Inflammatory Pancreatic Diseases, An Update: Digestive Diseases 2004

Inflammatory Pancreatic Diseases: An Update Editor Joachim Mössner, Leipzig 14 ﬁgures, 2 in color and 7 tables, 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

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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

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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

© 2004 S. Karger AG, Basel 0257–2753/04/0223–0235$21.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/ddi

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

Molecular Analysis of Pancreatic Juice

Dig Dis 2004;22:235–238

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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

Review Article Dig Dis 2004;22:239–246 DOI: 10.1159/000082795

The Stress Response of the Exocrine Pancreas Vuk Savkovic´a 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

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© 2004 S. Karger AG, Basel 0257–2753/04/0223–0239$21.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

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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

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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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241

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.

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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

Stress Response of the Exocrine Pancreas

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.

References 1 Iovanna JL, Keim V, Michel R, Dagorn JC: Pancreatic gene expression is altered during acute experimental pancreatitis in the rat. Am J Physiol 1991;261:G485–G489. 2 Fiedler F, Croissant N, Rehbein C, Iovanna JL, Dagorn JC, van Ackern K, Keim V: Acutephase response of the rat pancreas protects against further aggression with severe necrotizing pancreatitis. Crit Care Med 1998;26:887– 894.

244

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3 Dusetti NJ, Tomasini R, Azizi A, Barthet M, Vaccaro MI, Fiedler F, Dagorn JC, Iovanna JL: Expression proﬁling in pancreas during the acute phase of pancreatitis using cDNA microarrays. Biochem Biophys Res Commun 2000; 277:660–667. 4 Keim V, Rohr G, Stockert HG, Haberich FJ: An additional secretory protein in the rat pancreas. Digestion 1984;29:242–249.

5 Keim V, Iovanna JL, Dagorn JC: The acute phase reaction of the exocrine pancreas. Gene expression and synthesis of pancreatitis-associated proteins. Digestion 1994;55:65–72. 6 Dagorn JC: Acute phase reaction of the exocrine pancreas. Digestion 1997; 58(suppl 1): 50–2.:50–52. 7 Bodeker H, Keim V, Fiedler F, Dagorn JC, Iovanna JL: PAP I interacts with itself, PAP II, PAP III, and lithostathine/regIalpha. Mol Cell Biol Res Commun 1999;2:150–154.

Savkovic´ /Gaiser/Iovanna/Bödeker

8 Iovanna JL, Keim V, Nordback I, Montalto G, Camarena J, Letoublon C, Levy P, Berthezene P, Dagorn JC: Serum levels of pancreatitis-associated protein as indicators of the course of acute pancreatitis. Multicentric Study Group on Acute Pancreatitis. Gastroenterology 1994; 106:728–734. 9 Sarles J, Barthellemy S, Ferec C, Iovanna J, Roussey M, Farriaux JP, Toutain A, Berthelot J, Maurin N, Codet JP, Berthezene P, Dagorn JC: Blood concentrations of pancreatitis associated protein in neonates: Relevance to neonatal screening for cystic ﬁbrosis. Arch Dis Child Fetal Neonatal Ed 1999; 80:F118– F122. 10 Tribl B, Filipp D, Bodeker H, Yu P, Hammermuller I, McKerlie C, Keim V, Sibbald WJ: Pseudomonas pneumonia-mediated sepsis induces expression of pancreatitis-associated protein-I in rat pancreas. Pancreas 2004; 29: 33–40. 11 Heller A, Fiedler F, Schmeck J, Luck V, Iovanna JL, Koch T: Pancreatitis-associated protein protects the lung from leukocyte-induced injury. Anesthesiology 1999;91:1408–1414. 12 Dusetti NJ, Mallo G, Dagorn JC, Iovanna JL: Serum from rats with acute pancreatitis induces expression of the PAP mRNA in the pancreatic acinar cell line AR-42J. Biochem Biophys Res Commun 1994;204:238–243. 13 Dusetti NJ, Ortiz EM, Mallo GV, Dagorn JC, Iovanna JL: Pancreatitis-associated protein I (PAP I), an acute phase protein induced by cytokines. Identiﬁcation of two functional interleukin-6 response elements in the rat PAP I promoter region. J Biol Chem 1995; 270: 22417–22421. 14 Ortiz EM, Dusetti NJ, Vasseur S, Malka D, Bodeker H, Dagorn JC, Iovanna JL: The pancreatitis-associated protein is induced by free radicals in AR4-2J cells and confers cell resistance to apoptosis. Gastroenterology 1998; 114:808–816. 15 Livesey FJ, O’Brien JA, Li M, Smith AG, Murphy LJ, Hunt SP: A Schwann cell mitogen accompanying regeneration of motor neurons. Nature 1997;390:614–618. 16 Nishimune H, Vasseur S, Wiese S, Birling MC, Holtmann B, Sendtner M, Iovanna JL, Henderson CE: Reg-2 is a motoneuron neurotrophic factor and a signalling intermediate in the CNTF survival pathway. Nat Cell Biol 2000;2: 906–914. 17 Malka D, Vasseur S, Bodeker H, Ortiz EM, Dusetti NJ, Verrando P, Dagorn JC, Iovanna JL: Tumor necrosis factor alpha triggers antiapoptotic mechanisms in rat pancreatic cells through pancreatitis-associated protein I activation. Gastroenterology 2000;119:816–828. 18 Vasseur S, Folch-Puy E, Hlouschek V, Garcia S, Fiedler F, Lerch MM, Dagorn JC, Closa D, Iovanna JL: p8 improves pancreatic response to acute pancreatitis by enhancing the expression of the anti-inﬂammatory protein pancreatitis-associated protein I. J Biol Chem 2004; 279:7199–7207.

Stress Response of the Exocrine Pancreas

19 Zhang H, Kandil E, Lin YY, Levi G, Zenilman ME: Targeted inhibition of gene expression of pancreatitis-associated proteins exacerbates the severity of acute pancreatitis in rats. Scand J Gastroenterol 2004;39:870–881. 20 Mallo GV, Fiedler F, Calvo EL, Ortiz EM, Vasseur S, Keim V, Morisset J, Iovanna JL: Cloning and expression of the rat p8 cDNA, a new gene activated in pancreas during the acute phase of pancreatitis, pancreatic development, and regeneration, and which promotes cellular growth. J Biol Chem 1997;272:32360–32369. 21 Motoo Y, Iovanna JL, Mallo GV, Su SB, Xie MJ, Sawabu N: P8 expression is induced in acinar cells during chronic pancreatitis. Dig Dis Sci 2001;46:1640–1646. 22 Hoffmeister A, Ropolo A, Vasseur S, Mallo GV, Bodeker H, Ritz-Laser B, Dressler GR, Vaccaro MI, Dagorn JC, Moreno S, Iovanna JL: The HMG-I/Y-related protein p8 binds to p300 and Pax2 trans-activation domain-interacting protein to regulate the trans-activation activity of the Pax2A and Pax2B transcription factors on the glucagon gene promoter. J Biol Chem 2002;277:22314–22319. 23 Encinar JA, Mallo GV, Mizyrycki C, Giono L, Gonzalez-Ros JM, Rico M, Canepa E, Moreno S, Neira JL, Iovanna JL: Human p8 is a HMGI/Y-like protein with DNA binding activity enhanced by phosphorylation. J Biol Chem 2001; 276:2742–2751. 24 Vasseur S, Hoffmeister A, Garcia-Montero A, Mallo GV, Feil R, Kuhbandner S, Dagorn JC, Iovanna JL: p8-deﬁcient ﬁbroblasts grow more rapidly and are more resistant to adriamycininduced apoptosis. Oncogene 2002; 21: 1685– 1694. 25 Garcia-Montero AC, Vasseur S, Giono LE, Canepa E, Moreno S, Dagorn JC, Iovanna JL: Transforming growth factor beta-1 enhances Smad transcriptional activity through activation of p8 gene expression. Biochem J 2001; 357:249–253. 26 Vasseur S, Hoffmeister A, Garcia-Montero A, Barthet M, Saint-Michel L, Berthezene P, Fiedler F, Closa D, Dagorn JC, Iovanna JL: Mice with targeted disruption of p8 gene show increased sensitivity to lipopolysaccharide and DNA microarray analysis of livers reveals an aberrant gene expression response. BMC Gastroenterol 2003;3:25. 27 Ruley HE: Transforming collaborations between ras and nuclear oncogenes. Cancer Cells 1990;2:258–268. 28 Vasseur S, Hoffmeister A, Garcia S, Bagnis C, Dagorn JC, Iovanna JL: p8 is critical for tumour development induced by rasV12 mutated protein and E1A oncogene. EMBO Rep 2002;3:165–170. 29 Calvo EL, Mallo GV, Fiedler F, Malka D, Vaccaro MI, Keim V, Morisset J, Dagorn JC, Iovanna JL: Clusterin overexpression in rat pancreas during the acute phase of pancreatitis and pancreatic development. Eur J Biochem 1998;254:282–289.

Dig Dis 2004;22:239–246

30 O’Sullivan J, Whyte L, Drake J, Tenniswood M: Alterations in the post-translational modiﬁcation and intracellular trafﬁcking of clusterin in MCF-7 cells during apoptosis. Cell Death Differ 2003;10:914–927. 31 Lakins J, Bennett SA, Chen JH, Arnold JM, Morrissey C, Wong P, O’Sullivan J, Tenniswood M: Clusterin biogenesis is altered during apoptosis in the regressing rat ventral prostate. J Biol Chem 1998;273:27887–27895. 32 Criswell T, Klokov D, Beman M, Lavik JP, Boothman DA: Repression of IR-inducible clusterin expression by the p53 tumor suppressor protein. Cancer Biol Ther 2003; 2: 372– 380. 33 Leskov KS, Klokov DY, Li J, Kinsella TJ, Boothman DA: Synthesis and functional analyses of nuclear clusterin, a cell death protein. J Biol Chem 2003;278:11590–11600. 34 Santilli G, Aronow BJ, Sala A: Essential requirement of apolipoprotein J (clusterin) signaling for IkappaB expression and regulation of NF-kappaB activity. J Biol Chem 2003;278: 38214–38219. 35 Heller AR, Fiedler F, Braun P, Stehr SN, Bodeker H, Koch T: Clusterin protects the lung from leukocyte-induced injury. Shock 2003;20:166– 170. 36 Wagner AC, Weber H, Jonas L, Nizze H, Strowski M, Fiedler F, Printz H, Steffen H, Goke B: Hyperthermia induces heat shock protein expression and protection against cerulein-induced pancreatitis in rats. Gastroenterology 1996;111:1333–1342. 37 Strowski MZ, Sparmann G, Weber H, Fiedler F, Printz H, Jonas L, Goke B, Wagner AC: Caerulein pancreatitis increases mRNA but reduces protein levels of rat pancreatic heat shock proteins. Am J Physiol 1997;273:G937– G945. 38 Bhagat L, Singh VP, Song AM, van Acker GJ, Agrawal S, Steer ML, Saluja AK: Thermal stress-induced HSP70 mediates protection against intrapancreatic trypsinogen activation and acute pancreatitis in rats. Gastroenterology 2002;122:156–165. 39 Lee HS, Bhagat L, Frossard JL, Hietaranta A, Singh VP, Steer ML, Saluja AK: Water immersion stress induces heat shock protein 60 expression and protects against pancreatitis in rats. Gastroenterology 2000;119:220–229. 40 Groblewski GE, Grady T, Mehta N, Lambert H, Logsdon CD, Landry J, Williams JA: Cholecystokinin stimulates heat shock protein 27 phosphorylation in rat pancreas both in vivo and in vitro. Gastroenterology 1997; 112: 1354–1361. 41 Schafer C, Clapp P, Welsh MJ, Benndorf R, Williams JA: HSP27 expression regulates CCK-induced changes of the actin cytoskeleton in CHO-CCK-A cells. Am J Physiol 1999; 277:C1032–C1043. 42 Kubisch C, Dimagno MJ, Tietz AB, Welsh MJ, Ernst SA, Brandt-Nedelev B, Diebold J, Wagner AC, Goke B, Williams JA, Schafer C: Overexpression of heat shock protein Hsp27 protects against cerulein-induced pancreatitis. Gastroenterology 2004;127:275–286.

245

43 Schafer C, Williams JA: Stress kinases and heat shock proteins in the pancreas: Possible roles in normal function and disease. J Gastroenterol 2000;35:1–9. 44 Tomasini R, Samir AA, Vaccaro MI, Pebusque MJ, Dagorn JC, Iovanna JL, Dusetti NJ: Molecular and functional characterization of the stress-induced protein (SIP) gene and its two transcripts generated by alternative splicing. SIP induced by stress and promotes cell death. J Biol Chem 2001;276:44185–44192. 45 Jiang PH, Motoo Y, Iovanna JL, Pebusque MJ, Xie MJ, Okada G, Sawabu N: Tumor protein p53-induced nuclear protein 1 (TP53INP1) in spontaneous chronic pancreatitis in the WBN/ Kob rat: Drug effects on its expression in the pancreas. JOP 2004;5:205–216. 46 Tomasini R, Samir AA, Pebusque MJ, Calvo EL, Totaro S, Dagorn JC, Dusetti NJ, Iovanna JL: P53-dependent expression of the stress-induced protein (SIP). Eur J Cell Biol 2002; 81: 294–301. 47 Tomasini R, Samir AA, Carrier A, Isnardon D, Cecchinelli B, Soddu S, Malissen B, Dagorn JC, Iovanna JL, Dusetti NJ: TP53INP1s and homeodomain-interacting protein kinase-2 (HIPK2) are partners in regulating p53 activity. J Biol Chem 2003;278:37722–37729.

246

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48 Dusetti NJ, Jiang Y, Vaccaro MI, Tomasini R, Azizi SA, Calvo EL, Ropolo A, Fiedler F, Mallo GV, Dagorn JC, Iovanna JL: Cloning and expression of the rat vacuole membrane protein 1 (VMP1), a new gene activated in pancreas with acute pancreatitis, which promotes vacuole formation. Biochem Biophys Res Commun 2002;290:641–649. 49 Vaccaro MI, Grasso D, Ropolo A, Iovanna JL, Cerquetti MC: VMP1 expression correlates with acinar cell cytoplasmic vacuolization in arginine-induced acute pancreatitis. Pancreatology 2003;3:69–74. 50 Adler G, Rohr G, Kern HF: Alteration of membrane fusion as a cause of acute pancreatitis in the rat. Dig Dis Sci 1982;27:993–1002. 51 Jiang PH, Motoo Y, Vaccaro MI, Iovanna JL, Okada G, Sawabu N: Expression of vacuole membrane protein 1 (VMP1) in spontaneous chronic pancreatitis in the WBN/Kob rat. Pancreas 2004;29:225–230. 52 Ropolo A, Tomasini R, Grasso D, Dusetti NJ, Cerquetti MC, Iovanna JL, Vaccaro MI: Cloning of IP15, a pancreatitis-induced gene whose expression inhibits cell growth. Biochem Biophys Res Commun 2004;319:1001–1009.

53 Fiedler F, Mallo GV, Bodeker H, Keim V, Dagorn JC, Iovanna JL: Overexpression of the PC3/TIS21/BTG2 mRNA is part of the stress response induced by acute pancreatitis in rats. Biochem Biophys Res Commun 1998; 249: 562–565. 54 Samir AA, Ropolo A, Grasso D, Tomasini R, Dagorn JC, Dusetti N, Iovanna JL, Vaccaro MI: Cloning and expression of the mouse PIP49 (Pancreatitis Induced Protein 49) mRNA which encodes a new putative transmembrane protein activated in the pancreas with acute pancreatitis. Mol Cell Biol Res Commun 2000;4:188–193. 55 Kaiser AM, Saluja AK, Sengupta A, Saluja M, Steer ML: Relationship between severity, necrosis, and apoptosis in ﬁve models of experimental acute pancreatitis. Am J Physiol 1995; 269:C1295–C1304. 56 Rouault JP, Falette N, Guehenneux F, Guillot C, Rimokh R, Wang Q, Berthet C, Moyret-Lalle C, Savatier P, Pain B, Shaw P, Berger R, Samarut J, Magaud JP, Ozturk M, Samarut C, Puisieux A: Identiﬁcation of BTG2, an antiproliferative p53-dependent component of the DNA damage cellular response pathway. Nat Genet 1996;14:482–486.

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Review Article Dig Dis 2004;22:247–257 DOI: 10.1159/000082796

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

© 2004 S. Karger AG, Basel 0257–2753/04/0223–0247$21.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/ddi

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

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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-

Biochemical Parameters for Severity Stratiﬁcation of Acute Pancreatitis

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

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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 (<48 h) yes (<48 h) yes (<48 h)

no no no

ELISA RIA ELISA

22–25 27–29 30, 32

Leukocyte-derived proteases PMN-elastase yes (<48 h) Type II PLA2 yes (<48 h)

no yes

IA tr-FIA

71–74 76–78

Cytokines/chemokines IL-6 IL-8

yes (<48 h) yes (<48 h)

no autom. IA septic shock autom. IA

44–50, 82 45, 48, 51, 66, 85

Acute-phase proteins CRP SAA

yes (>48 h) yes (<48 h)

no no

autom. IA autom. IA

17–19, 23, 25, 37, 71, 72, 85 37, 39

Others PCT

yes (<48 h)

yes

semiautom. and fully autom. IA, dip-stick

53, 85–90

SS = Severity stratiﬁcation (<48 h = within 48 h of disease onset, >48 h = beyond 48 h of disease onset); IN = development of infected necrosis; IA = immunoassay; RIA = radioimmunoassay; ELISA = enzyme-linked immunosorbent assay; tr-FIA = time-resolved ﬂuoroimmunoassay.

Summary and Conclusion

A broad spectrum of parameters for a biochemical severity stratiﬁcation of acute pancreatitis have been suggested and can be classiﬁed into activated pancreas- and leukocyte-derived proteases, cytokines, chemokines, and acute-phase reactants. An ideal biochemical parameter for acute pancreatitis should accurately predict severity (development of necrosis and/or organ failure) during the early stages and indicate pancreatic infections within the later course of the disease. In addition, the ideal parameter should be simple in test performance, available under routine and emergency conditions, and carry low cost. Currently, no single parameter ever tested in acute pancreatitis meets all the respective criteria; however, there are some markers that come close to these requirements (table 1). For an early severity stratiﬁcation of acute pancreatitis within 48 h of disease onset TAP, CAPAP, PMN-elastase, SAA, IL-6, IL-8, and PCT have proven to be good candidates. As fully automated assays have become commercially available, IL-6, IL-8, SAA and PCT have shown

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most favorable results and are thus the most interesting parameters in this context. Severity stratiﬁcation beyond 48 h after onset of symptoms as well as monitoring the course of acute pancreatitis has been the major domain of the acute-phase proteins. Besides CRP, SAA and LBP may be useful markers to monitor disease progression and detecting complications such as organ failure. Whereas the development of pancreatic infections or sepsis cannot be reliably predicted by any of the acute-phase proteins PCT has emerged as the most promising parameter for this purpose followed by IL-8 which is a good candidate for monitoring septic multi-organ failure. All the respective parameters are available as fully automated tests, thus problems with assay practicability are no longer a point of issue. SAA and LBP do not add substantial new aspects over CRP to the severity stratiﬁcation and monitoring of acute pancreatitis. In contrast, PCT contributes to an improved biochemical assessment of early and late complications.

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References 1 Beger HG, Bittner R, Block S, Büchler M: Bacterial contamination of pancreatic necrosis. A prospective clinical study. Gastroenterology 1986;49:433–438. 2 Johnson CD, Abu-Hilal M, Members of the British Acute Pancreatitis Study Group: Persistent organ failure during the ﬁrst week as a marker of fatal outcome in acute pancreatitis. Gut 2004;53:1340–1344. 3 Isenmann R, Rau B, Beger HG: Early severe acute pancreatitis: Characteristics of a new subgroup. Pancreas 2001;22:274–278. 4 Dervenis C, Johnson CD, Bassi C, Bradley E, Imrie CW, McMahon MJ, Modlin I: Diagnosis, objective assessment of severity, and management of acute pancreatitis. Santorini Consensus Conference. Int J Pancreatol 1999; 25: 195–210. 5 Block S, Maier W, Bittner R, Büchler M, Malfertheiner P, Beger HG: Identiﬁcation of pancreatic necrosis in severe acute pancreatitis: Imaging procedures versus clinical staging. Gut 1986;27:1035–1042. 6 Larvin M, Chalmers AG, McMahon MJ: Dynamic contrast enhanced computed tomography: A precise technique for identifying and localizing pancreatic necrosis. BMJ 1990;300: 1425–1428. 7 Gerzof SG, Banks PA, Robbins AH, Johnson WC, Spechler SJ, Wetzner SM, Snider JM, Langevin RE, Jay ME: Early diagnosis of pancreatic infection by computed tomographyguided aspiration. Gastroenterology 1987; 93: 1315–1320. 8 Rau B, Pralle U, Mayer JM, Beger HG: The role of ultrasonographically guided ﬁne-needle aspiration cytology in the diagnosis of infected pancreatic necrosis. Br J Surg 1998; 85: 179– 184. 9 Trapnell JE: The natural history and prognosis of acute pancreatitis. Ann R Coll Surg Engl 1966;38:265–287. 10 Gorelick FS, Otani T: Mechanisms of intracellular zymogen activation. Baillieres Best Pract Res Clin Gastroenterol 1999;13:227–240. 11 Chiari H: Über die Selbstverdauung des menschlichen Pankreas. Z Heilkunde 1896;17: 69–96. 12 Frossard JL: Trypsin activation peptide (TAP) in acute pancreatitis: From pathophysiology to clinical usefulness. JOP 2001;2:69–77. 13 Halangk W, Krüger B, Ruthenbürger M, Stürzebecher J, Albrecht E, Lippert H, Lerch MM: Trypsin activity is not involved in premature, intrapancreatic trypsinogen activation. Am J Physiol Gastrointest Liver Physiol 2002; 282: G367–G374. 14 Büchler M, Malfertheiner P, Uhl W, Schölmerich J, Stockmann F, Adler G, Gaus W, Rolle K, Beger HG: Gabexate mesilate in human acute pancreatitis. German Pancreatitis Study Group. Gastroenterology 1993; 104: 1165– 1170.

