraded) excretion may be substantially underestimated [e.g., healthy individual excreting less than 25 mg of intact albumin per day may, based on these data, excrete more than 1300 mg of albumin-derived fragments daily (R12)]. Any tubular damage, or suppression of tubular lysosomal activity, may thus substantially increased albuminuria. Indeed, the antiproteinuric effect of ACE inhibitors may be caused by stimulated lysosomal protein degradation (due to suppressed production of TGF-β) and not only by decrease of glomerular capillary pressure, as is generally believed (R12). The potential role of disturbed protein tubular degradation in the severity of nephrotic proteinuria needs to be further clarified. Cubilin is a glycoprotein with a molecular weight of 460 kDa (A1, K21) with 27 CUB domains (subunits of complement C1r/C1s, epidermal growth factor (EGF),


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and bone morphogenetic protein), which serve as binding sites. Cubilin is expressed in the brush border of proximal tubular cells and in intracellular endocytic vacuoles (M4). Cubilin was originally identified in the ileum as a receptor playing an important role in the reabsorption of vitamin B12 and intrinsic factor and as a receptor for HDL and apolipoprotein A-I. Recently it has been demonstrated that cubilin serves as a receptor for albumin also in the proximal tubular cells (B7) and it is identical with the previously described receptor with high affinity to albumin. A cubilin domain important for albumin binding has not yet been identified. Dogs with disturbed cubilin expression in the brush border of the proximal tubule have impaired tubular endocytosis of albumin and suffer from albuminuria (F6). Cubilin lacks the transmembrane segment and binds with high affinity to the transmembrane endocytic albumin receptor megalin (M4), which has a molecular weight of about 600 kDa and serves as a coreceptor necessary for the endocytosis of cubilin and its ligands. Megalin belongs to the LDL receptor family (F1). Megalin binds many different ligands, for example, apolipoproteins, protein-bound vitamins, enzymes, peptides, hormones, and nonprotein drugs such as aminoglycosides and polymyxin B. Some of these megalin ligands are commonly used as markers of tubular proteinuria, for example, retinol-binding protein, vitamin D-binding protein, β2-microglobulin, and α1-microglobulin. Proteinuria in mice knocked out for the megalin gene is similar to that in patients with Fanconi syndrome. The direct affinity of megalin to albumin is, however, low, but in proteinuric states, megalin may serve as a low-affinity albumin receptor. The intracytoplasmic part of the megalin molecule contains a Src-like module and may possibly activate some protein kinases in tubular cells. After the dissociation of the ligand, the megalin–cubilin complex may be recycled to the apical membrane of the proximal tubule, whereas the majority of albumin is degraded in the lysosomes of the proximal tubule. Mice knocked out for the megalin gene have decreased tubular albumin reabsorption despite normal apical expression of cubilin. Antibodies directed against cubilin and megalin inhibit the reabsoption of albumin by isolated tubular cells (Z1). It is not clear whether defficiency of either cubilin or megalin may have a protective effect on tubulotoxicity and interstitial fibrosis induced by proteinuria. In rat tubular cells, inhibition of tubular albumin absorption by lysine resulted in the stimulation of the synthesis of the chemokine MCP-1 (W3). Similar studies with tubular cells with deficient expression of cubilin and megalin may contribute to the understanding of the role of tubular albumin reabsorption in the development of tubulointerstitial fibrosis. The putative role of other proteins, for example, immunoglobulins, or complement fragments also remains to be clarified. Human mutation of the cubilin gene results in Immerslund–Grasbeck disease (A3), characterized by gut malabsorption of vitamin B12 and tubular proteinuria (two thirds of which is albuminuria).

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Cubilin also binds light chains of immunoglobulins (B3), which are tubulotoxic as well. Further cubilin ligands may be transferrin and receptor-associated protein (RAP). RAP serves as an inhibitor of the binding of further ligands to megalin. The significance of the ability of cubilin to bind HDL and apolipoprotein A-I remains unclear. Despite nephrotic hyperlipidemia, plasma levels of HDL are normal in the majority of patients with nephrotic syndrome. Only in patients with long-lasting massive proteinuria may plasma levels of HDL, namely HDL2, be decreased due to its urinary loss (W5). Further study of the role of cubilin and megalin in proteinuric states may contribute to the understanding of the pathogenesis of proteinuriainduced tubulointerstitial fibrosis.

4. Biochemical Signs and Clinical Symptoms of Nephrotic Syndrome Nephrotic syndrome is a life-threatening disease. Adult patients may die of thromboembolic complications and children may die of infections, and persistent nephrotic syndrome confers a substantial risk of progression to end-stage renal failure. 4.1. HYPOPROTEINEMIA AND HYPOALBUMINEMIA IN NEPHROTIC SYNDROME In nephrotic syndrome, mainly proteins with lower molecular weight (to ca. 60 kDa, i.e., up to the size of albumin) are lost into urine, whereas filtration of larger molecules (with molecular weight higher than 200 kDa) is not markedly increased. Liver proteosynthesis is stimulated nonspecifically: Synthesis of both low-molecular-weight and high-molecular-weight proteins is increased. That is why a decrease of serum concentrations of low-molecular-weight proteins and, on the other hand, often an increase of serum concentrations of some highmolecular-weight proteins is typical for nephrotic syndrome. These changes in the composition of serum proteins play an important role in the pathogenesis of nephrotic hyperlipidemia, hypercoagulation, and thromboembolic complications. Long-lasting heavy proteinuria usually leads to negative nitrogen balance. The loss of lean body mass (up to 10–20% of dry body weight) may be masked by edema in nephrotic patients and may become apparent only after its removal. Hypoalbuminemia is a consequence of increased glomerular filtration of albumin. Loss of albumin is caused both by the loss of albumin into the urine (albuminuria) and by the reabsorption of albumin in the proximal tubule, which is mostly accompanied by its degradation. Increased tubular degradation of albumin may explain severe hypoalbuminemia in patients with only relatively moderate proteinuria, ca 3.5–4 g/day. Synthesis of albumin in the liver of nephrotic patients


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is increased, but may not compensate for the losses of albumin, and results in hypoalbuminemia. The severity of hypoalbuminemia is usually greater in elderly, malnourished patients with comorbidities with impaired capacity to increase liver proteosynthesis. Increased dietary protein intake usually does not improve the metabolism of albumin, as it stimulates dilatation of the glomerular afferent arteriole, increases the glomerular pressure, and can thus contribute to further increase of proteinuria. Low-protein diet can reduce proteinuria due to its effect on glomerular hemodynamics (constriction of the afferent arteriole), but at the same time it usually decreases protein synthesis in the liver, and in this way it can (mainly when applied for a longer time) further worsen the negative nitrogen balance. 4.2. HYPERLIPIDEMIA IN NEPHROTIC SYNDROME Hyperlipidemia (mainly hypercholesterolemia) is a regular part of nephrotic syndrome (K13, W6). Serum levels of cholesterol are often markedly elevated, usually above 10 mmol/L. However, in severely malnourished patients, normal or even decreased serum cholesterol level can be found. Serum levels of triacylglycerols fluctuate, from normal values to markedly elevated values (mainly in patients with proteinuria higher than 10 g/24 hr). There is a variable increase in plasma concentrations of very low density lipoproteins (VLDL, they correlate negatively with serum albumin level), intermediate-density lipoproteins (IDL), and LDL; however, plasma concentrations of HDL are usually normal (J3). Levels of lipoprotein(a) [Lp(a)] are also increased (W4). Remission of nephrotic syndrome or decrease of proteinuria may result in the decrease of plasma concentrations of Lp(a) (G2). Concentration of free fatty acids in serum is commonly decreased because they are normally bound to albumin and albumin is lost into the urine. The activity of lecithin cholesterol acyltransferase (LCAT) is usually decreased. Patients in the general population with a similar lipid profile to nephrotic subjects have a very high risk of accelerated atherosclerosis and its complications. The assumption that nephrotic hyperlipidemia is atherogenic was not easily confirmed. Cardiovascular risk is probably low in patients with short-term nephrotic syndrome entering remission either spontaneously or after immunosuppressive treatment. In other nephrotic patients with persistent unremitting nephrotic syndrome, the risk should be adjusted for the presence of other cardiovascular risk factors, for example, hypercoagulation, hypertension, or chronic renal failure. Based on available data, it is possible to conclude that in nephrotic syndrome (except for minimal change disease), the relative risk of myocardial infarction (after adjustment for age, sex, hypertension, and smoking) is 5.5 and the relative risk of death from a coronary event is 2.8 (O4). Long-lasting hyperlipidemia probably contributes also to the progression of chronic renal insufficiency (W5), possibly due to lipid (lipoprotein) deposits in

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glomeruli and in the renal interstitium. Renal damage was demonstrated in different experimental models of hyperlipidemia (dietary, genetic, and secondary). This effect seems to be pronounced in hypertensive animals (e.g., spontaneous hypertensive obese rats, Dahl salt-sensitive rats) and animals with preexisting renal damage (e.g., chronic nephrosis or remnant kidney in rats) (K13). In experimental animals, hypercholesterolemia may result in mesangial proliferation and sclerosis, focal glomerulosclerosis, and albuminuria (K8). Increased mesangial content of type IV collagen, laminin, and fibronectin was preceded by the influx of macrophages (K7) and prevented or ameliorated by hypolipidemic therapy (H1). The observed proliferation of mesangial cells may be stimulated by the intermediary metabolites of cholesterol synthesis (isoprenoids), for example, farnesylated p21 ras may mediate (through the activation of nuclear factor-κB (NF-κB) and NF-IL-6 and production of MCP-1 and IL-6) mitogenic signals provided by growth factors, for examples PDGF (O2). Statins may inhibit mesangial proliferation and decrease the expression of MCP-1 and IL-6 (O1). In nondiabetic patients with proteinuria, the rate of loss of renal function was almost twofold higher in patients with hypercholesterolemia and hypertriglyceridemia when compared to patients without marked hyperlipidemia (M1). The most significant association was demonstrated between elevated apo B levels and monthly decline in glomerular filtration rate (S4). Total and LDL cholesterol were also demonstrated to be independent risk factors for the progression of renal diseases in patients with type I diabetes (M5, K26). Interestingly, association between cholesterol and angiotensin II levels was found in diabetic patients (W2), suggesting a possible explanation for the association between hypercholesterolemia and progression of chronic renal failure. Small and short-term therapeutic studies with statins (K14) have not yet been able to demonstrate unequivocally the suggested positive influence (R1, S15) of hypolipidemic therapy on the progression of chronic renal failure. In the pathogenesis of nephrotic hyperlipidemia, both increased production and impaired catabolism of lipoproteins were demonstrated to play a role. Pathogenic mechanisms may be different in nephrotic patients with isolated hypercholesterolemia and in patients with combined hypercholesterolemia and hypertriglyceridemia (V6). Increased levels of apolipoproteins and rate-limiting enzymes of lipogenesis and their mRNAs have been demonstrated in the liver of nephrotic rats (V5), although increased liver cholesterol synthesis has not been confirmed in nephrotic patients (D9). Cholesterol synthesis in the liver probably does not change during antiproteinuric treatment (D9). The rate of synthesis of LDL apoprotein B is variable and depends on the presence or absence of hypertriglyceridemia (V6). Decreased catabolism of chylomicrons and VLDL has been demonstrated in nephrotic syndrome (D4). The fractional catabolic rate of apoliprotein B depends on the presence of hypertriglyceridemia: The fractional catabolic rate of


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apolipoprotein B is high in combined hyperlipidemia and low in isolated hypercholesterolemia (V6). Although hyperlipidemia may be partly reversed by the increase of plasma oncotic pressure with dextran infusion, decreased albumin and plasma oncotic pressure cannot fully explain nephrotic hyperlipidemia. In analbuminemic rats, lipid changes are different from those in nephrotic subjects (D4). There may be a direct causal link between proteinuria and lipid abnormalities because α1-acid glycoprotein isolated from urine of nephrotic patients may correct the impaired lipolysis of nephrotic rats (S16, K12). Thus, impaired lipoprotein metabolism may be caused by the loss of some regulatory substance into urine due to increased glomerular permeability. Hypercholesterolemia in the patients with persistent nephrotic syndrome should be treated. Statins are similarly effective as in nonnephrotic subjects and are able to reduce total and LDL cholesterol by about 30% (M2). Reduction of proteinuria by inhibitors of angiotensin-converting enzyme is also able to reduce LDL cholesterol (M2). Dietary therapy seems to be less effective. 4.3. PATHOGENESIS OF EDEMA IN NEPHROTIC SYNDROME According to the classical hypothesis, edema in nephrotic syndrome increases owing to the decrease of oncotic pressure in plasma due to hypoalbuminemia. Increased transsudation of the fluid to the extracellular space then leads to a decrease of the intravascular blood volume (so-called “underfilling”) with subsequent activation of the efferent renal sympathetic nerve activity, vasopresin production, activation of the renin–angiotensin–aldosterone system, and retention of sodium and water. In the case of long-lasting low plasma oncotic pressure, further transsudation of retained fluid to the extravascular space and formation of a transsudate (mainly ascites) occurs. This mechanism may play a role in some nephrotic children with minimal change disease and very high proteinuria (with albuminemia about 5 g/L). It has been demonstrated that children have really decreased plasma volume (V3, V4). Less pronounced hypoalbuminemia may not be accompanied by edema. Decrease of plasma oncotic pressure may lead to a temporary increase of the filtration of intravascular fluid into extravascular (interstitial) space. The increased interstitial pressure stimulates the increased flow of the lymph, which results in a decrease of the concentration of protein in the interstitial fluid and normalization of the gradient between the oncotic pressure of the plasma and the interstitial fluid. Subsequently, there is no driving force for further filtration of the intravascular fluid into the extravascular space. Patients with decreased intravascular blood volume should have hypotension, but many nephrotic patients (apart from nephrotics with minimal change disease) are hypertensive (K28). Adults with nephrotic syndrome were repeatedly demonstrated to have normal or increased (not decreased) plasma volume (G4).

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Plasma levels of atrial natriuretic factor reflecting the “effective” blood volume (filling of the central blood volume compartment) are elevated in nephrotic subjects (P8), suggesting circulatory “overflow” instead of underfilling. Diuretic response to the pharmacologic blockade of the renin–angiotensin system is limited (D10). Analbuminemic rats completely lacking albumin synthesis have no edema. Similarly, patients with urinary protein loss due to lymph fistula draining into the renal pelvis do not present with edema (P9). In patients with nephrotic syndrome entering into remission (i.e., with substantial decrease or disappearance of proteinuria) during immunosupressive therapy, urine flow rate rapidly increases and edema may disappear during several days, although hypoalbuminemia normalizes only within several weeks (K19). In contrast, during the initial stage of relapses in minimal change disease, urinary sodium retention precedes the development of massive proteinuria (V4). All these data support the hypothesis that primary sodium retention is independent of proteinuria. Further support for this view comes from experimental study in rats where only one kidney was exposed to puromycin aminonucleoside, which resulted in only unilateral nephrotic proteinuria. Only nephrotic kidney was demonstrated to have increased sodium reabsorption, suggesting that a local (primary sodium retention) and not a systemic factor (“underfilling” of the blood volume) plays a role (I1). In nephrotic rats micropuncture studies demonstrated that sodium delivery to the distal nephron is normal, suggesting the distal nephron as the place of avid sodium retention (I1). Decreased (not increased) sodium resorption in the proximal tubule was also demonstrated in nephrotic patients (G7). The cause of increased sodium resorption in the distal nephron in nephrotic subjects remains to be fully elucidated. Sodium retention may be partly reversed by renal denervation, suggesting the role of efferent renal sympathetic nerve activity. More attention has been paid to the impaired natriuretic response to the exogenous administration of atrial natriuretic peptide both in nephrotic animals (P6) and in humans (P7). Interestingly, in rats with unilateral nephrosis, blunted natriuretic response to atrial natriuretic peptide is confined to the proteinuric kidney (P6). Experimental studies (P6) demonstrated that binding of atrial natiuretic peptide to its receptor is unaltered, suggesting a postreceptor defect, illustrated by decreased urinary excretion of the second messenger of atrial natriuretic peptide, cyclic guanosine monophosphate (W9). Sodium retention in nephrotic animals can be reversed by the administration of specific phosphodiesterase inhibitors blocking the breakdown of cyclic guanosine monophosphate (V1). The putative relation between damage to the glomerular capillary wall resulting in massive proteinuria and decreased production of cyclic guanosinemonophosphate in response to atrial natriuretic peptide in the utmost part of the distal nephron (inner medullary collecting duct) remains unclear. In clinical practice, nephrotic edema may be further worsened by inadequate diuretic treatment resulting in temporary intravascular hypovolemia and/or by


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impaired renal perfusion caused by nonsteroidal antiinflammatory drugs (NSAIDs). In patients with increased renal sympathetic activity and increased plasma renin activity, renal perfusion is dependent on the increased renal production of vasodilatory prostaglandins, especially PGE2 and prostacyclin. Blocking of their production by NSAIDs may result in a decrease of renal plasma flow and glomerular filtration rate. Effective treatment of nephrotic edema requires adherence to a low-sodium diet (containing preferably only about 50 mmol of sodium per day) and administration of potent loop diuretics (e.g., furosemide), sometimes in high doses several times per day and/or in combination with thiazides and potassium-sparing diuretics (e.g., amiloride). Plasma ultrafiltration should be reserved for patients with refractory nephrotic edema and massive sodium and water retention. 4.4. THROMBOEMBOLIC COMPLICATIONS OF NEPHROTIC SYNDROME Thromboembolic complications represent the major life-threatening complications of nephrotic syndrome. It is believed that thromboembolic events occur in 10% of adult patients with nephrotic syndrome and in 2% of nephrotic children. Vein thromboses (often asymptomatic) are much more common than arterial ones. Arterial thromboses may be very dangerous, however, especially in elderly patients with atherosclerotic stenoses of coronary and peripheral arteries leading to acute vascular closure. Thrombotic diathesis clearly contributes (with hyperlipidemia) to the increased risk of coronary events in nephrotic subjects (O4). Renal vein thrombosis is clinically diagnosed in 8% of patients with nephrotic syndrome. Silent, unrecognized renal vein thrombosis may be much more common (10–50%), as proved with laboratory methods (e.g., Doppler sonography and venography of renal veins). Renal vein thrombosis (usually asymptomatic) is particularly common (20–30%) in patients with membranous nephropathy (B4) and may result in the loss of renal function. Pulmonary embolism remains clinically silent in about two thirds of affected persons and may be life-threatening. The hypercoagulable state of nephrotic subjects is further worsened with immobilization, hemoconcentration in patients with decreased intravascular volume (usually due to diuretic therapy), and corticosteroid therapy. Prophylactic anticoagulant therapy should be administered to high-risk patients, for example, patients with membranous nephropathy with nephrotic proteinuria and serum albumin level below 20 g/L. Patients with nephrotic syndrome often have markedly increased erythrocyte sedimentation rate (ESR) owing to high levels of fibrinogen. Thus, high ESR in nephrotic patients does not necessarily mean acute-phase reaction. The pathogenesis of the hypercoagulable state in nephrotic syndrome is complex because plasma levels of both fibrinolytic and regulatory proteins may be altered. Routine coagulation tests (Quick test and activated partial thromboplastin

