Inflammatory Pancreatic Diseases, An Update: Digestive Diseases 2004

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Inflammatory Pancreatic Diseases: An Update

Editor

Joachim Mössner, Leipzig

14 figures, 2 in color and 7 tables, 2004

Basel • Freiburg • Paris • London • New York • Bangalore • Bangkok • Singapore • Tokyo • Sydney

S. Karger Medical and Scientific Publishers Basel • Freiburg • Paris • London New York • Bangalore • Bangkok Singapore • Tokyo • Sydney

Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Drug Dosage The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.

All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center (see ‘General Information’). © Copyright 2004 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISBN 3–8055–7900–4

Vol. 22, No. 3, 2004

Contents

233 Editorial Mössner, J. (Leipzig)

Review Articles 235 Molecular Analysis of Pancreatic Juice: A Helpful Tool to Differentiate

Benign and Malignant Pancreatic Tumors? Teich, N.; Mössner, J. (Leipzig) 239 The Stress Response of the Exocrine Pancreas Savković, V.; Gaiser, S. (Leipzig); Iovanna, J.L. (Marseille); Bödeker, H. (Leipzig) 247 Laboratory Markers of Severe Acute Pancreatitis Rau, B.; Schilling, M.K. (Homburg/Saar); Beger, H.G. (Ulm) 258 Tropical Pancreatitis Tandon, R.K.; Garg, P.K. (New Delhi) 267 Pathogenesis of Pain in Chronic Pancreatitis Di Sebastiano, P.; di Mola, F.F.; Büchler, M.W.; Friess, H. (Heidelberg) 273 Mechanisms of Pancreatic Fibrosis Apte, M.V.; Wilson, J.S. (Sydney) 280 Endoscopic Therapy of Chronic Pancreatitis Mönkemüller, K.; Kahl, S.; Malfertheiner, P. (Magdeburg)

Original Paper 292 Chronic Parotitis: Not Another SPINKosis Gundling, F. (Leipzig/München); Reitmeier, F. (Hamburg); Tannapfel, A.; Schütz, A.; Weber, A. (Leipzig); Ussmüller, J. (Hamburg); Keim, V.; Mössner, J.; Teich, N. (Leipzig)

296 Author Index/Subject Index

© 2004 S. Karger AG, Basel Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Access to full text and tables of contents, including tentative ones for forthcoming issues: www.karger.com/ddi_issues

Dig Dis 2004;22:233–234 DOI: 10.1159/000082793

Editorial

This issue of Digestive Diseases is dedicated to some new and fascinating insights, especially in the pathogenesis of both acute and chronic pancreatitis. Bettina Rau, Schilling and Beger from Homburg/Saar and Ulm, Germany, present a literature review and their own data on the search for laboratory markers of acute pancreatitis with special regard to their clinical usefulness and test performance for stratifying severity and monitoring disease progression. Several parameters such as trypsinogen and procarboxypeptidase B activation peptide, polymorphonuclear lymphocyte-elastase, interleukin-6 and 8 (IL-8), serum amyloid A, and procalcitonin are obviously able to differentiate between mild and severe acute pancreatitis. Procalcitonin is a marker for predicting severe pancreatic infections. However, C-reactive protein still remains the standard as a fast and reliable marker of severity. Niels Teich and myself from Leipzig, Germany, discuss the still difficult differential diagnosis between pancreatic cancer and chronic pancreatitis, especially the early diagnosis of pancreatic cancer in preexisting chronic pancreatitis. The detection of specific tumor markers in pancreatic juice may be an attractive diagnostic tool, such as k-ras mutations, telomerase reactivation, or promoter methylation of the tumor suppressor genes, p16INK4a and p14ARF. The high specificity of molecular alterations in pancreatic cancer in some pilot studies is waiting for reproduction in large prospective trials, but has the potential to be a strong complementary marker of malignancy in patients with a pancreatic mass of uncertain origin.

© 2004 S. Karger AG, Basel 0257–2753/04/0223–0233$21.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/ddi

Vuk Savkovic´ , Gaiser, Iovanna and Bödeker from Leipzig, Germany, and Marseille, France, describe the acute-phase reactions during early pancreatic cellular injury. The exocrine pancreas reacts with well-coordinated changes in gene expression in order to prevent further progression of the disease. Rakesh Tandon and Pramod Garg from New Delhi, India, present their experience with tropical pancreatitis. This type of pancreatitis is characterized by pancreatic calcification and ductal dilatation in young malnourished patients who present with abdominal pain and/or diabetes. In about 50% of these patients mutations of an important inhibitor of trypsin, SPINK, are found. Pierluigi Di Sebastiano, Mola, Büchler and Friess from Heidelberg, Germany, describe our present knowledge on the pathogenesis of pain in chronic pancreatitis. Increased intraductal pressure as a result of single or multiple strictures and/or calculi is believed to be an important common cause of pain. Further causes include pancreatic fibrosis, interstitial hypertension and pancreatic ischemia. Additionally, extrapancreatic causes such as duodenal and common bile duct stenosis may lead to pain. The neurogenic inflammation hypothesis is supported by immunohistological reports. Neurotransmitters, such as substance P and its receptor, calcitonin generelated peptide and further neurotransmitters are increased in afferent pancreatic nerves. Minoti Apte and Jeremy Wilson from Sidney, Australia, contribute important experiments regarding the pathomechanisms of pancreatic fibrogenesis. Pancreatic fibrosis is an active process that may be reversible in the early stages. The identification and characterization of

pancreatic stellate cells indicate a key role for these cells in the fibrotic process. These cells can be activated by ethanol and its metabolites and by several factors that are upregulated during pancreatic injury including growth factors, cytokines and oxidant stress. Potential anti-fibrotic strategies such as antioxidants and cytokine inhibition have been assessed in experimental models only, but may gain therapeutic significance in human chronic pancreatitis. Klaus Mönkemüller, Kahl and Malfertheiner from Magdeburg, Germany, report on their experience with

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endoscopic therapies in chronic pancreatitis. In many cases endoscopy offers a definite therapy for pancreatic pseudocysts, pancreatic ascites and duct disruption. Endoscopic therapy is also useful in the short-term therapy of common bile duct strictures. However, a controversial discussion on whether the patient’s leading symptoms, namely pain, will be resolved in long-term is needed. As guest editor of this issue I would like to thank the authors for their contributions and I would be pleased if our readers find the selected articles interesting and rewarding. Joachim Mössner

Editorial

Review Article Dig Dis 2004;22:235–238 DOI: 10.1159/000082794

Molecular Analysis of Pancreatic Juice: A Helpful Tool to Differentiate Benign and Malignant Pancreatic Tumors? Niels Teich Joachim Mössner Medizinische Klinik und Poliklinik II, Universität Leipzig, Leipzig, Deutschland

Key Words Pancreatic cancer Chronic pancreatitis Pancreatic juice Molecular analysis

Abstract Chronic pancreatitis is an important predisposing condition leading to pancreatic carcinoma. As the differential diagnosis between these diseases may be difficult in 1 patient, the detection of specific tumor markers in pancreatic juice is an attractive diagnostic tool. Many studies have investigated tumor-mediated molecular alterations of the pancreatic juice, as k-ras mutations, telomerase reactivation, or promoter methylation of the tumor-suppressor genes p16INK4a and p14ARF. In this overview, we summarize these studies and conclude that molecular analysis of pancreatic juice is not useful for everyday care today. The high specificity of molecular alterations in pancreatic cancer in some pilot studies is waiting to be reproduced in large prospective trials, and has the potential to be a strong complementary marker of malignancy in patients with a pancreatic mass of uncertain dignity.

Introduction

Chronic pancreatitis is an important predisposing condition leading to pancreatic carcinoma [1]. However, the differential diagnosis between chronic pancreatitis and pancreatic carcinoma may be difficult in 1 patient. The accelerated development of high-fidelity ultrasound and magnet resonance tomographs enables the detection of very much smaller pancreatic tumors than in the past. While the sensitivity of the detection of small pancreatic tumors is enhanced, their specificity is not sufficient to differentiate the diagnosis of small pancreatic tumors [2, 3]. Direct tissue diagnosis is invasive and sometimes difficult to obtain. In this light, the detection of specific tumor markers in pancreatic juice, pancreatic brushings or duodenal aspirates could be attractive [4, 5]. Most clinical trials have been undertaken to investigate molecular markers of pancreatic cancer in pancreatic juice obtained by endoscopic retrograde cholangiopancreaticography (ERCP). Here we review important and innovative studies.