Biochemical Parameters for Severity Stratiﬁcation of Acute Pancreatitis

15 Hedström J, Sainio V, Kemppainen E, Haapiainen R, Kivilaakso E, Schröder T, Leinonen J, Stenman UH: Serum complex of trypsin 2 and alpha 1 antitrypsin as diagnostic and prognostic marker of acute pancreatitis: Clinical study in consecutive patients. BMJ 1996;313: 333–337. 16 McMahon M, Bowen M, Mayer A, Cooper F: Relation of alpha 2-macroglobulin and other antiproteases to the clinical features of acute pancreatitis. Am J Surg 1984;147:164–170. 17 Büchler M, Malfertheiner P, Schoetensack C, Uhl W, Beger: Sensitivity of antiproteases, complement factors and C-reactive protein in detecting pancreatic necrosis: Results of a prospective clinical study. Int J Pancreatol 1986; 1:227–235. 18 Wilson C, Heads A, Shenkin A, Imrie CW: Creactive protein, antiproteases and complement factors as objective markers of severity in acute pancreatitis. Br J Surg 1989; 76: 177– 181. 19 Dominguez-Munoz JE, Carballo F, Garcia MJ, Miguel de Diego J, Gea F, Yanguela J, de la Morena J: Monitoring of serum proteinase-antiproteinase balance and systemic inﬂammatory response in prognostic evaluation of acute pancreatitis. Results of a prospective multicenter study. Dig Dis Sci 1993;38:507–513. 20 Borgström A, Appelros S, Müller CA, Uhl W, Büchler MW: Role of activation peptides from pancreatic proenzymes in the diagnosis and prognosis of acute pancreatitis. Surgery 2002; 131:125–128. 21 Gudgeon AM, Heath DI, Hurley P, Jehanli A, Patel G, Wilson C, Shenkin A, Austen BM, Imrie CW, Hermon-Taylor J: Trypsinogen activation peptides assay in the early prediction of severity of acute pancreatitis. Lancet 1990; 335:4–8. 22 Tenner S, Fernandez-del Castillo C, Warshaw AL, Steinberg W, Hermon-Taylor J, Valenzuela JE, Hariri M, Hughes M, Banks PA: Urinary trypsinogen activation peptide (TAP) predicts severity in patients with acute pancreatitis. Int J Pancreatol 1997; 21: 105– 110. 23 Neoptolemos JP, Kemppainen EA, Mayer JM, Fitzpatrick JM, Raraty MGT, Slavin J, Beger HG, Hietaranta AJ, Poulakkainen PA: Early prediction of severity in acute pancreatitis by urinary trypsinogen activation peptide: A multicentre study. Lancet 2000;355:1955–1960. 24 Kemppainen E, Mayer J, Puolakkainen P, Raraty M, Slavin P, Neoptolemos JP: Plasma trypsinogen activation peptide in patients with acute pancreatitis. Br J Surg 2001; 88: 679– 680. 25 Johnson CD, Lempinen M, Imrie CW, Puolakkainen P, Kemppainen E, Carter R, McKay C: Urinary trypsinogen activation peptide as a marker of severe acute pancreatitis. Br J Surg. 2004;91:1027–1033.

Dig Dis 2004;22:247–257

26 Pezzilli R, Morselli-Labate AM, Barbieri AR, Plate L: Clinical usefulness of the serum carboxypeptidase B activation peptide in acute pancreatitis. JOP 2000;1:58–68. 27 Müller CA, Appelros S, Uhl W, Büchler MW, Borgström A: Serum levels of procarboxypeptidase B and its activation peptide in patients with acute pancreatitis and non-pancreatic diseases. Gut 2002;51:229–235. 28 Saez J, Martinez J, Trigo C, Sanchez-Paya J, Grino P, Company L, Laveda R, Penalva JC, Garcia C, Perez-Mateo M: A comparative study of the activation peptide of carboxypeptidase B and trypsinogen as early predictors of the severity of acute pancreatitis. Pancreas 2004;29:e9–e14. 29 Apppelros S, Petersson U, Toh S, Johnson C, Borgström A: Activation peptide of carboxypeptidase B and anionic trypsinogen as early predictors of the severity of acute pancreatitis. Br J Surg 2001;88:216–221. 30 Heath DI, Cruickshank A, Gudgeon AM, Jehanli A, Shenkin A, Imrie CW: The relationship between pancreatic enzyme release and activation and the acute-phase protein response in patients with acute pancreatitis. Pancreas 1995;10:347–353. 31 Rae D, Sumar N, Beechey-Newman N, Gudgeon M, Hermon-Taylor J: Type 1-prophospholipase A2 propeptide immunoreactivity is released from granulocytes. Clin Biochem 1995;28:71–78. 32 Mayer JM, Raraty M, Slavin J, Kemppainen E, Fitzpatrick J, Hietaranta A, Puolakkainen P, Beger HG, Neoptolemos JP: Severe acute pancreatitis is related to increased early urinary levels of the activation peptide of pancreatic phospholipase A2. Pancreatology 2002;2: 535–542. 33 Gabay C, Kushner I: Acute-phase proteins and other systemic responses to inﬂammation. N Engl J Med 1999;340:448–454. 34 Malle E, de Beer FC: Human serum amyloid A (SAA) protein: A prominent acute-phase reactant for clinical practice. Eur J Clin Invest 1996;26:427–435. 35 Schumann RR, Leong SR, Flaggs GW, Gray PW, Wright SD, Mathison JC, Tobias PS, Ulevitch RJ: Structure and function of lipopolysaccharide binding protein. Science 1990; 249:1429–1431. 36 Puolakkainen P, Valtonen V, Paananen A, Schröder T: C-reactive protein (CRP) and phospholipase A2 in the assessment of acute pancreatitis. Gut 1987;28:764–771. 37 Rau B, Steinbach G, Baumgart K, Gansauge F, Grünert A, Beger HG: Serum amyloid A versus C-reactive protein in acute pancreatitis: Clinical value of an alternative acute-phase reactant. Crit Care Med 2000;28:736–742. 38 Rau B, Steinbach G, Krüger CM, Baumgart K, Schilling M, Beger HG: Prognostic value of lipopolysaccharide-binding protein (LBP) in acute pancreatitis. Langenbecks Arch Surg 2003;388:181–188.

255

39 Mayer JM, Raraty M, Slavin J, Kemppainen E, Fitzpatrick J, Hietaranta A, Puolakkainen P, Beger HG, Neoptolemos JP: Serum amyloid A is a better early predictor of severity than Creactive protein in acute pancreatitis. Br J Surg 2002;89:163–171. 40 Rietschel ET, Kirikae T, Schade FU, Mamat U, Schmidt G, Loppnow H, Ulmer AJ, Zahringer U, Seydel U, DiPadova F, et al: Bacterial endotoxin: Molecular relationships of structure to activity and function. FASEB J 1994;8:217–225. 41 Wurfel MM, Kunitake ST, Lichenstein H, Kane JP, Wright SD: Lipopolysaccharide (LPS)-binding protein is carried on lipoproteins and acts as cofactor in the neutralization of LPS. J Exp Med 1994;180:1025–1035. 42 Norman J: The role of cytokines in the pathogenesis of acute pancreatitis. Am J Surg 1998; 175:76–83. 43 Makhija R, Kingsnorth AN: Cytokine storm in acute pancreatitis. J Hepatobiliary Pancreat Surg 2002;9:401–410. 44 de Beaux AC, Goldie AS, Ross JA, Carter DC, Fearon KC: Serum concentrations of inﬂammatory mediators related to organ failure in patients with acute pancreatitis. Br J Surg 1996;83:349–353. 45 Dugernier TL, Laterre PF, Wittebole X, Roeseler J, Latinne D, Reynaert MS, Pugin J: Compartmentalization of the inﬂammatory response during acute pancreatitis. Correlation with local and systemic complications. Am J Resp Crit Care Med 2003;168:148–157. 46 Mayer J, Rau B, Gansauge F, Beger HG: Inﬂammatory mediators in human acute pancreatitis: Clinical and pathophysiological implications. Gut 2000;47:546–552. 47 Brivet FG, Emilie D, Galanaud P: Pro- and anti-inﬂammatory cytokines during acute severe pancreatitis: An early and sustained response, although unpredictable of death. Crit Care Med 1999;27:749–755. 48 Chen CC, Wang SS, Lee FY, Chang FY, Lee SD: Proinﬂammatory cytokines in early assessment of the prognosis of acute pancreatitis. Am J Gastroenterol 1999;94:213–218. 49 Mentula P, Kylanpaa ML, Kemppainen E, Jansson SE, Sarna S, Puolakkainen P, Haapiainen R, Repo H: Plasma anti-inﬂammatory cytokines and monocyte human leucocyte antigen-DR expression in patients with acute pancreatitis. Scand J Gastroenterol 2004; 39: 178–187. 50 Heath DI, Cruickshank A, Gudgeon M, Jehanli A, Shenkin A, Imrie CW: Role of interleukin6 in mediating the acute-phase protein response and potential as an early means of severity assessment in acute pancreatitis. Gut 1993;34:41–45. 51 Messmann H, Vogt W, Holstege A, Lock G, Heinisch A, von Fürstenberg A, Leser HG, Zirngibl H, Schölmerich J: Post-ERP pancreatitis as a model for cytokine induced acutephase response in acute pancreatitis. Gut 1997; 40:80–85.

256

Dig Dis 2004;22:247–257

52 Chen CC, Wang SS, Lu RH, Lu CC, Chang FY, Lee SD: Early changes of serum proinﬂammatory and anti-inﬂammatory cytokines after endoscopic retrograde cholangiopancreatography. Pancreas 2003;26:375–380. 53 Kylänpää-Bäck ML, Takala A, Kemppainen EA, Puolakkainen PA, Leppäniemi AK, Karonen SL, Orpana A, Haapiainen RK, Repo H: Procalcitonin, soluble interleukin-2 receptor, and soluble E-selectin in predicting the severity of acute pancreatitis. Crit Care Med 2001;29:63–69. 54 Uehara S, Gothoh K, Handa H, Tomita H, Tomita Y: Immune function in patients with acute pancreatitis. J Gastroenterol Hepatol 2003;18:363–370. 55 Johnson CD: Platelet-activating factor and platelet-activating factor antagonists in acute pancreatitis. Dig Surg 1999;16:93–101. 56 Pezzilli R, Miniero R, Cappelletti O, Barakat B: Behaviour of serum interleukin-12 in human acute pancreatitis. Pancreas 1999; 18: 247–251. 57 Rau B, Baumgart K, Paszkowski AS, Mayer JM, Beger HG: Clinical relevance of caspase-1 activated cytokines in acute pancreatitis: High correlation of serum interleukin-18 with pancreatic necrosis and systemic complications. Crit Care Med 2001;29:1556–1562. 58 Chen CC, Wang SS, Lu RH, Chang FY, Lee SD: Serum interleukin 10 and interleukin 11 in patients with acute pancreatitis. Gut 1999; 45:895–899. 59 Johnson CD, Kingsnorth AN, Imrie CW, McMahon MJ, Neoptolemos JP, McKay C, Toh SKC, Skaife P, Leeder PC, Wilson P, Larvin M, Curtis LD: Double blind, randomised, placebo controlled study of a platelet activating factor antagonist, lexipafant, in the treatment and prevention of organ failure in predicted severe acute pancreatitis. Gut 2001; 48: 62– 69. 60 Dumot JA, Conwell DL, Zuccaro G Jr, Vargo JJ, Shay SS, Easley KA, Ponsky JL: A randomized, double blind study of interleukin 10 for the prevention of ERCP-induced pancreatitis. Am J Gastroenterol 2001;96:2098–2102. 61 Deviere J, LeMoine O, Van Laethem JL, Eisendrath P, Ghilain A, Severs N, Cohard M: Interleukin 10 reduces the incidence of pancreatitis after therapeutic endoscopic retrograde cholangiopancreatography. Gastroenterology 2001;120:498–505. 62 Rollins BJ: Chemokines. Blood 1997;90:909– 928. 63 Gross V, Andreesen R, Leser H-G, Ceska M, Liehl E, Lausen M, Farthmann EH, Schölmerich J: Interleukin-8 and neutrophil activation in acute pancreatitis. Eur J Clin Invest 1992; 22:200–203. 64 Pezzilli R, Billi P, Miniero R, Fiocchi M, Cappelletti O, Morselli-Labate AM, Barakat B, Sprovieri G, Miglioli M: Serum interleukin-6, interleukin-8, and 2-microglobulin in early assessment of severity of acute pancreatitis. Comparison with serum C-reactive protein. Dig Dis Sci 1995;40:2341–2348.

65 Pooran N, Indaram A, Singh P, Bank S: Cytokines (IL-6, IL-8, TNF): Early and reliable predictors of severe acute pancreatitis. J Clin Gastroenterol 2003;37:263–266. 66 Rau B, Steinbach G, Gansauge F, Mayer JM, Grünert A, Beger HG: The role of interleukin8 in the severity assessment of septic complications in necrotizing pancreatitis (abstract). Digestion 1997;58:11. 67 Bhatia M: Novel therapeutic targets for acute pancreatitis and associated multiple organ dysfunction syndrome. Curr Drug Targets Inﬂamm Allergy 2002;1:343–351. 68 Shokuhi S, Bhatia M, Christmas S, Sutton R, Neoptolemos JP, Slavin J: Levels of the chemokines growth-related oncogene a and epithelial neutrophil-activating protein 78 are raised in patients with severe acute pancreatitis. Br J Surg 2002;89:566–572. 69 Rau B, Baumgart K, Krüger CM, Schilling M, Beger HG: CC-Chemokine activation in acute pancreatitis: Enhanced release of monocyte chemoattractant protein-1 (MCP-1) in patients with local and systemic complications. Intensive Care Med 2003;29:622–629. 70 Pezzilli R, Ceciliato R, Barakat B, Corinaldesi R: Immune-manipulation of the inﬂammatory response in acute pancreatitis. What can be expected? JOP 2004;5:115–121. 71 Gross V, Schölmerich J, Leser HG Salm R, Lausen M, Ruckauer K, Schoffel U, Lay L, Heinisch A, Fahrtmann EH, et al: Granulocyte elastase in assessment of severity of acute pancreatitis: Comparison of with acute-phase protein C-reactive protein, alpha 1-antitrypsin and protease inhibitor alpha 2-macroglobulin. Dig Dis Sci 1990;35:97–105. 72 Uhl W, Büchler M, Malfertheiner P, Martini M, Beger HG: PMN-elastase in comparison with CRP, antiproteases, and LDH as indicators of necrosis in human acute pancreatitis. Pancreas 1991;6:253–259. 73 Dominguez-Munoz JE, Carbello F, Garcia MJ: Clinical usefulness of polymorphonuclear elastase in predicting the severity of acute pancreatitis. Results of a multicenter study. Br J Surg 1991;78:1230–1234. 74 Villanueva-Rodriguez A, Iglesias-Garcia J, Larino-Noia J, de Acosta MB, Iglesias-Canle J, Castroagudin JF, Dominguez-Munoz JE: Early prognostic evaluation of acute pancreatitis in clinical practice: the role of PMN-elastase (abstract). Pancreatology 2003;3:226. 75 Nevalainen T, Gronroos JM, Kortesuo PT: Pancreatic and synovial type phospholipases A2 in serum samples from patients with severe acute pancreatitis. Gut 1993;34:1133–1136. 76 Nevalainen TJ, Eskola JU, Aho AJ, Havia VT, Lovgren TN, Nanto V: Immunoreactive phospholipase A2 in serum in acute pancreatitis and pancreatic cancer. Clin Chem 1985; 31: 1116–1120. 77 Bird NC, Goodman AJ, Johnson AG: Serum phospholipase A2 activity in acute pancreatitis: An early guide to severity. Br J Surg 1989: 76:731–732.

Rau/Schilling/Beger

78 Mayer J, Rau B, Grewe M, Schoenberg MH, Nevalainen T, Beger HG: Secretory phospholipase A2 in patients with infected pancreatic necroses in acute pancreatitis. Pancreas 1998; 7:272–277. 79 Osborn L: Leukocyte adhesion to endothelium in inﬂammation. Cell 1990;62:3–6. 80 Kaufmann P, Smolle KH, Brunner GA, Demel U, Tilz GP, Krejs GJ: Relation of serial measurements of plasma-soluble intercellular adhesion molecule-1 to severity of acute pancreatitis. Am J Gastroenterol 1999; 94: 2412– 2416. 81 Powell JJ, Siriwardena AK, Fearon KCH, Ross JA: Endothelial-derived selectins in the development of organ dysfunction in pancreatitis. Crit Care Med 2001;29:567–572. 82 Wereszczynska-Siemiatkowska U, Dabrowski A, Siemiatkowski A, Mroczko B, Laszewicz W, Gabryelewicz A: Serum proﬁles of E-selectin, interleukin-10, interleukin-6, and oxidative stress parameters in patients with acute pancreatitis and nonpancreatic acute abdominal pain. Pancreas 2003;26:144–152.

Biochemical Parameters for Severity Stratiﬁcation of Acute Pancreatitis

83 Assicot M, Gendrel D, Carsin H, Raymond J, Guilbaud J, Bohuon C: High serum procalcitonin concentrations in patients with sepsis and infection. Lancet 1993;341:515–518. 84 Simon L, Gauvin F, Amre DK, Saint-Louis P, Lacroix J: Serum procalcitonin and C-reactive protein levels as markers of bacterial infection: A systematic review and meta-analysis. Clin Infect Dis 2004;39:206–217. 85 Rau B, Steinbach G, Gansauge F, Mayer JM, Grünert A, Beger HG: The potential role of procalcitonin and interleukin-8 in the prediction of infected necrosis in acute pancreatitis. Gut 1997;41:832–840. 86 Mandi Y, Farkas G, Takacs T, Boda K, Lonovics J: Diagnostic relevance of procalcitonin, IL-6, and sICAM-1 in the prediction of infected necrosis in acute pancreatitis. Int J Pancreatol 2000;28:43–51. 87 Riche FC, Cholley BP, Laisne MJC, Vicaut E, Panis YH, Lajeunie EJ, Boudiaf M, Valleur PD: Inﬂammatory cytokines, C reactive protein, and procalcitonin as early predictors of necrosis infection in acute necrotizing pancreatitis. Surgery 2003;133:257–262.

Dig Dis 2004;22:247–257

88 Ammori BJ, Becker KL, Kite P, Snider RH, Nylen ES, White JC, Barclay GR, Larvin M, McMahon MJ: Calcitonin precursors: Early markers of gut barrier dysfunction in patients with acute pancreatitis. Pancreas 2003; 27: 239–243. 89 Müller CA, Uhl W, Printzen G, Gloor B, Bischofberger H, Tcholakov O, Büchler MW: Role of procalcitonin and granulocyte colony stimulating factor in the early prediction of infected necrosis in severe acute pancreatitis. Gut 2000;46:233–238. 90 Kylänpää-Bäck ML, Takala A, Kemppainen E, Puolakkainen P, Haapiainen R, Repo H: Procacitonin strip test in the early detection of severe acute pancreatitis. Br J Surg 2001; 88: 222–227. 91 Steinbach G, Rau B, Debard AL, Javourez JF, Bienvenu J, Ponzio A, Bonfa A, Hubl W, Demant T, Külpmann WR, Buchholz J, Schumann G: Multicenter evaluation of a new immunoassay for PCT measurement on the Kryptor System. Clin Chem Lab Med 2004;42:440–449.

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Review Article Dig Dis 2004;22:258–266 DOI: 10.1159/000082797

Tropical Pancreatitis Rakesh K. Tandona Pramod K. Gargb a

Department of Gastroenterology, Pushpawati Singhania Research Institute for Liver, Renal and Digestive Diseases, and b All India Institute of Medical Sciences, New Delhi, India

Key Words Tropical pancreatitis Chronic pancreatitis Pancreatic calcification Diabetes, pancreatic SPINK1 mutation Endotherapy, pancreatic

Abstract Tropical pancreatitis is a special type of chronic pancreatitis that is seen mainly in tropical countries. The prevalence of tropical pancreatitis is about 126/100,000 population in southern India. It occurs usually in young people, involves the main pancreatic duct and results in large ductal calculi. The etiology is not known, but genetic mutations such as the SPINK1 gene mutation and environmental factors are likely causes. Clinically, 190% of patients present with abdominal pain. About 25% of patients develop diabetes which generally requires insulin for its control but is ketosis-resistant. Painless diabetes is another clinical presentation in some patients. Most patients develop malnutrition during the course of the disease. Steatorrhea is less common. Patients with tropical pancreatitis may develop pancreatic cancer as a longterm complication. The diagnosis can be established by plain radiography of the abdomen, ultrasonography, computerized tomography scan of the abdomen or endoscopic retrograde cholangiopancreatography. Management is directed towards relief from pain and control of diabetes and steatorrhea. Pain relief can be obtained

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by analgesics and enzyme supplementation with preparations rich in proteases. Endotherapy coupled with stone fragmentation by extracorporeal shock wave lithotripsy is an effective therapy for those who fail to respond to medical therapy. Surgical decompression of the main pancreatic duct by lateral pancreato-jejunostomy is reserved for patients with severe pain non-responsive to other forms of therapy. Copyright © 2004 S. Karger AG, Basel

Tropical pancreatitis (TP) is a type of chronic pancreatitis (CP) seen in tropical countries and is characterized by pancreatic calciﬁcation and ductal dilatation in a young malnourished patient who presents with abdominal pain and/or diabetes [1]. Initially described from Indonesia [2], it has been reported from many other tropical countries including India, Nigeria, Uganda, West Indies, Kenya, Sri Lanka, Madagascar and Zaire [3, 4], and has been called by a variety of names such as chronic calciﬁc pancreatitis of the tropics, juvenile TP syndrome, idiopathic chronic calciﬁc pancreatitis of the tropics, nonalcoholic TP and nutritional pancreatitis. However, the largest series has been described from south India by Geeverghese [5]. His monograph, based on his personal experience of treating and following up a large number of patients, provided the basic framework of our understanding and management approach to TP. Subsequent-

Rakesh K. Tandon, MD, PhD, FRCP (Edin) Department of Gastroenterology Pushpawati Singhania Research Institute for Liver, Renal and Digestive Diseases New Delhi (India) Tel. +91 11 26968304, Fax +91 11 26569559, E-Mail [email protected]

ly, there have been many reports of its occurrence in other parts of India (particularly Karnataka, Tamil Nadu, Orissa, Maharashtra and Delhi) [6], and more recently from China and other countries [7].