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time, APTT) are mostly normal. Plasma levels of many procoagulant proteins (mainly those with high molecular weight) are increased (e.g., fibrinogen, factor V, factor VII, von Willebrand factor, α 2-macroglobulin). Levels of several anticoagulant proteins are increased as well (e.g., protein C). Levels of prothrombin, factors IX, X, XI, and XII, and antithrombin III are unchanged or decreased, whereas the total antithrombin activity of plasma remains normal because of the rise in α 2-macroglobulin. Low levels of factor XII may be accompanied by prolonged APTT with no tendency to bleeding. Plasminogen concentrations have been found to be low in nephrotics, and protein C remains normal or raised, but free protein S activity is low. Recent studies suggest a major role for raised circulating levels of fibrinogen. Patients with nephrotic syndrome may also have thrombocytosis, increased platelet aggregability to different stimuli (adenosine diphosphate, thrombin, collagen, arachidonic acid, epinephrine), and increased levels of β-thromboglobulin (R1). 4.5. INFECTIONS IN NEPHROTIC SYNDROME Nephrotic patients (especially children) are prone to bacterial infections. Before antibiotics and corticosteroids were introduced into the therapy, pneumonia, peritonitis, and sepsis (usually caused by pneumococci) were the most frequent cause of death of nephrotic children with minimal change disease. Infections are more frequent in nephrotic children and after the age of 20; their prevalence markedly decreases because the majority of adults have antibodies against the capsular antigens of pneumococci. Infections remain an important complication of nephrotic syndrome in developing countries. In developed countries, nephrotic patients treated by immunosuppressive agents may frequently suffer from viral infections (mainly herpesvirus infections, e.g., cytomegalovirus and Epstein–Barr virus infections). The presence of edema and increased skin fragility (often the site of entrance of bacteria) are among the causes of increased risk of infections in nephrotic syndrome. Losses of immunoglobulin G and factor B (from the alternative pathway of the activation of complement) into the urine weaken the ability of the defense system to respond mainly to encapsulated microbes like pneumococci. The function of lymphocytes can be further weakened as a consequence of losses of zinc and transferrin into the urine. Weakening of the phagocytic function of macrophages has been described as well. 4.6. PROTEIN BINDING OF ENDOGENOUS AND EXOGENOUS SUBSTANCES Plasma levels of some hormones (thyroid and steroid hormones), vitamins (vitamin D metabolites), ions (iron, copper, and zinc) and drugs may be low in nephrotic subjects because of the low levels of protein-bound ligands (K11), as binding proteins are lost into the urine. Ligands also may be lost in the urine together with their


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binding proteins and their stores may be depleted. This may be of some clinical relevance in the case of vitamin D (G10). Hypoproteinemia may result in low levels of serum calcium, ceruloplasmin, and transferrin. Because losses of iron are at most 0.5–1.0 mg/24 hr, even with the heaviest proteinuria, other factors must operate to produce iron deficiency and microcytic hypochromic anemia. Although the copper-binding protein ceruloplasmin is lost in the urine in nephrotic subjects and its plasma levels are low, plasma and red cell copper concentrations are usually normal. Zinc circulates mainly bound to albumin and also to transferrin, and thus the reported reduction zinc concentration in plasma, hair, and white cells in nephrotic patients is not surprising. Vitamin D-binding protein and its associated vitamin are lost in nephrotic urine. Biochemical abnormalities in nephrotic patients (children and adults) include: hypocalcemia, both total (protein-bound) and ionized; hypocalciuria, reduced intestinal calcium absorption and negative calcium balance; reduced plasma 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol and, surprisingly, also 1,25-dihydroxycholecalciferol; and blunted response to parathormon (PTH) administration and increased PTH levels. Clinically, both osteomalacia and hyperparathyroidism have been described in nephrotic patients, more commonly in children than in adults, but bone biopsies are commonly normal, and clinically significant bone disease is very rare in nephrotic subjects. There is, however, evidence that patients with renal failure accompanied by nephrotic range proteinuria may be particularly prone to develop renal osteodystrophy. In fact, despite losses of thyroxin-binding globulin in the urine, proportional to total proteinuria and accompanied by the loss of bound T3 and T4, plasma concentrations of T4, T3, and thyroid stimulating hormone (TSH) are usually normal in nephrotic subjects. Sometimes T3 levels may be lowered and T4 levels slightly elevated with increased reverse T3, with normal TSH levels excluding the diagnosis of hypothyroidism. Hypoalbuminemia is to be taken into consideration also in hypothyroid patients on thyroxine substitution therapy. Cortisol-binding protein is also lost in the urine and plasma concentrations of cortisol may be also reduced. Many drugs are bound to circulating albumin. Hypoalbuminemia also increases plasma levels of free drugs, which may be counterbalanced by faster metabolism. Increased levels of the free drug may be clinically relevant as demonstrated for prednisolone (B6) and possibly warfarin. 4.7. CHANGES OF RENAL FUNCTION IN NEPHROTIC SYNDROME Patients with nephrotic syndrome can develop acute renal failure as a consequence of intravascular hypovolemia and/or sepsis with subsequent prerenal azotemia or acute tubular necrosis. Renal hypoperfusion in these patients can be potentiated by the administration of diuretics, inhibitors of angiotensin-converting

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enzyme, or nonsteroidal antirheumatic drugs. Acute renal failure can be also a consequence of bilateral thrombosis of renal veins or allergic interstitial nephritis (caused by nonsteroidal antirheumatic drugs or diuretics). Apart from nephrotic syndrome in minimal change disease, persistent nephrotic syndrome confers a substantial risk of progression to chronic renal failure. The risk of progression is usually related to the degree of proteinuria. Progression is moderate in patients with proteinuria lower than 2 g/24 hr, but is high in the patients with proteinuria higher than 5 g/24 hr.

4.8. PROTEINURIA AND PROGRESSION OF CHRONIC RENAL INSUFFICIENCY It has been known for a long time that patients with persistent massive proteinuria progress to chronic renal failure markedly more often than patients with low proteinuria (lower than 1 g/24 hr) (R7). Progression of chronic renal insufficiency does not correlate with the severity of the glomerular damage, but correlates tightly mainly with the severity of the tubulointerstitial damage. On one hand, proteinuria may be only a marker of the severity of the glomerular damage. On the other hand, the renoprotective effect of antiproteinuric drugs (e.g., inhibitors of angiotensin-converting enzyme) with no direct effect on the primary glomerular disease supports the view that the proteinuria has a direct pathogenic role in the mediation of tubulointerstitial damage. Direct tubular toxicity of proteinuria or interaction of tubular cells (or renal interstitium) with some specific components of the proteinuric urine, for example, albumin, transferrin, immunoglobulins and their fragments, components of complement, and chemotactic or growth factors, has been suggested (R7). As for albumin, apart from the tubulotoxic effect of structurally modified albumin, the antiapoptotic effect of albumin and its ligands on tubular cells has to be taken into consideration (I3). Albumin is the main plasma protein, with a molecular weight of about 69 kDa, and is important for normal plasma oncotic pressure and the transport of many biologically active substances, including free fatty acids, phospholipids (e.g., lysophosphatidic acid), prostanoids, heavy metals, steroid hormones, and vitamins. Albumin-bound lysophosphatidic acid serves as a survival factor for cultured mouse proximal tubular cells (L4). Lysophosphatidic acid is an exquisitely potent inhibitor of apoptosis, comparable with growth factors, for example, EGF. The influence of lysophosphatidic acid on the survival of tubular cells depends on the activation of phophatidylinositol 3-kinase (PI3K) with subsequent activation of Akt and pp70s6k. pp70s6k is a rapamycin-inhibited kinase, which plays an important role in the cellular proliferation. Lysophosphatidic acid also serves as a proliferation factor of mouse proximal tubular cells. Further albumin-bound factors important for the survival of the proximal tubular cells are phosphatidic acid


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and unsaturated fatty acids, for example, linoleic acid and oleic acid (K18). An apoptosis-inhibiting effect may be also mediated by delipidated albumin oxygen species, which may oxidize free amino groups of albumin, and albumin may then also react with reactive species formed by the peroxidation of membrane lipids. Moreover, albumin may (similar to ceruloplasmin and transferrin) bind transition metals (Fe2+ and Cu2+) and block the formation of the hydroxyl radical by the Fenton reaction (G12). The free sulfhydryl group in the cysteine in the position 34 of albumin plays an important role in the ability of albumin to neutralize reactive oxygen species. So, on one hand, albumin and albumin-bound phospholipids may serve as survival factors for tubular cells, whereas on the other hand, tubular cells may be activated and damaged by oxidatively modified albumin and albumin modified by carbonyl stress products, for example, advanced glycation end products (AGE) and advanced lipoperoxidation end products (ALE). AGEmodified proteins may activate its receptors (RAGE) on tubular epithelial cells with subsequent upregulation of NF-κB and peroxisome proliferator-activated receptor-γ (PPAR-γ ). This activation may result in tubular damage and interstitial fibrosis (I3). The relative contributions of different proteins and their ligands to proteinuriamediated tubular damage remain to be established. 5. Diagnosis of Nephrotic Syndrome Diagnosis of nephrotic syndrome depends on the identification of both the clinical signs (edema) and laboratory disorders (proteinuria, hypoproteinemia, hypoalbuminemia, hyperlipidemia). Lipid and coagulation abnormalities that also must be monitored are described in detail in the appropriate sections. Diagnostic follow-up in patients with proteinuria is demonstrated schematically in Fig. 2. Proteinuria >2 g/24 hr is usually of glomerular origin and renal biopsy should be performed. Proteinuria flour (1.00–3.54 TE/g) > trichome (1.74 TE/g) = bran (1.02–1.62 TE/g) (H7). The antioxidant activity of rye extracts was significantly correlated with their total content of monomeric and dimeric hydroxycinnamates. These data suggest that rye products may be a source of dietary phenolic antioxidants that may have potential health effects (A13, E3). In some cases food processing may actually increase its TAC. Formation of Maillard reaction products in a system containing gelatinized starch, glucose, lysine, and soybean oil increased substantially TAC of the mixture during heating at 100◦ C; TAC showed a further small increase during subsequent storage of the mixture at 25◦ C, paralleling the formation of Maillard reaction products (M9).

7. Total Antioxidant Capacity of Plants TAC of medical or culinary plants is of interest. In 12 medicinal herbs studied, TAC ranged from 1.88 to 22.30 µmol TE/g of fresh weight; highest TAC values were found for Catharanthus roseus, Thymus vulgaris, Hypericum perforatum, and Artemisia annua. TAC of 27 culinary herbs ranged from 2.35 to 92.18 µmol TE/g; the highest values were noted for Origanum × majoricum, O. vulgare ssp. hirtum, and Poliomintha longiflora (Z3). Anthocyanins (delphinidin 3-sambubioside and cyaniding 3-sambubioside) are the main contributors to TAC of petal extracts of roselle (Hibiscus sabdariffa L.), accounting for 51% of TAC of the extracts. About 24% of TAC was due to phenolic

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compounds (T5). TAC of 30 widely used medicinal plants of New Mexico ranged from 27 (root of Yucca sp.) to 972 µmol Trolox equivalents per gram dry weight (leaves of Ilex paraguensis). Of widely known species, Salvia officinalis had TAC of 442 and rose flowers had TAC of 804 µmol Trolox equivalents per gram. This study estimated TAC of dried plants extracted at 85◦ C for 10 min to simulate the preparation of tea (V6). Of 17 plants of southern Niger studied, 11 had a greater TAC than spinach leaves (14.3 µmol Trolox equivalents/g dry weight) and 14 had a greater TAC than potato flakes (7.1 µmol Trolox equivalents/g dry weight). This may be of importance because wild edible plants are consumed to some extent by people inhabiting western Sahel and other parts of sub-Saharan Africa. This “famine food” includes primarily leaves, fruits, flowers, and seeds of trees and shrubs. Generally, TAC of leaves was higher than that of fruits or seeds (C33). In fact, TAC of fresh plants was probably greater than measured in this study on aqueous extracts of dried plants. One should remember, however, that TAC of plant homogenates depends greatly on ecological conditions, state of growth, and season of collection (V9). The antioxidant activity of extracts of various edible plants has been estimated and compared. The list of species studied includes Achyrocline satureioides (Compositae), medicinal herb used in Argentina, Uruguay, Brazil, and Paraguay for its choleretic, antispasmodic, and hepatoprotective properties (D3); Helichrysum arenarium (C38); bark extracts of Anadenanthera macrocarpa (Fabaceae), Astronium urundeuva (Anacardiaceae), Mimosa verrucosa (Fabaceae), and Sideroxylon obtusifolium (Sapotaceae), four trees used as antiinflammatory agents in the Brazil (D4); various species of Salvia (S. candelabrum, S. ringens, S. tomentosa, S. nemorosa, S. glutinosa, and S. officinalis (Z4); Melissa officinalis (Labiatae) and Chelidonium majus (Papaveraceae) (V9), resinous exudates from Heliotropium species (L18); and a widely consumed tropical fruit guava (Psidium guajava) and its products (J2).

8. Antioxidant Activity of Individual Compounds TAC assays also may be useful for evaluation of antioxidant activities of isolated compounds and in structure–activity studies of antioxidants (T8). Comparison of antioxidant activities of a series of flavonoids and phenolic acids allowed the formulation of general rules governing the effective radical scavenging properties of these compounds, including the presence of o-dihydroxy structure in the B ring, which confers higher stability to the radical form and participates in electron delocalization of a 2,3 double bond in conjugation with a 4-oxo function and of 3and 5-OH groups with 4-oxo function (R16). Pannala et al. compared antioxidant properties of various flavonoids and studied the effects of B ring structure. They used stop-flow measurements of ABTS·+ reduction, measuring the reaction after

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0.1 and 3.0 sec, and found that compounds with a monophenolic B ring (e.g., apigenin, p-coumaric acid, pelargonidin) reacted rapidly (like Trolox), the reaction being complete within 0.1 sec, whereas compounds with a catechol-containing B ring (e.g., ferulic acid, epicatechin, delfinidin) showed a slow secondary phase after the fast reaction. Flavonoids with an unsubstituted B ring did not react within the time scale studied (P2). Another general conclusion concerned the dependence between the TEAC of flavonoids and their half-peak reduction potential Ep/2. Flavonoids that are efficient free radical scavengers, having TEAC ≥ 1.9 mM, were characterized by Ep/2 ≤ 0.2 V, in comparison with less efficient antioxidants with TEAC ≤ 1.5 mM and Ep/2 ≥ 0.2 V (R18). For a series of all-trans carotenoids, TEAC values were found to correlate quantitatively with computer-calculated ionization potentials. These correlations were observed when the ionization potential was calculated both as the negative of the energy of the highest occupied molecular orbital [−E(HOMO)] of the molecule and as the relative change in heat of formation upon the one-electron oxidation of the carotenoids (S24). Antioxidant activity of estrogens was compared on the basis of their activity to inhibit formation of ABTS·+ . Estriol, estrone, 17β-estradiol, and 17α-ethinylestradiol showed more than twofold higher activity than diethylstilbestrol, 2-hydroxyestradiol, and 4-hydroxyestradiol. Antioxidant activity of mestranol was negligible. On a molar basis, estrone was 2.43 times more effective than Trolox (R24). Antioxidant activities of caffeine (1,3,7-trimethylxanthine) and its main metabolites was compared. Caffeine, 1,7-dimethylxanthine, and 3,7-dimethylxanthine did not show any peroxyl radical-scavenging capacity at concentration up to 100 µM. However, the relative antioxidant activities (with respect to Trolox) of 1-methylxanthine and 1-methyluric acid were 0.82 and 0.58, respectively (L9). Thyroid hormones and their structural analogs showed lower DPPH-scavenging activity in comparison with butylated hydroxytoluene (BHT) as a standard compound. 3,5,3′ ,5′ -tetraiodothyroacetic acid, 3,3′ ,5′ -triiodo-L-thyronine, and thyroxine showed the highest antioxidant activity measured by DPPH reduction, 3,5,3′ 5′ tetraiodothyroacetic acid having over 20% of the activity of BHT (O5). Some drugs were found to have antioxidant activity in standard assays of TAC. Aminoguanidine was found to have an antioxidant activity, although three orders of magnitude lower than Trolox on a molar basis (C36). Dimethyl sulfoxide (DMSO), used as a solvent for many compounds, also shows antioxidant activity and delays ABTS oxidation (Y5). Interestingly, products of chlorination of quercetin with hypochlorous acid were found to have higher antioxidant activity (estimated by inhibition of ABTS oxidation) than the parent compound, with monochloroquercetin and dichloroquercetin, showing activity 1.76 times and 1.84 times, respectively, of that of quercetin (B13). TAC assays are also useful in the detection of antioxidants in chromatographic fractions obtained during fractionation of complex biological materials (B12, C2).

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9. Effect of Nutrition on Total Antioxidant Activity of Body Fluids TAC of body fluids (especially of blood plasma) has been increasingly broadly assayed as an index of the “redox status” of the body in healthy individuals and in various disease states. Many studies have been devoted to the effect of nutrition on TAC of blood plasma. These studies have often yielded inconsistent results, partly due to differences in the methods of assay and to different experimental conditions.