Copyright © 2004 S. Karger AG, Basel



Niels Teich, MD Universität Leipzig, Medizinische Klinik und Poliklinik II Philipp-Rosenthal-Strasse 27 DE–04103 Leipzig (Germany) Tel. +49 341 9712200, Fax +49 341 9712209, E-Mail [email protected]

The K-Ras Story

The k-ras gene is the locus for the c-k-ras proto-oncogene, lying on chromosome 12p12, and is about 45,000 bp in length. It encodes a 2.0-kb transcript which is highly conserved across species, and is translated into the p21ras protein. These proteins are located in the plasma membrane and could transduce growth and differentiation signals from activated receptors to protein kinases within the cell [6]. The wild-type K-ras gene encodes glycine (GGT) at codon 12, and the most common amino acid substitution is aspartic acid for glycine (46%), followed by valine (32%), arginine (13%), cysteine (5%), serine (1–2%), and alanine (!1%). More than 90% of pancreatic adenocarcinoma tissue sections harbor mutations in codon 12 of the k-ras gene [7]. The prevalence of this mutation in materials obtained by endoscopy ranges between 44 and 100% [8]. The detection rates differ between the materials obtained such as bile, pancreatic juice, secretin-stimulated pancreatic juice, pancreatic brushings or duodenal aspirates [9]. Initial enthusiasm deteriorated after the detection of k-ras mutations in a significant number of patients with chronic pancreatitis. In clinical routine this is the leading differential diagnosis to pancreatic cancer in a patient with a pancreatic tumor of unknown origin. As a consequence, the finding of a k-ras mutation is of limited specificity for pancreatic cancer. However, some groups have suggested a diagnostic advantage of this marker [8, 10], and several authors have suggested that the presence of a k-ras mutation in patients with chronic pancreatitis may be associated with a higher risk of malignant transformation [4, 11]. Today, little evidence supports this assumption. Queneau et al. [12] reported 2 of 10 patients with chronic pancreatitis in whom pancreatic carcinoma was discovered at an invasive stage at 7 and 17 months after detection of a K-ras mutation, versus none in 22 patients without the mutation (p ! 0.02). In contrast, two long-term follow-up studies refute these data. Furaya et al. [13] followed up 20 k-ras-positive patients with chronic pancreatitis over a mean period of 78 months, but no patient got pancreatic cancer. In accordance with this finding, Löhr et al. [14] reported that a K-ras gene mutation was found in 6 of 66 patients with chronic pancreatitis, but pancreatic neoplasm occurred in none of the mutation carriers over a mean follow-up period of 26 (4–54) months. Recently, using biopsy van Heek et al. [15] followed up 6 patients with a k-ras mutation over a mean period of 5 years and 5 months, but nobody got cancer.

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The prevalence of k-ras mutations in the pancreatic juice of patients with chronic pancreatitis is highly dependent on the detection method used. As expected, it rises with more sensitive and robust molecular biology techniques. In a previous analysis of different mutation-detecting methodologies, it was found that 8% of chronic pancreatitis patients before (n = 242) and 17% since 1997 (n = 532) were analyzed as carrying a k-ras mutation in pancreatic juice or tissue or duodenal fluid [16]. In a prospective evaluation of 358 consecutive patients who underwent ERCP, a 90% specificity and only 38% sensitivity were found for the detection of pancreatic cancer. This study, which closely resembles everyday care, shows that the search for a k-ras mutation is not appropriate to confirm or screen for pancreatic cancer [17]. In conclusion, the presence of a k-ras mutation is not specific enough to recommend its use in the clinical diagnosis of pancreatic cancer. Despite prevailing negative long-term studies with few patients, chronic pancreatitis patients with the k-ras mutation may be at an increased risk of developing pancreatic cancer than those patients without the mutation. Today, there is no clear consensus on the management and follow-up of these patients.

Telomerase Mutations

Telomerase is physiologically inactive in almost all somatic cells. Its activation in the course of tumor development stabilizes the telomeres and contributes to cell immortalization and subsequent proliferation [18]. Some studies investigated the presence and activity of telomerase in the pancreatic juice of patients with pancreatic cancer and chronic pancreatitis. The high specificity and sensitivity (91 and 84%, respectively) are hampered by the very low number of patients in all these studies (table 1). For the diagnosis of pancreatic cancer, telomerase activity in pancreatic juice may possibly be complementary to the K-ras mutation because it may decrease the rate of false-positive diagnosis [19]. Although it is interesting, there is no rationale in searching for telomerase presence or activity in pancreatic juice in clinical routine today. Promoter Methylation of p16INK4a and p14ARF Methylation of the promoters of tumor-suppressor genes, p16INK4a and p14ARF, will inactivate their tumor-suppressive function [23]. To investigate its diagnostic value in patients with chronic pancreatitis and pan-

Teich/Mössner

Table 1. Studies investigating telomerase activity and presence in chronic pancreatitis and pancreatic cancer [19–22] Method Suehara, 1997 Uehara, 1999 Myung, 2000 Seki, 2001

activity activity activity rtPCR

PaCa 9/12 8/10 11/12 15/17

cP 0/10 0/12 2/11 2/12

creatic cancer, Klump et al. [24] analyzed the pancreatic juice of 14 and 37 patients with these conditions, respectively. Despite its sensitivity for pancreatic cancer of only 43%, the pancreatic juice of cancer patients exclusively contained methylated promoters of the investigated tumor-suppressor genes [24]. Although this pilot study was restricted to a limited number of patients, the 100% specificity of this marker clearly outranges all radiological or laboratory markers of pancreatic cancer, and even cytology. Novel Targets for Aberrant Methylation To identify potential targets for aberrant methylation in pancreatic cancer, Sato et al. [25] analyzed global changes in the gene expression profiles of 4 pancreatic cancer cell lines after treatment with a demethylating agent and/or a histone deacetylase inhibitor. A substantial number of genes were induced 5-fold or greater. Within their comprehensive work, the methylation status of 3 genes (NPTX2, SARP2, and CLDN5) was examined in a large panel of specimens, and aberrant methylation of at least 1 of these 3 genes was detectable in 100% of the 43 primary pancreatic cancers and in 18 of 24 (75%) pancreatic juice samples obtained from patients with pancreatic cancer. Thus, a substantial number of genes are induced by 5Aza-dC treatment of pancreatic cancer cells, and many of them may represent novel targets for aberrant methylation in pancreatic carcinoma.

samples, at least one of these mutations could be detected [26]. Despite its low sensitivity and no specificity data, these experiments highlight cancer-specific DNA chips as a powerful technology. Whether it will be used in clinical routine is largely dependent on future multicenter studies with large patient cohorts and appropriate controls [26].

Future Perspectives

As radiologists and ultrasound experts are enabled to detect even tiny pancreatic tumors by enhanced technologies, it is the gastroenterologist’s difficult task to manage these patients. Today molecular technologies are of little value in everyday practice. However, tumor-derived molecular defects can be investigated in pancreatic juice and, as shown by the example of p16INK4a and p14ARF promoter methylation, may reach a 100% specificity [23]. Specificity seems to be the strength of molecular techniques, and is most pronounced in comparison with radiological techniques, the strength of which is high sensitivity. Its rational combination seems to be of value for the diagnosis and therapeutic advice of patients with a pancreatic mass of unknown origin. An unambiguous necessity is the evaluation of promising markers in prospective (multicenter) trials with large numbers of patients.

Somatic Mitochondrial Mutations Maitra et al. [26] suggested that somatic mitochondrial mutations are common in human cancers, and could be used as a tool for early detection of cancer. After arraying both strands of the entire human mitochondrial coding sequence on a chip, matched fluid samples (urine and pancreatic juice) obtained from 5 patients with bladder cancer and 4 with pancreatic cancer were investigated for cancer-associated mitochondrial mutations. In 6 of 9

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References 1 Lowenfels AB, Maisonneuve P, Cavallini G, Ammann RW, Lankisch PG, Andersen JR, Dimagno EP, Andren-Sandberg A, Domellof L: Pancreatitis and the risk of pancreatic cancer. International Pancreatitis Study Group. N Engl J Med 1993;328:1433–1437. 2 Kalra MK, Maher MM, Sahani DV, Digmurthy S, Saini S: Current status of imaging in pancreatic diseases. J Comput Assist Tomogr 2002;26:661–675. 3 Lopez Hanninen E, Amthauer H, Hosten N, Ricke J, Bohmig M, Langrehr J, Hintze R, Neuhaus P, Wiedenmann B, Rosewicz S, Felix R: Prospective evaluation of pancreatic tumors: accuracy of MR imaging with MR cholangiopancreatography and MR angiography. Radiology 2002;224:34–41. 4 Van Laethem JL, Vertongen P, Deviere J, Van Rampelbergh J, Rickaert F, Cremer M, Robberecht P: Detection of c-Ki-ras gene codon 12 mutations from pancreatic duct brushings in the diagnosis of pancreatic tumours. Gut 1995; 36:781–787. 5 Nakamura Y, Onda M, Uchida E: Analysis of K-ras codon 12 point mutations using duodenal lavage fluid for diagnosis of pancreatic carcinoma. Pancreas 1999;18(2):133–140. 6 Barbacid M: Ras genes. Annu Rev Biochem 1987;56:779–827. 7 Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M: Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 1988;53:549–554. 8 Boadas J, Mora J, Urgell E, Puig P, Roca M, Cusso X, Capella G, Lluis F, Farre A: Clinical usefulness of K-ras gene mutation detection and cytology in pancreatic juice in the diagnosis and screening of pancreatic cancer. Eur J Gastroenterol Hepatol 2001;13:1153–1159. 9 O’Mahony S, Sreedharan A: Does detection of K-ras mutations in pancreatic juice influence clinical decision making? Eur J Gastroenterol Hepatol 2001;13:1141–1142. 10 Lu X, Xu T, Qian J, Wen X, Wu D: Detecting K-ras and p53 gene mutation from stool and pancreatic juice for diagnosis of early pancreatic cancer. Chin Med J 2002; 115: 1632– 1636.