Epidemiology and Clinical Features

Since TP occurs most commonly in Kerala, we conducted an epidemiological study in one of its districts (Quilon) in the 1980s to discover the prevalence and characteristics of this disease in the community [8]. All 6,079 families living in the district of Quilon were visited and a total of 28,567 individuals (male:female ratio 1:1.08) were interviewed over a period of 1.5 years. Of these, 483 individuals were selected for a detailed study on the basis of the presence of abdominal pain (157 people), diabetes mellitus (266 people) or malabsorption/malnutrition (60 people). Based on those studies, a diagnosis of CP was established in 36 of 483 persons: 11 with predominantly abdominal pain; 19 with diabetes mellitus, and 6 with symptoms suggestive of malabsorption/malnutrition (table 1). Of these 36 patients, 28 had calciﬁc pancreatitis, whereas 8 had non-calciﬁc pancreatitis. Thus, the prevalence of CP in this community was 1: 793 (36/28,567; 126/100,000 population), whereas the prevalence of calciﬁc pancreatitis alone was 1: 1,020 (28/28,567; 98/100,000 population). This is in contrast to the estimated prevalence of CP of around 10–15/100,000 population in several Western industrialized countries and 45.4/100,000 population in Japan [9, 10]. Such a high prevalence of CP in India suggests that it is an endemic zone for CP and points towards a possible genetic and/or environmental factor as playing an important etiologic role. Of the 36 patients with TP in our survey, 13 were male and 23 were female (male:female ratio 1:1.8). The mean 8 SD age at the onset of symptoms was 23.9 8 12.0 years,

while at the time of diagnosis of TP it was 32.8 8 12.6 years. These demographic and clinical features in the 36 patients with TP were quite different from those described in patients with TP reporting to the hospitals and physicians’ ofﬁces, which formed the bulk of patients in earlier reports including those in Geeverghese’s [5] monograph (table 2). In all earlier hospital-based series, males far outnumbered females. None of the 36 patients in our community-based study consumed alcohol or had any other known cause of CP. The symptoms were much milder in our community-based patients than in hospitalbased patients described in different series. Thus, the symptoms among patients in the community were: abdominal pain (30.6%); diabetes mellitus (52.8%), and malabsorption (16.7%). In contrast, patients presenting to hospitals or family physicians have severe CP. Abdominal pain is the predominant symptom in these cases (approximately 80%) and calciﬁcation is seen in 80–90% of patients. Diabetes has generally been described as severe, often requiring more that 100 units insulin. However, the diabetes in our community-based patients could be controlled either by dietary changes and oral hypoglycemics or modest doses of insulin (!60 units/day). The clinical features of hospital-based TP patients also differ from those of alcoholic pancreatitis in many re-

Table 1. Diagnosis of chronic pancreatitis from the screening of 28,567 individuals in Kerala, India [8]

Symptom complex

Number of patients

Chronic pancreatitis calciﬁc

non-calciﬁc

Abdominal pain Diabetes mellitus Malabsorption

157 266 60

8 16 4

3 3 2

Total

483

28

8

Table 2. Tropical calciﬁc pancreatitis:

differences between clinical features in hospitalized and community-based patients [8]

Tropical Pancreatitis

Mean age, years (range) Sex, male:female Mean age at onset of symptoms Abdominal pain, % Steatorrhea, % Calciﬁcation, % Diabetes, % Insulin, units/day

Hospital patients

Community patients

21 (10–30) 1.6–3.8:1 childhood/adolescence 30–90 infrequent 80–90 80 60–200

29 (11–42) 1:1.8 23.9 years 30.6 16.7 52.8 61 only 50% needed <60

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259

Table 3. Differences in clinical features of tropical and alcoholic chronic pancreatitis

Clinical features

Tropical

Age at presentation, years Age at onset of pain, years Duration of pain, years Age at onset of diabetes, years Duration of diabetes, years Body mass index, kg/m2 Fasting plasma glucose, mg/dl Glycosylated hemoglobin, % Fecal chymotrypsin, U/g

32.6 22.9 8.2 30.2 4.7 18.9 190.9 10.4 4.6

Alcoholic p value 44.1 39.9 4.2 40.9 3.7 18.4 200.1 10.6 5.3

<0.001 <0.001 <0.001 <0.001 NS NS NS NS NS

Adapted from Chari et al. [54].

spects (table 3). Some of the special features of TP patients are a much younger age at onset, large ductal calculi, mainly in the head region, insulin-dependent but ketosis-resistant diabetes, and a high incidence of carcinoma of the pancreas [11]. Because these features were identiﬁed from hospital-based patients, the patients clearly belonged to the severe disease group. Indeed, 37.1% of TP patients required surgical treatment in one series. This is why Geeverghese [5] aptly described the natural history of calciﬁc TP as recurrent abdominal pain in childhood, diabetes at puberty and death in the prime of life.

How Is TP Different from Other Forms of CP?

The following features in a patient are characteristic of TP and distinguish it from other types of CP: (1) young age at onset; (2) residence in tropics; (3) nonalcoholic; (4) no other discernible cause of CP; (5) negative family history of pancreatitis; (6) large duct disease with ductal dilatation; (7) large pancreatic calculi predominantly in the head region; (8) presentation with chronic abdominal pain; (9) diabetes which requires insulin but is ketosisresistant, and (10) malnourishment.

Etiology

TP forms about 59% of all our patients with CP [7]. The etiology of TP is not known and is still considered idiopathic. However, certain potential etiological factors

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have been identiﬁed. Among them, genetic predisposition is most favored by recent data [7]. That and other factors are discussed below. Malnutrition Protein calorie malnutrition has long been suspected as a likely cause of TP because of the fact that the disease occurs predominantly in tropical countries where malnutrition is common and because reports from some of them, including India, Uganda and Nigeria, reveal that 80–90% of the subjects with calciﬁc pancreatitis come from the poor socioeconomic strata. Chronic protein undernutrition leads to structural as well as functional alterations in the pancreas. It also makes females more susceptible to pancreatic toxins. However, severe malnutrition is not associated with CP but with pancreatic atrophy and insufﬁciency [12, 13]. In a prospective study of 105 north Indian patients with CP, we found that the mean body mass index of patients was 22.89 8 3.28 which was similar to that of controls suggesting that malnutrition was not a cause of TP. On the other hand, 80% of patients lost weight following the onset of the disease as a consequence of poor intake and steatorrhea suggesting therefore that malnutrition was an effect and not a cause of CP [14]. Pancreatic Stasis Prolonged lack of food in stomach, and/or gastroenteritis and dehydration lead to stasis in the pancreas and blockage of the pancreatic duct by thickened mucus plugs, most of which get dislodged by the vigorous ﬂow of pancreatic juice in the convalescence phase when the patient takes protein-containing food. The sluggish pancreatic ﬂow produced by a protein-poor diet may not dislodge these mucus blocks. The plugs may enlarge due to repeated gastrointestinal infections and anorexia and ultimately undergo calciﬁcation. Such a setting is common in tropical countries and that may predispose to the development of TP [15]. However, this hypothesis has been discounted by the fact that the majority of patients with TP do not consume a signiﬁcantly protein-deﬁcient diet. Environmental Pancreatic Toxin The toxic hypothesis has been centered on consumption of cassava which contains a cyanogenic glycoside and is used liberally in southern India where TP is endemic [16]. This theory has also not found wide acceptance for the following reasons: (1) cassava does not feature in the diet of many people who develop TP; (2) there was no

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difference in cassava consumption between patients with TP and those without [17]; (3) patients with TP from northern India do not consume cassava, and (4) long-term cassava consumption did not produce diabetes or pancreatitis in a rat model [18]. Free Radical Injury The Manchester group [19] have shown that patients with alcoholic pancreatitis as well as other forms of CP including TP are deﬁcient in antioxidants and hence are more vulnerable to free radical injury. They have further shown that supplementation with antioxidants may result in a signiﬁcant decrease in analgesic requirements in patients with alcoholic pancreatitis [20]. A preliminary study from India has also shown that patients with TP do have increased free radical-mediated injury as evidenced by high levels of malondialdehyde and decreased antioxidant levels [21]. Autoimmune Mechanism An autoimmune mechanism causing TP has been postulated [5] on the basis of observations of the presence of round cells and eosinophilic inﬁltration in the pancreas, hypergammaglobulinemia and an alteration in cell-mediated immunity. However, strong evidence is not yet available to support this hypothesis.

tients with idiopathic CP, i.e. 2.5 and 11.5 times the expected frequency seen in the general population [26, 27]. Affected patients with CP were shown to have single-gene CFTR mutations and/or the 5T allele in intron 8 which resulted in a reduced activity of CFTR. In patients with typical cystic ﬁbrosis, there are severe mutations affecting both alleles; the result is pancreatic insufﬁciency caused by atrophy of the pancreas. On the other hand, a mutation affecting only one allele may result in diseases such as CP while retaining ‘pancreatic sufﬁciency’. In a preliminary study, we did not ﬁnd common CFTR gene mutations in patients with TP [28]. More recently, a mutation in the pancreatic secretory trypsin inhibitor (also known as serine protease inhibitor Kajal type 1 or SPINK1; N34S, chromosome 5) was found in 23% of patients with idiopathic pancreatitis versus 2% in the general population [29]. A similar mutation has been reported in Japanese patients with idiopathic pancreatitis [30]. SPINK1 inhibits trypsin within the pancreas but accounts for inactivation of only 20% of all activated trypsin [31]. It is therefore unlikely that the SPINK1 mutation alone will cause pancreatitis, but it might be a disease-modiﬁer lowering the threshold for pancreatitis [32]. A SPINK1 mutation has been found in 32–44% of TP patients in India [33, 34]. At present, intense search is on in many laboratories around the world to discover more mutations in patients with CP. There is every possibility that, in the near future, the genetic basis of CP will be further clariﬁed. Further genetic analyses are also urgently required in patients with TP.

Genetic Factors The landmark discovery by Whitcomb et al. [22] of a mutation in the gene for cationic trypsinogen on the long arm of chromosome 7 (7q35) in patients with hereditary pancreatitis veriﬁed the long-held belief that a genetic defect underlies hereditary pancreatitis. A lot of interest has recently been generated in the possibility that there may be a genetic basis for TP because of the following similarities between TP and hereditary pancreatitis: (1) both diseases affect young individuals; (2) calciﬁcation is very common in both, and (3) there is an increased risk of pancreatic cancer in both. Moreover, Indians born in Kerala but residing outside India continue to have an increased prevalence of TP [16]. An association of HLA DQ 9(A*0201-B*03003) has been shown with TP and diabetes (ﬁbrocalculous pancreatic diabetes, FCPD) [23]. However, one study from Bangladesh failed to show any mutation of the cationic trypsinogen gene among 13 patients with TP [24]. Another study did not ﬁnd a cationic trypsinogen gene mutation in 46 patients with FCPD [25]. Two groups demonstrated that the expected frequency of the cystic ﬁbrosis transmembrane conductance regulator (CFTR) gene mutation was much higher among pa-

The diagnosis of TP is based on a combination of clinical evaluation and imaging studies. In advanced disease, a plain ﬁlm of the abdomen or a contrast-enhanced computerized tomography may show pancreatic calciﬁcation and establish the diagnosis (ﬁg. 1, 2). In early cases, demonstration of ductal changes through endoscopic retrograde cholangiopancreatography or magnetic resonance cholangiopancreatography will establish the diagnosis (ﬁg. 3, 4). Pancreatic function tests are indeed the most sensitive for detecting the earliest changes in the exocrine pancreas [35], but they may be abnormal in any cause of

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Other Factors Parasitic infestation and other stray factors have been suggested but not proven in the causation of TP.

Diagnosis

261

CP-ERCP

Fig. 1. Plain radiograph of the abdomen showing a chain of calci-

ﬁcations all along the main pancreatic duct.

Fig. 3. Endoscopic retrograde cholangiopancreatography picture showing marked pancreatic ductal dilation.

Fig. 2. Contrast-enhanced computerized tomography showing ex-

Fig. 4. Magnetic resonance cholangiopancreatography showing the

tensive calciﬁcation in the pancreatic duct and parenchyma.

extensive ductal dilation.

pancreatic insufﬁciency, e.g. cystic ﬁbrosis and not necessarily in CP. Endoscopic ultrasonography has been touted as the most sensitive method of detecting the earliest changes of pancreatitis in the parenchyma, but its value remains to be established [36]. The gold standard for di-

agnosis is histopathology but that is rarely obtained unless the patient undergoes a pancreatic resection. Intraductal protein plugs with or without calciﬁcation and periductal ﬁbrosis with little inﬂammatory inﬁltration are characteristic features of TP (ﬁg. 5).

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Fig. 5. Histopathological slides showing intraductal protein plug (a) and periductal ﬁbrosis with very little inﬂammatory inﬁltration (b). Paucity of inﬂammatory inﬁltrate differentiates TP from alcoholic chronic pancreatitis which is characterized by signiﬁcant inﬂammatory inﬁltration.

Consequences of TP

TP can lead to endocrine and exocrine insufﬁciency like any other CP, the difference being that the degree of functional impairment is much more pronounced and early in TP compared with other forms of CP. Exocrine impairment leads to maldigestion and steatorrhea. Clinical steatorrhea is uncommon even in patients with advanced TP largely due to the restriction of fat consumption by the patients. Steatorrhea can be managed well with supplementation of oral pancreatic enzymes with a high lipase content. Endocrine insufﬁciency leads to pancreatic diabetes. Diabetes is present in 25–90% of patients with TP. Such a wide difference in the prevalence of diabetes is mainly due to the referral pattern. Patients presenting with diabetes as the major clinical problem are referred to diabetes clinics and data coming from such clinics often report a high prevalence of diabetes in patients with TP. On the other hand, diabetes is prevalent in about 31% of patients with TP in our gastroenterology clinic [7]. There are many special characteristics of diabetes in TP which are discussed below. Diabetes in TP Patients with TP develop diabetes during the course of the disease. Patients with calciﬁcation are more likely to develop diabetes. Overall, up to 60% of patients with TP may develop diabetes. Many patients with painless

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TP present primarily with diabetes. These patients are initially misdiagnosed as having insulin-dependent diabetes mellitus. FCPD is the term given to patients with painless calciﬁc pancreatitis with diabetes [37]. FCPD was earlier classiﬁed by the WHO as malnutrition-related diabetes mellitus as most of the patients with FCPD are malnourished. Diabetes in patients with TP is described as particularly severe, requiring high doses of insulin. Diabetes may be brittle in patients with TP with frequent episodes of hypoglycemia. This may be due to concomitant exocrine insufﬁciency. Patients with pancreatic diabetes usually require insulin for its control. However, the characteristic feature of the diabetes associated with TP is that patients with it are ketosis-resistant, i.e. even if insulin is withheld they do not develop ketosis. The possible reasons for ketosis resistance are better insulin reserve compared with insulin-dependent diabetes mellitus and low glucagon response to glucose load [37, 38]. It was believed that more than 90% of patients with TP would require insulin for the control of diabetes. However, the current experience has shown that up to one third of patients can be managed with oral hypoglycemic agents [7]. The insulin requirement was also thought to be very high (up to 100 units/day), but it has been shown that the majority of patients can be managed with regular doses of insulin. Patients with diabetes and TP may develop all the macro- and microvascular complications of poorly controlled diabetes if they survive long enough [37].

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263

Management

Medical treatment of TP is similar to that of any CP and is aimed at relieving pain and steatorrhea and controlling diabetes [1]. Pain Relief For pain relief initially non-opioid and later opioid analgesics are used. Another approach has been to use pancreatic enzymes (proteases) based on the understanding that delivering these enzymes in the duodenum could result in suppression of cholecystokinin and hence a decrease in pancreatic exocrine secretion. Their role in relieving pain is, however, questionable. The results of a meta-analysis of 6 randomized controlled trials showed no beneﬁt from enzyme therapy in relieving pain [39]. However, non-enteric coated pancreatic enzyme supplementation may relieve pain in patients with small pancreatic duct disease, idiopathic pancreatitis and in female patients [40–42]. The Asia-Paciﬁc consensus report on CP also suggests pancreatic enzymes and non-opioid analgesics as the initial therapy for pain relief in patients with CP [43]. Use of antioxidants has also been suggested recently for pain relief in CP. A combination of antioxidants containing at least 2 g methionine/day may help relieve pain if continued for about a year [20]. We have also shown that antioxidant supplementation relieves pain in TP [44]. Surgery is required predominantly for intractable pain in about a third of the patients. Its results are good only in patients with a dilated ductal system which is the case in the majority of TP patients. The most common operation performed is lateral pancreatojejunostomy (modiﬁed Puestow’s operation). In one of our studies, relief of pain was obtained in 90% of patients 3 months after the operation and this relief was long lasting (5 years) in 82% of patients [45]. Comparable results have been obtained from other centers in India [46]. Surgical drainage may also improve the control of diabetes in patients with TP [45]. The results of surgical drainage are less gratifying in CP in the Western world, as the predominant etiology there is alcohol abuse and the behavior of alcoholic CP may be different from that of TP. In occasional patients with pancreatitis restricted to one portion of the gland or in those patients with normal size ducts or in those having recurrent pain following lateral pancreato-jejunostomy, pancreatic resection or splanchnicectomy has been performed with fairly good results, but often with unavoidable exocrine and endocrine deﬁciencies or relapse [47].

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Endoscopic Therapy What has been achieved by surgery can now be done by endoscopy. Thus, the dilated pancreatic ductal system can be decompressed by endoscopic sphincterotomy and pancreatic ductal stone clearance by a combination of basketing and extracorporeal shock wave lithotripsy. These maneuvers have indeed yielded gratifying results. Various endoscopic series have reported 50–70% success for clearing the main pancreatic duct and 60–80% longterm pain relief with complications of !10% [48–50]. We have also found good results of endotherapy in 60% of patients with TP [51]. The results of endoscopic treatment are comparable with the surgical results, but the problem is that all endoscopic series have been case series and no controlled prospective trial is available. Furthermore, long-term results need to be interpreted in light of the fact that many patients get spontaneous relief from pain due to ‘burning out of the disease’ [52]. Thus, it is important to discover the true beneﬁt of endoscopic therapy in the long run. Randomized controlled studies comparing endoscopic and surgical treatment modalities are required. Until such time that these studies become available, however, most endoscopists would prefer a trial of endoscopic therapy before subjecting the patient to surgery if the medical therapy has failed, as the initial results of endoscopic therapy are encouraging and the patients prefer less invasive procedures. One study has recently been published which has shown comparable results of surgical and endoscopic treatment for pain relief in CP [53].

Conclusion

TP is a type of CP that occurs in the tropics and affects young patients. Its diagnosis is established by clinical evaluation and imaging, particularly plain ﬁlm of the abdomen, ultrasound and/or CT scan of the abdomen showing pancreatic calculi. Many etiological factors have been suspected, but genetic mutations appear as the most likely cause. Treatment is aimed at relieving pain and steatorrhea and controlling diabetes.

Tandon/Garg

References 1 Tandon RK: Tropical calciﬁc pancreatitis. J Gastroenterol Hepatol 1998; 13(suppl):S284– S248. 2 Zuidema PJ: Cirrhosis and disseminated calciﬁcation of the pancreas in patients with malnutrition. Trop Geogr Med 1959;11:70–74. 3 Sarles H, Payan H, Sahel J: Chronic pancreatitis, relapsing pancreatitis and calciﬁcations of pancreas. Gastroenterology 1976; 3: 1040– 1051. 4 Diabetes mellitus. Report of a WHO study group. World Health Organ Tech Rep Ser 1985;727:1–113. 5 Geeverghese PH: Pancreatic Diabetes: A Clinicopathological Study of Growth Onset Diabetes with Pancreatic Calculi. Bombay, Popular Prakastan, 1968. 6 Balakrishnan V: Chronic calciﬁc pancreatitis in the tropics. Ind J Gastroenterol 1984;3:65– 67. 7 Garg P, Tandon RK: Survey on chronic pancreatitis in the Asia-Paciﬁc region. J Gastroenterol Hepatol 2004;19:998–1004. 8 Balaji LN, Tandon RK, Tandon BN, Banks A: Prevalence and clinical features of chronic pancreatitis in southern India. Int J Pancreatol 1994;15:29–34. 9 Copenhagen pancreatitis study. An interim report from a prospective epidemiological multicentre study. Scand J Gastroenterol 1981;16: 305–312. 10 Lin Y, Tamakoshi A, Matsuno S, Takeda K, Hayakawa T, Kitagawa M, Naruse S, Kawamura T, Wakai K, Aoki R, Kojima M, Ohno Y: Nationwide epidemiological survey of chronic pancreatitis in Japan. J Gastroenterol 2000;35: 136–141. 11 Chari ST, Mohan V, Pitchumoni CS, et al: Risk of pancreatic carcinoma in tropical calcifying pancreatitis. An epidemiological study. Pancreas 1994;9:62–66. 12 Brooks SE, Golden MH: The exocrine pancreas in kwashiorkor and marasmus. Light and electron microscopy. West Indian Med J 1992; 41:56–60. 13 Balakrishnan V, Sauniere JF, Hariharan M, Sarles H: Diet, pancreatic function, and chronic pancreatitis in south India and France. Pancreas 1988;3:30–35. 14 Singh N, Sethi M, Garg PK, Joshi YK, Tandon RK: Malnutrition and chronic pancreatitis: A prospective study of cause and effect relationship. Indian J Gastroenterol 2002; 21(suppl): A42. 15 Barbezat GO, Hansen JDL: The exocrine pancreas and protein calorie malnutrition. Pediatrics 1968;42:77. 16 Narendranathan M: Chronic calciﬁc pancreatitis of the tropics. Trop Gastroenterol 1981; 2:40–45. 17 Narendranathn M, Cheriyan A: Lack of association between cassava consumption and tropical pancreatitis syndrome. J Gastroenterol Hepatol 1994;9:282–285.

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18 Mathangi DC, Deepa R, Mohan V, Govindaranjan M, Namasivayam A: Long-term ingestion of cassava (tapioca) does not produce diabetes or pancreatitis in the rat model. Int J Pancreatol 2000;27:203–208. 19 Braganza JM, Schoﬁeld D, Snehalatha C, Mohan V: Micronutrient antioxidant status in tropical compared with temperate-zone chronic pancreatitis. Scand J Gastroenterol 1993;28: 1098–104. 20 McCloy R: Chronic pancreatitis at Manchester, UK. Focus on antioxidant therapy. Digestion 1998;59(suppl 4):36–48. 21 Chouduary A, Garg PK, Tandon RK: The role of oxidative stress in tropical pancreatitis and effect of antioxidants supplementation on pain in patients with tropical pancreatitis (abstract). J Gastroenterol Hepatol 2001; 16(suppl): A132. 22 Whitcomb DC, Gorry MC, Preston RA, Furey W, Sossenheimer MJ, Urich CD, Martin SP, Gates LK Jr, Amann ST, Toskes PP, Liddle R, McGrath K, Umomo G, Post JC, Ehrich GD: Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat Genet 1996;14:141–145. 23 Sanjeevi CB, Kanungo A, Shtanvera A, Samal KC, Tripathi BB: Association with HLA class II alleles with different subgroups of diabetes mellitus in eastern India identify different associations with IDDM and malnutrition related diabetes. Tissue Antigen 1999;54:83–87. 24 Rossi L, Whitcomb DC, Ehrich GD, et al: Lack of R117H mutation in the cationic trypsinogen gene in patients with tropical pancreatitis from Bangladesh. Pancreas 1998;54:83–87. 25 Hassan Z, Mohan V, McDermott MF, et al: Pancreatitis in ﬁbrocalculous pancreatic diabetes mellitus is not associated with common mutations in the trypsinogen gene. Diabetes Metab Res Rev 2000;16:454–457. 26 Sharer N, Schwarz M, Malone G, Howarth A, Painter J, Super M, Braganza J: Mutations of the cystic ﬁbrosis gene in patients with chronic pancreatitis. N Engl J Med 1998; 339: 645– 652. 27 Cohn JA, Friendman KJ, Noone PG, Knowles MR, Silverman LM, Jowell PS: Relation between mutations of the cystic ﬁbrosis gene and idiopathic pancreatitis. N Engl J Med 1998; 339:653–658. 28 Makharia GM, Kabra M, Garg PK, Shastri SS, Tandon RK: Cystic ﬁbrosis transmembrane conductance regulator gene mutations (F508 and 3849+10KB C]T) in patients with chronic calciﬁc pancreatitis of tropics. J Gastroenterol Hepatol 2001;(suppl)16:A112. 29 Witt H, Luck W, Hennies HC, Classen M, Kage A, Lass U, Landt O, Becker M: Mutations in the gene encoding the serine protease inhibitor, Kazal type 1 are associated with chronic pancreatitis. Nat Genet 2000;25:213–216.