9.1. SHORT-TERM EFFECTS Many researchers succeeded in demonstrating discernible transient effects of meals on TAC of blood plasma. Consumption of 500 ml of cranberry juice (but not blueberry juice) induced an increase in blood plasma TAC, attaining a maximum after 60–120 min (P5). TAC of elderly women was also increased 0–4 hr after consumption of 240 g of strawberries, 1250 mg of ascorbic acid, or 240 g of raw spinach or drinking 300 ml of red wine. TAC of urine collected over 24 hr was also increased after consumption of vitamin C (by 45%), spinach (by 28%), and strawberries (by 10%) (C13). Consumption of 300 ml of red wine significantly increased TAC of blood plasma (by over 60%) 2–3 hr after the event. Ingestion of white wine, of lower TAC, evoked a smaller increase of plasma TAC (by 15–20%). No increase of TAC was noted after drinking the same volume of 10% water–alcohol solution (T7). In another study, increase in blood plasma TAC was noted after ingestion of green or black tea and alcohol-free red wine, but not white wine (S11) (Fig. 10). An increase of blood plasma TAC within 30 min after ingestion of not only red wine, but also 100 ml of malt whiskey, but not unmatured “new make” spirit, was also reported (D13). A significant increase in blood plasma TAC was observed after ingestion of a single dose of black or, especially, green tea (2 g of tea solids in 300 ml of water), peaking after about 60 min (L10). Other authors found much lower increases of blood plasma TAC, by 7.0% and 6.2%, respectively, 60 and 120 min after ingestion of 300 ml of green tea, and 12.0% and 12.7% after drinking 450 ml of tea (S29) or 4% after drinking 500 ml of green tea (B10). Effects of ingestion of 150 ml of tea were not significant (S29). Another study did not reveal any significant change of blood plasma TAC after drinking a high dose of black tea (M10). ABAP-induced formation of cholesteryl ester hydroperoxides in blood plasma was not affected by ingestion of 500 ml of black tea extract corresponding to six cups of tea (C23). Others found that ingestion of 300 ml of green or black tea increased blood plasma TAC, but addition of milk to tea abolished this effect. The mechanism of


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FIG. 10. Effect of drinking 300 ml of water, black tea, green tea, alcohol-free red wine, or alcohol-free white wine on TAC of blood plasma of volunteers. Data taken from Serafini et al. (S11). Mean values ± SD of results for five volunteers. TAC was measured by protection of phycoerythrin bleaching (G5). Stars indicate statistically significant differences.

this effect may involve binding of tea polyphenols to milk proteins causing reduction of their bioavailability (S10). Indeed, it was observed in model experiments that the antioxidant capacity of several components of green and black tea with α-, β-, and κ-casein or albumin is not additive; that is, a part of the total antioxidant capacity is masked by the interaction with proteins (A18). In other experiments, volunteers drank either black tea with milk or black tea alone at hourly intervals between 9 a.m. and 14 p.m. Subjects consuming no tea or tea with milk exhibited no significant change in blood plasma TAC across the 6 hr of the study day (9 a.m.–3 p.m). When the subjects consumed black tea without milk, TAC increased by 65% between 9 a.m. and 12 p.m., and at 3 p.m. was 76% higher than at 9 a.m. (L5). Using an in vitro model of the human gastrointestinal tract, it was found that TAC of “jejunal” dialyzates (subjected to enzymatic treatment equivalent to that encountered in the human stomach and jejunum) was the highest when green tea was introduced. When the green tea was mixed with whole milk or semiskimmed milk, TAC of the dialyzates decreased by about one half, and with skimmed milk by almost two thirds. Similar effects were noted for the black tea, but the initial TAC values were lower (K17). Ingestion of 225 g of fried onions brought about a slight increase in blood plasma TAC (up to 1.75 ± 0.10 mM after 2 hr and 1.76 ± 0.08 mM after 4 hr, as compared with the baseline of 1.70 ± 0.04 mM) (M11). Chocolate can also increase TAC of blood plasma. By 2 hr after ingestion of 80 g of semisweet (procyanidin-rich) chocolate, TAC of blood plasma increased by 31% (R10).

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An interesting (and controversial) question is the effect of eating a meal on TAC of blood plasma. When volunteers took a test meal consisting of “Milanese” meat (beef, egg, and breadcrumbs fried in maize oil) and fried potatoes, either with 400 ml of red wine or with an isocaloric aqueous ethanol solution, TAC of their blood plasma increased significantly 3 hr after the wine meal but not after the ethanol meal (N3). In a different experiment, volunteers who fasted overnight (12 hr) were then given a breakfast, a lunch, a snack, and a dinner. The meals were designed to contain negligible antioxidants. TAC significantly increased by up to 23% after the consumption of the lunch and the dinner. These results indicate that food intake, even if low in antioxidants, can increase the serum total antioxidant activity (C11). However, another study, made on diabetics, showed that plasma TAC significantly decreased during a meal. Consumption of red wine (300 ml) in the fasting state significantly increased TAC, whereas wine ingestion with a meal counterbalanced the decrease of TAC (C20). Yet other group found that in healthy subjects, a high-fat meal (1200 kcal, 90 g of fat, 46 g of protein, and 47 g of carbohydrates) did not affect blood plasma TAC (S6). 9.2. LONG-TERM EFFECTS Although a short-term modification of blood plasma TAC after ingestion of antioxidant-rich food apparently can be demonstrated, the effect of long-term nutritional intervention seems more doubtful. In one study, administration of 300 mg/day of α-tocopherol increased blood serum TAC by 25% after 7 days and by 32% after 14 days (V2). Administration of standardized Gingko biloba extract (300 mg/(kg day) for 5 days increased TAC of rat blood plasma. Complexation of the extract with phosphatidylcholine augmented this effect (C17). Subjects who consumed water spinach twice or more a week had higher mean TAC of blood plasma (W14). However, oral supplementation of ascorbic acid (500 mg/day for 2 weeks, then 1 g/day for the next 2 weeks) and ubiquinone (Q-10; 100 mg/day for 2 weeks and 300 mg/day for the following 2 weeks) in healthy volunteers did not affect significantly TAC of blood plasma or cerebrospinal fluid (L22). Similarly, a 3-week administration of 440 IU of α-tocopherol, 22,500 IU of β-carotene, and 620 mg of ascorbic acid did not affect TAC of blood plasma (H10). In another study, a fortnight administration of 60 mg or 6 g ascorbate daily brought about an increase of TAC of blood plasma, statistically significant with respect to placebo, but still very slight (12% and 10%, respectively) (A12). Supplementation for 6 weeks with ascorbic acid (500 mg daily) or RRR-α-tocopherol (73.5 mg daily) brought about statistically significant but minor increase in blood plasma TAC (by 5% and 3%, respectively) (H6). Diet rich in carotenoid supplement or with a spinach product failed to influence TAC after 3 weeks (C19). Similar results were found by other researchers


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(lack of effects of 2-week consumption of 330 ml of tomato juice, 330 ml of carrot juice, and 10 g of spinach powder) (B21). In another study, diet rich in fruits and vegetables (10 servings each day) did not affect TAC of young men and young women but significantly increased TAC of blood plasma of old (60–80 years) men and women (by about 7%) (C7). Subjects suffering from coronary artery disease were given two capsules containing 300 mg of grape procyanidin extracts (Leucoselect-phytosome) or placebo daily for 5 days. TAC of their blood serum was reported to increase on day 5 from 408.1 ± 22.9 to 453.3 ± 453.3 µM); however, samples were taken 1 hr postdose and most probably the results indicate a transient rather than a permanent effect (N10). In a 6-month randomized controlled intervention study, subjects with moderately increased cardiovascular risk factors (1) adhered to an advised diet, (2) performed controlled moderate exercise, (3) were subjected to both diet and exercise regime, or (4) were not subjected to any intervention. No significant alteration in blood serum TAC was observed in any group tested (R22). The consumption of tomato and tomato products has been inversely correlated with the development of some types of cancer and with blood plasma lipid peroxidation. Tomato consumption increases plasma lycopene and β-carotene concentration. Lycopene has been suggested to be responsible for the beneficial effects of tomato consumption because of its antioxidant and singlet-oxygen quenching capacities. Administration of tomato puree (25 g daily) for 14 days to volunteers whose diet was low in carotenoids and free from lycopene during the 7 previous days failed, however, to affect TAC of their blood plasma significantly (P7). However, another study pointed to an increase in TAC of blood plasma (from 0.93 ± 0.15 to 1.12 ± 0.18 mM) after consumption of 200 g of tomato soup and 230 g of canned tomatoes every day for 7 days. The increase in TAC of blood plasma took place when tomatoes were prepared with olive oil but not with sunflower oil (L8). Daily uptake of 5 mg of lycopene (gel capsules containing tomato oleoresin) during 1 week did not affect blood plasma TAC (B18). Drinking of 3 doses of apple and blackcurrent juice (750, 1000, and 1500 ml) for 1 week did not affect blood plasma TAC (Y4). Consumption of extra virgin olive oil (69 g per day) either rich or poor in phenols for 3 weeks did not affect significantly blood plasma TAC (V18). Systematic consumption of moderate amounts of alcohol does not seem to induce any discernible increase in blood plasma TAC. TAC of blood plasma of volunteers who drank 40 g alcohol/day for 3 weeks was not changed irrespective of whether the alcohol was drunk in the form of red wine, beer, or Dutch gin (V4). Another study in which volunteers drank 200 ml of red or white wine for 10 days gave similar results (S15). This effect may be dependent on the composition of the diet, however. In another experiment, volunteers in two groups of 21 male volunteers each followed either a Mediterranean diet or a high-fat diet for 3 months; during the second month, red wine was added isocalorically, 240 ml/day. TAC of

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blood plasma increased 28% above basal levels in the Mediterranean diet group, but not in the high-fat diet group (L11). The lack of long-term of effects of antioxidant supplementation on TAC of blood plasma is no surprise. If the normal level of ascorbate in blood plasma is about 50 µM and a dietary intervention succeeds in augmentating this level by 50% (i.e., up to 75 µM), this effect can hardly be discernible when measuring TAC of the order of 1–1.5 mM. This argument applies even more to tocopherol (mean plasma level of 20 µM) or β-carotene (0.9 µM) (R16). Another group of compounds which may nutritionally modify TAC of blood plasma is polyphenols. The possible contribution of polyphenolic components of food and beverages to the TAC of body fluids is a subject of controversy. It has been estimated that polyphenols are present in blood plasma at concentrations of 0.2–2 µM (P1). However, feeding rats a quercetin-augmented diet can increase the plasma levels of quercetin and its metabolites up to 10–100 µM (M27). No differences in blood plasma TAC were found after 30 days of feeding lowbirth-weight infants with a formula containing n-6 and n-3 long-chain polyunsaturated fatty acids (LCP) from purified phospholipids as compared with a group fed human milk (R1). Cyclic voltammetry of blood plasma of 2- to 4-month-old infants did not reveal any differences in the antioxidant capacity between breast-fed and modified cow milk formula-fed infants (G15). Renal dialysis patients fed semipurified, liquid formulas as a sole nutrition source for 3 weeks showed significantly decreased blood plasma TAC (D6). TAC of blood plasma of children with kwashiorkor, a severe edematous manifestation of malnutrition, was below 50% of that of healthy controls (F4). Smokers tended to have lower blood plasma TAC than nonsmokers, but the differences were not always significant statistically (D11, N11, P17). A 4-week period of abstinence of smokers from smoking was reported to increase TAC of their blood plasma by 42%, partially due to an increase in the level of plasma thiols. However, another study did not find differences in TAC of blood plasma of smoking and nonsmoking pregnant women (L6). Another experiment demonstrated that antioxidant supplementation of smokers (1 g of vitamin C, 600 mg of vitamin E, and 25 mg of β-carotene daily for 4 weeks) significantly increased TAC of blood plasma (by 28%) (G6). Among subjects receiving nicotine replacement therapy, TAC of plasma increased after 12 weeks in the group that quit smoking (1.43 vs 1.20 mM). In subjects who had only reduced the number of cigarettes smoked per day, differences in plasma TAC were not significant (P9). Feeding rats diet enriched with procyanidins complexed (1:3 w/w) with soybean lecithin (2.4%) for 3 weeks increased the TAC of their blood plasma (by 40% in young and by 30% in aged rats) (F1). Wistar rats fed a high-caloric, high-fat diet (chow supplemented with lard) and a high-caloric, normal-fat diet had decreased blood plasma TAC (by 8.8% and 9.0%, respectively) (B5). Intensive tocopherol supplementation of rats (20 g/kg diet of DL-α-tocopherol hydrogen succinate)


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for 3 weeks led to a considerable increase in blood plasma TAC (275 ± 27 vs. 181 ± 6 mM) (S23). TAC of blood plasma of rats fed a diet supplemented by gavage with phosphate-buffered saline or fish oil rich in ω-3 polyunsaturated fatty acids was decreased with respect to the control group and the decrease was much higher in the group fed a fatty acid-enriched diet (M23). Rats whose diet was supplemented with 5% dried oyster mushroom (Pleurotus ostreatus) fruiting bodies (used as a hypocholesterolemic agent) showed increased TAC of blood plasma (3.11 ± 0.18 vs. 2.69 ± 0.11 mM) but decreased TAC of liver homogenates (76 ± 1 vs. 99 ± 13 µmol/g tissue) (B16). Giving green tea to rats chronically intoxicated with ethanol significantly increased TAC of blood plasma of young rats (S20). Cyclic voltammetry studies of mice subjected to short-time (40-day) dietary restriction showed that this procedure modifies TAC of tissue homogenates. TAC of heart, kidney, and muscle was enhanced and that of liver and small intestine deteriorated, whereas TAC of brain did not change (D10). Iodide, an essential ingredient of a therapeutically used brine in some spas, was found to have antioxidant properties and to increase TAC of human serum at a concentration of 15 µM, which is quite comparable to the upper range of serum iodide levels achieved through balneotherapeutic intervention (W11).

10. Effect of Physical Exercise on Blood Plasma Total Antioxidant Capacity Data suggest that extensive physical exercise may increase blood plasma TAC. Long-term effects of systematic physical exercise are, however, controversial. Submaximal exercise (30 min) was reported not to alter blood plasma TAC significantly (A7). TAC of blood plasma was reported to increase immediately after a marathon run (by 25%) and this increase persisted 4 days later (by 12%) (L19). Similar effects (increase by 19%) were noted after a half-marathon (C29). Another study reported an increase in blood serum TAC by 22% during a 31-km run and by 16% during a marathon (V10). TAC of blood plasma was increased by 25% after a maximum aerobic exercise test and by 9% after a nonaerobic isometric exercise test (A8). Eccentric muscle exercise (70 maximal voluntary eccentric muscle actions on an isokinetic dynamometer, using the knee extensors of a single leg) did not affect blood serum TAC (C27). In another study, TAC increased after exhaustive aerobic (by 25%) and nonaerobic isometric exercise (by 9%) (A8). Intense 2-day dry land training by elite alpine ski racers (routine training) decreased. TAC of their blood plasma (S28). Military activities at moderate altitudes (about 3000 m above sea level) involving strenuous work (ca. 23 MJ/day) for up to 2 weeks did not affect TAC of blood plasma of male volunteers (C22). Adolescent (12–14 years) high-competition swimmers who trained more than 20 hr/week did not show any differences in blood plasma TAC with respect to controls who did

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not perform such intense physical exercise (S4). However, soccer players engaged in a regular training program had TAC of blood plasma 25% higher than controls (B20). In trained runners, a significant correlation was observed between peak VO2 and blood serum TAC (r = 0.365, p < 0.05) (C28). Intense 1-hr aerobic dance decreased TAC of saliva (A19). TAC of blood plasma of thoroughbred racehorses increased after a 1000-m race at maximum velocity (W9). A performance test of horses (two sessions of 200-m gallop at maximum speed, 18 hr of rest, then a 3000-m gallop at maximum power) resulted in a gradual increase of TAC of blood plasma of stallions. The same test performed after 70 days of training did not affect TAC of blood plasma of the animals, suggesting a metabolic adaptation including TAC homeostasis (A20).

11. Alterations in Total Antioxidant Capacity of Body Fluids and Tissue Homogenates in Diseases and Therapy This field, most interesting from a clinical point of view, is also rich in controversial reports. 11.1. BLOOD PLASMA Lower TAC of blood plasma was found for premature (25–30 weeks gestation) than term babies (1.21 ± 0.08 mM vs. 1.46 ± 0.07 mM). This difference was considerably decreased after 5 days (1.41 ± 0.09 mM vs. 1.50 ± 0.14 mM). TAC of blood plasma of preterm babies was also lower in comparison with mothers of term babies (1.25 ± 0.08 mM vs. 1.41 ± 0.07 mM) (R15). Lower TAC of preterm infants (gestational age of 27 ± 2 weeks) was confirmed by other authors. A correlation was found between TAC at birth and birth weight (G13). However, TAC of blood plasma of preterm infants (gestational age of 24–40 weeks) did not correlate with the outcome. TAC of these who died was higher than of those who survived (1.78 vs. 1.42 mM), but the difference was not significant statistically. The only significant difference in TAC measured at day 0 was between the infants who were oxygen-dependent (mean: 1.36 mM) and those who did not require oxygen on day 28 (mean: 1.64 mM) (D9). After correction for gestational age, cord serum TAC did not correlate with maternal smoking, preeclampsia, or chorioamnionitis (R21). Blood plasma TAC on day 10 was lower in neonates who developed bronchopulmonary dysplasia at 28 days of life (M25). This dependence was not confirmed by other authors, however (R21). In another study, 21 preterm infants (30 weeks) were divided into two groups according to their shortterm outcome, a; the good-outcome group with no signs of morbidity and the poor-outcome group with intraventricular hemorrhage and/or bronchopulmonary dysplasia and/or retinopathy. At day 3 the poor-outcome group had significantly


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higher TAC than the good-outcome group or the control group, mainly because of elevated uric acid concentration. By day 10 the TRAP decreased substantially in both groups (L24). TAC of blood serum in women with mild and severe preeclampsia and eclampsia was significantly lower than that of healthy pregnant women (controls), the mean percentage decreases amounting to 22%, 40%, and 59%, respectively (S14). Another study, however, found an increased TAC of blood plasma in preeclampsia (K4). Blood plasma of children with cystic fibrosis was found to have decreased TAC (by 16%) in spite of increased concentrations of ascorbic acid, uric acid, and thiol groups (L4). In another study TAC of children with cystic fibrosis was normal, but these children received vitamin supplementation in doses prescribed in international guidelines (α-tocopherol: 10 years, 200 mg daily; retinol: 2.5 mg daily; ascorbic acid: 100–200 mg daily) (M2). Other authors found TAC values for nonhospitalized patients (1.40 ± 0.20 mM) not different from laboratory control values (1.35 ± 0.11 mM), but greater than values for hospitalized patients (1.09 ± 0.17 mM). TAC in CF children correlated positively with anthropometric values (height, weight, body mass index) and pulmonary function (forced expiratory volume in 1 sec), but not with age (L3). No difference was found between TAC of blood plasma of patients with newly diagnosed noninsulin-dependent diabetes and healthy control subjects (A14). Another study found an age dependence in the changes of blood plasma TAC due to type 1 diabetes. There were no differences in TAC between prepubertal diabetic patients and the controls, but TAC was significantly decreased in adolescent diabetic patients compared to their controls (by 51%) and in young adult diabetic patients compared to their controls (by 60%) (T2). Other researchers also found decreased blood plasma TAC in type 1 diabetes (M8, V1, V15), accompanied by decreased urate levels. It was suggested that decreased TAC might explain, at least in part, the increased susceptibility of diabetic women to cardiovascular complications (M8). Type 2 diabetics were found to have blood plasma TAC decreased by 48% (patients with proteinuria) or 37% (patients without proteinuria) (O3). Other studies also demonstrated a significantly decreased blood plasma TAC in type 1 and type 2 diabetics (A4). TAC of blood serum in older (60–70 years) hyperglycemic patients (glucose > 200 mg/dl) was decreased by 18% with respect to that of healthy controls and only by 12% in normally glycemic diabetics (S12). Patients with active psoriasis were found to have significantly decreased TAC of blood plasma, whereas TAC of those with inactive psoriasis did not differ significantly from the controls (R20). However, another study found unchanged blood plasma TAC in psoriasis, in spite of increased (by 33%) concentration of uric acid (S13). Patients with hyperthyroidism due to Graves’ disease had TAC of blood plasma decreased by 17% (K10). Patients with ataxia telangiectasia were found to have