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11 Iguchi H, Sugano K, Fukayama N, Ohkura H, Sadamoto K, Ohkoshi K, Seo Y, Tomoda H, Funakoshi A, Wakasugi H: Analysis of Ki-ras codon 12 mutations in the duodenal juice of patients with pancreatic cancer. Gastroenterology 1996;110(1):221–226. 12 Queneau PE, Adessi GL, Thibault P, Cleau D, Heyd B, Mantion G, Carayon P: Early detection of pancreatic cancer in patients with chronic pancreatitis: Diagnostic utility of a Kras point mutation in the pancreatic juice. Am J Gastroenterol 2001;96:700–704. 13 Furuya N, Kawa S, Akamatsu T, Furihata K: Long-term follow-up of patients with chronic pancreatitis and K-ras gene mutation detected in pancreatic juice. Gastroenterology 1997; 113:593–598. 14 Löhr M, Muller P, Mora J, Brinkmann B, Ostwald C, Farre A, Lluis F, Adam U, Stubbe J, Plath F, Nizze H, Hopt UT, Barten M, Capella G, Liebe S: P53 and K-ras mutations in pancreatic juice samples from patients with chronic pancreatitis. Gastrointest Endosc 2001;53:734–743. 15 van Heek NT, Rauws EA, Caspers E, Drillenburg P, Gouma DJ, Offerhaus GJ: Long-term follow-up of patients with a clinically benign extrahepatic biliary stenosis and K-ras mutation in endobiliary brush cytology. Gastrointest Endosc 2002;55(7):883–888. 16 Löhr M, Maisonneuve P, Lowenfels AB: K-Ras mutations and benign pancreatic disease. Int J Pancreatol 2000;27(2):93–103. 17 Trumper L, Menges M, Daus H, Kohler D, Reinhard JO, Sackmann M, Moser C, Sek A, Jacobs G, Zeitz M, Pfreundschuh M: Low sensitivity of the ki-ras polymerase chain reaction for diagnosing pancreatic cancer from pancreatic juice and bile: A multicenter prospective trial. J Clin Oncol 2002;20:4331–4337. 18 Satyanarayana A, Manns MP, Rudolph KL: Telomeres, telomerase and cancer: an endless search to target the ends. Cell Cycle 2004; 3(9):1138–1150.

19 Myung SJ, Kim MH, Kim YS, Kim HJ, Park ET, Yoo KS, Lim BC, Wan Seo D, Lee SK, Min YI, Kim JY: Telomerase activity in pure pancreatic juice for the diagnosis of pancreatic cancer may be complementary to K-ras mutation. Gastrointest Endosc 2000;51:708–713. 20 Uehara H, Nakaizumi A, Baba M, Iishi H, Tatsuta M, Kitamura T, Ohigashi H, Ishikawa O, Takenaka A, Ishiguro S: Diagnosis of pancreatic cancer by K-ras point mutation and cytology of pancreatic juice. Am J Gastroenterol 1996;91:1616–1621. 21 Suehara N, Mizumoto K, Tanaka M, Niiyama H, Yokohata K, Tominaga Y, Shimura H, Muta T, Hamasaki N: Telomerase activity in pancreatic juice differentiates ductal carcinoma from adenoma and pancreatitis. Clin Cancer Res 1997;3(12 Pt 1):2479–2483. 22 Seki K, Suda T, Aoyagi Y, Sugawara S, Natsui M, Motoyama H, Shirai Y, Sekine T, Kawai H, Mita Y, Waguri N, Kuroiwa T, Igarashi M, Asakura H: Diagnosis of pancreatic adenocarcinoma by detection of human telomerase reverse transcriptase messenger RNA in pancreatic juice with sample qualification. Clin Cancer Res 2001;7(7):1976–1981. 23 Herman JG, Baylin SB: Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003;349(21):2042– 2054. 24 Klump B, Hsieh CJ, Nehls O, Dette S, Holzmann K, Kiesslich R, Jung M, Sinn U, Ortner M, Porschen R, Gregor M: Methylation status of p14ARF and p16INK4a as detected in pancreatic secretions. Br J Cancer 2003;88:217– 222. 25 Sato N, Fukushima N, Maitra A, Matsubayashi H, Yeo CJ, Cameron JL, Hruban RH, Goggins M: Discovery of novel targets for aberrant methylation in pancreatic carcinoma using high-throughput microarrays. Cancer Res 2003;63:3735–3742. 26 Maitra A, Cohen Y, Gillespie SE, Mambo E, Fukushima N, Hoque MO, Shah N, Goggins M, Califano J, Sidransky D, Chakravarti A: The Human MitoChip: A high-throughput sequencing microarray for mitochondrial mutation detection. Genome Res 2004; 14: 812– 819.

Teich/Mössner


The Stress Response of the Exocrine Pancreas Vuk Savkovicá Sebastian Gaisera Juan L. Iovannab Hans Bödekera a b

Medizinische Klinik und Poliklinik II, Universitätsklinikum Leipzig AöR, Leipzig, Germany; Centre de Recherche INSERM, Stress Cellulaire UMR 624, Marseille, France

Key Words Acute pancreatitis Stress response Exocrine pancreas Gene expression

Abstract Most attacks of acute pancreatitis display a self-limiting course. This suggests that pancreatic acinar cells may be able to protect themselves against cellular injury thus preventing further progression of the disease. In this review we describe several genes overexpressed in acute experimental pancreatitis which take part in the pancreatic stress response. We discuss the possible function of the pancreatitis-associated protein 1, the small nuclear protein p8, the glycoprotein clusterin, different heat shock proteins, the p53-dependent stress proteins TP53INP1 and TP53INP1, the vacuole membrane protein-1, as well as the interferon-inducible protein-15, the antiproliferative p53-dependent protein PC3/TIS21/BTG2, and the pancreatitis-induced protein-49. The implications of these proteins in pathophysiological processes like apoptosis regulation, regeneration, cell cycle and growth control, regulation of inflammation, and vacuole formation are discussed. Study of the function of stress proteins expressed in response to pancreatitis could widen our understanding of the pathophysiology of the disease and enable us to develop new rational therapeutic strategies.

Introduction

Most attacks of acute pancreatitis lead to a self-limiting disease suggesting that pancreatic cells are able to react against cellular injury in order to prevent further progression of the disease. This emergency programme, known as the pancreatic stress response, is characterised by a dramatic increase in the gene expression profile in acinar cells [1]. The expression of potentially harmful genes such as proteases is down-regulated whereas proteins with protective propensity, also known as stress proteins, are induced. Thus, modifications of the expression profile in the pancreas with acute pancreatitis may be a part of organ defence mechanism of the exocrine pancreas. This hypothesis was underlined by a study in which mild oedematous pancreatitis was induced with an aim to start the ‘emergency programme’ before inducing a necro-haemorrhagic pancreatitis. The induction of oedematous pancreatitis reduced the severity of necrotising pancreatitis along with mortality [2]. Clearly, explanations for the early events of acute pancreatitis call for identification of the hereby activated genes and likewise an understanding of their function. A grasp of these processes could lead to new and perhaps more effective therapeutic strategies for treating patients with acute pancreatitis. The pancreatic stress response has been characterised using miscellaneous approaches which have enabled a

Copyright © 2004 S. Karger AG, Basel



Hans Bödeker Medizinische Klinik und Poliklinik II Universitätsklinikum Leipzig AöR, Ph.-Rosenthal-Strasse 27 DE–04103 Leipzig (Germany) E-Mail [email protected]

comparison of gene expression profile between an afflicted and normal pancreas. In recent years, quantitative fluorescent cDNA microarray hybridisation allowed an investigation of the change in expression levels of several thousand genes after induction of pancreatitis. In addition, identification of the expressed sequence tags overexpressed in the pancreas during experimental pancreatitis revealed new proteins involved in the pathogenesis of pancreatitis [3]. In this review we focus on recent works describing proteins overexpressed in the acinar cells of the pancreas with acute pancreatitis.

expressing PAP-1 show significantly less apoptosis after exposure to TNF- [17]. PAP-1 hinders nuclear factor B (NFB) activation by TNF- in macrophages [18]. Consequently, expression of PAP-1 may influence invading leucocytes and therefore be able to negatively regulate the inflammatory response in acute pancreatitis. A recent article confirmed these data by showing that blockage of PAPs during acute pancreatitis by antisense targeting aggravated the severity of pancreatitis as well as the systemic inflammatory response [19]. In fact, expression of PAP-1 during acute pancreatitis has an anti-apoptotic and an anti-inflammatory effect.