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30 Kaneko K, Nagasaki Y, Furukawa T, Mizutamari H, Sato A, Masamune A, et al: Analysis of the human pancreatic secretory trypsin inhibitor (PSTI) gene mutation in Japanese patients with chronic pancreatitis. J Hum Genet 2001;46:293–297. 31 Rinderknecht H: Pancreatic secretory enzymes; in Go VLW, DiMagno EP, Gardner JD, Lebenthal E, Reber HA, Scheele GA (eds): The Pancreas: Biology, Pathobiology and Disease, ed 2. New York, Raven Press, 1993, pp 219–251. 32 Pfutzer RH, Barmada MM, Brunskil APJ, Finch R, Hart PS, Neoptolemos J, et al: SPINK1/PSTI polymorphisms act as disease modiﬁers in familial and idiopathic chronic pancreatitis. Gastroenterology 2000;119:615– 623. 33 Bhatia E, Choudhuri G, Sikora SS, Landt O, Kage A, Becker M, Witt H: Tropical calciﬁc pancreatitis: Strong association with SPINK1 trypsin inhibitor mutations. Gastroenterology 2002;123:1020–1025. 34 Chandak GR, Idris MM, Reddy DN, Mani KR, Bhaskar S, Rao GV, Singh L: Absence of PRSS1 mutations and association of SPINK1 trypsin inhibitor mutations in hereditary and non-hereditary chronic pancreatitis. Gut 2004; 53:723–728. 35 Homma T, Harada H, Koizumi M: Diagnostic criteria for chronic pancreatitis by the Japan Pancreas Society. Pancreas 1997;15:14–15. 36 Catalano MF, Lahoti S, Geenen JE, Hogan WJ: Prospective evaluation of endoscopic ultrasonography, endoscopic retrograde pancreatography, and secretin test in the diagnosis of chronic pancreatitis. Gastrointest Endosc 1998;48:11–17. 37 Mohan V, Nagalotimath SJ, Yajnik CS, Tripathy BB: Fibrocalculous pancreatic diabetes. Diabetes Metab Rev 1998;14:153–170. 38 Sidhu SS, Shah P, Prasanna BM, Srikanta SS, Tandon RK: Chronic calciﬁc pancreatitis of the tropics (CCPT): Spectrum and correlates of exocrine and endocrine pancreatic dysfunction. Diabetes Res Clin Pract 1995; 27: 127– 132. 39 Brown A, Hughes M, Tenner S, Banks PA: Does pancreatic enzyme supplementation reduce pain in patients with chronic pancreatitis: A meta-analysis. Am J Gastroenterol 1997;92: 2032–2035. 40 Slaff J, Jacobson D, Tillman CR, et al: Protease speciﬁc suppression of pancreatic exocrine secretion. Gastroenterology 1984;87:44–52. 41 Isaksson G, Ihse I: Pain reduction by an oral pancreatic enzyme preparation in chronic pancreatitis. Dig Dis Sci 1983;28:97–102. 42 Warshaw AL, Banks PA, Fernandez-Del Castillo C: AGA technical review: Treatment of pain in chronic pancreatitis. Gastroenterology 1998;115;765–776. 43 Tandon RK, Sato N, Garg PK: Chronic pancreatitis: Asia-Paciﬁc consensus report. J Gastroenterol Hepatol 2002;17:508–518.

265

44 Nandi B, Garg PK, Bhardwaj P, Prakash S, Tandon RK: Efﬁcacy of antioxidants for pain relief in patients with chronic pancreatitis: A randomized controlled trial. Ind J Gastroenterol 2002;21(suppl):A43. 45 Sidhu SS, Nundy S, Tandon RK: The effect of the modiﬁed Puestow procedure on diabetes in patients with tropical chronic pancreatitis: A prospective study. Am J Gastroenterology 2001;96:107–111. 46 Thomas PG, Augustine P, Ramesh H, Rangabashyam N: Observations and surgical management of tropical pancreatitis in Kerala and southern India. World J Surg 1990;14:32–42. 47 Izbicki RJ, Bloechle C, Knoefel WT, Rogiers X, Kuechler T: Surgical treatment of chronic pancreatitis and quality of life after operation. Surg Clin North Am 1999;79:913–944.

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48 Delhaye M, Vandermeeren A, Baize M, et al: Extracorporeal shock-wave lithotripsy of pancreatic calculi. Gastroenterology 1992; 102: 610–620. 49 Adamek HE, Jakobs R, Buttmann A, Adamek MU, Schneider AR, Riemann JF: Long term follow up of patients with chronic pancreatitis and pancreatic stones treated with extracorporeal shock wave lithotripsy. Gut 1999;45:402– 405. 50 Rosch T, Daniel S, Scholz M, et al: Endoscopic treatment of chronic pancreatitis: A multicenter study of 1000 patients with long-term follow-up. Endoscopy 2002;34:765–771.

51 Garg PK, Seth A, Gupta NP, Tandon RK: Endoscopic pancreatic sphincterotomy plus extracorporeal shock wave lithotripsy is effective treatment for tropical pancreatitis (abstract). J Gastroenterol Hepatol 2000;15(suppl):A204. 52 Ammann RW, Akovbinatz A, Largiader F, et al: Course and outcome of chronic pancreatitis. Gastroenterology 1984;86:820–828. 53 Dite P, Ruzicka M, Zboril V, Novotny I: A prospective, randomized trial comparing endoscopic and surgical therapy for chronic pancreatitis. Endoscopy 2003;35:553–558. 54 Chari ST, Mohan V, Jayanthi V, et al: Comparative proﬁles of alcoholic chronic pancreatitis and tropical chronic pancreatitis in Tamil Nadu, south India, Panceas 1992;7:52–58.

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Review Article Dig Dis 2004;22:267–272 DOI: 10.1159/000082798

Pathogenesis of Pain in Chronic Pancreatitis Pierluigi Di Sebastiano Fabio Francesco di Mola Markus W. Büchler Helmut Friess Department of General Surgery, University of Heidelberg, Heidelberg, Germany

Key Words Pancreatitis, chronic Intraductal pressure Fibrosis Interstitial hypertension Ischemia

Abstract The pathophysiology of pain in chronic pancreatitis (CP) is incompletely understood. Several hypotheses have been advanced, including pancreatic and extrapancreatic causes. The existence of different hypotheses to explain the genesis of pain in CP also reflects the different therapeutic approaches to pain in these patients. Increased intraductal pressure as a result of single or multiple strictures and/or calculi is believed to be a common cause of pain in CP patients with a dilated main pancreatic duct. Other suggested causes include pancreatic fibrosis, interstitial hypertension and pancreatic ischemia. Additionally, extrapancreatic causes like duodenal and common bile duct stenosis with scarring due to pancreatic inflammation are suggested as factors causing pain in CP. The ‘neurogenic inflammation’ hypothesis is a fascinating theory which is supported by different studies. Immunohistological reports have shown that the amount of neurotransmitters, such as substance P and its receptor, calcitonin gene-related peptide and other neurotransmitters, are increased in afferent pancreatic nerves and a correlation between pain and immune cell infiltration of the nerves has been reported in CP. In this review we will discuss the different pain hypotheses and will present the perspective that neuroimmune interaction is an important factor for pain generation in CP.

Introduction

Chronic pancreatitis (CP) is a progressive, destructive inﬂammatory process that ends in total destruction of the pancreas and results in malabsorption, diabetes mellitus, and severe pain. The etiology of CP is probably multifactorial, with about 65–70% of the cases being attributed to alcohol abuse. The remaining cases are classiﬁed as idiopathic CP (ICP; 20–25%), including tropical pancreatitis, which is a major cause of childhood CP in tropical regions, or unusual causes including hereditary pancreatitis, cystic ﬁbrosis (CF), and CP-associated metabolic and congenital factors [1, 2]. However, the most clinically relevant feature of CP is recurrent upper abdominal pain. Pain can be so intense and long-lasting that the follow-up care of patients is difﬁcult and frustrating [3, 4]. Many patients become addicted to narcotics. Several attempts have been made to evaluate pain in CP by using questionnaires and pain scales (e.g., a visual analog scale) [5, 6], but it is difﬁcult to get sufﬁcient data since intensity, radiation and duration are not constant. Several hypotheses have been advanced, including pancreatic and extrapancreatic causes. Three different typical pain proﬁles during the evolution of CP have been described: (a) repeated episodes of acute pancreatitis (acinar necrosis) in early stages; (b) spontaneous lasting pain relief in association with severe pancreatic dysfunction in late stage of uncomplicated CP, and (c) persistent severe pain (or frequent recurrent episodes of pain) usually in association with local complications such as pseudocysts, ductal hypertension or extrapancreatic complications

Copyright © 2004 S. Karger AG, Basel

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Pierluigi Di Sebastiano, MD Department of General Surgery, University of Heidelberg In Neuenheimer Feld 110, DE–69120 Heidelberg (Germany) Tel. +49 6221 56 4326, Fax +49 6221 56 4863 E-Mail [email protected]

such as partial obstruction of the common bile duct, peptic ulcer, and opiate addiction [7]. In this review we will discuss different pain hypotheses and will present the perspective that neurogenic inﬂammation plays a major role in pain generation in CP.

Extrapancreatic Pain

Bile duct stenosis and duodenal stenosis due to extensive pancreatic ﬁbrosis and inﬂammation have been considered extrapancreatic causes of pain [8, 9]. In 19.5% of 600 patients with CP, Becker and Mischke [10] described a pathological condition named ‘groove pancreatitis’. This is characterized by the formation of a scar plate between the head of the pancreas and the duodenum. A scar in the groove is said to lead to complications that are determined by the topography: disturbance in the motility of the duodenum, stenosis of the duodenum, and tubular stenosis of the common bile duct, occasionally leading to obstructive jaundice. These alterations are suggested to be responsible for several symptoms present in CP and for postprandial pain due to the compression of nerves and ganglia located between the pancreatic head and the duodenum [11].

atic dysfunction and calciﬁcations. However, the perception that the painful pancreas will burn itself out is not supported by other studies. The burn-out theory in CP has been questioned by epidemiological data which show that, despite pancreatic insufﬁciency, pain in many patients with CP continues the appearance of calciﬁcations, alcohol withdrawal or pancreatic surgery. It has been estimated that around 30% of the patients treated with decompressive surgery still exhibit recurrent attacks of pain [18]. In addition, octreotide, a somatostatin analog which strongly inhibits pancreatic secretion and therefore should interrupt this postulated pain cycle described above, failed to signiﬁcantly reduce the pain syndrome in many patients with chronic pancreatitis [19]. Ebbehoj [14] reported a direct relationship between pain intensity and intraductal pancreatic pressure before and after decompressive surgery. In contrast to this study, Manes et al. [17] found no relationship between pain score and pancreatic pressure, although the intrapancreatic pressure was positively correlated with ductal changes. Pancreatic pressure was signiﬁcantly higher in CP than in controls. Postoperatively, pancreatic pressure decreased by 15.3% in 4 patients with CP in whom pressure assessment was repeated after surgical decompression. They concluded that pancreatic parenchymal pressure is not closely related to pain in CP [17].

Pancreatic Pain

Increased Intrapancreatic Pressure Many investigators have related the origin of pain to increased pressure in pancreatic ducts and tissue [12–15]. The ductal hypertension hypothesis as an explanation for pain in CP is supported by observations that decompression of a dilated pancreatic duct or pseudocyst frequently relieves pain [16]. Pancreatic enzyme supplementation may also relieve pain in some CP patients due to the cholecystokinin-mediated feedback regulation of pancreatic exocrine secretion by the activity of proteases in the lumen of the small intestine [17]. According to this hypothesis, administration of pancreatic enzymes reduces hypercholecystokininemia in patients with CP, thus resulting in less stimulation of the pancreas, producing lower intraductal pressure and thereby reducing pain [17]. Interestingly, pancreatic insufﬁciency appearing several years after diagnosis of CP may be accompanied by a reduction or complete relief of pain, thus suggesting that the disease can burn itself out [8]. Amman et al. [11] observed pain relief a median of 4.5 years after onset. Pain relief was accompanied by a marked increase in pancre-

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Pancreatic Ischemia Another hypothesis suggests that pain is induced when increased pancreatic ductal and parenchymal pressure produce a compartment syndrome that causes ischemia [20]. This hypothesis is supported by experimental studies [21] showing that increased interstitial pressure correlates with decreased blood ﬂow in a feline model of chronic pancreatitis. These abnormalities were reversed by surgical incision of the gland and draining the pancreatic duct, but were minimally affected by stenting the pancreatic duct. This would suggest that incision of the gland may be more important in relieving pain than ductal drainage. Pancreatic Fibrosis CP is characterized by the presence of intra- and perilobular ﬁbrosis that leads to irreversible scarring. The pathogenesis of pancreatic ﬁbrogenesis is still unclear, but a common concept is that ﬁbrosis leads to increased intraductal pressure in the chronically inﬂamed pancreas and thereby to pain during the course of CP [22]. However, recent studies [23] revealed that the degree of pan-

Di Sebastiano/di Mola/Büchler/Friess

creatic ﬁbrosis has no signiﬁcant inﬂuence on pain generation since no correlation between the degree of ﬁbrosis and the intensity of pain could be demonstrated. Pancreatic Pseudocysts Pseudocysts of the pancreas can cause intense pain in CP patients. In the majority of the cases (60%) treatment with octreotide results in a reduction in size and in the eventual disappearance of the pseudocysts together with a reduction in pain [24]. Enlargement of pseudocysts, causing compression of adjacent structures, might be a mechanism for pain generation.

Inflammation in the Pancreas

Acute Inﬂammation In many patients recurrent attacks of acute inﬂammation lead to severe abdominal pain. The inﬂammatory process, involving activated enzymes and other injurious substances, could be responsible for pain generation. A recent report showed increased expression of the neurotropin, nerve growth factor (NGF), during the course of experimental acute pancreatitis in the rat [25]. In human CP neurotropin gene expression correlates with the intensity of pain [26]. Comparing these data we can speculate that similar pathogenetic mechanisms operate. However, this possibility should be investigated further. Neurogenic Inﬂammation Keith et al. [27] were the ﬁrst to suggest that neural and perineural alterations might be important in pain pathogenesis in CP. They concluded that pain severity correlated with the duration of alcohol consumption, pancreatic calciﬁcation, and with the percentage of eosinophils in perineural inﬂammatory cell inﬁltrates, but not with duct dilatation. A subsequent study demonstrated an increase in both the number and diameter of pancreatic nerve ﬁbers in the course of CP [28]. In tissue specimens from patients suffering from CP, foci of chronic inﬂammatory cells were often found surrounding the pancreatic nerves, which by electron microscopic analysis exhibit a damaged perineurium and invasion by lymphocytes. The changed pattern of intrinsic and possibly extrinsic innervation of the pancreas in CP suggested that there could be an upregulation of neuropeptides that usually populate those enlarged nerves. In fact, a further study showed [29] that there were striking changes in peptidergic nerves in CP. The changes consisted of an intensiﬁcation of immunostain-

Pathogenesis of Pain in Chronic Pancreatitis

ing for calcitonin gene-related peptide (CGRP) and substance P (SP) in numerous nerve ﬁbers. Because both these peptides are generally regarded as pain neurotransmitters, these ﬁndings provided evidence for direct involvement of pancreatic nerves in the long-lasting pain syndrome in CP. Subsequent reports [23, 30] revealed that the presence of growth-associated protein-43 (GAP43), an established marker of neuronal plasticity, directly correlated with the pain scores in patients with CP. GAP43 is a neuronal protein known to be involved in the development of axonal growth cones and presynaptic terminals, and mRNA and protein levels of GAP-43 are increased after neuronal lesions. In the chronically inﬂamed human pancreas, enzymatic and double ﬂuorescence immunohistochemistry revealed a signiﬁcant expression of GAP-43 in the majority of pancreatic nerve ﬁbers. These immunohistochemical ﬁndings correlated with clinical and pathological ﬁndings in CP patients, including the parenchyma-ﬁbrosis ratio and the degree of perineural immune cell inﬁltration. Furthermore, a strong relationship with individual pain scores was present. The inﬁltration of pancreatic nerves by immune cells is signiﬁcantly related to pain intensity, whereas pain scores do not correlate with the degree of pancreatic ﬁbrosis or with the duration of the disease. The demonstration of a direct relationship between the degree of perineural inﬂammation and the clinical pain syndrome strongly supports the hypothesis of ‘neuroimmune interaction’ as an important, if not predominant, factor in pain generation in CP patients. An interesting question concerns the mechanisms that contribute to the enlargement of pancreatic nerves. A recent study analyzed the expression of nerve growth factor (NGF) and one of its receptors (TrkA) in patients suffering from CP [26]. NGF belongs to the neurotropin family and plays a role in neuroblast proliferation and neuronal maturation, affecting neuronal phenotype and maintaining neuronal survival. NGF signaling is mediated via binding high- and low-afﬁnity receptors. TrkA is present in dorsal root and peripheral ganglia cells of primary sensory nerves, and is involved in signal transduction of noxious stimuli and tissue injury. Inﬂammation results in an elevation of NGF levels in different diseases. Interestingly, NGF may itself have cytokine-like functions; it can modify mast-cell, macrophage and B-cell functions, but may also activate TrkA located on sensory and sympathetic nerve ﬁbers innervating the site of inﬂammation, thus modulating neuroimmune interactions. In CP tissue samples, NGF and TrkA mRNA expression are markedly increased and enhanced in pancreatic nerves and ganglia. Comparison of the molecular ﬁndings

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269

Fig. 1. Substance P pathway in chronic pancreatitis.

with clinical parameters revealed a signiﬁcant relationship between NGF mRNA levels and pancreatic ﬁbrosis and acinar cell damage and between TrkA mRNA levels and pain intensity. These ﬁndings indicate that the NGF/ TrkA pathway is activated in CP and that this activation might inﬂuence nerve growth and the pain syndrome, most probably by modulating the sensitivity of NGF-independent primary sensory neurons through increasing channel and receptor expression [26]. Similar results, showing a positive correlation with pain intensity and frequency in patients suffering from CP, were reported for brain-derived neurotropic factor (BDNF) gene expression, a member of the neurotropin family [31]. In addition, upregulated NGF might inﬂuence the pain syndrome in CP patients by regulating transcription and synthesis of SP and CGRP, as well as through the release of histamine. The neuropeptide SP is the main tachykinin involved in neural transmission of sensory information, smooth muscle contraction, nociception, sexual behavior and possibly wound healing and tissue regeneration [32, 33]. SP has wide-ranging functional effects, including the cross-talk between nervous and immune systems by acting through its speciﬁc receptor, neurokinin-1 (NK-1R). A recent report by Shrikande et al. [34] demonstrated a signiﬁcant correlation between NK-1R and clinical-pathological ﬁndings in CP patients. In CP samples, NK-1R mRNA expression and protein were localized mainly in

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nerves, ganglia, blood vessels, inﬂammatory cells and occasionally in ﬁbroblasts. A signiﬁcant relationship between NK-1R mRNA levels and intensity, frequency and duration of pain in CP patients was reported. The expression of NK-1R in inﬂammatory cells and blood vessels also points to a cross-talk between immunoreactive SP nerves and inﬂammatory cells and blood vessels, and further supports the existence of a neuroimmune interaction that probably inﬂuences the pain syndrome and chronic inﬂammatory changes in CP. The exact mechanisms that are involved in the interaction between inﬂammatory cells and nerves and ganglia – neuroimmune cross-talk – are not yet fully clariﬁed. Different cytokines have been shown to interact with SP in various paradigms for pain and inﬂammation. SP directly stimulates the release of interleukin-8 (IL-8) from macrophages. IL-8 release generates hyperalgesia by stimulation of post-ganglionic sympathetic neurons. A significant increase in IL-8 mRNA was reported in CP tissue samples [35]. IL-8 was present mainly in macrophages surrounding the enlarged pancreatic nerves, in remaining acinar cells and often in ductal cells. IL-8 mRNA expression was positively correlated with the inﬂammatory score and the presence of ductal metaplasia in CP tissue samples. The reported ﬁndings in the literature on the interaction of SP and IL-8, in combination with what was reported in CP, suggest that the increased mRNA expression of IL-8 in CP could in part be mediated by SP released from sensory pancreatic nerves. In addition, the release of IL-8 from the remaining exocrine pancreatic parenchyma suggests the fascinating hypothesis of an intrinsic maintenance of the inﬂammatory response after the ﬁrst damage to the pancreatic gland, thus sustaining progression and evolution of the disease (ﬁg. 1).

Experimental Models

The majority of the data discussed above are based on studies of human tissue specimens obtained from patients with CP, and the absence of a valid animal model of CP creates a lack of knowledge in understanding the pathogenesis of pain in CP. Animal models for CP are difﬁcult to develop, since it is difﬁcult to mimic the typical clinical and morphological features. Established models focus on different aspects of CP, but do not combine alterations observed in humans. One approach to research pancreatic pain is to study the phenomenon of referred hypersensitivity, typical of visceral pain. These

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ﬁndings were exploited to develop two recently described rat models of pancreatic pain. Vera-Portocarrero et al. [36] induced ‘persistent’ pancreatitis by systemic application of dibutylin dichloride in rats and showed an increase in withdrawal events after von Frey ﬁlament stimulation of the abdomen and decreased withdrawal latency after thermal stimulation during a period of 7 days, indicating a ‘sensitized nociceptive’ state accompanied by increased levels of substance P, but not CGRP levels in the spinal cords. However, a major question remains about the clinical relevance of rat models. Chemically induced inﬂammatory models may not completely reproduce all aspects of human pancreatic pathology, including changes in the duct and gross destruction of the architecture of the pancreatic lobules. Nevertheless, these models represent useful systems to investigate molecular changes in the pancreas and the afferent nervous system that contribute to pain in pancreatitis and to test potential novel analgesics.