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decreased blood plasma TAC (R9). HIV patients showed increased blood plasma TAC, by 30% in the asymptomatic group and by 57% in the AIDS group. Increased catalase and lactate dehydrogenase activities in blood serum were also found in the patients and were attributed to tissue damage (R11). Surprisingly, another study found a decrease of TAC of blood plasma by 40% in HIV-infected persons (C21). In a prospective study, TAC was found to be increased in persons with subclinical carotid atherosclerosis, due to the increase urate concentration. It was suggested that this effect represent a means to counteract oxidative damage related to atherosclerosis (N7); in any case, increased TAC may be a bad prognostic with respect to the risk of atherosclertosis. A decreased TAC of blood plasma and of hemolyzates was reported in alcoholic liver disease (H2). Decreased TAC was observed in acute pancreatitis (W10). TAC of blood plasma is increased in patients with Gilbert syndrome due to high bilirubin levels (bilirubin is a good antioxidant) (V16). However, it does not seem that this increased bilirubin level (and enhanced TAC) can protect against coronary heart disease, as suggested by some authors (V19). Blood plasma TAC was considerably lowered in patients with Crohn’s disease [1.11 ± 0.28 vs. 1.34 ± 0.26 mM (G4)]. Patients with hyperphenylalaninemia and phenylketonuria consuming a proteinrestricted diet have decreased blood plasma TAC (by about 14%) (V3). Blood plasma TAC was significantly lower in subjects with severe hyperhomocysteinemia compared with their parents and healthy control subjects (M24). Some authors found elevated TAC of blood plasma in hemodialysis patients (D1, N6), but most found it decreased in patients undergoing regular hemodialysis. The hemodialysis procedure itself was shown to decrease blood plasma/serum TAC by 11% (C30) and 15% (D1). Supplementation of hemodialysis patients with carnitine (15 mg/kg intravenously, three times weekly, after each hemodialysis session for 6 months) significantly increased their blood plasma TAC (from 1.65 ± 0.09 to 2.06 ± 0.17 mM). This change was accompanied by decrease in indices of oxidative stress (V14). Following kidney transplantation, TAC increased, reaching the level of healthy controls at the end of the first week after surgery (L20). TAC was significantly decreased in children with nephrotic syndrome controls (0.84 ± 0.14 vs. 1.21 ± 0.62 mM), probably due to the low intake of vital antioxidants (Z1). Other authors also found decreased (by 32%) blood plasma TAC in persons with nephrotic syndrome (D7). However, TAC of blood plasma was increased (by 18%) in chronic renal failure patients due to increased concentration of uric acid (C30). Blood plasma TAC was not altered in patients with Alzheimer’s disease, vascular dementia, Parkinson’s disease and dementia, or Parkinson’s disease (F6, F10). However, another study found decreased blood plasma TAC in Alzheimer patients (by 24%) (R12). TAC was decreased in blood plasma of children with Down syndrome (by 26%) (C18). Epileptic patients receiving phenytoin showed decreased TAC of blood sera, apparently due to the oxidative stress induced by the drug (M3).


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Plasma TAC was decreased in patients with schizophrenia and inversely correlated with symptom severity during the drug-free condition. There were no significant differences between on- and off-haloperidol treatment conditions indicating that this effect cannot be attributed to neuroleptic treatment (Y3). Lower TAC of blood plasma was found in persons with chronic fatigue syndrome who showed magnesium deficiency (M7). Blood plasma TAC was decreased in lung cancer patients by 10% (E4). No correlation was found between cigarette consumption (in pack-years) and blood plasma TAC, although such a correlation (negative) was found for uric acid and ascorbate (E4). However, no differences in blood plasma TAC was found comparing children at the age of 3–16 years who had been diagnosed as suffering from cancers: malignant bone tumors, malignant brain tumors, lymphoma malignum, malignant liver tumors, and malignant germ cell tumors (P16). Decreased TAC of blood plasma was found in patients with colorectal cancer (S21). TAC of blood plasma of patients who underwent cerebral hemisphere infarction was significantly decreased; no such change was noted in cerebrospinal fluid (L12). TAC of blood plasma was generally decreased in young (22–40 years) survivors of acute myocardial infarction, suggesting that it may constitute a risk factor for coronary heart disease (F3). However, no change in TAC of blood serum was found in patients with angiographically defined coronary atherosclerosis (D8). No difference in mean levels of TAC was observed between those with and without hypertension or cardiovascular disease. It was concluded on this basis that measurement of plasma TAC as a risk factor in epidemiologic studies of cardiovascular diseases may have limited use (W14). Coronary artery bypass was found to decrease blood plasma TAC (T1). Similarly, a brief episode of myocardial ischemia due to elective coronary angioplasty on the left anterior descending coronary artery decreased TAC of blood plasma in the great cardiac vein after 1 and 5 min of the angioplasty; TAC returned to normal after 15 min (B22). Another study found lower TAC in the great cardiac vein than in aorta aortic levels before baloon inflation, and its further decrease after 1 min (R19). Postoperative systemic inflammatory response, measured by the severity of pulmonary injury (edema) following elective aortic surgery, was found to show a negative correlation with blood plasma TAC before surgery. After surgery, TAC was lower in patients with pulmonary edema (C35). Other authors found decreased blood plasma TAC following major abdominal surgery (L15). TAC (and uric acid levels) of blood plasma was decreased significantly 24 hr after laparoscopic cholecystectomy. These results suggest that free radicals generated at the end of a laparoscopic procedure, possibly as a result of an ischemia–reperfusion phenomenon induced by the inflation and deflation of the pneumoperitoneum, consume blood plasma antioxidants (G9).

TABLE 12 TOTAL ANTIOXIDANT CAPACITY OF HUMAN BODY FLUIDS IN VARIOUS PATHOLOGIESa Control value (Trolox equivalents)

Value in patients (Trolox equivalents)

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Disease or condition

Material

Acute myocardial infarction Chronic lymphocytic leukemia Chronic renal failure Coronary atherosclerosis Critically ill patients Cystic fibrosis Epilepsy, patients treated with phenytoin Graves’ disease Hemodialysis HIV infection HIV infection

Blood serum Blood serum Blood plasma Blood serum Blood serum Blood plasma Blood serum

2.04 ± 0.20 mMb 2.04 ± 0.20 mMb 1.870 ± 0.01 mM 1.52 ± 0.25 mM 1.69 ± 0.20 mM 580 ± 79 µM 1.73 ± 0.20 mM

1.73 ± 0.18 mMb 1.68 ± 0.19 mMb 2.20 ± 0.02 mM 1.53 ± 0.20 mM 1.05 ± 0.26 mM 488 ± 34 µM 1.58 ± 0.10 mM

Benzoate oxidation (K13) Benzoate oxidation (K13) ORAC (C6) ABTS (M18) TAS (Randox) TRAP (W8) TAS (Randox)

K13 K13 C30 D8 D1 L4 M3

Blood plasma Blood serum Blood plasma Blood plasma (herparin) Blood plasma Blood plasma Blood plasma Blood plasma

830 ± 4 µM 1.69 ± 0.20 mM 269 ± 81 µM 280 ± 49 µM

690 ± 80 µM 1.43 ± 0.11 mM 161 ± 97 µM Asymptomatic: 365 ± 40 µM; AIDS syndrome: 440 ± 60 µM 1.52 ± 0.13 mM 0.40 ± 0.20 mM 1.143 ± 0.181 mM 1.14 ± 0.01 mM (postmenopausal without hot flushes); 0.90 ± 0.01 mM (postmenopausal with hot flushes) 3.31 ± 0.29 mM 1.52 ± 0.14 mM 1.721 ± 0.345 mM

TAS (Randox) TAS (Randox) ABTS/peroxidase Chemiluminescence (L17)

K10 D1 C21 R11

TAS (Randox) ABTS/myoglobin ABAP/luminol TAS (Randox)

V3 F4 E4 L7

ORAC (R-phycoerythrin) TAS (Randox) ABAP/R-phycoerythrin

D7 V3 G6

1.281 ± 0.297 mM

ABAP/luminol

N11

Hyperphenylalaninemia Kwashiorkor Lung cancer Menopause

Nephrotic syndrome Phenylketonuria Smokers supplemented with Vit C (1 g), Vit E (600 mg), β-carotene (25 mg, 4 weeks) Smokers

1.76 ± 0.19 mM 0.87 ± 0.21 mM 1.273 ± 0.199 mM 1.49 ± 0.02 mM (premenopausal)

Blood plasma Blood plasma Blood plasma

4.88 ± 0.50 mM 1.76 mM ± 0.19 mM 1.349 ± 0.249 mM (smokers given placebo)

Blood plasma

1.440 ± 0.284 mM

Method

Ref.

G6

ABAP/luminol (L17)

T4

Blood plasma Blood plasma

2.70 ± 0.50 mM 270 ± 0.50 mM

1.70 ± 0.50 mM 1.40 ± 0.50 mM

ABTS·+ decolorization ABTS·+ decolorization

O3 O3

Cerebrospinal fluid Seminal plasma

100.8 ± 24.2 µMb

53.3 ± 18.7 µMb

Benzoate oxidation (K13)

K13

1.654 ± 0.094 mM

Enhanced chemiluminescence

P4

Seminal plasma Seminal plasma Seminal plasma Seminal plasma

1.654 ± 0.115 mM

With leukocytospermia: 0.860 ± 0.193 mM; without leukocytospermia: 0.915 ± 0.065 mM 1.015 ± 0.079 mM


P3

1.443 ± 0.105 mM

0.939 ± 0.107 mM

Enhanced chemiluminescence (W10)

H9

1.443 ± 0.105 mM

1.186 ± 0.097 mM


H9

Fertile men: 2.2 ± 0.6 mM

Randox kit

O2

1.654 ± 0.115 mM

Infertile, with pathogens: 0.7 ± 0.4 mM; infertile, no pathogens: 1.1 ± 0.6 mM 1.174 ± 0.058 mM


P3

1.654 ± 0.115 mM

1.026 ± 0.150 mM


P3

1.95 ± 0.29 mMc

1.91 ± 1.10 mMc

ABTS oxidation, kinetic (Y5)

K5

Blood plasma

1.288 ± 0.350 mM

Systemic inflammatory response syndrome Type 2 diabetes Type 2 diabetes with proteinuria Meningitis

Blood plasma

Chronic prostatitis

Idiopathic infertility 265

ABAP/R-phycoerythrin

803 ± 31 µM

Smokers: 0.963 ± 0.369 mM; ex-smokers: 1.363 ± 0.473 mM 606 ± 20 µM

Smokers

Incidental varicocele Infertile varicocele Leukocytospermia

Varicocele Varicocele with infection Renal diseases including chronic renal failure

Seminal plasma Seminal plasma Urine

Oxygen-radical-absorbing capacity; ABTS, 2,2′ -azinobis(3-ethylbenzthiazoline-6-sulfonic acid); TAS, total antioxidant status; TRAP, total peroxylradical-trapping antioxidant capability of plasma; ABAP, 2,2′ -azobis(2-amidopropane). b Uric acid equivalents. c Ascorbic acid equivalents. a ORAC,

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Prime solutions used during cardiopulmonary bypass, prepared with either pasteurized human albumin or fresh-frozen plasma, were found to have low TAC, which may increase oxidative stress in neonates undergoing cardiopulmonary bypass (M26). TAC of patients subjected to surgery (abdominal or breast cancer operations) showed remarkable stability, showing small changes (increase or decrease) immediately after and 6 hr after surgery and normalizing 12 hr after surgery (M28). TAC of blood sera of critically ill patients (due to endocarditis, severe pulmonary edema, pneumonia, or respiratory failure) was significantly decreased (by 38% on the average) (D1). Another study, however, found increased TAC in critically ill patients, especially in nonsurvivors. Apparently, increased blood plasma TAC reflects renal dysfunction in this case (M1). Systemic inflammatory response syndrome (SIRS) due to various causes (sepsis, acute pancreatitis, major burns, trauma, major operation, immune disorders) brought about a significant decrease of blood plasma TAC (by 25% on the average). During the development of SIRS, TAC decreased progressively up to day 14 in survivors and increased in nonsurvivors (T4). Some data concerning TAC of body fluids in various diseases are given in Table 12. Welders chronically exposed to chromium did not show altered blood plasma TAC (E2). Total body irradiation, a routine preconditioning procedure for treatment of leukemia and aplastic anemia before bone marrow transplantation, decreased TAC of blood plasma by 36%, as estimated by cyclic voltammetry (C26). TAC was found to decrease by about 40% during chemotherapy of patients with various hematologic malignancies with busuflan, VP-16, and cyclophosphamide (D12). The controversial procedure of blood ozonation was reported to decrease blood plasma TAC by 20% (B17). Treatment of hypercholesterolemic patients with bezafibrate (600 mg/day) for 1 month decreased TAC of their blood serum (G16). Propofol anesthesia decreased TAC of blood plasma of patients by 9.5%; this effect was caused by hemodilution because mean hemoglobin concentration of the blood decreased accordingly (S26). On the other hand, therapy of essential arterial hypertension with dihydropyridine or calcium channel antagonists (felodipine, amlodipine or lercanidipine) for 10 weeks led to a significant increase of blood plasma TAC (by 42%) (D5). Therapy with gliclazide, a sulfonylurea hypoglycemic drug, enhanced blood plasma TAC (O1). TAC of plasma of blood anticoagulated with CPDA-1 fluid (containing citrate, phosphate, dextrose, and adenine) and stored under blood-banking conditions at 3◦ C showed decreased TAC after the day 12 of storage. A decrease of TAC by approximately 25% was observed on day 16, and a decrease by over 30% was noted on days 20 and 25 (J4).


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11.2. OTHER BODY FLUIDS AND TISSUE HOMOGENATES TAC of lacrimal fluid decreased with the progress of primary open-angle glaucoma. Blood plasma TAC decreased significantly in the third far-advanced stage. A course of total antioxidant vitamin therapy normalized plasma TAC even in patients with far-advanced glaucoma, whereas the lacrimal TAC did not normalize (M4). Tracheobronchial aspirate fluid in mechanically ventilated preterm infants correlated positively with mean daily fractional inspired oxygen concentration and was very low in babies not requiring oxygen therapy (V13). Blister fluid obtained from patients with cutaneous thermal injury had TAC 24% lower than that of blood serum. This may reflect oxidative stress and consumption of antioxidants in the blister site (H8). However, TAC of blood serum of severely burned patients was increased in 42% of the patients (mean value of TAC was elevated by 11% in the whole group of burned patients) (F2). In women there was a negative correlation between the number of amalgam restorations or total amalgam surface (P13) or salivary levels of mercury (released from dental amalgam surface) and TAC of saliva (P14). A significant correlation between Hg and TAC was evident in both male and female subjects. A significant negative correlation between TAC and Hg levels or number of amalgam restorations or amalgam surface was evident in women. A similar negative correlation was found between blood plasma level of mercury, related to the number of amalgam restorations, and TAC of blood plasma (P12). No differences were found in the TAC of saliva between smokers and nonsmokers (K11, Z2). TAC of saliva of hemodialysis patients was higher before dialysis than in healthy individuals and rapidly decreased toward normal values following the dialytic procedure (M13). TAC of peritoneal fluid was significantly lower in women with unexplained infertility (0.49 ± 0.21 mM) than in fertile patients (0.67 ± 0.24 mM) and or patients with tubal infertility (0.76 ± 0.26 mM) (P15). Infertile men were found by most researchers to be more likely than fertlile ones to have depressed TAC of seminal plasma (L13, W10). Patients with varicocele, idiopathic infertility, and varicocele associated with infection showed decreased TAC of seminal plasma (H9, P3) (Table 12). Leukocytospermia was found to be associated with decreased TAC of seminal plasma. Infertile men with leukocytospermia and pathogens had TAC of seminal plasma decreased by about 60% and those with leukocytospermia and no pathogens had decreased TAC by about 50% (O2). TAC of seminal plasma was considerably lowered in patients with chronic prostatitis, in both the group with leukocytospermia and that without leukocytospermia (P4), and was decreased in asthenozoospermic and oligoasthenozoospermic hyperviscous ejaculates, regardless of sperm count, suggesting that a severe impairment of seminal antioxidative capacity could be associated with semen hyperviscosity (S18). However, some authors found no differences between normozoospermic

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and asthenozoospermic patients attending intracytoplasmic sperm injection programs, finding TAC of seminal plasma not to correlate with the outcome of the treatment and to be independent of age, smoking status, or increased leukocyte count in the sperm of the patients (J5). Comparison of TAC of seminal plasma of men who underwent vasectomy reversal showed that there was no statistically significant difference between those who recovered fertility (1.96 ± 0.22 mM) and those who remained infertile (1.53 ± 0.17 mM) (K9). Fast TRAP assay of human seminal plasma was a good predictor of pregnancy after in vitro fertilization; semen with a fast TRAP 99% O2) decreased TAC of rat lung homogenates (cytosol fraction) by 26%. TAC of the acid-soluble fraction was even more depressed (by 40%). Feeding rats before the experiment with vitamin E-rich diet (500 IU/kg of vitamin E acetate) or diet containing blueberry extract (2.5%) but not spinach extract (0.85%) attenuated this effect of hyperoxic exposure (C14). Methanol intoxication of rats (3 g/kg) was found to decrease TAC of their liver homogenates (S19).