Pancreatitis-Associated Protein-1 p8

The pancreatitis-associated protein (PAP) was identified in 1984 by an early proteomic approach comparing pancreatic juice from rats with acute experimental pancreatitis and healthy controls [4]. In fact, PAP-1 is a member of a protein family called PAPs/regs (abbreviation of regenerating protein) with several members in different species [for review, see 5, 6]. Different members of the PAP family are concomitantly expressed and apparently form homo- and heterodimers [7]. These interactions may influence the function of the PAPs. PAP-1 serum levels may be utilised as a biological marker of pancreatitis [8] and as an indicator of cystic fibrosis when screening newborns [9]. PAP-1 is not only expressed in response to pancreatitis but also in systemic infections [10] and could indicate pancreatic dysfunction in septic patients. High serum levels limit systemic complications of acute pancreatitis by reducing leukocyte-induced lung injury [11]. The fact that PAP-1 could be induced in cell culture by the serum of rats with acute pancreatitis but not from healthy animals [12] led to more extensive studies on its mechanism of regulation and functions. PAP-1 expression is induced in the pancreatic acinar AR4–2J cell by interleukin-6 (IL-6) and dexamethasone and is explained by the presence of two IL-6 response elements in the PAP-1 promoter [13]. Expression of PAP-1 is also induced in acinar cells by oxidative stress leading to an enhanced resistance against apoptosis induced by free radicals [14]. Interestingly, PAP-1, referred to as reg-2 in these publications, also has anti-apoptotic properties in neurons. PAP-1/reg-2 is produced by regenerating motoneurons, it stimulates growth of Schwann cells and is therefore asserted as an obligatory neurotrophic factor [15, 16]. Also PAP-1 is induced in acinar cells by tumour necrosis factor- (TNF-) through a pathway which includes the mitogen-activated protein kinase-1 (MEK1). Cells over-

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p8 mRNA is strongly, rapidly and transiently activated in pancreatic acinar cells during the acute phase of pancreatitis [20]. The 8-kDa protein p8 is also expressed in developing pancreas and in chronic pancreatitis [21] and in some pancreatic cancers. The fact that p8 is also expressed in pancreatic cancer suggests that p8 may be a link between inflammation and neoplastic pancreatic diseases. Functional studies revealed that p8 acts as a transcription cofactor which binds the transcription factor p300 along with the regulatory Pax2 trans-activation domain interacting protein [22]. DNA binding by p8 is strongly enhanced by phosphorylation of serine/threonine residues [23]. Generation of p8-deficient mice allowed profound studies of p8 function. p8-deficient fibroblasts grow more rapidly and are more resistant against apoptosis induced by cytostatic drugs [24]. Transforming growth factor- (TGF-) is a central cytokine in chronic as well as acute pancreatitis. An important transducer of TGF- signalling is the transcription factor family of Smad. Studies on p8-deficient fibroblasts show that it is crucial for TGF--induced transcriptional activity of Smad [25]. Pancreatitis induced in p8-deficient mice leads to a more severe course of the disease as shown by measuring lipase and amylase serum levels, myeloperoxidase activity in the pancreas, and histological scores. Interestingly, expression of PAP-1 is lessened in p8-deficient animals in acute pancreatitis indicating that p8 transcriptional activity is needed for PAP-1 transcription [18]. Furthermore, lipopolysaccharides (LPSs), strong inducers of cellular stress, induced expression of p8. The fact that p8 is both a co-transcription factor as well as a stress-responsive gene suggested that p8 might mediate the LPS-induced stress response. Supporting this hypothesis, DNA microarray analysis revealed that a lack of p8

Savkovic´ /Gaiser/Iovanna/Bödeker

leads to aberrant gene expression in response to the endotoxin. Treatment with LPS likewise resulted in higher serum levels of TNF- and higher mortality in p8-deficient animals. In the pancreas and liver p8-deficient mice displayed increased amounts of myeloperoxidase and hydroperoxide. Both are markers of neutrophil tissue infiltration and indicators of oxidative stress [26]. p8 is expressed in several types of human cancer. In fact, p8 expression seems to be crucial for tumour progression in metastasis. To examine the role of p8 in cancer growth a model of mouse embryonic fibroblasts transfected with the adenoviral oncogene E1A along with a mutated RAS oncoprotein was used to induce malignant transformation [27]. Importantly, targeted disruption of the p8 gene completely hindered the E1A/RAS-induced malignant transformation of fibroblasts as estimated in vitro by soft agar assays and tumour formation in nude mice [28]. Taken together, p8 seems to be a stress-induced transcriptional cofactor which influences tumorigenesis as well as inflammation.

Clusterin

Clusterin is highly expressed in the course of acute experimental pancreatitis as well as during pancreatic development [29]. Clusterin is found in the pancreatic juice from the inflamed pancreas and its expression could be shown in acinar and some ductal cells by in situ hybridisation. Clusterin is produced through a complex biogenesis that leads to different isoforms attained either by differential splicing or post-transcriptional modifications [30, 31]. Full-length, fully glycosylated clusterin is a secretory protein with anti-apoptotic functions. This form is mainly expressed in exocrine pancreas [29] and its expression can be suppressed by p53 [32]. A shortened splice variant, called nuclear clusterin [33], and probably also a low glycosylated form transcribed from the fulllength mRNA are pro-apoptotic [30]. In pancreatic cells full-length clusterin is expressed in response to diverse stimuli which also induce apoptosis [29]. Experiments with stable transfected AR4-2J cells show that clusterin protects from apoptosis by stressors which mimic cellular stress in acute pancreatitis. Clusterin seems also to have an anti-inflammatory effect since the protein diminishes NFB activation in AR4-2J cells after supramaximal cerulein stimulation [Savkovic´ et al., in preparation]. This finding is consistent with enhanced NFB in clusterindeficient fibroblasts [34]. Furthermore, clusterin-deficient mice show strongly elevated lipase and amylase se-

Stress Response of the Exocrine Pancreas

rum values suggesting a protective role for clusterin in acute pancreatitis [Savkovic´ et al., in preparation]. In addition, clusterin seems to be able to attenuate systemic complications of acute pancreatitis. For example clusterin is able to protect the lung from leukocyte-induced lung injury [35]. Therefore, clusterin has anti-apoptotic and anti-inflammatory function in the exocrine pancreas.

Heat Shock Proteins

Heat shock proteins (HSPs) are part of the cellular stress machinery and therefore among the usual suspects for stress responsive genes in acute pancreatitis. Indeed, several groups have already described overexpression of different members of the HSP protein family. HSP70 expression induced by hyperthermia in pancreatic lobules in vitro and whole body hyperthermia protects against cerulein-induced pancreatitis [36]. Inopportunely, there are also contradictory data about HSP70 since induction by cerulein causes elevated mRNA levels but is not followed by elevated protein expression [37]. The role of HSP70 in experimental pancreatitis could be clarified by antisense targeting experiments. Administration of antisense-HSP70 oligonucleotides abrogated the effect of whole body hyperthermia-induced protection against cerulein-induced pancreatitis and premature trypsin activation [38]. Induction of HSP60 by water immersion stress also may protect against cerulein-induced pancreatitis by reduction of trypsin activation [39]. Another member of the HSP family, the small HSP27, is phosphorylated under cholecystokinin (CCK) stimulation in the rat pancreas depending on the mitogen-activated protein kinase-activated protein kinase-2 pathway [40]. Studies of CCK-A receptor expression in Chinese hamster ovary cells demonstrate that CCK-induced phosphorylation of HSP27 regulates actin polymerisation [41]. Indeed, the protective effect of human HSP27 depends on its phosphorylation as shown by experiments with transgenic mice expressing wild-type HSP27 and the non-phosphorylatable HSP27-mutant [42]. HSP70 and HSP60 appear to be protective due to inhibition of trypsin activation, while the protective role of HSP27 may be caused by its influence on the cytoskeleton [for review, see 43].

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TP53INP1 and TP53INP1

TP53INP1s were identified in a quantitative cDNA microarray approach comparing gene expression between inflamed and normal pancreas [44]. The proteins are two splice variants of a gene initially called stress-induced protein (SIP). TP53INP1 and TP53INP1 are 18- and 27-kDa isoforms which are overexpressed in acute pancreatitis. In situ hybridisation showed restriction of TP53INP1 expression to the acinar cells in the inflamed pancreas. TP53INP1s are also expressed in the WBN/ Kob rat model of spontaneous chronic pancreatitis [45]. Confocal microscopy of tagged TP53INP1 shows a nuclear distribution of the proteins. Overexpression of TP53INP1 and TP53INP1 induced apoptosis as measured by the colony-forming assay and TUNEL. Furthermore, the typical morphological characteristics of apoptosis have been described [44]. TP53INP1s are induced by a variety of cellular stressors like UV radiation, mutagenic stress, ethanol, heat shock and oxidative stress. This stress-induced expression is p53-dependent, leading to the name TP53INP1 (tumour protein 53-induced nuclear protein-1) [46]. p53-dependent apoptosis is promoted by the homeodomain interacting protein kinase-2 (HIPK2) which binds to p53 and can phosphorylate the key regulator of apoptosis and cell cycle regulation. TP53INP1s interact physically with p53 as well as HIPK2. This interaction regulates p53 transcriptional activity on p53-target genes like p21, mdm2, pig3 and bax [47]. Therefore, TP53INP1s are stress-induced splice variants of a gene which is activated and regulates p53. Like p8, TP53INP1s may be a link between inflammatory and neoplastic disease in the pancreas.