Conclusion

The mechanism for the generation and continuation of chronic pain in CP remains a major clinical challenge. The recent concept of neuropeptides released from enteric and afferent neurons, and their functional interactions with inﬂammatory cells, might play a key role. The most interesting ﬁndings in CP is the presence of a spatial relationship between peptidergic neurons and inﬂammatory cells. Furthermore, there is the intriguing possibility of functional interaction among neuropeptides, immune cells, cytokines and nerve growth factors [37, 38]. A correlation between those molecular data and pain has been demonstrated in CP and the present information provides evidence for neuroimmune cross-talk in the pathogenesis of pain and inﬂammation in CP. If validated, these ﬁndings may have major implications for the pathogenesis of pain in CP and will provide novel targets for therapy. However, multifactorial elements are involved and this may well explain why all patients do not respond to the same treatment modality.

References 1 Steer ML, Waxman I, Freedman S: Chronic pancreatitis. N Engl J Med 1995; 332: 1482– 1490. 2 Di Sebastiano P, di Mola FF, Friess H, Büchler MW: Chronic pancreatitis: The perspective of pain generation by neuroimmune interaction. Gut 2003;6:906–910. 3 Beger HG, Büchler M, Malfertheiner P (eds): Standards in Pancreatic Surgery. New York, Springer, 1993, pp 41–46. 4 Warshaw AL, Banks PA, Fernandez-Del Castillo C: AGA Technical review: Treatment of pain in chronic pancreatitis. Gastroenterology 1998;115:765–776. 5 Beger HG, Krautzberger W, Bittner R, Büchler M: Duodenum-preserving resection of the head of the pancreas in patients with severe chronic pancreatitis. Surgery 1985; 97: 467– 473. 6 Glasbrenner B, Adler G: Evaluating pain and the quality of life in chronic pancreatitis. Int J Pancreatol 1997;22:163–170. 7 Jensen AR, Matzen P, Malchow-Moller A, Christoffersen I: Pattern of pain, duct morphology and pancreatic function in chronic pancreatitis: A comparative study. Scand J Gastroenterol 1984;19:334–338. 8 Lankisch PG, Lohr-Happe A, Otto J, Creutzﬁeldt W: Natural course of chronic pancreatitis. Pain, exocrine and endocrine pancreatic insufﬁciency and prognosis of the disease. Digestion 1993;54:148–155.

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9 Levy P, Lesur G, Belghiti J, Fekete F, Bernades P: Symptomatic duodenal stenosis in chronic pancreatitis: A study of 17 cases in a medical surgical series of 306 patients. Pancreas 1993; 8:563–567. 10 Becker V, Mischke U: Groove pancreatitis. Int J Pancreatol 1991;10:173–182. 11 Amman RW, Muellhaupt B, Zürich Pancreatitis Study Group: The natural history of pain in alcoholic chronic pancreatitis. Gastroenterology 1999;116:1132–1140. 12 Manes G, Pieramico O, Uomo G: Pain in chronic pancreatitis: Recent pathogenetic ﬁndings. Minerva Gastroenterol Dietol 1992; 38: 137–43. 13 Bradley EL 3d: Pancreatic duct pressure in chronic pancreatitis. Am J Surg 1982; 144: 313–316. 14 Ebbehoj N: Pancreatic tissue ﬂuid pressure and pain in chronic pancreatitis. Dan Med Bull 1992;39:128–133. 15 Ebbehoj N, Borly L, Bulow J, Rasmussen SG, Madsen P, Matzen P, Owre A: Pancreatic tissue ﬂuid pressure in chronic pancreatitis. Relation to pain, morphology, and function. Scand J Gastroenterol 1990;25:1046–1051. 16 Ebbehoj N, Borly L, Madsen P, Matzen P: Pancreatic tissue ﬂuid pressure during drainage operations for chronic pancreatitis. Scand J Gastroenterol 1990;25:1041–1045. 17 Manes G, Büchler M, Pieramico O, Di Sebastiano P, Malfertheiner P: Is increased pancreatic pressure related to pain in chronic pancreatitis? Int J Pancreatol 1994;15:113–117.

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18 Beger HG, Schlosser W, Friess HM, Büchler MW: Duodenum-preserving head resection in chronic pancreatitis changes the natural course of the disease: A single-center 26-year experience. Ann Surg 1999;230:512–519. 19 Malfertheiner P, Mayer D, Büchler M, Dominguez-Munoz JE, Schiefer B, Ditschuneit H: Treatment of pain in chronic pancreatitis by inhibition of pancreatic secretion with octreotide. Gut 1995;36:450–454. 20 Reber HA, Karanjia ND, Alvarez C, Widdison AL, Leung FW, Ashley SW, Lutrin FJ: Pancreatic blood ﬂow in cats with chronic pancreatitis. Gastroenterology 1992;103:652–659. 21 Karanjia ND, Widdison AL, Leung F, Alvarez C, Lutrin FJ, Reber HA: Compartment syndrome in experimental chronic obstructive pancreatitis: Effect of decompressing the main pancreatic dust. Br J Surg 1994;81:259–264. 22 di Mola FF, Friess H, Martignoni ME, Di Sebastiano P, Zimmermann A, Innocenti P, Graber H, Gold LI, Korc M, Büchler MW: Connective tissue growth factor is a regulator for ﬁbrosis in human chronic pancreatitis. Ann Surg 1999;230:63–71. 23 Di Sebastiano P, Fink T, Weihe E, Friess H, Innocenti P, Beger HG, Büchler MW: Immune cell inﬁltration and growth-associated protein 43 expression correlate with pain in chronic pancreatitis. Gastroenterology 1997; 112: 1648–1655. 24 Gullo L, Barbara L: Treatment of pancreatic pseudocysts with octreotide. Lancet 1991;338: 540–541.

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25 Toma H, Winston J, Micci MA, Shenoy M, Pasricha PJ: Nerve growth factor expression is up-regulated in the rat model of L-arginine-induced acute pancreatitis. Gastroenterology 2000;119:1373–1381. 26 Friess H, Zhu ZW, di Mola FF, Kulli C, Graber HU, Andren-Sandberg Å, Zimmermann A, Korc M, Reinshagen M, Büchler MW: Nerve growth factor and its high afﬁnity receptor in chronic pancreatitis. Ann Surg 1999;230:615– 624. 27 Keith RG, Keshavjee SH, Kerenyi NR: Neuropathology of chronic pancreatitis in humans. Can J Surg 1985;28:207–211. 28 Bockman DE, Büchler M, Malfertheiner P, Beger HG: Analysis of nerves in chronic pancreatitis. Gastroenterology 1988;94:1459–1469. 29 Büchler M, Weihe E, Friess H, Malfertheiner P, Bockman E, Müller S, Nohr D, Beger HG: Changes in peptidergic innervation in chronic pancreatitis. Pancreas 1992;7:183–192.

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30 Fink T, Di Sebastiano P, Büchler M, Beger HG, Weihe E: Growth associated protein-43 and protein gene product 9.5 innervation in human pancreas: Changes in chronic pancreatitis. Neuroscience 1994;63:249–266. 31 Zhu ZW, Friess H, Wang L, Zimmermann A, Büchler MW: Brain-derived neurotrophic factor (BDNF) is upregulated and associated with pain in chronic pancreatitis. Dig Dis Sci 2001; 46:1633–1639. 32 Di Sebastiano P, Weihe E, di Mola FF, Fink T, Innocenti P, Friess H, Büchler MW: Neuroimmune appendicitis. Lancet 1999; 354: 461– 466. 33 Weihe E, Nohr D, Müller S, Büchler M, Friess H, Zentel HJ: The tachykinin neuroimmune connection in inﬂammatory pain. Ann NY Acad Sci 1991;632:283–295.

34 Shrikande S, Friess H, di Mola FF, Tempia A, Conejio-Garcia JR, Zhu Z, Zimmermann A, Büchler MW: NK-1 receptor gene expression is related to pain in chronic pancreatitis. Pain 2001;91:209–217. 35 Di Sebastiano P, di Mola FF, Di Febbo C, Baccante G, Porreca E, Innocenti P, Friess H, Büchler MW: Expression of interleukin-8 (IL8) and substance P in human chronic pancreatitis. Gut 2000;47:423–428. 36 Vera-Portocarrero L, Lu Y, Westlund K: Nociception in persistent pancreatitis in rats: Effects of morphine and neuropeptide alterations. Anesthesiology 2003;98:474–484. 37 Stanisz AM, Stanisz JA: Nerve growth factor and neuroimmune interactions in inﬂammatory diseases. Ann NY Acad Sci 2000; 917: 268–72. 38 Levi-Montalcini R, Skaper SD, Dal Toso R, Petrelli L, Leon A: Nerve growth factor: From neurotrophin to neurokine. Trends Neurosci 1996;19:514–20.

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Review Article Dig Dis 2004;22:273–279 DOI: 10.1159/000082799

Mechanisms of Pancreatic Fibrosis M.V. Apte J.S. Wilson Pancreatic Research Group, University of New South Wales, Sydney, Australia

Key Words Pancreatic fibrosis Stellate cells, pancreatic Chronic pancreatitis

Abstract Pancreatic fibrosis, a characteristic histopathological feature of chronic pancreatitis, is no longer considered an epiphenomenon of chronic injury, but an active process that may be reversible in the early stages. The identification and characterization of pancreatic stellate cells (PSCs) in recent years has had a significant impact on research into pancreatic fibrogenesis. Accumulating evidence from both in vivo studies (using human pancreatic sections and experimental models of pancreatic fibrosis) and in vitro studies (using cultured pancreatic stellate cells) indicates a key role for activated PSCs in the fibrotic process. These cells are now known to be activated by ethanol and its metabolites and by several factors that are upregulated during pancreatic injury including growth factors, cytokines and oxidant stress. Based on this knowledge, potential antifibrotic strategies such as antioxidants and cytokine inhibition have been assessed in experimental models. Studies are also underway to characterise the signaling pathways/molecules responsible for mediating PSC activation, in order to identify potential therapeutic targets for the inhibition of PSC activation, thereby preventing or reversing the development of pancreatic fibrosis. Copyright © 2004 S. Karger AG, Basel

© 2004 S. Karger AG, Basel 0257–2753/04/0223–0273$21.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/ddi

Introduction

Pancreatic ﬁbrosis is a constant histopathological feature of chronic pancreatitis of varying aetiologies, even though the distribution of ﬁbrosis may differ according to the cause, as reported in a recent article by Kloppel et al. [1]. Thus, alcoholic chronic pancreatitis has been shown to be characterised by inter (peri)-lobular ﬁbrosis, hereditary pancreatitis by periductal ﬁbrosis, autoimmune pancreatitis by periductal and interlobular ﬁbrosis and obstructive chronic pancreatitis by intralobular ﬁbrosis. Fibrosis is generally deﬁned as the accumulation of excessive amounts of extracellular matrix proteins in a tissue. In health, the normal architecture of the pancreas is maintained by a delicate balance between extracellular matrix (ECM) synthesis and degradation. In pathological states, this balance is signiﬁcantly disturbed leading to the deposition of excessive quantities of ECM proteins and the development of ﬁbrosis. It is now generally accepted that ﬁbrosis is not a mere end-product of chronic injury, but an active, dynamic process that may be reversible in the early stages. An understanding of the mechanisms responsible for the development of ﬁbrosis has the potential to lead to the development of therapeutic strategies to prevent or retard the ﬁbrotic process. Research into pancreatic ﬁbrogenesis is a rapidly expanding ﬁeld, one that was given signiﬁcant impetus by the recent development of methods to isolate and culture pancreatic stellate cells (PSCs). These methods provided a much-needed in vitro tool to characterise the morphology and function of these cells and evidence

Assoc. Prof. Minoti Apte Pancreatic Research Group Room 463, Level 4, Health Services Building, Liverpool Hospital Campbell Street, Liverpool, NSW 2170 (Australia) Tel. +61 2 9828 4931, Fax +61 2 9828 4970, E-Mail [email protected]

accumulated over the past 5 years indicates a key role for PSCs in the production of pancreatic ﬁbrosis [2–5]. PSCs are located in the immediate vicinity of pancreatic acini and have long cytoplasmic processes that encircle the basal aspect of acinar cells. In their quiescent (normal) state, PSCs store vitamin A (as evidenced by the characteristic, rapidly fading blue-green ﬂuorescence emitted by the cells on exposure to UV light) in the form of lipid droplets in the cytoplasm [2, 3]. Electron microscopy reveals a prominent rough endoplasmic reticulum, collagen ﬁbrils and vacuoles (lipid droplets) surrounding a central nucleus [3]. PSCs can be identiﬁed using immunostaining techniques for selective markers including desmin (a cytoskeletal intermediate ﬁlament), glial ﬁbrillary acidic protein, neural cell adhesion molecule and the neurotrophin nerve growth factor [2, 6]. The ability to culture PSCs has enabled a detailed study of their morphology and function. Phase contrast microscopy of PSCs in early culture (!48 h), reveal ﬂattened polygonal cells with abundant vitamin A-containing lipid droplets in their cytoplasm. The cells stain positive for markers such as desmin and glial ﬁbrillary acidic protein in a pattern characteristic of cytoskeletal proteins. After culture on uncoated plastic for 48 h, PSCs assume an activated, myoﬁbroblast-like phenotype, readily identiﬁed by the expression of -smooth muscle actin (SMA, a cytoskeletal protein) in the cells. Numerous biological functions of PSCs have now been well studied. These include their ability to (i) proliferate [4, 7]; (ii) migrate [8]; (iii) synthesise and secrete the extracellular matrix proteins that make up ﬁbrous tissue (collagen, laminin, ﬁbronectin) [2], and (iv) synthesise and secrete matrix-degrading enzymes (matrix metalloproteinases, MMPs) and their inhibitors (tissue inhibitors of metalloproteinases) [9]. The capacity of PSCs to not only synthesise but also degrade ECM proteins suggests that, in health, PSCs play an important role in the maintenance of normal architecture of the pancreas by regulating ECM turnover.

Role of PSCs in Fibrogenesis

Studies investigating the role of PSCs in pancreatic ﬁbrogenesis have used both in vivo and in vitro approaches. The in vivo approach has involved examination of pancreatic tissue from patients with chronic pancreatitis (predominantly alcohol-induced) and from animal models of pancreatic ﬁbrosis, while the in vitro approach has involved the study of the response of cultured PSCs to activating factors.

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In vivo Studies Immunohistochemical and histological examination of pancreatic sections from patients with chronic pancreatitis has been reported by two recent studies by Haber et al. [10] and Casini et al. [11]. Both studies provide evidence to support a role for PSCs in human pancreatic ﬁbrosis. They have established the following. (i) Collagen is the predominant extracellular matrix protein in areas of ﬁbrosis, as indicated by the Sirius red stain for collagen. (ii) Areas that stain positive for collagen also exhibit positive immunostaining for SMA, suggesting the presence of activated PSCs in ﬁbrotic areas. (iii) SMA-positive cells in ﬁbrotic areas are the only cells that exhibit positive staining for messenger RNA for collagen (as assessed by in situ hybridization) indicating that activated PSCs may be the principal source of the collagen deposited in the ﬁbrotic pancreas (ﬁg. 1). (iv) Pancreatic acinar cells adjacent to areas of ﬁbrosis exhibit strongly positive staining for transforming growth factor- (TGF, a known proﬁbrogenic growth factor for stellate cells), while such staining is absent in acinar cells remote from bands of ﬁbrosis. These observations suggest that TGF secreted by pancreatic acinar cells may have a paracrine effect on PSCs, leading to increased collagen synthesis by the cells. TGF staining is also apparent in spindle-shaped cells in the ﬁbrotic bands. (v) Fibrotic areas of the pancreas exhibit signiﬁcant staining for 4-hydroxynonenal (a lipid peroxidation product), indicating increased oxidant stress in the vicinity of activated PSCs. This is an important observation given the in vitro ﬁndings that PSCs respond to oxidant stress by activation (detailed below). (vi) Expression of the receptor for platelet-derived growth factor (PDGF) is signiﬁcantly increased (at both mRNA and protein levels) in areas of ﬁbrosis in chronic pancreatitis. In view of the fact that PDGF is a potent mitogenic and chemotactic factor for stellate cells, increased PDGF receptor expression on the PSCs may be one of the mechanisms responsible for the increased numbers of PSCs observed in ﬁbrotic areas. While human studies have provided important insights into the presence of activated PSCs in pancreatic ﬁbrosis, they are limited by their point-in-time nature. In contrast, experimental models of pancreatic ﬁbrosis have enabled chronological observations, thereby allowing assessment of early changes in PSCs during the development of pancreatic injury. Several rodent models of pancreatic ﬁbrosis have been described in the literature. Rat models include: (i) trinitrobenzene sulfonic acid injection

Apte/Wilson

Fig. 1. Dual staining of human chronic pancreatitis section for SMA and procollagen 1I mRNA. Parafﬁn section of the pancreas from a patient with chronic pancreatitis stained for SMA (a marker of activated stellate cells) using immunostaining techniques and for procollagen 1I mRNA using in situ hybridisation. Spindle-shaped cells in ﬁbrotic areas exhibited staining for both factors. Line diagram in the inset indicates the pattern of staining: nuclear staining for procollagen 1I mRNA, and cytoplasmic staining for -SMA. These ﬁndings indicate that SMA-positive cells are the predominant source of collagen in ﬁbrotic areas in chronic pancreatitis.

into the pancreatic duct [10]; (ii) intravenous injection of an organotin compound dubutyltin chloride [12]; (iii) spontaneous chronic pancreatitis in WBN/Kob rats [13, 14]; (iv) severe hyperstimulation obstructive pancreatitis (involving supramaximal cerulein by intraperitoneal injection + bile-pancreatic duct ligation) [15]; (v) repeated by intraperitoneal injections of a superoxide dismutase inhibitor [16]; (vi) intragastric high dose alcohol administration + repeated cerulein injections [17, 18], and (vii) chronic alcohol administration (liquid diet) with repeated cyclosporin and cerulein injections [19]. Mouse models of pancreatic ﬁbrosis that have examined the role of PSCs include: (i) transgenic mice overexpressing TGF [20], and (ii) repetitive pancreatic injury by repeated injections of supramaximal cerulein [21]. Using immunohistochemistry, in situ hybridization and histology, the above studies have examined the effects on PSCs over the time-course of injury. In general, the ﬁndings indicate that PSCs are activated early in the course of the injury and are the predominant source of collagen in ﬁbrotic areas (conﬁrming the results observed in human studies). Increased numbers of PSCs in areas of ﬁbrosis have also been demonstrated in the above animal models. Interestingly, in the trinitrobenzene sulfonic

In vitro Studies The ﬁndings of in vivo studies discussed above suggest that during pancreatic injury, PSCs proliferate, transform into their activated phenotype and synthesise increased amounts of ECM proteins leading to the development of pancreatic ﬁbrosis. In order to identify the speciﬁc factors responsible for PSC activation during pancreatic injury, researchers have turned to in vitro studies using cultured PSCs. In general, the selection of putative activating factors for study has been based upon current knowledge of in vivo events during pancreatic injury, such as the production of oxidant stress and the release of growth factors and cytokines including TGF, PDGF, tumour necrosis factor- (TNF) and the interleukins 1, 6 and 8 (IL1, IL6 and IL8). In addition, factors that may have direct toxic effects on PSCs have also been examined. These include ethanol and its metabolites acetaldehyde and fatty acid ethyl esters. PSC activation in response to exposure to the above factors

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acid model [10], receptor expression for the mitogen PDGF was also found to be increased, providing a possible mechanism for the proliferation of PSCs in ﬁbrotic areas (this ﬁnding concurs well with that described by Casini et al. [11] in human chronic pancreatitis).

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MMP2

Densitometer units (% of control)

72 kD

250 200 150 100 50 0 Control

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1.0

TGFβ1 (ng/ml)

Fig. 2. Effect of TGF1 on MMP2 secretion by pancreatic stellate cells. A representative Western blot is shown for MMP2 expression in cells incubated for 24 h with either culture medium alone (control) or with TGF1 (0.5 or 1 ng/ml). Densitometry of all Western blots (n = 5 separate cell preparations) showed a signiﬁcant increase in MMP2 levels in PSCs incubated with 0.5 and 1 ng/ml TGF1 compared to controls (*p ! 0.05).

in vitro has been assessed using at least one or more of a number of parameters such as cell proliferation, SMA expression, ECM protein synthesis, matrix degradation via the production of MMPs, loss of vitamin A stores, cell migration, cytokine release and contractility. The ﬁndings of these studies may be summarized as follows. (i) PDGF is a potent mitogenic and chemotactic factor for PSCs [8, 22, 23]. (ii) TGF is a potent ﬁbrogenic cytokine inducing the synthesis and secretion of collagen, ﬁbronectin and laminin by PSCs [4, 7]. TGF also increases the production of MMP2 by PSCs (ﬁg. 2) [9]. Since MMP2 degrades basement membrane collagen, it is hypothesised that the consequent loss of normal collagen (matrix) facilitates the deposition of abnormal (ﬁbrillar) collagen, thereby promoting ﬁbrosis. (iii) The proinﬂammatory cytokines TNF, IL1 and IL6 induce PSC activation as assessed by one or more of the following indicators – proliferation, SMA expression and collagen synthesis [24]. (iv) Exposure to a pro-oxidant complex such as iron sulphate/ascorbic acid (which increases oxidant stress

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within PSCs) leads to their activation as indicated by increased SMA expression and collagen synthesis. Importantly, this oxidant stress-induced activation is prevented by the antioxidant -tocopherol (vitamin E) [25]. (v) Ethanol itself directly activates PSCs, most likely due to its oxidation to acetaldehyde (via the enzyme alcohol dehydrogenase (ADH) which has been shown to be active in PSCs) and the subsequent generation of oxidant stress within the cell [25]. Of note is the fact that ethanol can cause activation of PSCs from their quiescent phase and does not require the cells to be pre-activated to exert its stimulatory effects [25]. This is an important ﬁnding suggesting that, in vivo, PSC activation may occur early during chronic alcohol intake even in the absence of necro-inﬂammation. Perpetuation of this activation may occur during ethanol-induced necroinﬂammatory episodes leading to the development of ﬁbrosis. The non-oxidative ethanol metabolite fatty acid ethyl ester has not been shown to affect PSC proliferation or activation, although it has been reported to stimulate speciﬁc signaling molecules within PSCs (see below) [26]. (vi) In addition to the effects of exogenous activating factors on PSCs via paracrine pathways, it is now established that PSCs are capable of activation via autocrine pathways. PSCs synthesise and secrete cytokines such as TGF and IL1 [27, 28]. Moreover, the production of these cytokines can be stimulated by exogenous compounds such as ethanol, acetaldehyde and TGF itself [29]. These ﬁndings suggest that once activated, PSCs are capable of perpetuation of activation even in the absence of the initial trigger factors (ﬁg. 3). This phenomenon may represent one of the mechanisms responsible for progression of chronic pancreatitis despite the cessation of the initial insult (alcohol and/or acute ﬂare). Signalling Pathways in PSCs In recent years, there has been increasing interest in the evaluation of signaling mechanisms that may mediate the observed effects of various factors on PSCs. In general, cellular signalling involves ligand binding to speciﬁc receptors or direct effects of cellular stressors which leads to a cascade of phosphorylation of signalling molecules. The phosphorylated effector protein of the pathway then translocates to the nucleus and inﬂuences the transcription of speciﬁc genes that regulate cellular function. It is hoped that the identiﬁcation and characterisation of responsible signaling molecules will allow the therapeutic targeting of speciﬁc pathways so as to prevent or reverse PSC activation and thereby prevent or retard the ﬁbrotic process.