12. Points of Concern Although there is no doubt that TAC is a good measure of total content of antioxidants in food and beverages, it cannot say anything about the bioavailability of these compounds. More important from a clinical point of view is the question of whether TAC can be a valid biomarker of the antioxidant status of the body. A considerable variation of TAC values of blood plasma (and other body fluids) for healthy persons can be seen from data reported by various authors (Table 8). One reason for these discrepancies is the diversity of assay methods employing different oxidants (in the inhibition assays) and indicators. There may be additional reasons for these discrepancies. Due to differences in reactivities of the

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contributions of individual antioxidants to TAC, results of measurements will depend on detailed reaction conditions. Effects of small changes in the assay conditions are sometimes problematic. Trolox equivalent antioxidant capacities (TEAC) found in a comparative study for ascorbic acid differed threefold (0.99 ± 0.09 vs. 0.31 ± 0.03) between two assays of by ABTS·+ reduction; the only differences between the assays concerned the method of ABTS·+ preparation (oxidation with MnO2 vs. potassium persulfate) and time of reaction (S7). Antioxidant activity may also be a nonlinear function of concentration of some antioxidants, that is, TAC values may decrease with increasing concentration of the compound studied (M21, W7). In some methods TAC was reported to be not proportional to the dilution (or volume introduced) of blood plasma (C9, S8, W6). This effect may be due to binding of ABTS·+ (and other indicators) to plasma proteins, especially albumin (M17), or binding of some sample components to indicator proteins (O4) Therefore, analysis of the same volume of the fluids studied can be recommended; nevertheless, comparison of results obtained under different conditions can hardly be done in some cases. Substances that show antioxidant activity in one assay do not necessarily exhibit it in another tests. This statement refers, among others, to the reduction of ABTS·+ , a compound promiscuous in its reactions. It has been pointed out that broadening of the definition of an antioxidant to “any substance that inhibits oxidative damage to a target” (or reduces an indicator) “under the assay conditions being used” is prone to the danger that “every chemical in the laboratory could then be classified as either an antioxidant and/or a prooxidant on the basis of assays that have little biological meaning” (H4). Another potentially important factor may be the storage of the samples. Usually the samples are collected over some time period and stored at −80◦ C under the tacit assumption that the storage does not affect the valued measured. No change in the cyclic voltammetry pattern, reflecting the antioxidant content, was found after 6month storage of blood plasma at −70◦ C (C24). Plasma concentrations of retinol, β-carotene, and α-tocopherol were not considerably affected by storage at −70◦ C for at least 4 years (C32). Ascorbate may be prone to oxidation during storage; its contribution to TAC of blood plasma is not high, but may be more critical in other material. Data have been published recently pointing to a considerable drop of TAC (assayed with ABAP and R-phycoerythrin) during the first several days of storage at −80◦ C (down to ca. 60% of the initial values) (G5, G6). More attention should be paid to the stability of TAC during sample storage and, if possible, only fresh samples should be analyzed. In the case of tissue homogenates, the procedures of preparation and storage of the material can be expected to be of even greater importance. How well do the results of TAC determinations by different methods coincide? One group found no correlation between results by ORAC and TEAC and by FRAP and TEAC and only a weak correlation between ORAC and FRAP (r = 0.35,


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p = 0.019) when studying the same group of persons (C9). Usually the correlation is much better, but it is always far from perfect (P8). A more general reservation concerns the biological relevance of TAC. It should be noted that TAC is not a clearly defined quantity. Each oxidant has some spectrum of action and some specificity. Therefore, the contribution of various antioxidants to TAC measured against various oxidants will differ (R5, R6, R8) and the same refers to the indicator. Ascorbate is very efficient in scavenging peroxyl radicals, whereas transferrin and ceruloplasmin are efficient in prevention of metal-dependent oxidations. Uric acid is efficient in scavenging nitrogen dioxide while of little importance in preventing reactions of hypochlorous acid (H4). TAC should be measured against oxidants that are most relevant biologically. But is it possible to define them? Many researchers would agree that the hydroxyl radical is the most dangerous, and therefore most important, biological oxidant. However, it is so reactive that TAC against hydroxyl radical should roughly correspond to the content of organic matter (nevertheless, sources of hydroxyl radical are used in some TAC assays). Peroxyl radicals formed during decomposition of azo initiators under aerobic conditions are convenient oxidants. In spite of similarities to peroxyl radicals, which may be of biological importance, they can hardly be assumed to be biologically important oxidants. The same argument refers to other commonly used oxidants. Is the TAC against peroxyl radicals a valid measure of the ability of the material tested to counteract oxidation by peroxynitrite or hypochlorite? Is TAC, representing mainly the second line of antioxidant defense (interception of reaction of uncontrolled oxidation), as important as specific antioxidant mechanisms of the first line of antioxidant defense (preventing the formation of reactive oxygen species)? If so, one should perhaps expect high TAC values of biological fluids of crucial importance such as seminal plasma. Instead, TAC of blood plasma and seminal plasma is comparable with that of urine and much lower than that of feces (Tables 8 and 10). Increase in TAC is not always a good prognostic; it may simply indicate an initial response to oxidative stress, as with concentrations of individual antioxidants and activities of antioxidant enzymes, or when it is due to disturbances in uric acid metabolism. Because uric acid is the main determinant of TAC of blood plasma, TAC increases in situations when the concentration of urate is increased, for example, in metabolic disorders and kidney failure. TAC is increased in urine from renal transplant recipients with delayed graft function (S16). Ischemia of small intestine leads to an increase in TAC of rat blood serum, which is maximal (almost twofold) immediately after termination of 45-min ischemia (S22). TAC of blood plasma of rats poisoned with a high dose of carbon tetrachloride (1200 mg/kg, intraperitoneal injection, measurement 16 hr after injection) was significantly (over twofold) increased (K1). These apparently paradoxical effects can be explained, however, by release of antioxidants from cells undergoing necrosis. Increase in TAC after intensive physical exercise also may be a marker of tissue

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damage or increased metabolic generation of uric acid rather than of mobilization of antioxidant defense. TAC of blood plasma in rats fed an ethanol-supplemented diet increases, although ethanol is known to induce oxidative stress. The effect is due to ethanolinduced purine degradation and increase in the level of uric acid (G2). TAC of blood plasma in critically ill patients with renal dysfunction is augmented, again due to increase in uric acid level (M1). Caloric restriction, a procedure known to improve the “redox status” and prolong the life span of mammals, decreases TAC of rat serum (C12). The dominant significant contribution of uric acid to TAC of blood plasma, obscuring changes in the concentrations of other antioxidants, makes TAC less sensitive to changes in the concentrations of other antioxidants. Moreover, uric acid may be a prooxidant in some situations (S3) and a harmful metabolite in other cases. Attempts have been made to calculate or measure the urate-independent fraction of TAC (P20).

13. Conclusions In spite of all these concerns and limitations, TAC is an easily and increasingly broadly used parameter employed in clinical studies and in food science, useful in comparison of the antioxidant content of body fluids, cell and tissue homogenates, food, and beverages. Although comparison of data obtained in different laboratories is difficult, estimation of TAC under standardized conditions allows for detection of changes in the content of antioxidants in blood plasma induced by ingestion of nutritional oxidants and antioxidants. The TAC changes in many diseases and may have a prognostic value in some of them (a higher TAC does not always mean a good prognosis). Interpretation of results of TAC determination still poses questions, indicating the need for further studies, but this simple assay may join the list of standard laboratory analyses of blood and seminal plasma, other body fluids, and tissue biopsies. REFERENCES A1. Abuja, P. M., and Albertini, R., Methods for monitoring oxidative stress, lipid peroxidation and oxidation resistance of lipoproteins. Clin. Chim. Acta 306, 1–17 (2001). A2. Adamson, G. E., Lazarus, S. A., Mitchell, A. E., Prior, R. L., Cao, G., Jacobs, P. H., Kremers, B. G., Hammerstone, J. F., Rucker, R. B., Ritter, K. A., and Schmitz, H. H., HPLC method for the quantification of procyanidins in cocoa and chocolate samples and correlation to total antioxidant capacity. J. Agric. Food Chem. 47, 4184–4188 (1999). A3. Aejmelaeus, R. T., Holm, P., Kaukinen, U., Metsä-Ketelä, T. J. A., Laippala, P., Hervonen, A. L. J., and Alho, H. E. R., Age-related changes in the peroxyl radical scavenging capacity of human plasma. Free Radic. Biol. Med. 23, 69–75 (1997).


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LYMPHOID MALIGNANCIES: IMMUNOPHENOTYPIC ANALYSIS Amy Chadburn and Sheshadri Narayanan Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, New York

1. Introduction: Immunophenotypic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Background: Immunophenotypic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Immunohistochemistry: Antigen Retrieval. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Flow Cytometry: Basic Operational Concepts of a Flow Cytometer . . . . . . . . . . . 2.4. Comparison: Immunohistochemistry and Flow Cytometery . . . . . . . . . . . . . . . . . 3. Normal Lymphoid Cell Development and Antigen Expression . . . . . . . . . . . . . . . . . . . 3.1. Background. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Normal B Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Normal T Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Neoplastic Lymphoid Cell Antigen Expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Background. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. B Cell Neoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. T/ NK Cell Neoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Hodgkin Lymphoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Selected Biochemical Consequences of Neoplastic Transformation. . . . . . . . . . . . . . . . 5.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Methodological Considerations: Monoclonal Gammopathies . . . . . . . . . . . . . . . . 5.3. Multiple Myeloma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Essential Monoclonal Gammopathy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Waldenstrom’s Macroglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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analysis is employed clinically in order to obtain diagnostic, prognostic, and, more recently, treatment information. Some of the uses of immunophenotyping include determination of cell lineage, clonality (particularly with respect to lymphoid lesions), stage of differentiation, and expression of specific gene products (particularly those of oncogenes and tumor suppressor genes, which are involved in the pathogenesis of the disease) and identification of viruses and other organisms. More recently, with the development of monoclonal antibody therapy, such as Rituximab directed against the CD20 antigen (C24, H1) and drugs directed to specific gene products, such as Gleevac to the BCR-ABL fusion protein (M19), immunophenotyping has become crucial for determining optimal treatment options for individual patients. In addition, the identification of specific abnormal gene products expressed by tumor cells (such as p53 in mantle cell lymphoma) gives the clinician and patient important prognostic information, allowing for better informed clinical decision making and biologically directed therapeutic decisions (G6, H18, L16). Nowhere in the clinical arena is immunophenotyping a more integral part of diagnosis, prognosis, and therapy than in lymphoid proliferations. Furthermore, as exemplified by both the REAL (revised European and American Classification of Lymphomas) (H11) and the WHO (World Health Organization) (J2) classifications of lymphomas, routine morphology alone is insufficient for the diagnosis of a lymphoid proliferation. Correlation of morphology, immunophenotype, and in some cases genotype, in addition to the clinical findings are necessary for arriving at a specific and accurate diagnosis. The versatility of immunophenotypic analysis has enhanced its usefulness. Immunophenotyping can be performed on a variety of specimens including cryopreserved tissue sections, routinely preserved tissue sections, cells in suspension, tissue-touch preparations (“touch preps”), fine-needle aspiration smears, cytology thin preps and smears, as well as bone marrow aspirate smears. With the evolution of medical science and technical ability the number and types of antigens that can be detected in each type of preparation have greatly increased. Furthermore, as an additional result of these technical and scientific advances, immunophenotyping, particularly of routinely fixed tissue sections, can be performed reliably in many laboratories. Although the majority of diagnostic pathologists rely on immunohistochemistry for the immunophenotypic analysis of lesions, hematopathologists employ both immunohistochemistry, primarily of tissue sections, and immunofluorescence flow cytometry of cells in suspension to characterize hematopoietic lesions. Each of these techniques has specific advantages and disadvantages depending on the desired diagnostic information, but they are in many ways complementary. However, the basic principles of immunophenotyping are similar for both methods.

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2. Background: Immunophenotypic Analysis 2.1. GENERAL An antigen is a substance that stimulates antibody production. The antigenic determinant or epitope is the structural part of an antigen that reacts with the antibody (M23, T1). Because most antigens are relatively complex proteins, carbohydrates, and/or lipids they have may have several antigenic determinants and thus may be capable of inducing and reacting with more than one antibody. The recognition of the antigenic determinant by the antibody is based not only on the amino acid sequence or the type of carbohydrate, but also on the three-dimensional electrophysical structure of the antigen as a whole (M23, T1, T8). The structure of an individual antigen is variably susceptible to tissue handling procedures. Fixation, type of fixation, length of fixation, temperature, and autolysis among other things alter the ability of an antibody to detect a specific antigenic determinant (K6, M23, T1, T8). For example, whereas some antigens are well “preserved” in formalinfixed tissues (such as the epitope detected by the antibody L26 directed to the CD20 antigen), others are altered or destroyed (such as the epitope detected by the antibody B1, which is also a CD20 antigen). “Clusters of differentiation” (CD) are groups of antigens recognized by antibodies with similar reactivities (B13). For example, the monoclonal antibodies L26 and B1 exhibit a similar pattern of reactivity and are clustered together as CD20 (N1, Z1), whereas the antibodies Leu22, MT-1, and DFT-1 are clustered as CD43 (C23, S29) based on their comparable immunoreactivity patterns. Antibodies are immunoglobulins (Ig) that bind to antigens (K6, M23, T1, T8). There are five classes of antibodies, IgM, IgD, IgG, IgA, and IgE; however, only IgG, and to a lesser extent IgM, antibodies are used for immunophenotyping. IgG has a Y-shape structure and is composed of two heavy chains and two light chains linked by disulfide bonds. The V-shaped portion of the Y-shaped immunoglobulin contains the variable regions. The amino acid sequence in the variable regions determines the specificity of the antibody for an antigen (B17, M23, T8). The binding strength of an antibody to a specific antigen (i.e., affinity) is a result of both “functional” and “intrinsic” affinities (B17). The higher the affinity, the better the antibody, because the antibody is more likely to remain bound to the antigen throughout the staining procedure. The intrinsic affinity of the antibody to the antigen is dependent on the tertiary structures of both the antibody and the antigen-binding sites and includes hydrogen bonding, electrostatic and van der Waals forces, and hydrophobicity. This intrinsic affinity is a function of the amino acid sequence in the hypervariable region of the immunoglobulin. The intrinsic affinity of a specific antibody–antigen binding interaction is defined by the affinity constant K = [AbAg complex]/[Ab] × [Ag]. Thus, antibodies with different

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hypervariable regions (i.e., composed of different amino acid sequences) can recognize identical antigens, but because of structural differences resulting in different tertiary structure and “charge” structure, have different affinities (B17, K6, M23, T1, T8). “Functional” affinity relates to the time required for the antibody to reach equilibrium with an antigen in the tissue or on cells in suspension. The faster an antibody reaches a plateau of maximum staining, the greater is the functional affinity (B17). Antibodies used for immunohistochemistry and immunoflow cytometry are either polyclonal or monoclonal antibodies. Polyclonal antibodies, while reacting to a given antigen, detect different epitopes on this antigen with variable affinity. Polyclonal antibodies are made by different cells and are usually produced by immunizing animals. Monoclonal antibodies, on the other hand, are made by a single clone of cells (usually a hybridoma) and are identical in their reactivity with the antigen to which they are made (B17, K6, M23, T1, T8). Both types of antibodies have specific advantages and disadvantages. In general polyclonal antibodies are more sensitive because (1) they are more likely to react with molecules that do not contain repetitive antigenic determinants and (2) they consist of multiple antibodies with variable specificities capable of reacting with multiple different epitopes of a given antigen. However, because they are a heterogeneous mix of antibodies, which includes those to various epitopes of the desired antigen as well as those to any impurities in the immunization material, polyclonal antibodies are associated with a relatively high degree of background staining. This can be lessened with adsorption procedures and, if possible, antibody dilution. Furthermore, although polyclonal antibodies are relatively easy to produce, there is variation in antibody binding affinity between antibody “lots” which can lead to inconsistent staining results unless each separate lot is carefully titered (B17, K6, M23, T1, T8). In contrast, monoclonal antibodies are less sensitive, but more specific. Thus, there is less cross-reactivity than with polyclonal antibodies. However, cells expressing low levels of a specific antigen or lacking the specific antigenic epitope may not be detected (K6, T1, T8). Antibodies may be termed “primary,” “secondary,” “tertiary,” etc., depending on when they are employed in the immunostaining procedure. The primary antibody is directed against the antigen under study, whereas the secondary antibody is directed against the primary antibody, the tertiary antibody is directed against the secondary antibody, and so forth (B19, T1, T8). In general an antibody is directed against the species in which the previous antibody was developed or, as in the case of primary antibodies used in the clinical setting, against human (B19, M23, T1). For example, if the primary antibody were a mouse anti-human antibody, then the secondary antibody would be directed against a mouse immunogobulin. Depending on the procedure employed, the primary, secondary, or tertiary antibody is conjugated to an enzyme, which reacts with a chromogen that can be visualized


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when using a standard light microscope or to a fluoresceinated compound, which can be detected by a flow cytometer or visualized using a fluorescent microscope. In some cases the antibody may be conjugated to biotin (biotinylated). Because biotin readily binds to avidin, which can bind to more biotin molecules that are labeled with an enzyme, this results in significant amplification of the signal. When the label is directly attached to the primary antibody, as in flow cytometry, no additional antibodies against antibodies (i.e., secondary or tertiary antibodies) are used (B19, M23, T1, T8). Labels are (or result in) “visually” identifiable materials. The most commonly used labels in immunohistochemistry are enzymes. Although fluoresceinated compounds are also used in immunohistochemistry, they are more frequently as used antibody labels in flow cytometry. Labels may be conjugated directly to antibodies, either the primary antibody, as is often the case in flow cytometry, or to “secondary” or “tertiary” antibodies. In immunohistochemistry the most common label is horseradish peroxidase, although calf intestinal alkaline phosphatase is also often used. The enzyme labels are used in combination with a variety of chromogens, including 3-amino-9-ethylcarbazoel (AEC), 4-chloro-1-napthol, and 3,3′ -diaminobenzidine tetrahydrocholoride (DAB). These chromogens, when incubated with an enzyme, result in a stable, colored reaction product that can be visualized through a light microscope allowing for morphologic identification of individual immunoreactive cells. DAB is currently the most frequently used chromagen and results in a permanent, alcohol-insoluble dark-brown reaction product that does not dissolve in xylene-based mounting mediums (B18, M23, T1). The fluoresceinated compounds, although used in some special types of immunohistochemical cases, are primarily used in flow cytometry (M20, M23). The most commonly used fluorescent labels are fluorescein isothiocyanate (FITC) and phycoerythrin; however, other compounds such as tetramethylrhodamine isothiocyanate, Cy-5, and Texas red have also been used. Antibodies conjugated to different fluoresceinated compounds can be used simultaneously to detect different antigens on the same cell. However, care must be taken to (1) match the excitation wavelength of the label(s) to the wavelengths of the light sources as well as (2) ensure that the different labels emit energy at sufficiently different wavelengths so as to be recognized as separate events. Unlabeled primary antibodies can also be detected by using a fluoresceinated generic secondary antibody, such as a FITC-conjugated sheep anti-mouse antibody (K22, M20). A variety of techniques (Fig. 1), which use a variable combination of antibody steps, enzyme labels, and chromogens, are employed in immunohistochemistry. The most common immunohistochemical methods are the PAP (peroxidase– antiperoxidase), ABC (avidin–biotin) peroxidase or alkaline phosphatase, and the APAAP (alkaline phosphatase–anti-alkaline phosophatase) techniques; however,

FIG. 1. Some of the methods used for immunostaining.