Vacuole Membrane Protein-1

The rat vacuole membrane protein-1 (VMP1) was cloned from a cDNA library made with polyadenylated mRNA from rat pancreas with acute pancreatitis [48]. The single copy gene gives rise to 3 different splice variants of 1.9, 2.7 and 3.5 kb. In different tissues the level of expression of the three splice variants vary. In the normal pancreas VMP1 is poorly expressed. Within the course of experimental pancreatitis the 1.9- and the 2.7-kb splice variant are concomitantly induced while the 3.5-kb variant could not be detected. In the developing rat pancreas VMP1 is expressed until day 11 postpartum. In situ hybridisation showed that expression of VMP1 is restricted to the acinar cells of the inflamed pancreas. In

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transfection experiments with tagged VMP1 followed by subcellular fractioning VMP1 was detected in the membrane fraction. Interestingly, overexpression of VMP1 leads to vacuole formation, an important event in the pathophysiology of acute pancreatitis, and VMP1 is integrated in the membrane of these vacuoles [48]. In addition, VMP1 expression correlates with vacuolisation in the inflamed pancreas [49]. Until now vacuole formation has been interpreted as an ‘accident’ in pancreatitis, probably caused by breakdown of the intracellular trafficking and potentially leading to formation of fusion vesicles in which trypsin activation is taking place and autodigestion may start [50]. However, the fact that VMP1 is expressed as early as 1 h after the induction of pancreatitis suggests that vacuole formation might be an active mechanism in pancreatitis rather than an ‘accident’. Induction of apoptosis is a prominent feature associated with VMP1 expression following vacuole formation in the pancreas with pancreatitis. Overexpression of VMP1 further leads to morphological evidence of apoptosis and a dramatically reduced number of clones in the colony-forming assay [48]. Accordingly, apoptosis and VMP1 overexpression are concomitant events in animal models of acute experimental pancreatitis as well as chronic pancreatitis [51].

Interferon-Inducible Protein-15

The interferon (IFN)-inducible protein-15 (IP15) was identified in a microarray-based experiment as an expressed sequence tag overexpressed during the acute phase of pancreatitis [52]. The gene codes for a putative transmembrane protein of 137 amino acids. The normal pancreas shows poor IP15 expression, but the protein is strongly activated after induction of an experimental pancreatitis. Induction starts as early as 1 h after initiation of pancreatitis and peaks at 9 h. Expression is limited to acinar cells as shown by in situ hybridisation of the inflamed pancreas. IP15 mRNA expression is also evident in the developing pancreas. Interestingly, IP15 was also inducible by LPS as well as systemic infection with Salmonella enteritidis. The IP15 gene contains an IFN-responsive element which leads to strong induction of IP15 after treatment with IFN- in cell culture experiments. A perinuclear, vesicle-like distribution of tagged IP15 was detected by confocal microscopy. The anti-proliferative effect of IFN suggested that IP15 might have an effect on cell proliferation. Indeed, stable transfection with IP15 resulted in a significant reduction in cellular growth rate.


Expression of IP15 resulted in reduced number of colonies in the colony-forming assay. The effect of IP15 expression is anti-proliferative but not pro-apoptotic since the rate of cell death was not changed in transfection experiments [52]. Therefore, IP15 may decrease or delay cell growth during the early phase of acute pancreatitis.

PC3/TIS21/BTG2 and PIP49

The PC3/TIS21/BTG2 protein [53] and the pancreatitis-induced protein-49 (PIP49) [54] are both strongly expressed during the acute phase of experimental pancreatitis. PC3/TIS21/BTG2 is an anti-proliferative p53-dependent component of the cellular DNA damage-response pathway and has anti-apoptotic functions. Expression of PIP49 is restricted to acinar cells in the inflamed pancreas. The function of this putative transmembrane protein remains elusive.

Conclusion

The exocrine pancreas reacts with a well-coordinated change in gene expression to acute pancreatitis in order to prevent further progression of the disease. This stress response of pancreatic acinar cells displays several interesting features, and understanding the underlying mechanisms can widen our knowledge of the pathophysiology of pancreatitis. Apoptosis Regulation Regulation of apoptosis was identified as an important factor in acute pancreatitis. Kaiser et al. [55] compared 5 different animal models of pancreatitis and found a negative correlation between apoptosis and the severity of the disease. During the acute phase of pancreatitis several apoptosis-regulating proteins are activated. While PAP-1 [14], clusterin [Savkovic´, in preparation] and PC3/TIS21/BTG2 [53] are anti-apoptotic, TP53INP1s and VMP1 promote cell death [44, 48]. Therefore, apoptosis regulation in acute pancreatitis seems to be finely controlled. We may speculate that the balance between pro- and anti-apoptotic effectors might depend on the context of any cell. When cellular stresses for the acinar cell exceed a certain threshold, apoptosis may be induced to avoid necrosis and chaotic liberation of potentially harmful substances like activated proteases. Programmed cell death allows degradation of intracellular proteins and controlled phagocytosis by macrophages. This putative


mechanism may explain that the great majority of attacks of pancreatitis are mild and self-limiting. Only when proapoptotic effectors fail to control cell death in time, necrosis may become overwhelming and lead to a fatal course of the disease. Regenerating Processes in Pancreatitis The fact that several stress-response genes of the pancreas [20, 29, 48, 52] are also expressed during the development of the pancreas (table 1) may explain the features of the restitutio ad integrum, the full recovery after pancreatitis. Pancreatic cells which overcome the acute phase of pancreatitis regress to a pluripotent cell phenotype in so-called tubular complexes. The expression of the genes mentioned probably reflects the regression and following re-differentiation of the pancreatic cells. Cell Cycle and Growth Regulation Several stress-response genes identified in acute pancreatitis interfere with cell cycle and growth regulation. TP53INP1 and TP53INP1 functionally interact with p53 and the p53-regulating protein HIPK2. p53 is an important gatekeeper in the cell cycle and growth regulation as well as in apoptosis [47]. Expression of PC3/TIS21/ BTG2 as well as the TP53INP1s are regulated in a p53dependent manner [46, 56]. Expression of the anti-apoptotic form of clusterin is suppressed by p53 [32]. Overexpression of p8 is implicated in cell growth arrest. The presence of p8 increases the p53 expression level and its transactivating capacity. On the other hand, p53 is a negative transactivator of p8 [24]. The fact that p8 is essential for malignant transformation suggests that p8 acts as prooncogene [28] and therefore expression of p8 in chronic pancreatitis may be a mitogenic trigger in the context of this pre-malignant disease. Regulation of Inflammation The exocrine pancreas reacts on systemic malfunctions and likewise influences systemic complications of the pancreatitis. Stress-induced pancreatic proteins can regulate the inflammatory response and complications in acute pancreatitis. PAP-1 [18] and clusterin [Savkovic´, in preparation] display anti-inflammatory features in the exocrine pancreas. Both molecules seem to be able to diminish systemic complications of pancreatitis like leukocyte-induced lung injury [11, 35]. p8 influences a great number of stress-induced genes at the transcriptional level [26]. This transcriptional co-factor seems to hinder an uncontrolled inflammatory systemic response in the liver, lung and pancreas.

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Table 1. Regenerating processes in pancreatitis

Protein

Biological effect

Expression in Subcellular developing pancreas distribution

PAP-1

Anti-apoptotic Anti-inflammatory

–

Secretory

p8

Co-transcription factor Obligatory for mitogenic effect of oncogenes E1A/RAS

+

Nuclear

Clusterin

Anti-apoptotic Anti-inflammatory Anti-apoptotic form suppressed by p53

+

Secretory Cytoplasmic? Nuclear?

HSP70 and HSP60

Protection against trypsin activation

?

Cytoplasmic

HSP27

Regulation of the actin cytoskeleton

?

Cytoplasmic

TP53INP1

Co-factor of p53 Pro-apoptotic p53-dependent

?

Nuclear

VMP1

Induces vacuole formation Pro-apoptotic

+

Transmembrane Associated with vacuoles

IP15

Anti-proliferative Induced by interferon-

+

Transmembrane Perinuclear

PC3/TIS21/BTG2

Anti-apoptotic p53-dependent

?

Unknown

PIP49

Unknown

?

Transmembrane

Vacuole Formation in Pancreatitis The expression of VMP1 may question the central theory about the induction of pancreatitis. It has been proposed that breakdown of intracellular trafficking leads to fusion of great intracellular vesicles in which premature activation of trypsin may take place [50]. This theory supports the idea that vacuole formation is a passive event that the acinar cells suffer. However, contrary to this hypothesis is the fact that pancreatic acinar cells express

VMP1 which strongly suggests that vacuole formation is an active feature during acute pancreatitis [48, 52]. In conclusion, the identification of stress-induced genes activated during the acute phase of pancreatitis and the characterisation of their function widen our understanding of the disease and may finally lead to the development of new rational therapeutic concepts in pancreatitis.