Apte/Wilson

One of the major pathways that regulates cell functions such as protein synthesis, cell differentiation and cell division is the mitogen-activated protein kinase (MAPK) pathway [30]. Consequently, this pathway has been the focus of attention of a number of recent studies. It is now established that the effects of ethanol, acetaldehyde and oxidant stress on PSCs are mediated by activation of all 3 classes of the MAPK pathway, namely extracellular signal-regulated kinase (ERK1/2), p38 kinase and c-jun amino terminal kinase (JNK) [23, 31, 32]. Most recently, it has been demonstrated that ethanol and acetaldehyde also activate two signaling molecules upstream of the MAPK cascade, phosphatidylinositol 3-kinase (PI3K) and protein kinase C [33]. With respect to the growth factors, it has been reported that PDGF-induced PSC proliferation is mediated by the ERK pathway [23], while PDGF-induced PSC migration is a function of the PI3K pathway [34]. However, it is important to note that cross-talk exists between PI3K and ERK, so that modulation of one pathway is usually associated with a change in the function of the other [32]. TGF has been shown to exert its proﬁbrogenic effect on PSCs via the intracellular signaling mediator SMAD2 [35]. This cytokine is also known to increase its own mRNA expression in PSCs in an autocrine manner, a process that has been shown to be regulated by the ERK pathway [35]. Recent studies have suggested a role for the peroxisome proliferator-activated receptor (PPAR, a ligand-activated transcription factor which controls cellular growth and differentiation), in PSC activation [36, 37]. Notably, the PPAR ligand troglitazone has been reported to inhibit PDGF-induced and culture-induced activation of PSCs [14].

Activating factors Cytokines

Perpetuation of activation

Pancreatic fibrosis

Fig. 3. Perpetuation of PSC activation. Activation of PSCs by factors, such as cytokines, oxidant stress, ethanol and its metabolites via paracrine mechanisms, leads to increased endogenous production of cytokines by PSCs themselves, which exert their effects on the cells via autocrine pathways. This may lead to perpetuation of the activated state of PSCs even in the absence of the initial trigger factors.

As indicated by the above discussion, there is increasing evidence to support a key role for PSCs in ﬁbrogenesis. However, in contrast to the large body of work related to PSC activation, the processes that may prevent or reverse such activation have only recently begun to attract some attention. In vitro studies have shown that antioxidants such as vitamin E and N-acetylcysteine can prevent oxidant stress- or ethanol-induced PSC activation [25, 38]. Inhibition of relevant signaling pathways using speciﬁc inhibitors has also been shown to prevent PSC activation [31–34, 37]. Most recently, interesting data have been reported with regard to the role of vitamin A in the induction of

PSC quiescence. As noted earlier, loss of vitamin A stores is an invariable association of PSC activation. A recent study by McCarroll et al. [39] demonstrates that activated PSCs revert to a quiescent state when incubated with retinol (vitamin A) or its metabolites all trans retinoic acid and 9-cis retinoic acid. Treated cells re-acquire vitamin A lipid droplets in their cytoplasm and exhibit a more rounded shape without the long cytoplasmic projections characteristic of transformation. The signiﬁcance of vitamin A-induced quiescence may be particularly relevant to the setting of ethanol-induced PSC activation. It is well established that the metabolic pathways of retinol and ethanol are very similar [40, 41]. Both compounds are converted to their aldehydes via retinol dehydrogenase (RolDH) and ADH, respectively, and subsequently to retinoic acid and acetic acid (via retinaldehyde dehydrogenase and aldehyde dehydrogenase, respectively). RolDH and ADH can utilise both retinol and ethanol as substrates [41]. Studies in the liver have shown that ethanol competitively inhibits the conversion of retinol to retinaldehyde [42]. The resulting decrease in retinoic acid levels is thought to mediate activation of hepatic stellate cells. McCarroll et al. [39] have demonstrated that retinol supplementation can signiﬁcantly inhibit ethanol-related PSC activation (ﬁg. 4). These ﬁndings suggest that vitamin A may be a potentially useful candidate as an antiﬁbrogenic agent in alcoholic pancreatic ﬁbrosis.

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Densitometer units (% of control)

Collagen I

150 kD

180 160 140 120 100 80 60 40 20 0 Control

E50

Rol

E50+Rol

Fig. 4. Effect of retinol supplementation on ethanol-induced collagen production by PSCs. A representative Western blot is shown for collagen type-I expression in PSCs incubated for 48 h with either culture medium alone (control), ethanol 50 mM (E50), retinol 10 M (Rol) or ethanol 50 mM + retinol 10 M (E50+Rol). Densitometry of all Western blots (n = 3 separate cell preparations) showed a signiﬁcant increase in collagen expression in PSCs incubated with ethanol compared to controls: * p ! 0.005. Retinol alone signiﬁcantly inhibited collagen expression compared to controls: ** p ! 0.005. In addition, retinol signiﬁcantly inhibited the ethanolinduced increase in collagen expression in PSCs: E50 vs. E50+Rol, # p ! 0.005.

Antiﬁbrotic therapeutic strategies have been assessed predominantly in experimental models of pancreatic ﬁbrosis (although not all studies have examined PSC function). A detailed discussion of all such strategies is beyond the scope of this review. Brieﬂy, the approaches used have

included antioxidants (vitamin E [43], oxypurinol [44]), TGF suppression (using TGF-neutralizing antibodies [45] or a herbal medicine Saiko-keishi-to [46]), TNF inhibition (using a TNF antibody [47], soluble TNF receptors or an inhibitor of TNF production, pentoxifylline [44]) and modulation of signaling molecules (using troglitazone, a PPAR ligand known to inhibit PDGFinduced PSC activation [14]). Given our current knowledge of PSC biology and function, it would not be unreasonable to presume that the beneﬁcial effects of the above approaches are mediated via an inhibition of PSC activation in the pancreas. In summary, our understanding of the ﬁbrogenic process in the pancreas has been signiﬁcantly improved in recent years with the characterization of the biology of PSCs. Both in vivo and in vitro evidence supports a key role for these cells in pancreatic ﬁbrosis. In health, PSCs most likely function as regulators of normal pancreatic architecture by maintaining a ﬁne balance between ECM synthesis and degradation. During pancreatic injury, PSCs are exposed to increased levels of growth factors, cytokines, and cellular stressors such as oxidant stress and toxic ethanol metabolites. The consequent activation of PSCs (which may be further perpetuated via autocrine pathways) leads to an imbalance between ECM synthesis and degradation due to the synthesis of excessive amounts of ECM proteins – a process that eventually manifests as pancreatic ﬁbrosis.

References 1 Kloppel G, Detlefsen S, Feyerabend B: Fibrosis of the pancreas: The initial tissue damage and the resulting pattern. Virchows Arch 2004; 445:1–8. 2 Apte MV, Haber PS, Applegate TL, Norton ID, McCaughan GW, Korsten MA, Pirola RC, Wilson JS: Periacinar stellate shaped cells in rat pancreas – Identiﬁcation, isolation, and culture. Gut 1998;43:128–133. 3 Bachem MG, Schneider E, Gross H, Weidenbach H, Schmid RM, Menke A, Siech M, Beger H, Grunert A, Adler G: Identiﬁcation, culture, and characterization of pancreatic stellate cells in rats and humans. Gastroenterology 1998; 115:421–432. 4 Schneider E, Schmid-Kotsas A, Zhao J, Weidenbach H, Schmid RM, Menke A, Adler G, Waltenberger J, Grunert A, Bachem MG: Identiﬁcation of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells. Am J Physiol Cell Physiol 2001;281: C532–C543.

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5 Apte MV, Wilson JS: Stellate cell activation in alcoholic pancreatitis. Pancreas 2003;27:316– 320. 6 Apte MV, Park S, Phillips PA, Santucci N, Goldstein D, Kumar RK, Ramm GA, Büchler M, Friess H, McCarroll JA, Keogh G, Merrett N, Pirola R, Wilson JS: Desmoplastic reaction in pancreatic cancer: Role of pancreatic stellate cells. Pancreas 2004;29:179–187. 7 Apte MV, Haber PS, Darby SJ, Rodgers SC, McCaughan GW, Korsten MA, Pirola RC, Wilson JS: Pancreatic stellate cells are activated by proinﬂammatory cytokines: Implications for pancreatic ﬁbrogenesis. Gut 1999;44: 534–541. 8 Phillips PA, Wu MJ, Kumar RK, Doherty E, McCarroll JA, Park S, Pirola RC, Wilson JS, Apte MV: Cell migration: A novel aspect of pancreatic stellate cell biology. Gut 2003; 52: 677–682.

9 Phillips PA, McCarroll JA, Park S, Wu M-J, Korsten MA, Pirola RC, Wilson JS, Apte MV: Pancreatic stellate cells secrete matrix metalloproteinases – Implications for extracellular matrix turnover. Gut 2003;52:275–282. 10 Haber P, Keogh G, Apte M, Moran C, Pirola R, McCaughan G, Korsten M, Wilson J: Activation of pancreatic stellate cells in human and experimental pancreatic ﬁbrosis. Am J Pathol 1999;155:1087–1095. 11 Casini A, Galli A, Pignalosa P, Frulloni L, Grappone C, Milani S, Pederzoli P, Cavallini G, Surrenti C: Collagen type I synthesized by pancreatic periacinar stellate cells (PSC) co-localizes with lipid peroxidation-derived aldehydes in chronic alcoholic pancreatitis. J Pathol 2000;192:81–89. 12 Emmrich J, Weber I, Sparmann GH, Liebe S: Activation of pancreatic stellate cells in experimental chronic pancreatitis in rats. Gastroenterology 2000;118:A166.

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13 Ohashi K, Kim JH, Hara H, Aso R, Akimoto T, Nakama K: WBN/Kob rats. A new spontaneously occurring model of chronic pancreatitis. Int J Pancreatol 1990;6:231–247. 14 Shimizu K, Shiratori K, Kobayashi M, Kawamata H: Troglitazone inhibits the progression of chronic pancreatitis and the proﬁbrogenic activity of pancreatic stellate cells via a PPAR gamma-independent mechanism. Pancreas 2004;29:67–74. 15 Murayama KM, Barent BL, Gruber M, Brooks A, Eliason S, Brunt EM, Smith GS: Characterization of a novel model of pancreatic ﬁbrosis and acinar atrophy. J Gastrointest Surg 1999; 3:418–425. 16 Matsumura N, Ochi K, Ichimura M, Mizushima T, Harada H, Harada M: Study on free radicals and pancreatic ﬁbrosis – Pancreatic ﬁbrosis induced by repeated injections of superoxide dismutase inhibitor. Pancreas 2001; 22: 53–57. 17 Tsukamoto H, Towner SJ, Yu GS, French SW: Potentiation of ethanol-induced pancreatic injury by dietary fat. Induction of chronic pancreatitis by alcohol in rats. Am J Pathol 1988; 131:246–257. 18 Uesugi T, Froh M, Gabele E, Isayama F, Bradford BU, Ikai I, Yamaoka Y, Arteel GE: Contribution of angiotensin II to alcohol-induced pancreatic ﬁbrosis in rats. J Pharmacol Exp Ther 2004;17:17. 19 Gukovsky I, Lugea A, Cheng J, French B, E RN, French SW: Model of chronic alcoholic pancreatitis. Gastroenterology 2002;122:A93. 20 Vogelmann R, Ruf D, Wagner M, Adler G, Menke A: Effects of ﬁbrogenic mediators on the development of pancreatic ﬁbrosis in a TGF-beta1 transgenic mouse model. Am J Physiol Gastrointest Liver Physiol 2001; 280: G164–G172. 21 Neuschwander-Tetri BA, Burton FR, Presti ME, Britton RS, Janney CG, Garvin PR, Brunt EM, Galvin NJ, Poulos JE: Repetitive self-limited acute pancreatitis induces pancreatic ﬁbrogenesis in the mouse. Dig Dis Sci 2000;45: 665–674. 22 Apte MV, Keogh GW, Wilson JS: Chronic pancreatitis: Complications and management. J Clin Gastroenterol 1999;29:225–240. 23 Jaster R, Sparmann G, Emmrich J, Liebe S: Extracellular signal regulated kinases are key mediators of mitogenic signals in rat pancreatic stellate cells. Gut 2002;51:579–584. 24 Mews P, Phillips P, Fahmy R, Korsten M, Pirola R, Wilson J, Apte M: Pancreatic stellate cells respond to inﬂammatory cytokines: Potential role in chronic pancreatitis. Gut 2002; 50:535–541. 25 Apte MV, Phillips PA, Fahmy RG, Darby SJ, Rodgers SC, McCaughan GW, Korsten MA, Pirola RC, Naidoo D, Wilson JS: Does alcohol directly stimulate pancreatic ﬁbrogenesis? Studies with rat pancreatic stellate cells. Gastroenterology 2000;118:780–794.

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26 Masamune A, Kikuta K, Satoh M, Suzuki N, Shimosegawa T: Fatty acid ethyl esters activate activator protein-1 and mitogen-activated protein kinases in rat pancreatic stellate cells. Pancreatology 2004;4:311. 27 Apte M, Keating J, Phillips P, Friess H, Büchler M, Korsten M, Pirola R, McCaughan G, Wilson J: Endogenous expression of proinﬂammatory cytokines and nerve growth factor by pancreatic stellate cells – Implications for ﬁbrosis and neural changes in chronic pancreatitis. J Gastroenterol Hepatol 2001;16:A114. 28 Shek FW, Benyon RC, Walker FM, McCrudden PR, Pender SL, Williams EJ, Johnson PA, Johnson CD, Bateman AC, Fine DR, Iredale JP: Expression of transforming growth factor1 by pancreatic stellate cells and its implications for matrix secretion and turnover in chronic pancreatitis. Am J Pathol 2002; 160: 1787–1798. 29 Apte M, Keating J, Phillips P, Friess H, Büchler M, Pirola R, McCaughan G, Korsten M, Wilson J: Endogenous expression of proinﬂammatory cytokines and nerve growth factor by pancreatic stellate cells – Implications for ﬁbrosis and neural changes in chronic pancreatitis. Pancreas 2001;23:428. 30 Lopez-Ilasaca M: Signaling from G-proteincoupled receptors to mitogen-activated protein (MAP)-kinase cascades. Biochem Pharmacol 1998;56:269–277. 31 McCarroll JA, Phillips PA, Park S, Doherty E, Pirola RC, Wilson JS, Apte MV: Pancreatic stellate cell activation by ethanol and acetaldehyde: Is it mediated by the mitogen-activated protein kinase signaling pathway? Pancreas 2003;27:150–160. 32 McCarroll JA, Phillips PA, Kumar RK, Park S, Pirola RC, Wilson JS, Apte MV: Pancreatic stellate cell migration: Role of the phosphatidylinositol 3-kinase (PI3-kinase) pathway. Biochem Pharmacol 2004;67:1215–1225. 33 McCarroll J, Phillips P, Santucci N, Pirola R, Wilson J, Apte M: Alcoholic pancreatic ﬁbrosis: Role of the phosphatidylinositol-3 kinase (PI3-K) and protein kinase C (PKC) pathways in pancreatic stellate cells. J Gastroenterol Hepatol 2004, in press. 34 Masamune A, Kikuta K, Satoh M, Kume K, Shimosegawa T: Differential roles of signaling pathways for proliferation and migration of rat pancreatic stellate cells. Tohoku J Exp Med 2003;199:69–84. 35 Ohnishi H, Miyata T, Yasuda H, Satoh Y, Hanatsuka K, Kita H, Ohashi A, Tamada K, Makita N, Iiri T, Ueda N, Mashima H, Sugano K: Distinct roles of SMAD2-, SMAD3-, and ERK-dependent pathways in transforming growth factor-beta1 regulation of pancreatic stellate cellular functions. J Biol Chem 2004; 279:8873–8878. 36 Masamune A, Kikuta K, Satoh M, Sakai Y, Satoh A, Shimosegawa T: Ligands of peroxisome proliferator-activated receptor-gamma block activation of pancreatic stellate cells. J Biol Chem 2002;277:141–147.

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37 Masamune A, Kikuta K, Satoh M, Suzuki N, Shimosegawa T: Protease-activated receptor2-mediated proliferation and collagen production of rat pancreatic stellate cells. J Pharmacol Exp Ther 2004;14:14. 38 Kikuta K, Masamune A, Satoh M, Suzuki N, Shimosegawa T: 4-Hydroxy-2,3-nonenal activates activator protein-1 and mitogen-activated protein kinases in rat pancreatic stellate cells. World J Gastroenterol 2004; 10: 2344– 2351. 39 McCarroll JA, Phillips PA, Santucci N, Pirola R, Wilson J, Apte M: Vitamin A induces quiescence in culture-activated pancreatic stellate cells – Potential as an anti-ﬁbrotic agent? Pancreas 2003;27:396. 40 Han CL, Liao CS, Wu CW, Hwong CL, Lee AR, Yin SJ: Contribution to ﬁrst-pass metabolism of ethanol and inhibition by ethanol for retinol oxidation in human alcohol dehydrogenase family – Implications for etiology of fetal alcohol syndrome and alcohol-related diseases. Eur J Biochem 1998;254:25–31. 41 Duester G: Families of retinoid dehydrogenases regulating vitamin A function: Production of visual pigment and retinoic acid. Eur J Biochem 2000;267:4315–4324. 42 Sauvant P, Sapin V, Abergel A, Schmidt CK, Blanchon L, Alexandre-Gouabau MC, Rosenbaum J, Bommelaer G, Rock E, Dastugue B, Nau H, Azais-Braesco V: PAV-1, a new rat hepatic stellate cell line converts retinol into retinoic acid, a process altered by ethanol. Int J Biochem Cell Biol 2002;34:1017–1029. 43 Gomez JA, Molero X, Vaquero E, Alonso A, Salas A, Malagelada JR: Vitamin E attenuates biochemical and morphological features associated with development of chronic pancreatitis. Am J Physiol Gastrointest Liver Physiol 2004;287:G162–G169. 44 Pereda J, Sabater L, Cassinello N, GomezCambronero L, Closa D, Folch-Puy E, Aparisi L, Calvete J, Cerda M, Lledo S, Vina J, Sastre J: Effect of simultaneous inhibition of tnf-alpha production and xanthine oxidase in experimental acute pancreatitis: The role of mitogen activated protein kinases. Ann Surg 2004;240: 108–116. 45 Menke A, Yamaguchi H, Gress TM, Adler G: Extracellular matrix is reduced by inhibition of transforming growth factor beta1 in pancreatitis in the rat. Gastroenterology 1997;113:295– 303. 46 Su SB, Motoo Y, Xie MJ, Taga H, Sawabu N: Antiﬁbrotic effect of the herbal medicine Saiko-keishi-to (TJ-10) on chronic pancreatitis in the WBN/Kob rat. Pancreas 2001;22:8–17. 47 Hughes CB, Gaber LW, Mohey el-Din AB, Grewal HP, Kotb M, Mann L, Gaber AO: Inhibition of TNF alpha improves survival in an experimental model of acute pancreatitis. Am Surg 1996;62:8–13.

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Review Article Dig Dis 2004;22:280–291 DOI: 10.1159/000082800

Endoscopic Therapy of Chronic Pancreatitis Klaus Mönkemüller Stefan Kahl Peter Malfertheiner Otto-von-Guericke Universität, Universitätsklinikum Magdeburg, Magdeburg, Deutschland

Key Words Pancreatic duct Nerve entrapment, pancreatic Pancreatitis, management Inflammation, chronic pancreatic Fibrosis, peripancreatic

Abstract We present an overview of endoscopic therapies for chronic pancreatitis (CP) and its associated conditions. It is evident that endoscopy can be a definite therapy for pancreatic pseudocysts, pancreatic ascites and pancreatic duct (PD) disruption. Endoscopic therapy has also been useful in the short-term and medium therapy of common bile duct strictures due to CP, the best results being obtained if there are no calcifications in the head of the pancreas. Although most experts agree that obstruction to the outflow of pancreatic juice and the resulting increased pressure within the main PD is one of the major factors contributing to pain and that endoscopic therapy has been proven effective to remove stones as well as to dilate PD strictures and place stents across the PD, there is no convincing evidence from randomized trials that the patient’s dominant symptom of CP, i.e. pain, is resolved in an appropriate and long-term fashion. We believe that there are other factors which are important in the etiology of chronic pain such as pancreatic inflammation and peripancreatic fibrosis with result-

© 2004 S. Karger AG, Basel 0257–2753/04/0223–0280$21.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/ddi

ing nerve entrapment around the gland. The reader is reminded that endoscopic therapy is associated with significant and important complications, therefore appropriate patient selection and patient information are of paramount importance. Nevertheless, it is important to consider that one advantage of endoscopic management of CP is that it is less invasive as compared with surgery, often effective for years, does not hinder further surgery, and can be repeated. Finally we want to emphasize that there are many valid surgical, radiological and endoscopic techniques to treat the complications of CP. Therefore, the approach to CP and its complications should be by a multidisciplinary team of gastroenterologists, surgeons, radiologists, endoscopists and pain specialists. Copyright © 2004 S. Karger AG, Basel

Introduction

Chronic pancreatitis (CP) results in a gradual loss of pancreatic function leading to malabsorption, weight loss and metabolic problems such as diabetes mellitus [1–3]. From the subjective clinical perspective abdominal pain is the dominant clinical problem in patients with CP [3– 7]. It can be due to pseudocysts, as well as strictures and stones in the pancreatic duct (PD) [6–11]. Although not

Peter Malfertheiner Universitätsklinikum Magdeburg, Otto-von-Guericke Universität Leipziger Strasse 44, DE–39112 Magdeburg (Germany) Tel. +49 391 6715162, Fax +49 391 6715159 E-Mail [email protected]

Fig. 1. Male patient with calciﬁcation in pancreatic head prior to (a) and 1 year after (b) stent therapy. Treatment failed to relief CBD obstruction.

conclusively demonstrated, most experts agree that obstruction to the outﬂow of pancreatic juice and the resulting increased pressure within the main PD is one of the major factors contributing to pain [1–10]. Not less important in the etiology of chronic pain appears to be the chronic pancreatic inﬂammation and peripancreatic ﬁbrosis with resulting nerve entrapment around the gland [1–3, 11, 12]. These factors may be the most important explanation for the origin of pain in these patients and could explain why endoscopic interventions are not generally associated with long-lasting relief of the patient’s symptoms. The aims of endoscopic therapy are to alleviate pain by reducing the pressure within the ductal system, to drain pancreatic pseudocysts, to improve pancreatic head common bile duct (CBD) strictures which have resulted from calcifying CP and to reestablish anatomical continuity of the disrupted PD [1, 7, 9, 13]. Success rates for stone extraction and stenting of strictures are high in specialized centers that employ experienced endoscopists, but pain often recurs during long-term follow-up [14–20]. For chronic pain believed to be the result of peripancreatic nerve entrapment, endoscopic ultrasound (EUS)-guided nerve blockage may also be an appropriate endoscopic method to alleviate pain [21]. The reader is advised that there are many valid surgical, radiological and endoscop-

Endoscopic Therapy of Chronic Pancreatitis

ic techniques to approach CP and its complications [1, 3, 7, 8, 15, 19, 22]. There are very few prospective data comparing these methods among themselves [23]. Also, there is a lack of long-term data regarding outcome after endoscopic therapy of CP [24]. Prospective data on radiologic or surgical techniques are also scarce. But one advantage of endoscopic management of CP is that it is less invasive as compared with surgery, often effective for years, does not hinder further surgery, and can be repeated [25–36]. The aim of this article is not to endorse the endoscopic approach to CP but to describe the techniques, indications, complications and results of endoscopic therapy of CP and its associated conditions.