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newer polymeric systems, which result in a larger signal (such as the EnVision system), are becoming more popular. Depending on the type of tissue, the type of tissue fixation, and the antigen under study, one of these techniques may be preferable over the others (B19, H22, K22, M23, N5, T1, T7).

2.2. IMMUNOHISTOCHEMISTRY: ANTIGEN RETRIEVAL Prior to the 1970s for all practical purposes immunohistochemical staining could only be performed on frozen and not routinely fixed tissue sections. Although some aspects of the immunoarchitectural pattern of the tissue and the relative number of immunoreactive cells could be evaluated using frozen tissue sections, individual immunoreactive cells, easily seen in routinely fixed tissues, as well as some architectural features are difficult to identify in frozen tissue sections. Morphologic detail of the tissue is retained by tissue preservation or “fixation,” which, depending on the fixative, is usually brought about by cross-linkage, as with formalin, or coagulative denaturation, as with ethanol, of proteins. The various fixation processes, however, also destroy, alter, or “mask” many tissue antigens (F7, M23, T1). Prior to the 1970s only a small number of antigens, due to this effect of tissue fixation, were detectable by immunostaining in routinely processed tissue sections. In an effort to expand the number of antigens detectable in paraffin tissue sections, Huang in 1975 treated formalin-fixed tissue with enzyme digestion (H23) prior to immunohistochemical staining. Although enzyme pretreatment “unmasked” or “retrieved” some antigens altered by formalin fixation, the immunoreactivity of the majority of antigens in routinely processed tissue sections was not significantly improved (L7). However, in 1991, based on studies by FraenkelConrat and associates (F16 –F18), Shi et al. described an antigen retrieval method using high-temperature heating of routinely processed tissue sections prior to immunostaining (S13). Although the heat retrieval method in this initial report included microwaving dewaxed tissue sections in a heavy metal solution, subsequent modifications in heating (i.e., using a pressure cooker, autoclave, vegetable steamer, or water bath) and in buffer solutions with respect to pH (both high and low), molarity (usually ranging from 1 to 10 mM), and composition (i.e., ethylenediaminetetraacetic acid and citrate) have been described and used with success (C8, E4, M22, M23, N15, S12, T2). Furthermore, enzyme digestion and heat retrieval in combination can be used for antigen retrieval. Antigen retrieval has widely expanded the number of antigens that can be detected in paraffin tissue sections and/or has decreased the amount of antibody necessary to detect a specific antigen. In addition to antigen retrieval, antibodies were developed to fixation-reisistant epitopes. Therefore, in many situations, it is possible to use almost exclusively paraffin tissue sections for clinical immunophenotyping, preserving frozen tissue for molecular-genetic studies.


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2.3. FLOW CYTOMETRY: BASIC OPERATIONAL CONCEPTS OF A FLOW CYTOMETER Flow cytometry measures cells moving in fluid (M20). In flow cytometry optical and fluorescence measurements of cells suspended in fluid are made as they pass single file through a beam of monochromatic light. The physical characteristics (i.e., “intrinsic” properties) of each cell can be determined by the pattern of light scattered; this does not require immunostaining (Fig. 2). The number of cells expressing a specific antigen and the number of antigens expressed by a cell are determined by counting the number of cells emitting energy and by counting the number of photons emitted by an individual cell following light activation; this requires immunostaining using a fluoresceinated labeled antibody (L6, M20). The photons scattered or emitted by each cell are separated into their different constituent wavelengths by a series of mirrors and filters landing on specific detectors that convert light energy to electrical impulses (“analog” signals). The analog signals are subsequently converted to “digital” signals (numbers) that are tabulated as a frequency distribution in either a histogram or a dot plot (Fig. 3). The numbers (i.e., signals) obtained for each cell are proportional to the amount of light scattered (reflecting size and granularity) and emitted [reflecting the number of a specifically expressed antigen(s) as detected by a labeled antibody(ies)]. Furthermore, based on light scattering and light emission, specific populations of cells can be analyzed by preferential gating. Although the exact mechanics of the flow cytometer are beyond the scope of this review, fluidics, particularly maintaining a single-cell laminar flow through the flow chamber (“cell”), optics, and computer system/electronics are all crucial components (L6, M20). 2.4. COMPARISON: IMMUNOHISTOCHEMISTRY AND FLOW CYTOMETERY The choice of immunophenotypic analysis technique of a specimen in general, and of lymphoid lesions specifically, depends on the type and amount of available tissue and the question that the pathologist and clinician wish to answer. As listed in Table 1, both immunohistochemistry and flow-cytometric analysis have specific advantages and disadvantages with respect to type and amount of information that can be garnered from their application. FIG. 2. Measurement of intrinsic characteristics of cells by flow cytometry: Forward scatter (size) and side scatter (granularity). The laser light is deflected by cells in both a forward direction (forward scatter) and at 90◦ angles (side scatter), indicating size and granularity, respectively. (1) Small lymphocytes, as in B-CLL/SLL, are relatively small agranular cells that do not reflect much light in either the forward direction or at 90◦ . This cell population “falls” in the lower left corner of the dot plot. (2) In contrast, large, somewhat more granular cells, as in acute myelogenous leukemia, deflect a significant amount of light with respect to both forward and side scatter, resulting in the population being somewhat farther to the right (larger) and higher (more granular) than the small lymphocyte population.

FIG. 3. Flow cytometery histogram and dot plot. (1) Analysis of expression of a single antibody can be evaluated using a histogram. The farther to the right on the x axis, the larger is the number of a given antigen expressed by the cells. Thus, peak A corresponds to the essentially antigen-negative population and peak B corresponds to the antigen-positive population. The areas under the peaks are proportional to the number of positive cells. (2) Using a dot plot, the simultaneous analysis of the expression of more than one antigen can be performed. Similar to the histogram, the farther to the right on the x axis and to the top on the y axis corresponds to more antigen expression by the cells. The population in the lower left quadrant (c) contains the negative cell population (in this case essentially no negative cells are present). The lower right quadrant (d) contains a small population that only expresses CD33. The upper left quadrant (a) contains essentially no cells expressing CD34, and the upper right quadrant (b) shows a large population of cells expressing both CD33 and CD34.


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TABLE 1 COMPARISON OF TECHNIQUES OF IMMUNOPHENOTYPIC ANALYSIS: FLOW CYTOMETRY VS. IMMUNOHISTOCHEMISTRY Flow cytometry Highly sensitive Quantitative Analysis of a large number of individual cells Rapid, can be done in a few hours

Cells in suspension, i.e., bone marrow aspirates, peripheral blood, effusions, etc. Analysis of specific cell populations by selective gating (size, granularity, antigen expression) Easy and specific determination of expression of multiple surface antigens Wide spectrum of antibodies

No architecture Cannot identify an individual immunoreactive cell Can only easily detect surface antigen expression; cytoplasmic and nuclear antigen expression requires permibilization of membranes Poor tumor cell viability can result in false-negative results Less biologically “safe,” more biohazard precautions

Immunohistochemistry Sensitive, but not as sensitive as flow cytometry Qualatative, except for a few antigens (i.e., image analysis) Analysis of a mixture of cells Faster than in the past, but still requires cutting of tissue; also, dependent on specimen and differences in techniques Can be done on cytospins of cell suspensions or on smears, but more difficult to do and interpert Not possible

Double staining can be done; however, determination of surface expression of more than one antigen is very difficult; cytoplasmic and surface or nuclear and surface antigen expression relatively easy Wide spectrum of antibodies; for frozen tissue sections relatively similar as for flow, but sometimes less useful because of sensitivity; for paraffin tissue sections less, but with antigen retrieval the gap is narrowing Architecture identifiable and sometimes highlighted by immunostaining Individual immunoreactive cells can be identified Surface, cytoplasmic, and nuclear antigen expression can be detected

Can often determine immunophenotype (i.e., B vs. T) of necrotic cells In the case of paraffin-embedded tissue, biologically “safer”

3. Normal Lymphoid Cell Development and Antigen Expression 3.1. BACKGROUND Lymphoid neoplasms in both the REAL and the WHO classifications are defined as specific entities based on morphology, immunophenotype, genotype, and clinical characteristics (H11, J2). These neoplastic entities, however, tend to mimic their

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normal counterparts, particularly with respect to morphology and immunophenotype and to a lesser degree genotype. Thus, to understand and accurately employ immunophenotypic analysis in the diagnosis and characterization of lymphoid proliferations a basic understanding of the stages of lymphoid differentiation and antigen expression is necessary. Although our understanding of lymphoid cell differentiation has significantly expanded in the last 10 years, it is not completely delineated, particularly with respect to the T cell lineage. 3.2. NORMAL B CELL DIFFERENTIATION The B cell progresses through several stages of development: progenitor B cells, pre-pre-B cells, pre-B cells, immature B cells, resting mature (“naive”) B cells, activated/differentiating B cells (which include centroblasts, centrocytes, and memory cells), and plasma cells (Table 2) (A12, D21, F12, M12, N1, N2, S28). Precursor B lymphoblasts are the earliest B cells and include cells within the first three stages of differentiation (i.e., progenitor B cell, pre-pre-B cell, and preB cell). These cells, thought to originate in the bone marrow from pluripotential lymphoid precursor cells, express terminal deoxynucelotidyl transferase (TdT), an enzyme that polymerizes the addition of deoxyribonucleotides to DNA (H22). The earliest recognizable precursor B lymphoblast (progenitor B cell) expresses only a limited number of surface antigens, including the class II antigen HLA-DR and the progenitor marker CD34; these cells may express the B cell antigen receptor component CD79 in the cytoplasm. With further differentiation, the cells undergo immunoglobulin heavy-chain, then light-chain gene rearrangement and express other B cell-related antigens such as CD19, CD10, CD20, cytoplasmic µ (cµ), and cytoplasmic CD22 (cCD22). Pre-B cells, which have an intact, fully

TABLE 2 B CELL DIFFERENTIATION: IMMUNOPHENOTYPIC PROFILES OF NORMAL B LYMPHOBLASTS

TdT CD19 CD20 CD22 CD79 CD10 HLA-DR CD34 Misc

Progenitor B

Pre-Pre-B

+ − − − See below − + + Maybe cCD79a

+ + See below Cyto Cyto + + + sCD20 late in stage

Pre-B + + + Cyto + + + See below Maybe CD34; cµ


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TABLE 3 IMMUNOPHENOTYPE OF BENIGN B CELLSa

CD19 CD20 CD22 CD10 BCL-2 BCL-6 CD38 CD138 sIg cIg Misc

Naive

Centroblast

Centrocyte

Memory

Plasma cell

+ + − − + − − − Usually MD − Some express CD5

+ + + + − + − − − − Ki-67+; undergo IgH class switching

+ + + + − + − − + −

+ + + − + − − − M, not D −/+

+ − − − + − + + − + IgG > IgA; others rare

a Limited

immunophenotypic profile.

rearranged immunoglobulin heavy-chain gene (S5), lose TdT, CD34, CD10, and cµ and express CD21 and surface immunoglobulin M (sIgM) to become immature B cells; a subset of these cells also expresses CD5 (D10, K18). These cells progressively increase surface CD22 and sIgD expression and decrease sIgM expression to become naive (resting mature) B cells (A12, D21, F12, K15, M12, N1, N2, S28). Naive B cells, which are antigen- “naive,” express HLA-DR, CD19, CD20, CD22, CD21, CD79, sIgM, and sIgD and often CD5, leave the bone marrow, and circulate in the peripheral blood and populate the primary follicles and mantle cell zones (Table 3) (I2, K11, M2). After encountering an antigen and aided by costimulatory signals they undergo blast transformation, proliferate, and migrate into the center of the primary follicle, where, by association with the CD21-positive, CD35-positive follicular dendritic cells (FDCs), they form a germinal center. On average germinal centers are formed by three B cells, which proliferate to give rise to (10–15) × 104 cells (L15). These germinal center B cell blasts (known as “centroblasts”) are rapidly proliferating (Ki-67-positive) cells that lack sIg and the antiapoptotic protein BCL2, but express CD10 and the transcription repressor factor BCL6 (Fig. 4; see color insert). Centroblasts often undergo “class switching” to change from IgM production to that of a higher affinity immunoglobulin, usually either IgG or IgA (B1, H6, L13). These cells “mature” to centrocytes (small cleaved cells). Centrocytes, which still are BCL2-protein-negative, but are no longer significantly proliferating, express sIg of variable affinity for the stimulating antigen; those cells, with insufficient affinity for the antigen, die by apoptosis. Centrocytes with sufficient affinity bind the antigen (trapped on the FDCs) and through

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a complex set of interactions between FDCs and T cells, reexpress BCL2 (so advoiding apoptosis), turn off BCL6 expression, and differentiate into plasma cells or memory B cells (C6, H6, L13, L14, M1, M2, P14). Memory B cells usually express sIgM without sIgD, and the pan-B cell antigens CD19, CD20, CD22, and CD79, but lack expression of CD5, CD10, and BCL-6. They usually are present at the edge of the follicle in the marginal zone (D17, S19, V2, V5). Plasma cells lack sIg, but express cytoplasmic immunoglobulin (cIg), usually cIgG or cIgA. These cells in general express the pan-B cell antigens CD19 and CD79a as well as CD38 and CD138, but lack expression of CD56 (B4, G8, H7, R10). 3.3. NORMAL T CELL DIFFERENTIATION T cell development, similar to B cell ontogeny, can also be divided into stages based on antigen expression (Table 4): extrathymic precursor, prothymocyte, immature thymocyte, common thymocyte, mature thymocyte, and mature peripheral T cell. However, while the early stages of T cell development are well characterized, the later stages of T cell differentiation, that is, those of the mature peripheral T cell, have not been fully delineated (C3, F12, H13, K15, K16, K20, R6–R8,V3, V4). Furthermore, although natural killer (NK) cells are closely related to T cells (S21), the stages of differentiation of this lymphoid cell population are not completely understood.

TABLE 4 T CELL DIFFERENTIATION: IMMUNOPHENOTYPIC PROFILES OF THYMOCYTES AND THYMOCYTE PRECURSORS

TdT HLA-DR CD34 CD7 CD2 CD3 CD5 CD4 CD8 CD1 TCRαβ Misc

Precursor

Pro-Thymocyte

Immature thymocyte

Common thymocyte

Mature thymocyte

+ + + − − −

+ + + + +/− Cyto +/−

+ − − + + Cyto

+ − − + + Cyto

− − − − − Extrathymic

− − − − − Cortical thymus

+ − − − − Cortical thymus

variably + − − + + +; Cyto variably+ + + + − + Medullary thymus

+ +; See below +; See below + − Cortical thymus; double positive T cells (CD4/CD8)


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The earliest T cells, as with B cells, are found in the bone marrow. These pluripotential extrathymic precursor lymphoid cells express TdT, HLA-DR, and CD34. Prothymocytes, based on the additional expression of CD7, generally thought to be the first T cell-lineage-associated antigen expressed during development (H13, P15, V3), are the earliest identifiable T cells. Within the bone marrow, some of the prothymocytes begin to produce and express cytoplasmic CD3 epsilon (cCD3ε) (C3, V4); some may also express CD2. Subsequently the prothymocytes migrate from the marrow to the thymus. In the thymus, based on the ability to recognize and bind to peptide sequences of the major histocompatability complex (MHC) displayed by the thymic epithelium, a proportion of the primitive T cells is selected for development. Those thymocytes that either strongly react or do not bind to these MHC peptide sequences are eliminated (D10). Apoptosis or programmed cell death eliminates approximately 99% of primitive T cells that migrate to the thymus (N6). In the cortical region of the thymus the prothymocytes lose expression of HLA-DR and CD34 and acquire expression of additional antigens to differentiate into immature thymocytes and then into TdT-positive, CD1-positive common thymocytes, which express surface CD2, CD5, and CD7, and cCD3. The common thymocytes are “double-positive” cells expressing both CD4 and CD8. These cells further differentiate into either CD4-positive, CD8-negative or CD4-negative, CD8-positive cells, express the complete TCR-CD3 surface membrane complex, and lose CD1 to become mature (“medullary”) thymocytes. These medullary thymocytes variably express TdT and cCD3. With further maturation, these cells completely lose expression of cCD3 and TdT and enter the peripheral circulation and tissues as mature helper/inducer CD4-positive or cytotoxic/suppressor CD8-positive T cells (C3, H13, K16, R20, R6–R8, V4). Although T cells can be divided into two populations (helper/inducer and cytotoxic/suppressor) based on CD4 and CD8 expression, T cells can also be divided into two populations based on the structure of the antigen receptor complex, either α/β or γ /δ, associated with the CD3 antigen (D10). The γ /δ T cells represent approximately 5% of all T cells, lack expression of CD5, CD4, and often CD8, are thought to be associated with a more primitive type of immune response, and are usually found in the splenic red pulp and epithelial sites (A15, C26, T6). The α/β T cells usually express all four pan-T cell antigens (CD2, CD3, CD5, and CD7) and either CD4 or CD8. The CD4-positive cells can be further divided based on cytokine secretion: Th1 cells, which secrete interleukin-2 (IL-2) and interferon γ and “help” macrophages and other T cells, and Th2 cells, which secrete IL-4, IL-5, IL-6, and IL-10 and “help” B cells in antibody production (D10, D11). Natural killer cells express some antigens that are also expressed by cytotoxic T cells. NK cells are usually CD2-, cCD3-, CD16-, and CD56-positive and can express CD7, CD8, and CD57. Furthermore, NK cells and some cytotoxic T cells also express cytotoxic proteins such as perforin, granzyme B, and T cell intracellular antigen 1 (TIA-1) (B22, C12, F8, J1, K21, Y2).