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Laboratory Markers of Severe Acute Pancreatitis B. Raua M.K. Schillinga H.G. Begerb a b

Department of General, Visceral, and Vascular Surgery, University of the Saarland, Homburg/Saar, and Department of General Surgery, University of Ulm, Ulm, Germany

Key Words Pancreatitis, acute Trypsinogen activation peptide Procarboxypeptidase B activation peptide Interleukins Serum amyloid A Procalcitonin

a fast, reliable, and cost-effective assessment of severity in acute pancreatitis. PCT substantially contributes to an improved stratification of patients at risk to develop major complications and deserves routine application. Copyright © 2004 S. Karger AG, Basel

Abstract Background: A large array of parameters has been proposed for the biochemical stratification of severity and prediction of complications in acute pancreatitis. However, the number of accurate and readily available variables for routine application is still limited. Methods: The literature was reviewed for laboratory markers of acute pancreatitis with special regard to their clinical usefulness and test performance for stratifying severity and monitoring disease progression. Results: Several parameters, such as trypsinogen and procarboxypeptidase B activation peptide, PMN-elastase, interleukin-6 (IL-6) and 8 (IL-8), serum amyloid A (SAA), and procalcitonin (PCT), can differentiate between mild and severe acute pancreatitis within 48 h of disease onset with favorable diagnostic accuracy. Because fully automated assays have become available, IL-6, IL-8, PCT, and SAA are the most interesting parameters in this respect. For monitoring disease progression beyond 48 h, acute-phase proteins, IL-6, IL-8, and PCT are valuable markers. PCT is the first biochemical variable for predicting severe pancreatic infections and overall prognosis throughout the course of acute pancreatitis with high sensitivity and specificity. Conclusions: Among all the biochemical variables available, C-reactive protein is still the standard for



Introduction

Since the early 1980s major advances in our understanding of the natural history of acute pancreatitis with the identification of relevant prognostic factors [1–3] have driven management toward a multidisciplinary approach. Hence, it has been well recognized that immediate and goal-directed treatment significantly influences the further course and outcome of this disease [4]. As a consequence, early and reliable diagnosis of complications has become a compelling issue for clinicians. The introduction of contrast-enhanced computed tomography (CE-CT) for the detection of necrosis [5, 6] as well as guided fine-needle aspiration (FNA) techniques to differentiate sterile necrosis from pancreatic infections [7, 8] have been the cornerstones of improved management of this disease. However, although being highly accurate and reliable, neither CE-CT nor FNA are universally available, carry the risk of potential complications, and constitute considerable cost factors. In the mid 1960s the first evidence arose showing that acute pancreatitis is reflected by abnormalities of many serum/plasma variables [9]. During the following decades much effort was put into the search for biochemical pa-

Priv.-Doz. Dr. med. Bettina Rau Department of General, Visceral, and Vascular Surgery, University of the Saarland Kirrberger Strasse, DE–66421 Homburg/Saar (Germany) Tel. +49 6841 16 22630, Fax +49 6841 16 23132 E-Mail [email protected]

Fig. 1. Schematic overview of the inflammatory cascade in acute pancreatitis. Activation of various leukocyte subsets and endothelium at the local site of injury leads to the release of pro- and anti-inflammatory cytokines, chemokines, and other mediators. An overt and sustained activation of proinflammatory mediators leads to systemic inflammatory response syndrome (SIRS) which may further proceed to multiorgan dysfunction syndrome (MODS), infected necrosis and sepsis.

rameters which allow early stratification of patients at risk of developing complications such as necrosis, infection of necrosis, septic complications or organ failure. Beyond the potential to predict disease severity many of these parameters were found to be determinants of disease progression and subsequent complications in the pathomechanisms of acute pancreatitis (fig. 1). Although a still increasing array of potentially useful parameters is currently available, their large-scale clinical use is often hampered by time-consuming and cost-intensive assay procedures. Referring to the pathophysiological background the most important biochemical parameters for severity stratification and monitoring of acute pancreatitis are discussed below.

Pancreatic Proteases and Antiproteases

Since the mid 1980s, a key role in the pathophysiology of acute pancreatitis has been attributed to the proteaseantiprotease imbalance. Hence, trypsinogen activation is believed to be one of the earliest pathophysiological events which triggers a cascade of other pancreatic proenzymes such as chymotrypsinogen, type-I prophospholipase A2, procarboxypeptidase B, or proelastase in acute

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pancreatitis [10]. According to the ‘autodigestion’ theory of Chiari [11] more than 100 years ago, premature trypsinogen activation within the acinar cells has been found in various experimental models of acute pancreatitis [10, 12]. Subsequently, significant amounts of trypsinogen and other proteases have been measured in the interstitial space as well as in the systemic circulation with a positive correlation to the extent of pancreatic tissue destruction and overall disease severity [12]. However, trypsinogen activation is only a temporary event in acute pancreatitis and most recent experimental studies have questioned the prevailing opinion of its dominating pathophysiological role [13]. However, these findings would at least in part explain the failure of antiprotease therapy in clinical acute pancreatitis [14]. Biochemical severity stratification by means of proteases and antiproteases released from the pancreas during acute pancreatitis has been the subject of numerous studies. Antiproteases The role of antiproteases as biochemical markers of severity has been addressed by several clinical studies. The trypsin-2-1-antitrypsin complex in serum has been shown to be superior to trypsinogen-2, C-reactive protein (CRP) and amylase in diagnosing acute pancreatitis and

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could differentiate between severe and mild attacks within 12 h after hospital admission [15]. The 2-macroglobulin is another antiprotease, which binds to pancreatic proteases such as trypsin or elastase. Because the protease-antiprotease complex is rapidly degraded and eliminated from the systemic circulation by macrophages, a rapid decrease in 2-macroglobulin levels is observed during severe attacks of acute pancreatitis [16–19]. Although early disease prediction and severity stratification may be possible by means of antiproteases, neither the diagnostic accuracy nor the assay procedures are appropriate for use under routine conditions. Activation Peptides The synthesis of digestive enzymes as inactive proenzymes represents an important defense mechanism protecting the pancreas from autodigestion. Upon activation by the duodenal brush-border enzyme enterokinase, a low-molecular-weight (!10 kD) peptide, the so-called activation peptide is cleaved off and the biologically active site of the enzyme is exposed. Trypsinogen-activation peptide (TAP), carboxypeptidase B activation peptide (CAPAP), and the phospholipase activation peptide (PLAP) account for the most important activation peptides in acute pancreatitis. The results of several studies clearly indicate that measuring activation peptides is superior to that of leaking proenzymes such as trypsinogen2 to predict severity [20]. Trypsinogen Activation Peptide TAP is by far the most extensively investigated activation peptide in acute pancreatitis. TAP is known to be disease-specific, not influenced by the underlying etiology of acute pancreatitis and is detectable in the systemic circulation as well as in urine [21–23]. The clinical usefulness of this parameter has been investigated by three multicenter trials. An US-American trial showed that urinary TAP achieved a sensitivity of 100% and a specificity of 85% in predicting a severe attack of acute pancreatitis within 48 h of disease onset [22]. Two more recent European multicenter trials showed somewhat less favorable results with a sensitivity of 58–62% and a specificity of 73% within 24 h [23, 24] which increased to a sensitivity of 83% and a specificity of 72% 48 h following onset of symptoms [23]. On the other hand, overall accuracy rates of urinary TAP in predicting a severe attack did not exceed 75% even 48 h after the onset of acute pancreatitis, which were also achieved by clinical scoring systems [23]. Unfortunately, the very early burst-like secretion of TAP with a rapid consecutive decline makes discrimination

Biochemical Parameters for Severity Stratification of Acute Pancreatitis

between severe and mild cases no longer possible after 72 h [22–25]. Therefore, monitoring the progression of the disease to severe organ failure or septic complications which usually develops beyond 48 h after symptom onset is not possible. In addition, the current ELISA technique prohibits analysis of this parameter in the daily laboratory routine. The development of a semiquantitative strip test may overcome this problem which, however, has not been evaluated so far. Carboxypeptide Activation Peptide The activation peptide CAPAP is a diagnostic marker for acute pancreatitis and has been found to correlate with disease severity as well [26–29]. CAPAP can be measured in plasma and urine and is more stable than TAP due to its larger size [27]. As observed for TAP, the highest diagnostic accuracy in predicting pancreatic necrosis is obtained by measuring this activation peptide in urine with accuracy rates of about 90%. Unfortunately, CAPAP levels also rapidly decline and are thus not useful in depicting severe cases in the later course of the disease [29]. The CAPAP assay is currently available as radioimmunoassay only, which prohibits an introduction of this parameter to clinical routine analysis. Phospholipase A2-Activating Peptide PLAP, also termed PROP, is the activation peptide of pancreatic phospholipase A2 (PLA2). Measurement of PLAP/PROP was initially designed as an indirect approach to assess PLA2 activity [30]. Unlike the other two activation peptides PLAP is not only released from the pancreas but also from activated neutrophils [31]. This adds an interesting aspect to the assessment of this activation peptide in a way that urinary PLAP levels correlated with the systemic inflammatory response as well [30]. A recent multicenter trial could show that urinary PLAP/PROP provides reasonable discrimination between mild and severe attacks achieving a sensitivity of 71% and a specificity of 59% within 48 h of symptom onset [32]. However, urinary PLAP/PROP was of no value in predicting remote organ failure due to the rapid decline in the further course of the disease. Surprisingly, no correlation between urinary TAP and PLAP/PROP values was observed. Considering the lower accuracy of urinary PLAP/PROP compared to TAP and the time-consuming assay procedure, future clinical application of this parameter seems to be unlikely. On the basis of the published literature there is no doubt that assessment of pancreatic protease activation peptides are a valid markers for an early severity stratifi-

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cation of acute pancreatitis. This may be of specific interest for specialized centers whenever early severity stratification for clinical trials and improved inter-institutional comparison of patients is an issue. However, from an economical and practical standpoint a large scale clinical application of TAP, CAPAP or PLAP/PROP will be unlikely. Because most patients with acute pancreatitis are admitted or referred beyond the 48-hour diagnostic window after disease onset, the general need for very early markers of severity has to be questioned. Even if the development of an ‘immunostick‘ for a combined assessment of activation peptides will be developed in the future, the clinical use of these parameters will probably remain a scientific one due to the limited indication and therefore persisting high cost.