Indications for Endoscopic Therapy

The observation that drainage of the main PD (MPD) in selected cases of severe calcifying CP has a radical beneﬁt on pain reduction supports the hypothesis that pain is mainly due to obstruction of the MPD [4–7]. Therefore, endoscopic therapy has become a valuable alternative to surgery for attempting to decompress the MPD [1, 3–9]. Endoscopic therapy is indicated to dilate strictures in the PD, place plastic stents into the PD and remove stones (ﬁg. 1, 2) [8, 9, 11, 29]. Pancreatic pseudocysts can also

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Fig. 2. Female patient with long-lasting alcoholic chronic pancreatitis. Outﬂow obstruction was thought to be a main cause of recurrent episodes of pain. After stent insertion, ESWL and stone removal she had uneventful course of the disease for 18 months. After this short interval she again had recurrent attacks and painful disease until surgery.

be drained endoscopically, using transpapillary, cystogastrostomy or cystoduodenostomy approaches, or a combination of the three, with success rates of up to 90% and long-term resolution of the pseudocyst of up to 80% [30– 36]. The indications for pseudocyst drainage include pain, gastric outlet or biliary obstruction, infection, bleeding, and intra-abdominal leakage [33–36]. Biliary obstruction due to ﬁbrosis in the region of the pancreatic head can lead to jaundice, cholangitis and cholestasis, all of which are indications for endoscopic intervention [37– 46]. Other complications of CP, such as pancreatic ﬁstulas, pancreatic ascites and disrupted PD are also indications for endotherapy [47–49]. Contraindications for endoscopic therapy are the same as for any endoscopic procedure.

Endoscopic Techniques and Complications

Endoscopic Pancreatic Sphincterotomy The cornerstone of therapeutic pancreatic endoscopy are PD cannulation and endoscopic pancreatic sphincterotomy (EPS). There are three methods to cut the major

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papilla pancreatic sphincter. The ﬁrst method involves the use of a standard pull-type sphincterotome, with or without a guide wire [26, 28, 37, 50–57]. The sphincterotome is inserted into the PD and oriented along the axis of the PD, which usually is in the 1- to 2-o’clock position. The cutting wire is then extended into the duct for about 6–7 mm, and a 5- to 10-mm sphincterotomy is then performed preferably using a pure cutting current to prevent deep ductal injury and pancreatitis [26, 28, 29, 50–55]. The second technique involves the use of a needle-knife to cut over a previously placed pancreatic stent [12, 51, 52]. The reason to leave a stent in the PD after the EPS is to prevent pancreatitis [50]. It is preferable to use an unﬂapped stent with a diameter of 5–7 french, but any small pancreatic stent is appropriate [12, 50, 52]. This small diameter unﬂapped stent is reported to fall out of the PD in 50% of cases within the next 7–10 days [52]. Stents which remain inside should be removed within 10 days of endoscopy in order to avoid PD damage [50, 52]. The disadvantage of this technique with stent insertion is that it requires a second endoscopic retrograde cholangiopancreatography (ERCP) in at least 50% of patients. The third technique involves the performance of the EPS

Mönkemüller/Kahl/Malfertheiner

with either of the above techniques (sphincterotome or needle-knife) over a stent but after a biliary sphincterotomy has been performed. When using this technique careful attention needs to be given to the direction of the cut, as the distorted anatomy of the major papilla muscle ﬁbers has been disrupted by the previous biliary sphincterotomy. In such circumstances the cut needs to be directed towards the 2- to 4-o’clock position [8, 12, 29]. It is noteworthy that most authorities recommend a pure cutting current for EPS, but most studies on EPS have used a blended type current with variable wattage (40– 70 W) [50–57]. Minor papilla (MiP) EPS is most commonly performed in the setting of pancreas divisum [50, 52]. The technique of MiP EPS is similar to that of major papilla EPS with the exception that the direction of the cut is towards the 10- to 12-o’clock position and that the size of the sphincterotomy is limited to 4–6 mm [50]. The size of the MiP sphincter is much smaller and its anatomy less well known. Therefore it is highly recommended to exercise caution when cutting this sphincter and limiting the size of the cut to 4–6 mm. Many endoscopists prefer the use of a stent and needle-knife to better gauge the direction of the cut [58]. A useful modiﬁcation to this approach has been described by Wilcox and Mönkemüller [58]. These authors reported on 11 patients in whom MiP EPS was performed after a guide wire had been placed into the dorsal PD and served as a guide, so that an incision could then be safely be made with a needle-knife [50]. This technique is especially useful when deep cannulation of the MiP is not possible using a catheter or sphincterotome or when it is not possible to place a stent through the MiP into the dorsal PD. Complications following MiP EPS range from 10 to 35%, the most common being pancreatitis, which is usually mild to moderate, but severe pancreatitis and perforation as well as deaths have been reported [12, 50, 52]. The endoscopist needs to be aware that complications after EPS occur in 5–30% of patients [3–5, 9, 11, 12, 17, 26, 28, 44, 50, 53, 54]. Kozarek et al. [17] were the ﬁrst to publish signiﬁcant experience with EPS. They reported on 56 patients, 54 of whom had CP, who underwent endoscopic PD sphincterotomy [9]. Acute complications noted in 10% of patients included exacerbation of pancreatitis and cholangitis. Chronic complications included induction of asymptomatic ductal changes in 16%, thought to be related to endoprosthesis placement, and stenosis of the sphincterotomy site in 14%, requiring repeated endoscopic or surgical sphincter section [17]. Esber et al. [54] reported a complication rate for EPS in 236

Endoscopic Therapy of Chronic Pancreatitis

consecutive patients. The authors used a pull-type sphincterotome (40 W of blended current) in 113 patients and needle-knife over a pancreatic stent (same current) in 90 patients. Post-procedure pancreatitis occurred in 14%, being severe in 3% and moderate in 21% [54]. In another large series of EPS, sphincterotomy-related complications were observed in 7 of the 171 patients (4.1%), including 3 cases of bleeding, 3 patients with mild pancreatitis, and 1 with retroduodenal perforation [28]. Ell et al. [53] also collected the data prospectively on all patients undergoing EPS for CP between January 1989 and September 1996. Patients were followed by clinical investigation and blood sample analysis at 4, 24, and 48 h after EPS. EPS was performed in 118 patients with CP. Ninety-four patients (80%) underwent guide wire-assisted EPS, and 24 patients (20%) underwent needle-knife EPS. In total, EPS was successful in 116 patients (98%). The complication rate was 4.2% (4 cases of moderate pancreatitis, 1 severe bleeding, no deaths) [53]. As mentioned previously, many experts have recommended the placement of a pancreatic drainage to limit the incidence of post-sphincterotomy pancreatitis [12, 50, 52, 56, 57]. In a retrospective study Elton et al. [55] evaluated the use of PD drainage after EPS. The authors performed 164 EPS on 160 patients during a 5-year period. They compared EPS done with overnight nasopancreatic catheter placement with those done with stenting or no drainage. Of the 164 sphincterotomies, 98 were done with overnight nasopancreatic drainage, 50 with stent placement, and 16 with no drainage. Complications (all pancreatitis) were signiﬁcantly more frequent in the group with no drainage (12.5%) as compared with those with drainage (0.7%) [55].

Dilation and Stenting of Pancreatic and Biliary Strictures

Both PD and CBD strictures are common complications of CP [1–11, 12, 37–46]. These strictures tend to be very ﬁbrotic. Dilation of these strictures with a balloon does not generally result in a long-term response [12, 50]. Therefore placement of stents into the PD or CBD across these strictures is generally advocated [7–16]. PD strictures can be isolated, diffuse, multiple, or associated with dilation of the PD, stones, ductal leak or with PD sphincter stenosis. The technique of stent placement into the PD is similar to that of biliary stenting. Pancreatic stents come in a wide variety of sizes and designs. Pancreatic stents are generally straight, with multiple side holes to

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283

Table 1. Endoscopic therapy of chronic pancreatitis

Author/year

n

Kozarek [48], 1998 17 Cremer et al. [8], 76 1990 Binmoeller et al. 74 [29], 1995 Bahsin et al. [51], 30 1997 Rösch et al. [65], 1,211 2002

Improved Morbidity Follow-up pain, % % months 76 94

12 5

8 37

74

–

90

10

16

66

13

60

3–12

Techniques of Pancreatic Stone Removal

facilitate drainage from the PD branches [1, 3, 50]. The caliber of these stents ranges from 3 to 10 french, and the length is generally from 1 to 12 cm or more. Flaps or barbs are located on both ends, one end or none. Flapped stents should be employed when there is a plan to leave the stent over a prolonged period and not only for a few days [50, 52]. In contrast to biliary stents with larger diameters, pancreatic stents tend to occlude more rapidly. Most stents occlude by 8 weeks, and 5- to 7-french stents tend to occlude by week 4 [59]. Interestingly, even if stents become totally occluded, pancreatic juice drainage still occurs around the stent [50]. The indications for pancreatic stents include: prophylaxis against ERCP-induced pancreatitis; to perform over-the-stent needle-knife EPS; to heal PD disruption; to localize pancreatic stones before ESWL; to dilate PD strictures; for transpapillary pseudocyst drainage, and therapy or prevention of PD sphincter stenosis [1–11, 33–40]. Of major concern when deciding to place a stent into the PD are reports of ductal and parenchymal changes occurring due to the stent [60]. Therefore, PD stents should not be used for periods longer than 3–4 weeks in normal glands. The extent of damage by stents and the maximal duration of stents in the PD in patients with CP has not been clearly established. Morgan et al. [61] evaluated the response to PD stenting in patients with radiologically conﬁrmed CP. Twenty-ﬁve consecutive patients had 40 stent placement episodes. In 28 (70%) of the 40 episodes, the MPD caliber increased or was unchanged after stenting; pain improved in 20 (71%) of 28 episodes. Pain improved in 6 (50%) of 12 patients with smaller ducts after stenting. Stent patency was documented upon retrieval in 34 episodes; most stents were occluded. Stent-induced strictures developed in 18% of 40

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stent episodes. These results suggest that MPD caliber after endoscopic stenting is not a good indicator of pain response or stent patency. MPD was often larger, and even with stent occlusion, patients’ symptoms were frequently improved [61, 62]. Nevertheless, caution is always advised when using stents and to leave them in the PD only if there is clear therapeutic beneﬁt.

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Endoscopic removal of obstructing PD stones is difﬁcult to achieve using standard techniques employed for bile duct stones, due to the frequently associated strictures, stone composition and stone impaction [12, 50, 52, 62, 63]. Rarely, stones ﬂoating freely inside the dilated PD or impacted in the region of the head of the pancreas can be removed with a Dormia basket or balloon [12, 63]. But in general, pancreatic stones are difﬁcult to remove without previous mechanical or extracorporeal shock wave lithotripsy (ESWL) [63]. ESWL is the method of choice to fragment calciﬁed pancreatic stones [12, 50]. For patients with noncalciﬁed radiolucent stones, ESWL is usually not necessary [12]. The reader is referred to excellent reviews to study the details of ESWL [12, 62]. In essence, there are two main types of ESWL machines, electromagnetic or hydraulic which deliver the shock waves at various electrical powers and intervals [12, 62, 63]. Also, today it is possible to deliver ESWL via the endoscope into the PD. A pulsed-dye laser lithotripter requires visual control of a laser ﬁber which is directed via the pancreatoscope or directly applied via ﬂuoroscopic control [12]. However, these techniques of intraductal ESWL remain anecdotal and its use is limited to a few expert centers for pancreatic diseases [12]. The long-term follow-up data and our own clinical experience disclosing that pain recurs frequently in patients with CP, demonstrate that stone removal alone is not enough therapy for chronic calcifying pancreatitis.

Clinical Results of Endoscopic Therapy for CP

There are very limited prospective data comparing radiologic, surgical and endoscopic therapies for CP and its associated complications. To our knowledge, there is only one prospective study comparing surgery versus endoscopy for the therapy of CP [23]. Dite et al. [23] invited 140 consecutive patients with PD obstruction and pain to participate in a randomized trial comparing endother-

Mönkemüller/Kahl/Malfertheiner

apy and surgery. Of the 140 eligible patients, only 72 agreed to be randomized. Surgery consisted of resection (80%) and drainage (20%) procedures, while endotherapy included sphincterotomy and stenting (52%) and/or stone removal (23%). In the entire group, the initial success rates were similar for both groups, but at the 5-year follow-up, complete absence of pain was more frequent after surgery (37 vs. 14%) [23]. In the randomized subgroup, results were similar (pain absence 34% after surgery vs. 15% after endotherapy, relief 52% after surgery vs. 46% after endotherapy). The increase in body weight was also greater by 20–25% in the surgical group, while new-onset diabetes developed with similar frequency in both groups (34–43%), again with no differences between the results for the whole group and the randomized subgroup [23]. In a retrospective review of records on all patients who had EPS during a 4-year period, Okolo et al. [64] evaluated whether the use of EPS resulted in a symptomatic response and clinical improvement, deﬁned as greater than 50% reduction in the magnitude of pain. Of 55 patients who underwent EPS after a median follow-up of 16 months, 60% of all patients reported improvement in pain scores. Complications of pancreatic sphincterotomy included pancreatitis in 5 patients (9%), bleeding in 2 (3.6%) and early stent occlusion in 5 patients (9%) [64]. Dite et al. [26] performed EPS in 42 patients with CP and abdominal pain. Treatment led to the disappearance of or a signiﬁcant decrease in the epigastric pain in 85.7% patients shortly after the treatment; in 47.1% of patients the painless period lasted for a further 24 months after the therapy. An increase in body weight of about 2 kg occurred in 53% of the treated subjects during the 2 years following therapy [26]. Elton et al. [55] reported on their experience with 164 EPS. Pancreatic sphincterotomy was effective when used as primary therapy, with 64% of patients so treated experiencing complete and long-lasting resolution of symptoms after the procedure [55]. In the largest multicenter experience of endoscopic therapy for CP reported to date, Rösch et al. [65] investigated 1,211 patients with painful CP and ductal obstruction due to either strictures or stones. Follow-up data were available for 1,018 (85%) patients. The median follow-up was 4.9 years. The long-term success of endotherapy was 86% in the entire group, and 65% in the intention-to-treat analysis. One quarter of the patients ended up undergoing pancreatic surgery for the relief of symptoms [65]. Table 1 provides data on several important studies of endoscopic therapy for CP.

Endoscopic Therapy for Pancreatic Strictures Despite being used for more than 2 decades, pancreatic stent therapy for PD strictures or stones remains controversial [24–27]. Kozarek et al. [9, 17] were among the ﬁrst to describe experience with the placement of stents into the PD. In their ﬁrst report, they described 17 patients, 9 with acute relapsing pancreatitis and 8 with CP, who had drain or stent placement for hypertensive PD sphincter, dominant ductal stenosis, duct disruption, or pseudocyst. Six patients continued long-term stent placement with a marked reduction in chronic pain or attacks of recurrent pancreatitis. All 6 pseudocysts resolved initially and 1 recurred and required surgery [9]. Over a 9-year period, Binmoeller et al. [29] treated 93 patients with narcotic-dependent pain due to CP and with a dominant PD stricture visualized by ERCP with sent drainage. The stents were exchanged according to symptoms, and removed if the stricture was judged to be adequately dilated after stenting. Sixty-nine patients (74%) reported complete (n = 46) or partial (n = 23) pain relief at 6 months. Stents were removed in 49 patients after a mean of 15.7 months; during a mean follow-up of 3.8 years, 36 patients remained pain-free, and 13 had a relapse of pain (11 were retreated by endoscopic drainage (ED) and subsequently became pain-free) [29]. Smits et al. [44] studied the long-term safety and efﬁcacy of pancreatic stenting in 51 patients with CP and persistent pain with dominant strictures in the PD who were treated with plastic pancreatic stents. Patients were followed for a median of 34 (range 6–128) months. Nine of the 49 patients (82%) had clinical improvement. Stents were removed in 22 of the 40 patients with persistent beneﬁcial response in all (median follow-up 28.5 months). Stent dysfunction occurred in 27 of the 49 patients (55%) and was successfully treated by exchanging the stent [44]. Ponchon et al. [66] also evaluated a standardized protocol of endoscopic stenting of the PD. Twenty-three patients with abdominal pain due to CP and stricture of the distal main PD were treated according to the following protocol: after balloon dilation of the stenosis, a 10-french stent was placed into the main PD and then exchanged every 2 months, the total duration of drainage being 6 months. Use of analgesics could be discontinued in 17 patients (74%) on termination of drainage, and in 12 patients (52%) 1 year later [66].

Endoscopic Therapy of Chronic Pancreatitis

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Endoscopic Therapy of Pancreatolithiasis Endoscopic removal of obstructing duct stones is difﬁcult to achieve. Endoscopic therapy is usually combined with ESWL [11, 12, 18, 67]. Dumonceau et al. [11] eval-

285

uated 70 new patients who underwent pancreatic sphincterotomy and attempted stone removal. Complete ductal clearance of the calculi was obtained in 50% of the cases. Fifty-four percent of all patients with painful CP did not experience any pain recurrence within 2 years [11]. Several uncontrolled trials have shown that fragmentation of stones with ESWL facilitates their endoscopic removal. In one of the most recent studies, Brand et al. [67] prospectively evaluated the effectiveness of ESWL and endotherapy for patients with PD stones. Forty-eight consecutive patients with stones not extractable with a Dormia basket were included. EPS was routinely performed in all patients. A median of 13 ESWL sessions and 22,100 shock waves were required. Twelve patients required balloon dilation of a PD stricture and 27 a plastic stent. Ductal drainage was achieved in 75% of patients. After a median follow-up of 7 months signiﬁcant pain relief was achieved in 45% of patients [67]. ESWL can also be useful if endoscopic therapy fails. Costamagna et al. [63] evaluated 35 patients with severe CP who were treated by ESWL for endoscopically irretrievable obstructive stones. Fragmentation of stones was obtained in all cases while complete clearance and decompression of PD were obtained in 26 of 35 (74.3%) and in 30 of 35 (85.7%) cases, respectively [63].