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4. Neoplastic Lymphoid Cell Antigen Expression 4.1. BACKGROUND In the early 1990s it became clear that the schemes in use for lymphoma classification were inadequate. The development of new techniques employed in the biological sciences had greatly increased our understanding of the human genome, normal cellular functions, and the pathobiology of disease. Because of the nature of lymphoid cells and the relative ease with which they can be studied, significant inroads had been made with respect to the understanding of normal lymphoid cell development and the pathogenesis of lymphomas. These advances highlighted the fact that lymphoma classification could no longer rely only on morphologic characteristics of the tumor cells, but required the integration of morphology, immunophenotype, genotype, and clinical features of the disease process as well as an understanding of normal lymphocyte development and function to accurately define neoplastic lymphoid processes. With this background an international group of hematopathologists (the International Lymphoma Study Group; ILSG) developed in 1994 a lymphoma classification, the REAL classification (revised European and American classification of lymphoid neoplasms), which defined lymphoid tumors as biologic entities based on morphologic, immunophenotypic, genotypic, and, in some instances, clinical characteristics. This REAL classification established the framework for the updated World Health Organization (WHO) Classification of Tumours (J2). Furthermore, based on the known morphology, immunophenotype, and genotype of specific lymphoid neoplasms it is possible in some instances to identify the specific counterpart in normal B and T cell development (H11, J2). Because of the integrative nature of lymphoma classification, a discussion of the immunophenotyping of lymphoid neoplasms must also include discussion of the other main criteria, that is, morphology and genotype. However, because morphology is highly dependent on prompt, optimal fixation and therefore is not consistent and molecular-genetic studies are labor-intensive, lengthy, specialized procedures, the diagnosis and classification of lymphoid lesions in most pathology laboratories relies heavily on immunophenotypic findings. Basic immunophenotypic criteria helpful in the diagnosis of lymphoid proliferations are listed in Table 5. Lymphoid neoplasms are divided into three main categories: B cell neoplasms, T/NK cell neoplasms, and Hodgkin lymphoma. The B and T/NK cell neoplasms are further divided into precursor B and mature B cell neoplasms and into precursor T and mature T/NK cell neoplasms, respectively. The mature B cell and T/NK neoplasms consist of a variety of different neoplastic lymphoid entities, which can be grouped based on primary clinical presentation or based on median survivals without treatment reflecting biologic behavior. However, due to the large number of separate entities, only the more common lesions will be discussed. For information concerning the entities not discussed here or for more in-depth information, the


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TABLE 5 BASIC IMMUNOPHENOTYPIC CRITERIA FOR THE DIAGNOSIS OF LYMPHOID NEOPLASMS B cell lymphoma Expression of κ or λ not both Anomalous expression of a T cell antigen T cell lymphoma Loss of a pan-T cell antigen Abnormal CD4/CD8 only suggestive Classical Hodgkin lymphoma Expression of CD30 and CD15 Lack of expression of CD45 B cell-antigen expression variable Precursor lymphoblastic leukemia/lymphoma TdT-positive

reader is advised to consult the World Health Organization (WHO) Classification of Tumours (J2) and Knowles’s Neoplastic Hematopathology (K19). 4.2. B CELL NEOPLASMS B cell neoplasms based on morphology, immunophenotype, and genotype often, but not always, correspond to stages of B cell development. However, some B cell lymphomas either do not clearly correspond to a specific developmental stage (i.e., appear to be of variable origin such as chronic lymphocytic leukemia/small lymphocytic lymphoma) or do not appear to be related to any known stage of normal development (i.e., hairy cell leukemia) (H10). 4.2.1. Pecursor B Lymphoblastic Leukemia/Lymphoma (B-ALL/ LBL) These lymphoid neoplasms are TdT- and HLA-DR positive. The majority of the cases also express CD19 and cytoplasmic CD79a (cCD79a). In many cases the tumor cells are CD10- and CD24-positive. There is variable expression of CD20 and cCD22/CD22. Most of the cases are also CD99-positive (D28, H11, K18, R11). The degree of differentiation of the B lymphoblasts and the genetic composition of the tumor cells greatly influences prognosis in B-ALL/LBL. The tumor cells of early precursor B-ALL/LBLs are TdT-, HLA-DR-, CD19-, cCD22-, and cCD79apositive. In the common B-ALL/LBLs the malignant cells express CD10 in addition to the markers expressed by the early precursor tumors. In the most “mature” B-ALL/LBLs, known as pre-B-ALL/ LBLs, the cells express cytoplasmic µ (cµ). Usually B-ALL/LBLs do not express surface immunoglobulin (sIg), however, sIg expression does occur in rare cases (K18). A variety of cytogenetic abnormalities

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are associated with B-ALL/LBL. B-ALL/LBLs that are hyperdiploid (with greater than 51 chromosomes) are associated with a good prognosis, whereas those tumors with a BCR-ABL [t(9;22)(q34;q11.2)], an AF4/MLL [t(4;11)(q21;q23)], or other MLL (11q23) translocations are associated with a bad prognosis. The B-ALL/LBLs containing the t(4;11) are usually TdT-positive, CD19-positive, and HLA-DR-positive, but lack expression of CD10; many cases are also CD15positive (i.e., are early precursor B-ALL/LBLs) (A9, B3, L5, P1, P18, R2, R4). In general B-ALL/LBLs that express cµ and contain a t(1;19) are considered to be associated with a poor prognosis. However, with intensive and specific therapy this may not always be the case (A9, C28, L3). The t(12;21)(p13;q22) translocation (TEL-AML1;ETV6-AML1) is associated with a relative good prognosis. These B-ALL/LBLs tend to be CD20-negative, but often express the myeloid-associated antigen CD13 (A5, B20, D15). Most patients with B-ALL/LBL present with bone marrow involvement with secondary involvement of the peripheral blood. Only a small number of patients present initially with disease in lymph nodes or in extranodal sites such as the skin (B25, K18). 4.2.2. Mature B Cell Neoplasms In the WHO classification the entities that comprise the mature B cell neoplasms may be grouped based on primary clinical presentation: predominantly disseminated lymphoma/leukemias, primarily extranodal lymphomas, and primarily nodal lymphomas (Table 6). However, lymphomas also exhibit different biologic behavior and can be thought of as indolent, aggressive, or highly aggressive tumors (H10, H11). However, in practical terms most predominantly disseminated lymphoma/leukemia neoplasms are indolent lymphomas; some primarily extranodal and primarily nodal lymphomas are also clinically indolent lymphomas. Morphologically, the majority of the indolent B cell lymphomas are composed of small relatively bland appearing lymphocytes. Although theoretically the different neoplasms are composed of cells with characteristic morphologic features, in practice the cells are relatively similar morphologically and, particularly in tissue that is not optimally preserved, may be indistinguishable when examined using a light microscope. Based on characteristic antigen expression profiles as determined by immunophenotypic studies, these morphologically similar, clinically similar lesions can usually be divided into separate entities. Furthermore, the morphologic separation of many of the primarily extranodal lymphomas from reactive infiltrates is also extremely difficult. However, based on antigen expression profiles (Table 6) and immunoarchitectural features, this problem has been considerably lessened. Although some of the large-cell aggressive B cell neoplasms have yet to be reproducibly divided into smaller, more biologically specific groups, DNA microarrary technology suggests that this will be possible in the future based on genetic and immunophenotypic characteristics (A8).

TABLE 6 IMMUNOPHENOTYPE OF SELECTED B CELL LYMPHOMASa CLL/SLL CD19 CD20 CD22 CD5 CD10 CD23 CD43 BCL-1 BCL-6 CD138 cIg Misc

+ + −/ft+ + − + + − − − Occ. Atypical: CD38+; FMC7 neg.

MCL

FL

MZL/MALT

SMZL

HCL

BL

DLCL

+ + + + − Usu − + + − − − p53: prognosis

+ + + − + − − − + − − CD21+ FDC MW: smooth edges; BCL-2 + follicle centers

+ + + Rare − − −/+ − − − −/+ CD21+ FDC MW: fragmented edges; CK: LELs

+ + + − − − −/+ − − − −/+ IgD+

+ + + − − − − − − − −

+ + + − + − Some − + − − Ki-67>90%

+ + + − See text − − − See text − −

TRAP+, DBA.44+, CD11c, CD25+, CD103+, HC-2+

PCM + − − − − − − Some − + + Some CD56+

aCLL/SLL, Chronic lymphocytic leukemia/small lymphocytic lymphoma; MCL, mantle cell lymphoma; FL, follicular lymphoma; MZL/MALT, marginal zone lymphoma/mucosa-associated lymphoid tisuse lymphoma; SMZL, splenic marginal zone lymphoma; HCL, hairy cell leukemia; DLCL, diffuse large-cell lymphoma; PCM, plasma cell myeloma; BCL-1, cyclin D1; FDC MW, Follicular dendritic cell meshworks; CK, cytokeratin; LELs, lymphoepitheilal lesions.

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4.2.2.1. Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/ SLL). These mature B cell neoplasms are predominantly disseminated lymphoma/leukemias, which are clinically indolent. It is postulated that they correspond to the recirculating CD5-positive, CD23-positive naive B cells that are found in the peripheral blood, primary follicles, and the follicular mantle cell zones (I2, M3). However, recently it has been found that there are two apparent categories of CLL/SLL based on the presence or absence of somatic mutations in the immunoglobulin genes, which correlate with morphology, antigen expression, and clinical behavior. Cases with somatic mutations, suggesting that the malignant cells are memory B cells, exhibit the typical CLL/SLL morphology, that is, small, round lymphocytes with clumped nuclear chromatin and a scant amount of cytoplasm. Furthermore, genetic microarray analysis shows a gene expression profile that more closely matched memory than naive B cells (K13). These cases of CLL/SLL less often express CD38 and are associated with a more indolent clinical course. Other cases, which do not contain somatic mutations, are presumably naive B cells. These cases are usually composed of morphologically “atypical” CLL/SLL cells, which have irregular nuclear contours and somewhat more basophilic cytoplasm. These cells more frequently express CD38, more often contain a trisomy 12, and are associated with a more aggressive clinical course (D1, H2, K12, M18). In spite of these differences, however, both types of CLL/SLL are more closely related to each other than to other B cell neoplasms and in general exhibit similar immunophenotypic profiles (D2, K12, M18). Because many cases of CLL/SLL involve the peripheral blood, immunophenotypic analysis is often performed by flow cytometry; however, similar results can be obtained by immunostaining of frozen tissue sections. Classically the malignant cells express the pan-B cell antigens CD19, CD20 (weak), CD22 (very weak or absent), and CD79a and faintly express monoclonal sIg of the IgM or of the IgM and IgD heavy-chain isotype(s). In addition, these cells express CD5 and CD23 and usually faintly express the adhesion molecule CD11c. These cells lack expresion of CD10, which is expressed by follicle center cells as well as by many B lymphoblasts, and in general also lack expression of FMC7 (Table 6) (F15, K8, M18). Immunostaining of paraffin tissue sections involved by CLL/SLL shows similar immunophenotypic findings as found by flow cytometry or frozen tissue section staining (Fig. 5; see color insert), namely that the tumor cells are usually CD20-, CD79a-, CD5-, and CD23-positive and CD3- and CD10-negative. Some antigens, however, such as CD19 and FMC7, are not yet detectable in paraffin tissue sections, whereas sIg expression (as with most B cell lymphomas) is often difficult to identify (F15, M18, S30). Furthermore, antigen expression is fixative-dependent. For example, CD5 expression is extremely difficult to detect in B5-fixed tissues (S30) and in some laboratories CD23 expression is identified in less than 50% of


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cases (G2, S15). This lack of CD23 expression may be due to improper fixation (personal observation). Other antigens, however, are more easily identified in paraffin tissue sections and often complement the results of flow cytometry. Furthermore, these additional markers may be crucial in confirming the diagnosis of CLL/SLL and separating it from the other indolent and/or disseminated lymphoid neoplasms. BCL-1 and BCL-6 are both expressed in the nucleus and thus the expression of these two gene products are easier to identify in paraffin tissue sections. Immunostaining for BCL-1 and BCL-6 is important because, although cyclin D1 (BCL-1) is expressed by mantle cell lymphomas and BCL-6 is expressed by follicular lymphomas, neither is expressed in CLL/SLL. Furthermore, although CLL/SLL tumor cells are usually CD43-positive, the percentage of cases positive for this antigen varies depending on the CD43 antibody clone used for immunostaining. For example, the Leu22 antibody (L60 clone; Becton-Dickinson, Mt. View, CA) detects more cases of CLL/SLL than the MT-1 antibody (B8, C16, D4, F11, K35, N13, S29, S30, Z3, Z4). Expression of CD38 and of the proliferation-associated antigen Ki-67/MIB-1 are important prognostic indicators in CLL/SLL. Typically only a small number of CLL/SLL cells are Ki-67-positive (D13); however, cases of chemotherapyresistant CLL/SLL have a higher percentage of Ki-67-positive cells in the peripheral blood (p < 0.05) (A16). Furthermore, expression of CD38 by greater than 30% of the malignant cells is associated with higher stage of disease, unresponsiveness to chemotherapy, and shorter survival (D6, D27, H3, H14, I1). Patients with p53 protein-positive CLL/SLL also have a poor prognosis with a shorter treatment-free interval from diagnosis, a poorer response to therapy, and decreased survival time (C27). A small percentage of CLL/SLLs will transform to high-grade lymphoma (Richter’s syndrome). Morphologically these cases usually either resemble diffuse large-cell lymphoma or Hodgkin lymphoma. In these latter cases the large, transformed cells express the immunophenotype of Reed–Sternberg cells and often show evidence of infection with the Epstein–Barr virus; in some of the diffuse large-cell lymphoma cases the immunophenotype of the tumor cells is identical to that of the original CLL/SLL (K30, O1, R14). Although many of these aggressive lymphomas have evolved from the low-grade CLL/SLL, molecular-genetic studies suggest that some may represent an unrelated tumor (G3, M14, O1). 4.2.2.2. Mantle Cell Lymphoma (MCL). Although MCL is considered an indolent lymphoma, the median survival is less than for the other indolent tumors. The disease is primarily nodal; however, the peripheral blood is involved in about 25% of cases. The benign counterpart is unknown (S31). Mantle cell lymphoma cells are generally small to medium-sized lymphoid cells with variably irregular nuclear contours and a scant amount of cytoplasm. Morphologically, MCL cells can be difficult to distinguish from those of CLL/SLL,

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follicular lymphoma, and marginal zone lymphoma. Immunophenotypically, MCLs are positive for the pan-B cell antigens CD19, CD20, CD22, and CD79a, express bright monoclonal surface immunoglobulin, usually sIgM or sIgM and sIgD, and are CD5-positive (Table 6; Fig. 6; see color insert). In contrast to CLL/SLL, MCL cells lack expression of CD23 (in most cases) and usually express FMC7, while in contrast to follicular lymphoma, MCL cells lack expression of CD10 and BCL-6 (F11, K8, K34, M18, S30, X1, Z3). The tumor cells are also usually positive for CD43 in paraffin tissue sections (S1, S29, S30, Z3). In addition, the tumor cells are often associated with loose, ill-defined CD21- or CD35-positive follicular dendritic cell meshworks (S30). In contrast to all other sIg-positive mature B cell neoplasms, MCL cells more often express the lambda immunoglobulin light-chain determinant in comparison to the kappa light chain (H11, K8). In comparison to most other B cell lymphomas, MCL is relatively unique based on overexpression the cell cycle protein cyclin D1, which can be identified immunophenotypically in the nuclei of the tumor cells in formalin-fixed paraffin tissue sections (B8, D3, K34, S30, S33, V7, Y4, Z4). The overexpression of the cyclin D1 protein is due to the characteristic genetic translocation found in MCL, t(11;14), which juxtaposes the BCL-1 (PRAD1, CYCLIN D1) gene on chromosome 11 next to the immunoglobulin heavy-chain gene on chromosome 14 (R12, W7, W8). The median survival of patients with MCL is 3–5 years. However, if the patients develop additional abnormalities with respect to other cell cycle proteins, such as p53, p16, and p18, including structural alterations, the median survival decreases to approximately 18 months. Immunodetection of p53, p21, and p16 (all of which can be identified in paraffin tissue sections) is associated with more aggressive disease and a poor prognosis (L4, P12). In addition, a high proliferation rate, which can be determined by immunostaining for Ki-67, is also associated with a poor prognosis (A13, B21, S32). 4.2.2.3. Follicular Lymphoma (FL). These tumors are indolent, primarily nodal, lymphomas and are composed of a mixture of centrocytes and centroblasts. These lymphomas are graded based on the number of centroblasts, which are the proliferating cells. Immunophenotypically, the follicular lymphoma cells are CD19-, CD20-, CD22-, and CD79a-positive B cells. The tumors cell usually express sIg (IgM with or without IgD, IgG, or IgA); a few, however, are sIg-negative. The tumors cells are usually CD10-positive and BCL-6-positive (Table 6; Fig. 7; see color insert). In contrast to CLL/SLL and MCL, FL tumor cells are usually CD5- and CD43negative; only rare cases of grade III follicular lymphoma (those lymphomas composed predominantly of centroblasts) are CD43-positive (H12, K4, L2, N8, R1). Follicular lymphoma cells, when forming nodules or follicles, are associated with CD21-positive follicular dendritic cell (FDC) meshworks (Fig. 7). These meshworks are often relatively tight and complete; fragmentation of the FDCs


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tends to occur first in the center of the meshworks leaving the edges relatively intact (A3, D5, S9). The tumor cells, in general, express the antiapoptotic protein BCL-2 (Fig. 7). This finding is in distinct contradiction to normal germinal center B cells, which are BCL-2-negative (Fig. 4), and thus the overexpression of BCL-2 by B cells forming follicles is diagnostic of follicular lymphoma. The abnormal expression of the BCL-2 protein is due to t(14;18), where the bcl-2 gene on chromosome 18 is translocated to chromosome 14. Therefore, bcl-2 gene transcription comes under the control of the immunoglobulin heavy-chain gene promoter resulting in overexpression of the antiapoptotic BCL-2 protein. This overexpression of the BCL-2 protein gives the tumor cells a survival advantage. The percentage of FL cases that are BCL-2-positive varies based on the cytologic grade, with greater than 90% of grade I, greater than 80% of grade II, but only about 75% of grade III FL being BCL-2-positive. However, in primary cutaneous FL, BCL-2 is often not overexpressed by the malignant cells (A3, B12, D5, H19, K24, L1, M25, N9, N12, N16, P5, V8, Y3). 4.2.2.4. Marginal Zone Lymphoma. Three indolent neoplastic B cell entities, nodal marginal zone lymphoma (MZL), extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), and splenic marginal zone lymphoma (SMZL)/splenic lymphoma with villous lymphocytes (SLVL), are assumed to be derived from a related post-germinal center B cell (H10, M5). The tumors are composed of morphologically variable small to medium-sized lymphocytes exhibiting centrocytic, monocytoid, immunoblastic, centroblastic, and/or plasmacytic differentiation. The circulating SLVL cells have relatively abundant cytoplasm and villous projections (I7–I9). Furthermore, although these subtypes, SMZL, MZL, and MALT lymphoma, tend to present in either spleen, lymph nodes, or extranodal sites, respectively, they can also occur in other sites (C4, D25). 4.2.2.4.1. Splenic marginal zone lymphoma (SMZL). These tumors involve both the white and the red pulp of the spleen. In the white pulp the neoplastic cells surround or replace the germinal centers, often with effacement of the mantle cell zone, and infiltrate the marginal zone. In the red pulp the tumor cells form nodules and sheets and infiltrate the sinuses. The neoplastic lymphocytes may circulate and involve the bone marrow (H5, I9). Immunophenotypically, the neoplastic cells express B cell-associated antigens such as CD20 and CD79a and monotypic surface immunoglobulin of the IgM and IgD isotypes (C4, I6, M17, M26, V5). The tumors, which exhibit plasmacytic differentiation, may also express monotypic cytoplasmic immunoglobulin. They also appear to be IRTA-1-positive (C7). The cells in general lack expresion of CD5, CD10, CD23, CD43, CD103, HC-2, and BCL-6 and are negative for cyclin D1, excluding the diagnoses of CLL/SLL, mantle cell lymphoma, follicular lymphoma, and hairy cell leukemia (Table 6) (C4, D17, I6, L2, M17, M15, M16). The tumor cells are associated with a low proliferation rate based on Ki-67 expression (I6).