Acute-Phase Proteins

Acute-phase proteins constitute a family of inflammatory proteins which are predominantly synthesized in the liver in response to various infectious and non-infectious stimuli [33]. The most famous and well-established member is CRP; more recently serum amyloid A protein (SAA) [34] and lipopolysaccharide (LPS)-binding protein (LBP) [35] are further members which accomplished the spectrum of acute-phase reactants sharing an essential feature for large scale use in the daily routine application: all of them have become available as fully automated immunoassays. C-Reactive Protein Severity stratification of acute pancreatitis by CRP has a long tradition and still represents the ‘gold standard’ new biochemical parameters have to compete with. Numerous adequately powered studies have proven the benefits of CRP determinations in acute pancreatitis over the past two decades [17–19, 23, 25, 36–38]. The practicability of the assay procedure, the cost and the overall availability have rendered CRP as a widely established means for both severity stratification and monitoring the course of the disease. CRP is the parameter of choice to differentiate necrotizing from interstitial edematous acute pancreatitis with a diagnostic accuracy of more than 80%. In this respect, the cutoff level is 150 mg/l within the first 48 h after onset of symptoms according to the most recent consensus conference [4]. As well documented for all acute-phase proteins, CRP is not useful in predicting infectious complications such as infected necrosis or pancreatic abscess. Another shortcoming of CRP is the rela-

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tively long delay of its induction with systemic peak values at 72–96 h after disease onset [17, 37, 38], thus making very early severity assessment impossible. Serum Amyloid A SAA proteins comprise a family of apolipoproteins which are, comparable to CRP, mainly synthesized in the liver in response to cytokines following an acute-phase stimulus such as physical injury or infection [33, 34]. In contrast to CRP and LBP, several potential functions have been suggested; however, no definite physiological role has been established for SAA so far [34]. As an alternative acute-phase reactant for the severity stratification of acute pancreatitis only two adequately powered studies have been published so far [37, 39]. A common finding of both studies was an earlier release with a wider dynamic range of SAA than observed for CRP. However, both studies are not quite comparable because they differ in endpoint analysis and assay techniques applied. The multicenter study found that SAA was a better early predictor of severe acute pancreatitis than CRP using a conventional ELISA technique [39]. Our study could not show any advantage of SAA over CRP in stratifying severity at any time point during the course of acute pancreatitis by using a fully automated assay technique [37]. Further studies will be needed to define a convincing clinical benefit of SAA over CRP determinations to justify the still higher cost of this alternative acute-phase reactant. Lipopolysaccharide-Binding Protein LPS is a constituent of the outer coat of gram-negative bacteria and the strongest inducer of systemic inflammatory response and sepsis. However, LPS does not affect the host directly, but activates immunocompetent cells to produce a variety of proinflammatory mediators [40]. Monocyte/macrophage activation by LPS is dependent on the presence of LBP, a class-1 acute-phase protein. LBP is one of the most important cofactors involved in mediating the systemic host response to LPS [35] on the one hand and contributing to the host’s defense mechanisms against sepsis by promoting neutralization of LPS [41] on the other. Hence, only one study has evaluated this parameter in acute pancreatitis using a fully automated immunoassay technique [38]. LBP concentrations were uniformly elevated in acute pancreatitis and correlated well with the overall disease severity. The quantitative systemic release of LBP was lower, but revealed similar dynamics as CRP with a maximum increase around the 4th day after onset of symptoms. However, LBP did

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not show a correlation with the development of septic complications. As observed for SAA, LBP did not offer any clear advantage over CRP for stratifying severity in acute pancreatitis. Among the acute-phase proteins CRP still remains the gold standard in predicting severity beyond 48 h after onset of acute pancreatitis. This readily available, fast and inexpensive test is still the reference parameter among the indicators of necrosis. Obvious shortcomings of CRP and other acute-phase reactants are the fact that none of them provides any reliable discrimination of patients at risk of developing infectious complications from those not at risk.

Cytokines

Cytokines are a family of low-molecular-weight proteins which have been extensively investigated in inflammatory conditions including acute pancreatitis. Currently, there is no more doubt about the detrimental role of many cytokines in promoting local tissue destruction and mediating distant organ complications [42, 43]. More than a decade ago the first clinical reports on the role of cytokine measurements in acute pancreatitis appeared in the literature and still continue to address this topic. The development of fast and fully automated assay techniques have overcome the problem of the conventional ELISA measurements so that cytokine determinations could be introduced to routine laboratories and have accomplished the spectrum of biochemical parameters for the severity stratification of many inflammatory diseases. Tumor Necrosis Factor- and Interleukin-1 In contrast to their outstanding pathophysiological impact [42, 43], both of the so-called ‘first-order’ proinflammatory cytokines, tumor necrosis factor- (TNF-) and interleukin-1 (IL-1) play no role as biochemical markers for the severity assessment of acute pancreatitis. In the clinical setting TNF- measurements are difficult, because they are substantially hampered by intermittent TNF- release and a short plasma half-life of less than 20 min. Binding of TNF- to its receptor complex on target cells renders these cells non-responsive to further stimulation by shedding the TNF/TNF receptor complex which is subsequently released into the systemic circulation. The soluble TNF receptor complex is more stable than the cytokine itself and thus easier to measure. A difference between mild and severe pancreatitis as well as between the presence and absence of pancreatitis-associ-


ated organ failure has been demonstrated [44, 45]. Similar observations have been made for IL-1, which shows an early and transient increase in most severe cases only [45–48]. The IL-1 receptor antagonist (IL-1ra) is thought to reflect in vivo IL-1 activity and was found to correlate with severe acute pancreatitis complicated by organ failure [45–47, 49]. Interleukin-6 IL-6 is the principle cytokine which induces the synthesis of acute-phase proteins such as CRP, SAA, LBP and many others. Systemic concentrations of IL-6 have been found to be early and excellent predictors of severity. A large number of clinical studies have uniformly shown that IL-6 is dramatically increased in complicated attacks [45–52]. The rise of IL-6 concentrations generally occurs 24–36 h earlier than that of CRP with significantly elevated levels as long as complications persist. In the ‘human model’ of endoscopic retrograde cholangiopancreatography (ERCP)-induced acute pancreatitis, the very early peak of IL-6 could be nicely demonstrated in patients who developed clinical and/or laboratory signs of post-ERCP pancreatitis [51, 52]. IL-6 measurements have already been introduced as a routine parameter in many laboratories and represent an easy and rapid means to select patients at risk of developing severe disease. Other Cytokines There are still a growing number of other pro- and antiinflammatory cytokines such as soluble IL-2 receptor [46, 53, 54], platelet-activating factor [55], IL-12 [54, 56], IL18 [57], IL-10 [45–47, 49, 52, 58] or IL-11 [49, 58], which are of distinct scientific interest as far as the pathophysiology of severe acute pancreatitis is concerned. Moreover, the pathophysiological role of some parameters provided the rationale for subsequent clinical trials in instances of platelet-activating factor [59] and IL-10 [60, 61]. Most of these cytokines provide very good discrimination between severe and mild courses; however, they are currently of no clinical relevance as biochemical markers in the daily clinical routine.

Chemokines

Chemokines are a family of small (8–10 kDa), inducible, secreted cytokines with chemotactic and activating effects on different leukocyte subsets, thus providing a key stimulus for directing leukocytes to the areas of injury [62]. Chemokines can be subdivided on a structural

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basis into the CXC and the CC subfamily which also determines their biological activity: while a subgroup of the CXC chemokines, such as IL-8, epithelial neutrophil-activating protein-78, and growth-related oncogene- are potent neutrophil chemoattractants and activators, the CC chemokines comprising monocyte chemoattractant protein-1, 2 and 3, macrophage inflammatory protein-1 and 1, regulated on activation, normal T-cell expressed and secreted (RANTES), and Eotaxin predominantly affect monocytes [62]. Interleukin-8 IL-8 is the most well-known member of the CXC-chemokine family and responsible for neutrophil chemoattraction, degranulation, and release of neutrophil elastase. Among patients with acute pancreatitis IL-8 has been shown to be an early prognostic marker of disease severity within the first day after onset of symptoms [48, 63–65] with a rapid decease after 3–5 days. Thus, IL-8 reveals obvious parallels to IL-6 as prognostic marker in acute pancreatitis. However, our group described an even more interesting aspect of IL-8 assessment. In patients with necrotizing pancreatitis who developed septic multiorgan failure during the later stages of the disease, IL-8 has proven as an excellent marker for monitoring this lifethreatening complication [66]. As for IL-6, a fully automated assay is available for IL-8. Thus the use of this chemokine for disease monitoring has become possible on a daily routine basis; however, the relatively high cost still prohibits a large-scale application of both IL-6 and IL-8 in clinical practice. Other Chemokines A number of further chemokines are currently under investigation. Hence, their role in acute pancreatitis is still a pathophysiological rather than a diagnostic one [67]. In 2 cohort studies the course of different CXC and CC chemokine members has been analyzed so far. In one of the studies the CXC chemokine members, epithelial neutrophil-activating protein-78 and growth-related oncogene- have been found to be very early and accurate predictors of severity in acute pancreatitis [68]. The second study was performed by our group and could show that the development of remote organ failure in acute pancreatitis was closely associated with a dramatic elevation in the CC chemokine monocyte chemoattractant protein-1 in the systemic circulation [69]. Although the first clinical results are very promising, no appropriate assays are available for a fast and easy measurement of the respective chemokines.

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Leukocyte-Derived Enzymes/Proteases

The activation of different leukocyte subsets has been well recognized as an important mechanism in the development of diseases severity and pancreatitis-associated organ failure [70]. Whereas the mononuclear leukocyte subset is the predominant source for the release of cytokines and chemokines, activated polymorphonuclear leukocytes (PMNs) release a number of proteases. Beyond their pathophysiological importance several PMN-derived proteolytic enzymes have been described as good biochemical markers for the severity stratification of acute pancreatitis. Polymorphonuclear Elastase PMN elastase is a proteolytic enzyme which is synthesized and released from infiltrating neutrophils invading the pancreas only few hours after the first evidence of intrapancreatic acinar cell damage. Accordingly, enhanced systemic release of PMN elastase is an early feature in clinical acute pancreatitis as well, with peak values even before CRP and other parameters begin to rise [63, 71– 73]. In a multicenter trial PMN elastase reached sensitivity and specificity rates of more than 85% in predicting severe acute pancreatitis [73]. Concentrations rapidly decline in patients with an uneventful recovery, while a persistent elevation of this enzyme was observed in nonsurvivors [71]. Hence, the PMN elastase test has not been adopted into routine laboratories because of problems with the assay and the reproducibility of the test results. Very recently, a new, routinely applicable assay has been developed which has obviously overcome the previous disadvantages [74]. However, as already a number of excellent parameters are available for a fast and accurate early severity stratification of acute pancreatitis, the fate of PMN elastase measurement remains questionable. Phospholipase A2 Besides type-I PLA2, which is of pancreatic origin, type-II or synovial-type PLA2 is secreted by activated neutrophils [75]. Whereas type-I PLA2 is of no prognostic value, synovial-type PLA2 provides good discrimination between severe and mild attacks of acute pancreatitis throughout the course of the disease. In several studies immunoreactive PLA2 was found to reflect the clinical severity [46, 76], and even better results were obtained if the catalytic activity of this enzyme was measured [36, 75, 77]. Interestingly, a more recent study has outlined a new diagnostic aspect of immunoreactive type-II PLA2 in acute pancreatitis: the course of type-II PLA2 concen-

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trations closely correlated with the development of pancreatic infections in patients with necrotizing pancreatitis [78]. Unfortunately, no assay for routine clinical analysis has ever been developed for measuring type-II PLA2. Therefore, this interesting and potentially useful parameter continues to play a role in scientific respect only.

Adhesion Molecules

Cell adhesion molecules are expressed on vascular endothelial cells and leukocytes in response to proinflammatory cytokines such as TNF-, IL-1 and IL-8, and cause leukocyte adhesion, margination and migration. Members of the adhesion molecule family include selectins, integrins, and intercellular adhesion molecules (ICAMs) [79]. The pathophysiological role of adhesion molecules in acute pancreatitis has been convincingly shown in the experimental setting [67]. In contrast, so far only a few studies have addressed the role of adhesion molecules in clinical acute pancreatitis. The adhesion molecules ICAM-1 and E-selectin were found to be significantly increased in severe acute pancreatitis during early stages of the disease [80–82]. Moreover, in a very recent study E-selectin remained markedly elevated in severe attacks throughout the entire observation period of 10 days after hospital admission [82]. In contrast, Eselectin levels failed to differentiate mild from severe acute pancreatitis within 24 h after hospital admission in a Finnish study [53]. Despite the proven pathophysiological implications the clinical usefulness of soluble adhesion molecules for the severity stratification or monitoring of acute pancreatitis remains uncertain unless further studies prove the opposite.

Procalcitonin

Procalcitonin (PCT) is the inactive 116-amino-acid propeptide of the biologically active hormone calcitonin with a long half-life in the systemic circulation. Since its first description in 1993 [83] an extensive number of reports have largely confirmed that PCT is the first biochemical variable which closely correlates with the presence of bacterial or fungal infections and sepsis [84]. It is well known that necrotic infection is a major complication in the course of acute pancreatitis and has a major impact on management and outcome [1, 4]. In the absence of a valid clinical or biochemical parameter, guided fine-needle aspiration (FNA) has been the only means for


an early and accurate diagnosis of infected necrosis during the past decades [7, 8] and still represents the standard new methods have to compete with. In a cohort study comprising 51 patients with acute pancreatitis we found a highly significant correlation of elevated PCT levels and the subsequent development of infected necrosis. At a cutoff level of 1.8 ng/ml PCT was able to predict this complication with a sensitivity and specificity of more than 90% [85]. This observation was confirmed by subsequent studies [86–88], a most recent trial reported a negative predictive value of 91% within the first 3 days after hospital admission by combining PCT and IL-6 [87]. However, opposite results have been obtained by another group who could not demonstrate a correlation between PCT levels and subsequent infection of pancreatic necrosis [89]. Besides controversies in predicting septic complications PCT has been shown to be an accurate means for early severity stratification in acute pancreatitis [53, 85–89]. Moreover, in two large Finnish studies PCT was able to predict subsequent organ failure with a sensitivity of 94% and a specificity of at least 73% already 24 h after hospital admission [53, 90]. Even by using a semiquantitative PCT strip test severe acute pancreatitis could be predicted with a sensitivity of 92% and a specificity of 84% at 24 h and all patients with evolving organ failure were correctly identified [90]. PCT determinations are mainly performed as semi-automated assays; however, a semiquantitative strip test is an attractive alternative for a fast and easy PCT determination under emergency conditions. Recently, a fully automated assay has been developed which carries the same precision and enables analysis of samples within 30 min [91]. A European-wide, multicenter trial on the clinical value of PCT in predicting septic complications in severe acute pancreatitis and peritonitis has just been closed. A total of 5 surgical centers enrolled 104 patients with severe acute pancreatitis and 82 patients with peritonitis in whom PCT was monitored on a real-time basis for up to 3 weeks after study inclusion. The final results of this trial will be published soon and demonstrate an excellent diagnostic accuracy of PCT in predicting severe infected necrosis and overall prognosis in acute pancreatitis. On the basis of the data available at present PCT is one of the most promising parameters for early severity stratification as well as monitoring the course of acute pancreatitis. In terms of the assay technique, PCT meets all demands to be run under clinical routine and emergency conditions.

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Table 1. Clinical value of biochemical parameters in predicting severity and/or infected necrosis/septic shock in patients with acute pancreatitis based on results of multicenter trials or at least two adequately powered clinical studies [4]

Parameter

SS

IN

Assay

References

Pancreatic proteases TAP CAPAP PLAP/PROP

yes (

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Phobias (Diseases and Disorders)

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Inflammatory Pancreatic Diseases, An Update: Digestive Diseases 2004

Pancreatic Cancer - An Update (Digestive Diseases, 1)

Handbook of Digestive Diseases

Pelvic Inflammatory Disease (Deadly Diseases and Epidemics)

Animal Models of Human Inflammatory Skin Diseases

Medicine - Inflammatory Diseases of the Brain

Gene Therapy for Autoimmune and Inflammatory Diseases

Diseases

Neuroprotection in Brain Ischemia: An Update (Cerebrovascular Diseases 2006)

Western Diseases: An Evolutionary Perspective

Nutrition in Gastrointestinal and Liver Diseases (Special Issue: Digestive Diseases 2003, 3)

Digestive Involvement in Systemic Autoimmune Diseases (Handbook of Systemic Autoimmune Diseases, Volume 8)

Alcohol And the Gastrointestinal Tract: Special Issue: Digestive Diseases 2005

Allergic Diseases

Colonic Diseases

Colonic Diseases

Neurodegenerative Diseases

Pleural Diseases

Diseases and Disorders - AIDS (Diseases and Disorders)

Diseases and Disorders - SARS (Diseases and Disorders)

Rickettsial Diseases

Surgical Treatment of Pancreatic Diseases (Updates in Surgery)

New Developments in the Management of Gastric Cancer (Digestive Diseases 2004)

Small animal ear diseases: an illustrated guide

Small Animal Ear Diseases: An Illustrated Guide

Phobias (Diseases and Disorders)

Tropical Diseases in Travelers

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