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Although a small number of cases of SMZL progress to large-cell lymphoma, in contrast to CLL/SLL and MCL, this progression is not usually associated with p53 mutations or overexpresion. However, the cases that progressed were associated with a higher proliferation rate based on Ki-67 expression (C2). Patients with SLVLs that contain p53 mutations, some of which can be detected by immunostaining, do have, however, significantly worse survival (G9). 4.2.2.4.2. Nodal marginal zone lymphoma (MZL). These tumors may show a variable pattern of lymph node infiltration including parafollicular, perisinusal, nodular, and diffuse. Residual reactive follicles may show infiltration by the neoplastic cell population (S30). These lymphomas are immunophenotypically similar to extranodal marginal zone lymphomas of the MALT type. The neoplastic cells are monotypic B cells that express pan-B cell antigens such as CD20 and CD79a. In contrast to SMZL cells, MZL cells usually express heavy-chain immunoglobulin of the IgM, IgG, or IgA isotypes and are usually IgD-negative; however, some cases are reported to express IgD similar to SMZL (C4, I8). In cases with plasmacytic differentiation, tumor cells may exhibit monotypic cytoplasmic immunoglobulin expression. The neoplastic cells lack expression of CD5, CD10, CD23, and cyclin D1; some cases, however, are CD43-positive (Table 6). The cells are FMC7-positive and CD23-negative (A4, D4, D17, G1, K34, L2, X1, Y3). Although the immunophenotypic profile of the neoplastic cells is helpful in diagnosing MZL, there is no clearly defined marker specific for MZL cells. However, a recent study suggests that IRTA-1 may possibly be such a marker because it is commonly expressed by MZL lymphomas (including SMZL and MALT lymphomas) and rarely by other lymphoma entities (C7). Thus, the immunoarchitectural features of the specimen are often crucial in reaching the diagnosis of MZL. Immunostaining for CD21 and CD35, which identify FDCs, often shows extensive fragmentation of the edges of follicular meshworks. These FDC meshworks are “broken down” by the infiltrating neoplastic MZL cells. The neoplastic cells, which are usually BCL-2-positive, can be identified in sequential immunostained or double-immunostained sections in the areas of FDC fragmentation. The BCL-2-positive MZL cells infiltrate among the BCL-2-negative residual benign germinal center cells resulting in an increased number of BCL-2-positive cells in the follicle center. Furthermore, immunostaining for both CD10 and BCL-6 shows areas of negativity in the involved germinal centers; these areas of negativity are foci of MZL (F11, L1, X1). 4.2.2.4.3. Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT Lymphoma). These tumors often arise in the marginal zone of the MALT, but extend into the interfollicular area and, in epithelial tissues, extend into the epithelium, forming lymphoepithelial lesions (I4, I7, I10). Although frequently diagnosed in the stomach, MALT lymphomas occur in other sites such as the salivary glands and skin (B2, H21). Many MALT lymphomas appear to be associated with chronic inflammatory situations. For example, gastric MALT lymphomas


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are highly associated with Helicobacter pylori infection (E1, I4, I7, W12). Furthermore, patients with autoimmune diseases such as Sjogren’s syndrome and Hashimoto’s thyroiditis are also at an increased risk for MALT lymphoma (B27, I3, I5, I7, R13). Immunophenotypically, MALT lymphomas, similar to nodal MZL, are usually IgM-, IgA-, or IgG-positive; they do not express IgD (C4, I5, I7). The malignant cells are CD20-, CD79a-positive B cells that lack expression of CD10, CD23, BCL-6, and cyclin D1; greater than 80% of cases are also BCL-2-positive. Many cases are CD43-positive; the malignant cells may also express CD21 and CD35 (Table 6). MALT lymphomas, in general, lack expression of CD5; however, rare cases have been found to be CD5-positive (B2, C4, D17, F10, I4, I7, I10, L1, L2, T4). Most of the MALT lymphomas also express IRTA-1 (C7). Cases that exhibit plasmacytic differentiation may express monotypic cytoplasmic immunoglobulin (B2, H21, I4, I5, I7). Similar to nodal MZL, immunoarchitectural features are helpful in identifying MALT lymphoma. Infiltration of CD21 or CD35 follicular dendritic cell meshworks by CD10-negative, BCL-6-negative, BCL-2-positive MALT lymphoma cells and the presence of lymphoepithelial lesions as identified by immunostaining for cytokeratin and B cell markers (Fig. 8; see color insert) are immunoarchitectural features characteristic of MALT (I4, I7, I10, W11, W12). However, lymphoepithelial lesions can also be seen in reactive processes particularly in thryoid, stomach, lung, and salivary glands (I4). In gastric MALT, the identification of H. pylori, which can be facilitated by immunostaining for the organism, may direct treatment because some cases of gastric MALT lymphoma can be cured by antibiotic therapy (C17, W10). Recent studies have shown that MALT lymphomas are associated with t(11;18) (q21;q21) and t(1;14)(q22;q23) (D24, L11, L12, R9, Y9, A2, D23, L11, W13). The latter translocation is associated with overexpression of the BCL-10 gene product in the nucleus. Although BCL-10 is expressed in gastric MALT lymphomas without the t(1;14), the presence of the t(11;18) and expression of BCL-10 is associated with higher stage disease (D23, L11). Furthermore, MALT lymphomas confined to the stomach (stage IE) with t(11;18) do not respond to H. pylori eradication (D24, L10, Y7). 4.2.2.5. Hairy Cell Leukemia (HCL). This B cell malignancy primarily involves the red pulp of the spleen (not white pulp), peripheral blood, and bone marrow and is composed of medium-sized, bland-appearing lymphocytes with relatively abundant cytoplasm including “hairy” cytoplasmic projections. Immunophenotypically, HCL cells are CD19-, CD20-, CD22-, CD79a-positive B cells, which express relatively bright monotypic surface immunoglobulin (IgM, IgMD, IgG, or IgA). They are CD5-, CD10-, and CD23-negative, but brightly express CD11c, CD25, CD103, HC-2, and FMC7 (A4, D15, G1, M15–M17). In paraffin tissue sections, immunostaining for tartrate-resistant acid phosphatase (TRAP), DBA.44, and CD25 in addition to a B cell marker is helpful in identifying

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the tumor cells particularly in treated cases as well as in cases where no bone marrow aspirate is available (Table 6; Fig. 9; see color insert) (H20, S3, Y6). 4.2.2.6. Burkitt’s Lymphoma (BL). The classic cases of this highly aggressive lymphoma are composed of relatively monotonous medium-sized cells. At low power the tumor has a “starry-sky” appearance due to the large number of tingible body macrophages (macrophages containing debris); numerous mitotic figures are present. There are three clinical forms of the disease: endemic (which occurs in equatorial Africa and New Guinea), sporadic (which occurs worldwide) and immunodeficiency-associated (occurs primarily in HIV-positive patients). Virtually all the endemic, approximately 25–40% of the immunodeficiency-associated, and a variable number of the sporadic cases are positive for the Epstein–Barr virus (EBV) (H4, K17, M21, R5, S20). All cases have a translocation involving the c-MYC gene on chromosome 8 (G10, M21, N7, P3, S14, S18, Y5). The BL tumor cells are positive for CD19, CD20, CD22, CD79a, CD10, and BCL-6 and express monotypic light-chain surface immunoglobulin of the IgM heavy-chain isotype. The cells lack expression of CD5 and CD23; in addition, they are BCL-2-negative. Many of the cases are CD43-positive (C10). The majority of the endemic cases are CD21-positive (Table 6). CD21 detects the C3d receptor, which is also the receptor for EBV. Nearly 100% of the cells are Ki-67-positive, indicative of their high proliferation rate (B23, D17, H11, H24, L1, M6, M21, S20). 4.2.2.7. Diffuse Large-Cell Lymphoma (DLCL). These clinically aggressive tumors may present in lymph nodes or in extranodal sites (H11). They can occur in patients of all ages (A1, A14). Most cases arise de novo; however, some may represent transformation from a low-grade lymphoma. Immunophenotypically, DLCLs usually express pan-B cell antigens (Table 6). However, some cases lack expression of one or more of these antigens (C10, K5, S10). The cells may express sIg and/or cytoplasmic immunoglobulin; the latter is usually seen in cases that morphologically show plasmacytic differentiation (D18, S25). Some cases express CD30 (P13). Approximately one third of DLCLs are CD10-positive (range 25–60%) (D17, K9, O3, U1, X1), and approximately 80% are BCL-6-positive (D17, H13, K9, O4, P14, S16). Approximately 55% (45–80%) of cases express BCL-2 (K9, K25, M27). The DLCLs only rarely express CD138 (K9). IRF4/MUM1, however, is frequently expressed in DLCL (F3). Approximately one third of cases express CD43, including some cases that lack expression of all pan-B cell antigens (C10, P6, K5, N10, S10). In these “CD43only” cases it is important to do additional studies, including immunoglobulin and T cell-receptor-chain gene rearrangement studies, to determine cell lineage because this antigen can be expressed by myeloid tumors as well as by T and B cell neoplasms (K5, S10). In addition, some B cell DLCLs will express the T cell-associated antigen CD45RO. This, however, occurs only rarely and usually in cases of HIV-related lymphoma (G4).


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Occasional DLCLs are CD5-positive. These cases may be either de novo CD5positive large-cell lymphomas or represent cases that have transformed from either CLL/SLL or MCL; the transformed MCL cases are BCL-l-immunopositive, whereas many of the transformed CLL/SLL cases are CD23-positive (B14, D26, K30, M13, Y1). CD5-positive de novo DLCL is a more aggressive disease process and is associated with decreased survival (H8, Y1). Cases that overexpress BCL-2 are also associated with a worse prognosis including lower remission rates and shorter survival times (B5, K25, K26, X1). However, in general DLCLs that are germinal-center-cell-like (such as those tumors that express CD10 and/or BCL-6) have a better prognosis (A8, B5). 4.2.2.8. Plasma Cell Myeloma (PCM). There are several plasma cell neoplasms: plasma cell myeloma (including the variants nonsecretory myeloma, smoldering myeloma, indolent myeloma, and plasma cell leukemia), plasmacytoma (solitary and extrameduallary), immunoglobulin deposition diseases (primary amyloidosis, systemic light/heavy-chain deposition disease), osteosclerotic myeloma (POEMS syndrome), and heavy-chain disease (gamma, mu, and alpha); in addition there are precursor lesions (monoclonal gammopathy of undetermined significance, MGUS). The diagnosis of plasma cell myeloma, the most common plasma cell neoplasm, is based on clinical, radiologic, and pathologic findings. Plasma cell myeloma is a multifocal neoplasm, which primarily involves the bone marrow and is characterized by lytic bone lesions, pathologic fractures, bone pain, hypercalcemia, and monoclonal protein in the serum (G7, G8). The plasma cells in plasma cell myeloma lack expression of sIg, but express monotypic cIg (K31). The tumor cells usually express IgG or, less commonly, IgA. Expression of the other heavy-chain immunoglobulins is rare. In about 15% of cases only immunoglobulin light chains are produced (Bence–Jones myeloma). Most myeloma plasma cells lack expression of CD20 and CD22, but express CD79a. Although normal plasma cells are CD19-positive and express PAX-5, myeloma plasma cells are CD19-negative and lack expression of PAX-5. Because the PAX-5 gene is a regulator of CD19 transcription, the lack of PAX-5 expression may be responsible for the lack of CD19 expression by the myeloma cells (H7, L8, M7). In addition, myeloma plasma cells express CD38 and CD138 and may express CD56 (Table 6). CD56, also known as neural cell adhesion molecule (NCAM), is not expressed by normal plasma cells and can be used to separate myeloma plasma cells (which can be positive) from those in MGUS (E2, H7, L8, L9, V1). Furthermore, the expression of CD56 correlates with the presence of lytic bone lesions in myeloma patients (E2). Myeloma plasma cells, like normal plasma cells, often express antigens normally expressed by nonhematopoeitic cells such as epithelial membrane antigen (EMA). They may also be CD30-positive; however, the pattern of CD30 expression is homogeneous within the cytoplasm compared to the membrane-Golgi positivity of Reed–Sternberg cells in Hodgkin lymphoma (B15, P7, S8). Multiple genetic

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abnormalities have been identified in myeloma including structural alterations in chromosomes 1, 9 (thought to be related to lack of PAX-5 expression), 11, 14, and 17. Structural abnormalities in the p53 gene on chromosome 17 have been identified in up to 25% of cases and are associated with a poorer prognosis. The t(11;14) results in overexpression of cyclin D1, which is also associated with a poor prognosis. The t(4;14) results in overexpression of fibroblast growth factor receptor-3 (FGF-R3), which has been found to contribute to disease progression (C18, C19, D14, D22, K23, M7, S4, V6). 4.3. T/NK CELL NEOPLASMS T/NK (natural killer) cell lymphomas are much less common in Western countries and much less understood than the B cell lymphomas. Thus, the discussion of these entities will be much briefer. In addition, a significant number of the T/NK lymphomas, such as enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, subcutaneous panniculitis-like T cell lymphoma, blastic NK cell lymphoma, T cell granular lymphocytic leukemia, and adult T cell leukemia/ lymphoma, will not be discussed in this review. The reader is advised to consult the recent World Health Organization Classification of Tumours (J2). 4.3.1. Precursor T Cell Lymphoblastic Leukemia/ Lymphoma (T-ALL/LBL) These tumors are composed of lymphoblasts of T cell origin. The disease occurs most frequently in teenage males, who present with a mediastinal mass with or without peripheral blood and bone marrow involvement (K18). The T cell lymphoblasts are TdT-positive and most frequently exhibit expression of the pan-T cell antigen CD7. The tumor cells exhibit variable expression of other antigens normally expressed by normal thymocytes including CDl, CD2, CD5, CD4, and CD8; some cases are both CD4- and CD8-positive. Most T-ALL/ LBLs lack expression of sCD3, however, the cells exhibit cytoplamic expression of this pan-T cell antigen (M24, R6, W2). Some cases may be CD10positive and/or CD79a-positive (P8, W2). The tumor cells may also express the myeloid-associated antigens CD13 and CD33 (Q1, Y8). Some cases, particularly the CD4-positive/CD8-positive T-ALL/ LBLs, are also BCL-6-positive. Many also abnormally express the antiapoptotic proteins BCL-2 and BCL-X(L). These findings suggest that these genes and gene products may contribute to the pathogenesis of this disease (H25). 4.3.2. Mature T/NK Cell Lymphomas These tumors include all T cell neoplasms that are derived from postthymic T cells and, because of immunophenotypic and apparent biologic similarities, include the natural killer (NK) cell lymphomas.


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4.3.2.1. Mycosis fungoides (MF). This mature T cell lymphoma presents in the skin and is composed of small and medium-sized lymphocytes with cerebriform nuclei, which infiltrate the epidermis and dermis. Sezary syndrome is an aggressive variant of MF characterized cliniopathologically by erythroderma, lymphadenopathy, and circulating malignant cells (W9). The malignant cells in MF are usually positive for CD2, CD3, βf1 (detecting the β chain of the αβ portion of the T cell-receptor complex), CD5, and CD4; most cases lack expression of CD7 (P6, W9). However, because some benign T cell infiltrates in the skin often show decreased CD7 expression, this may or may not be helpful (M29, R3). Some cases may be CD8-positive. Some patients may develop/transform to tumor stage MF/large-cell lymphoma. In these instances the tumor cells often express activation-associated antigens such as CD25, CD30, and CD71; some cases may also gain expression of CD15 (C9, H9, W5). In addition, the tumor cells may exhibit a different T cell-antigen phenotype usually characterized by loss of additional pan-T cell antigens (W9). 4.3.2.2. Cutaneous Anaplastic Large-Cell Lymphoma (Cutaneous ALCL). These lesions occur primarily in adults as localized skin lesions. In some instances the lesions spontaneously regress (B9, B10, K3, K10). These lesions should not be confused with systemic anaplastic large-cell lymphomas because these primary cutaneous lesions are associated with much less aggressive clinical course and a 90% 5-year survival (B7, K3, P2). In addition, CD30-positive compared to CD30negative primary cutaneous large-cell lymphomas have a better prognosis, although some of these CD30-positive cases may not be true primary cutaneous ALCLs (B9, B10). Immunophenotypically, cutaneous ALCL cells are usually CD30-positive and express pan-T cell antigens, most frequently CD2, and are usually CD4-positive. In addition, they usually express CD43 (B9, B10, K3, W6). In contrast to systemic ALCL, cutaneous ALCL cells less commonly express EMA (C15, K10); cutaneous ALCLs often are positive for cytotoxic cell-associated antigens such as granzyme B, perforin, and TIA-1 (B22, K32). Again in contrast to the systemic ALCL cases, cutaneous ALCL cases are negative for the ALK-1 protein (Table 7) (B16). 4.3.2.3. Anaplastic Large-Cell Lymphoma (ALCL). These tumors are composed of CD30-positive pleomorphic cells. There are several variants: common (accounting for about 70% of cases), lymphohistiocytic (10%), small cell (

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