Biliary Strictures Associated with CP

Biliary strictures are a known complication of chronic calciﬁc pancreatitis occurring in approximately 10–30% of these patients [37–46]. These strictures are responsible for painless cholestasis, obstructive jaundice, fat malabsorption due to bile acid deﬁciency, recurrent cholangitis, secondary biliary cirrhosis and possibly chronic abdominal pain [41–45]. Early interventions to relieve these stenoses will prevent the development of complications [37– 45]. Although surgical decompression of the biliary tree by anastomosis of the gallbladder or common duct to the small intestine or bilioenteric anastomosis completely relieves symptoms and allows liver function to improve signiﬁcantly, data are emerging showing that endoscopy has favorable excellent short and intermediate results [37–46]. Endoscopic placement of biliary stents is an effective initial treatment for jaundice and cholangitis caused by CBD strictures secondary to CP; however, the role of endoscopic treatment for long-term management of these strictures is less clear [38]. Interest in endoscopic therapy remains because surgery also has its drawbacks, increased perioperative morbidity and mortality, and re-

286

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currence due to anastomotic strictures or failed sphincteroplasty [22, 45, 68]. The issue of an increased incidence of cholangiocarcinoma in bilioenteric anastomosis needs also be deﬁned [68]. There are many studies evaluating the use of endotherapy for biliary strictures [37–46, 69–72]. Most studies have evaluated short- and medium-term responses, but few have addressed long-term responses (15-year outcomes). In addition to the clear indications for decompression of biliary strictures such as cholangitis, jaundice and stones, there has been a concern that abdominal pain is also due to these strictures [45]. Kahl et al. [69] recently presented the data of a prospective study including a total of 61 patients with CBD strictures caused by CP of alcoholic etiology who were treated by endoscopic stent insertion for 1 year with scheduled stent exchanges. ED was successful in all cases, with complete resolution of jaundice during the 1-year follow-up period. Pain scores, the number of patients requiring analgesics, the amount of analgesics required, and the type of analgesic medication did not change following treatment of CBD stricture. Successful ED of biliary obstruction had no inﬂuence on the pain pattern in patients with CP [69]. Farnbacher et al. [70] published their retrospective experience with 31 CP patients with CBD stenoses. In total, 101 endoprostheses were implanted endoscopically, exchanged after 3 8 2 months, and removed after 10 8 8 months. All jaundiced patients showed immediate improvement of cholestasis after drainage. Complete regression of stenosis and pre-stenotic dilation was accomplished only in 13%; dilation remained unchanged in 10%, and even showed progression in 22%. Cholestatic parameters remained normal in all patients with complete normalization of the CBD [70]. A retrospective evaluation was made of the long-term results of endoscopic stenting in 58 patients with benign biliary strictures due to CP [71]. Immediate relief of jaundice and cholestasis was achieved in all patients after endoscopic stent insertion. Median follow-up was 49 months. Sixteen (28%) of the 58 patients had regression of the biliary stricture and permanent removal of the stent. Forty-two patients had persistent biliary stricture: 26 had continued stenting and 16 underwent surgical procedures. Postoperative relief of jaundice was achieved in 15 of the 16 patients [71]. In another retrospective study, 39 patients with CP and symptomatic CBD stenoses underwent endoscopic stenting. After a median stenting time of 9 (range 1–144) months, 46% of the patients demonstrated regression of the stricture and clinical improvement, 26% required further stenting, and 28% were referred to sur-

Mönkemüller/Kahl/Malfertheiner

gery. Thirty-one percent demonstrated complete clinical recovery of the stricture as well as 10.2% a complete, radiologically veriﬁed stricture regression in a median follow-up of 58 months [40]. In one of the largest prospective studies published to date, Kahl et al. [69] described their experience with 61 patients with symptomatic CBD strictures caused by alcoholic CP who were treated by endoscopic stent insertion for 1 year with scheduled stent changes every 3 months. Initial ED was successful in all cases, with complete resolution of obstructive jaundice. After 1 year from the initial stent insertion, the obstruction was resolved in 19 patients (31.1%) and stents were removed without any need of additional procedures. During a median followup of 40 (range 18–66) months, 16 patients had no recurrence of symptomatic CBD stricture (long-term success rate 26.2%). Of 45 patients who needed deﬁnitive therapy, 12 patients (19.7%) were treated with repeated plastic stent insertion, 3 (4.9%) with insertion of a metal stent, and 30 patients (49.2%) underwent surgery. Among the variables tested, calciﬁcation of the pancreatic head was the only factor that was found to be of prognostic value (ﬁg. 1). Of 39 patients with calciﬁcation of the pancreatic head, only 3 (7.7%) were successfully treated with a 1-year period of plastic stent therapy, whereas in 13 of 22 patients (59.1%) without calciﬁcation, this treatment was successful [42]. Vitale et al. [41] evaluated the combination of balloon dilation prior to stent placement as a potential long-term treatment for these strictures. Twenty-ﬁve patients underwent sphincterotomy, balloon dilatation of the stricture, and then placement of a polyethylene stent (7–11.5 french). Stents were exchanged at 3- to 4-month intervals to avoid the complications of clogging and cholangitis. Twenty of the 25 patients did not have any recurrence of stricture for a mean of 32 months [41]. Another approach to CBD strictures associated with CP is to place as many stents as possible across the CBD stricture. Pozsar et al. [39] used this strategy in 29 patients. Biliary sphincterotomy, dilation of the stricture, and insertion of plastic biliary stents (7.5–10 french) were performed. Patients were scheduled for elective stent changing/restenting at 3-month intervals or any time when it was urgently indicated. Eighteen patients (60%) had complete radiologic and serologic recovery after a mean of 21.1 months. There were 3 deaths (10%): 1 for an unrelated cause and 2 with septic shock of biliary origin [39]. In selected patients with CP in whom conventional plastic stenting fails and in whom surgery is contraindicated or declined, insertion of a biliary self-expanding

Endoscopic Therapy of Chronic Pancreatitis

metal stent (SEMS) may be a valuable treatment option [37, 72–75]. The exact role of endoscopic stenting with SEMS as deﬁnitive treatment for CBD stricture due to CP has not yet been clearly deﬁned. Deviere et al. [37] treated 20 patients, 11 of whom had been treated previously with plastic endoprostheses. All had persistent cholestasis, 7 patients had jaundice, and 3 overt cholangitis. Endoscopic stent placement was successful in all cases. After a mean follow-up of 33 months, the stent lumen remained patent and functional, except in 2 patients who developed epithelial hyperplasia within the stent resulting in recurrent biliary obstruction 3 and 6 months after placement. One of these patients ultimately required surgical drainage [37]. Van Berkel et al. [72] treated 13 patients with CP with SEMS for benign biliary strictures. After a mean follow-up period of 50 months (range 6 days to 86 months), 9 patients (69%) were successfully treated with SEMS therapy. SEMS treatment was not successful in 4 patients (due to stent migration in 1 case and occlusion in 3). Of the successfully treated patients, 4 required either balloon dilation or placement of a new stent. The mean patency period of the SEMS was 60 months [72].

ED of Pancreatic Pseudocysts

ED can be accomplished via the papilla of Vater (transpapillar or transampular) or via the gastrointestinal wall (transmural), either through the stomach (cystogastrostomy) or through the duodenum (cystoduodenostomy) [30–36, 62, 76–79]. Before attempting ED drainage of a pseudocyst it is very important to review a CT of the abdomen. If a pseudocyst is more than 1 cm away from the gastrointestinal wall, ED should not be attempted. The technique of transmural pancreatic ﬂuid (TP) drainage is essentially the same as when placing a stent into the biliary tract. ED may be achieved by placing a stent (5 or 7 french, but up to 10 french) into the PD. The transmural approach is selected based on the site of maximal extrinsic compression of the pseudocyst on the stomach or duodenum [36]. This site may be predicted by CT and conﬁrmed endoscopically. Even if there is no clear communication with the collection, some endoscopists always perform a TP drain with a stent into the PD. Before attempting ED of a pseudocyst, antibiotic prophylaxis should be instituted [62–76]. When possible we prefer to drain the pseudocysts transmurally with the help of EUS. Multiple techniques to drain pancreatic pseudocysts transmurally have been described [30–36]. Most pseudocysts can be successfully

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287

Table 2. Endoscopic therapy of pancreatic pseudocysts

Author/year

n

TP

CD

CG

Initial success Complications

Kozarek et al. [9], 1989 Cremer et al. [8], 1990 Smits et al. [32], 1995 Barthet et al. [81], 1995 Catalano et al. [35], 1995 Binmoeller et al. [29], 1995 Mönkemüller et al. [36], 1998 Cunningham et al. [82], 1999

18 33 37 30 21 53 97 76

16 0 12 30 21 31 5 46

0 22 10 0 0 10 36 15

0 11 7 0 0 6 56 15

88 96 65 77 76 89 95 85

localized endoscopically by visualization of a bulge into the lumen which results from extrinsic compression by the pancreatic pseudocyst. The collection is initially punctured through the ‘bulge’ in the gastrointestinal luminal wall using an injection catheter. Howell et al. [76] have recommended the injection of up to 5 ml of contrast into the pseudocyst cavity in order to localize it under ﬂuoroscopic control and have termed this method ‘endoscopic needle localization or ENL’. Once the appropriate site is chosen, a cystoenterostomy is created with a variety of endoscopic instruments which include the standard needle-knife, pre-cut sphincterotome, exposed end of a polypectomy snare, laser, a double-channel ﬁstulotome or the Seldinger technique [30–36, 76]. When using the Seldinger technique, a wire is placed through the needle into the cavity, and the catheter is removed [36]. A biliary or small duodenal balloon (!12 mm diameter) is then placed over the wire, through the opening into the cystoenterostomy and then it is fully inﬂated [36]. It is very important to establish a ‘large’ cystogastrostomy opening so that the material can ﬂow freely into the gut lumen. The cystoenterostomy is then kept patent by the insertion of 1 to 4 or 5 double 10-french pigtail stents. If pus or necrotic debris is present inside the collection, then placement of a nasopancreatic catheter for irrigation is essential in order to mobilize the material from the collection. Routinely we perform a follow-up CT 4 weeks after the initial ED. It is well documented that in the presence of pseudocysts which compress against the gastrointestinal wall, the mucosa tends to become edematous and inﬂamed, therefore the risk of bleeding is theoretically increased. Some authorities recommend the routine use of EUS before ED of pseudocysts. Endosonography is coming to be regarded as essential before endoscopic therapy, since it can precisely identify the best and safest point at which to per-

288

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– 6 35 13 5 11 15 2

form the cystoenterostomy. Other experts perform ED solely with the use of EUS [77, 78]. EUS has the advantage that it can be used even when there is no bulging of the cyst into the digestive tract [77–79]. There are now several studies demonstrating the success of ED for pseudocysts occurring in the setting of chronic and acute pancreatitis [30–36]. The advantage of ED over percutaneous methods is internalization of drainage and subsequent avoidance of pancreatico-cutaneous ﬁstulae. Additionally, pancreatography performed at the time of attempted ED allows assessment of the pancreatic ductal system for correctable leaks, strictures, or intraductal stones. The results of the ED approach are similar to those of surgical drainage, with similarly high technical success rates and low recurrence and complication rates. There are to date no randomized trials comparing treatment modalities for drainage of pancreatic pseudocysts. Table 2 shows some of the largest series of ED drainage of pancreatic pseudocysts. The incidence of major hemorrhage or perforation after transmural drainage of pseudocysts ranges from 6 to 34% in experienced hands [30–36, 80]. Smits et al. [32] reported an incidence of complications of 30% after transgastric puncture and suggested that the transduodenal approach was a safer route of access into pseudocysts. Long-term follow-up of endoscopic treatment of chronic pseudocysts has shown recurrence rates for endoscopic transgastric drainage to be approximately 20%, and transduodenal drainage 9% [32]. Giovannini et al. [78] have one of the largest experiences using EUS as the sole method for ED of pancreatic pseudocysts. Using an interventional echo endoscope with a linear curved array transducer, the authors reported their experience with 35 patients who had pseudocysts with a mean diameter of 7.8 (4–12) cm. Most pseudocysts were located in the tail of the pancreas. The EUS instrument used was the FG 38X endoscope manufactured by

Mönkemüller/Kahl/Malfertheiner

Pentax-Hitachi. Successful ED was accomplished in 99% of cases. Surgery was performed in the 2 other patients. Concerning the pancreatic pseudocysts, placement of an 8.5-french stent was successful in 10 patients and of a nasopancreatic drain in 5 patients. 15% have relapsed over a mean follow-up of 27 (6–48) months. Seifert et al. [77, 79] have also reported success using a new largechannel echo endoscope which allows a one-step approach using either a 7- or 10-french stent-over-needle. These studies and others demonstrate that internal drainage of pancreatic pseudocysts and abscesses exclusively performed with an echo endoscope is a safe and efﬁcient method, and it can be added to the therapeutic armamentarium of the therapeutic endoscopist [77–79].

Miscellaneous: Fistulas, Complete Disruption of MPD and Pancreatic Ascites

Internal pancreatic ﬁstulas (IPF) are an uncommon but well-recognized complication of CP that are associated with signiﬁcant morbidity and mortality. Because of their low incidence, management is still controversial. In a study from Brazil IPF was identiﬁed in 11 (7.3%) of 150 patients with CP. The presentation was pancreatic ascites in 9 patients and pleural effusion in 2 cases [14]. Endoscopic placement of a transpapillary PD stent was performed in 4 patients who presented partial MPD stricture or disruption; surgical therapy was performed in 2 patients exhibiting complete MPD obstruction or disruption. Stents were removed 3–6 weeks after initial placement. IPF resolved in 10 of 11 patients (90.9%) within 6 weeks [14]. Complete disruption of the main PD is an unusual event in the course of acute or CP [9, 13]. Endoscopic management has already proven effective in the treatment of partial PD ruptures [12, 50, 52]. Kozarek [48] treated 18 patients with total PD disruptions, including 14 with deﬁnable ﬂuid collections; they were treated with

transpapillary PD drains or stents. Twelve of these patients had undergone a previous percutaneous or surgical pancreatic drainage procedure or both, and 8 had longterm drainage tubes in chronic ﬁstulous tracts. Transpapillary catheters could be placed across the ductal disruption or directly into the ﬂuid collection in each case, and 16 of 18 patients had resolution of the disrupted PD. Twelve of 14 ﬂuid collections resolved. Complications were limited to mild exacerbation of pancreatitis symptoms in 2 patients and 2 patients who developed subsequent stent occlusion leading to recurrent pancreatitis (1 patient) or recurrent duct blow-out with pseudocyst (1 patient). At a median follow-up of 16 months, 7 patients ultimately required surgery for ongoing pancreatic pain or residual/recurrent ﬂuid collection. The transpapillary treatment of ongoing pancreatic ductal disruption with or without ﬂuid collection has the potential to obviate surgery in some patients, change an urgent surgical procedure into an elective one, or even assist the surgeon in the performance of intraoperative pancreatography. Management of pancreatic ascites with conservative medical therapy or surgery has met with limited success. Decompression of the pancreatic ductal system through transpapillary stent placement, an alternative strategy, has been reported in only a handful of cases of pancreatic ascites. Bracher et al. [49] reported on 8 cases of pancreatic ascites managed endoscopically. A 5- or 7-french transpapillary PD stent was placed as the initial drainage procedure. Pancreatic ascites resolved in 7 of 8 patients (88%) within 6 weeks. Ascites resolved in the 8th patient, a poor candidate for surgery, following placement of a 5-mm expandable metallic pancreatic stent. No infections, alterations in ductal morphology, or other complications related to stent placement were noted. There was no recurrence of pancreatic ascites or duct disruption at a mean follow-up of 14 months. Endoscopic therapy (i.e. TP PD drainage) should be routinely considered in the initial management of patients with pancreatic ascites.

References 1 Godil A, Chen YK: Endoscopic management of benign pancreatic disease. Pancreas 2000; 20:1–13. 2 Hammarstrom LE, Stridbeck H, Ihse I: Endoscopic drainage in benign pancreatic disease: Immediate and medium term outcome. Eur J Surg 1997;163:577–589.

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3 Lehman GA, Sherman S, Hawes RH: Endoscopic management of recurrent and chronic pancreatitis. Scand J Gastroenterol 1995; 208(suppl):81–89. 4 Linder S, Engstrom CF, von Rosen A, Wiechel KL: Endoscopic clearance of the pancreatic duct in chronic pancreatitis with severe pain. Surg Endosc 1993;7:37–41.

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5 Laugier R, Renou C: Endoscopic ductal drainage may avoid resective surgery in painful chronic pancreatitis without large ductal dilatation. Int J Pancreatol 1998;23:145–152.

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6 Pap A, Topa L, Berger Z, Flautner L, Varro V: Pain relief and functional recovery after endoscopic interventions for chronic pancreatitis. Scand J Gastroenterol 1998; 228(suppl):98– 106. 7 Bockman DE, Büchler M, Malfertheiner P, Beger HG: Morphology of nerves in chronic pancreatitis and the interrelationship with inﬂammatory tissue; in Beger HG, Büchler M, Ditschuneit H, Malfertheiner P (eds): Chronic Pancreatitis. Berlin, Springer, 1990, vol 10, pp 77–82. 8 Cremer M, Deviere J, Delhaye M, Vandermeeren A, Baize M: Non-surgical management of severe chronic pancreatitis. Scand J Gastroenterol 1990;175:77–84. 9 Kozarek RA, Patterson DJ, Ball TJ, Traverso LW: Endoscopic placement of pancreatic stents and drains in the management of pancreatitis. Ann Surg 1989;209:261–266. 10 Prinz RA, Greenlee HB: Pancreatic duct drainage in chronic pancreatitis. Hepatogastroenterology 1990;37:295–300. 11 Dumonceau JM, Deviere J, Le Moine O, Delhaye M, Vandermeeren A, Baize M, Van Gansbeke D, Cremer M: Endoscopic pancreatic drainage in chronic pancreatitis associated with ductal stones: Long-term results. Gastrointest Endosc 1996;43:547–555. 12 Deviere J, Delhaye M, Cremer M: Pancreatic duct stones management. Gastrointest Endosc Clin N Am 1998;8:143–180. 13 Deviere J, Bueso H, Baize M, Azar C, Love J, Moreno E, Cremer M: Complete disruption of the main pancreatic duct: Endoscopic management. Gastrointest Endosc 1995;42:445–451. 14 Chebli JM, Gaburri PD, de Souza AF, Ornellas AT, Martins Junior EV, Chebli LA, Felga GE, Pinto JR: Internal pancreatic ﬁstulas: Proposal of a management algorithm based on a case series analysis. J Clin Gastroenterol 2004; 38: 795–800. 15 Vijungco JD, Prinz RA: Management of biliary and duodenal complications of chronic pancreatitis. World J Surg 2003; 27: 1258– 1270. 16 Costamagna G, Mutignani M: Metal stents in common bile duct strictures secondary to chronic pancreatitis: A new endoscopic treatment for an old problem. Endoscopy 2004;36: 450–452. 17 Kozarek RA, Ball TJ, Patterson DJ, Brandabur JJ, Traverso LW, Raltz S: Endoscopic pancreatic duct sphincterotomy: Indications, technique, and analysis of results. Gastrointest Endosc 1994;40:592–598. 18 Robert JY, Bretagne JF, Darnault P, Raoul JL, Siproudhis L, Heresbach D, Gosselin M: Endoscopic treatment and extracorporeal lithotripsy in chronic calcifying pancreatitis. Preliminary results in 16 patients. Gastroenterol Clin Biol 1993;17:797–803. 19 Bassi C, Falconi M, Caldiron E, Salvia R, Sartori N, Valerio A, Butturini G, Contro C, Casetti L, Marcucci S, Pederzoli P: To what extent is surgery superior to endoscopic therapy in the management of chronic pancreatitis? Ital J Gastroenterol Hepatol 1998;30:571–579.

290

Dig Dis 2004;22:280–291

20 Frey CF, Suzuki M, Isaji S: Treatment of chronic pancreatitis complicated by obstruction of the common bile duct or duodenum. World J Surg 1990;14:59–69. 21 Schmulewitz N, Hawes R: EUS-guided celiac plexus neurolysis – Technique and indication. Endoscopy 2003;35:S49–S53. 22 Hakaim AG, Broughan TA, Vogt DP, Hermann RE: Long-term results of the surgical management of chronic pancreatitis. Am Surg 1994;60:306–308. 23 Dite P, Ruzicka M, Zboril V, Novotny I: A prospective, randomized trial comparing endoscopic and surgical therapy for chronic pancreatitis. Endoscopy 2003;35:553–558. 24 Conwell DL: Motion-pancreatic endoscopy is useful for the pain of chronic pancreatitis: Arguments against the motion. Can J Gastroenterol 2003;17:61–63. 25 Delhaye M, Matos C, Deviere J: Endoscopic management of chronic pancreatitis. Gastrointest Endosc Clin N Am 2003;13:717–742. 26 Dite P, Zboril V, Cikankova E: Endoscopic therapy of chronic pancreatitis. Hepatogastroenterology 1996;43:1633–1637. 27 Tocchi A, Costa G, Lepre L, Liotta G, Mazzoni G, Sita A: The long-term outcome of hepaticojejunostomy in the treatment of benign bile duct strictures. Ann Surg 1996; 224: 162– 167. 28 Jakobs R, Benz C, Leonhardt A, Schilling D, Pereira-Lima JC, Riemann JF: Pancreatic endoscopic sphincterotomy in patients with chronic pancreatitis: A single-center experience in 171 consecutive patients. Endoscopy 2002;34:551–554. 29 Binmoeller KF, Jue P, Seifert H, Nam WC, Izbicki J, Soehendra N: Endoscopic pancreatic stent drainage in chronic pancreatitis and a dominant stricture: Long-term results. Endoscopy 1995;27:638–644. 30 Beckingham IJ, Krige JE, Bornman PC, Terblanche J: Endoscopic management of pancreatic pseudocysts. Br J Surg 1997; 84: 1638– 1645. 31 Kozarek RA, Ball TJ, Patterson DJ, Freeny PC, Ryan JA, Traverso LW: Endoscopic transpapillary therapy for disrupted pancreatic duct and peripancreatic ﬂuid collections. Gastroenterology 1991;100:1362–1370. 32 Smits ME, Rauws EA, Tytgat GN, Huibregtse K: The efﬁcacy of endoscopic treatment of pancreatic pseudocysts. Gastrointest Endosc 1995; 42:202–207. 33 De Palma GD, Galloro G, Puzziello A, Masone S, Persico G: Endoscopic drainage of pancreatic pseudocysts: A long-term follow-up study of 49 patients. Hepatogastroenterology 2002; 49:1113–1115. 34 Sharma SS, Bhargawa N, Govil A: Endoscopic management of pancreatic pseudocyst: A longterm follow-up. Endoscopy 2002;34:203–207. 35 Catalano MF, Geenen JE, Schmalz MJ, Johnson GK, Dean RS, Hogan WJ: Treatment of pancreatic pseudocysts with ductal communication by transpapillary pancreatic duct endoprosthesis. Gastrointest Endosc 1995;42:214– 218.

36 Mönkemüller KE, Baron TH, Morgan DE: Transmural drainage of pancreatic ﬂuid collections without electrocautery using the Seldinger technique. Gastrointest Endosc 1998; 48: 195–200. 37 Deviere J, Cremer M, Baize M, Love J, Sugai B, Vandermeeren A: Management of common bile duct stricture caused by chronic pancreatitis with metal mesh self expandable stents. Gut 1994;35:122–126. 38 Ng C, Huibregtse K: The role of endoscopic therapy in chronic pancreatitis-induced common bile duct strictures. Gastrointest Endosc Clin N Am 1998;8:181–193. 39 Pozsar J, Sahin P, Laszlo F, Forro G, Topa L: Medium-term results of endoscopic treatment of common bile duct strictures in chronic calcifying pancreatitis with increasing numbers of stents. J Clin Gastroenterol 2004; 38: 118– 123. 40 Eickhoff A, Jakobs R, Leonhardt A, Eickhoff JC, Riemann JF: Endoscopic stenting for common bile duct stenoses in chronic pancreatitis: Results and impact on long-term outcome. Eur J Gastroenterol Hepatol 2001;13:1161–1167. 41 Vitale GC, Reed DN Jr, Nguyen CT, Lawhon JC, Larson GM: Endoscopic treatment of distal bile duct stricture from chronic pancreatitis. Surg Endosc 2000;14:227–231. 42 Kahl S, Zimmermann S, Genz I, Glasbrenner B, Pross M, Schulz HU, McNamara D, Schmidt U, Malfertheiner P: Risk factors for failure of endoscopic stenting of biliary strictures in chronic pancreatitis: A prospective follow-up study. Am J Gastroenterol 2003; 98: 2448– 2453. 43 Stabile BE, Calabria R, Wilson SE, Passaro E Jr: Stricture of the common bile duct from chronic pancreatitis. Surg Gynecol Obstet 1987;165:121–126. 44 Smits ME, Badiga SM, Rauws EA, Tytgat GN, Huibregtse K: Long-term results of pancreatic stents in chronic pancreatitis. Gastrointest Endosc 1995;42:461–467. 45 Warshaw AL, Schapiro RH, Ferrucci JT Jr, Galdabini JJ: Persistent obstructive jaundice, cholangitis, and biliary cirrhosis due to common bile duct stenosis in chronic pancreatitis. Gastroenterology 1976;70:562–567. 46 Eckhauser FE, Knol JA, Strodel WE, Achem S, Nostrant T: Common bile duct strictures associated with chronic pancreatitis. Am Surg 1983;49:350–358. 47 Kuo Y, Wu C: The role of ERCP in pancreatic ascites. Dig Dis Sci 1994;39:1143–1146. 48 Kozarek RA: Endoscopic therapy of complete and partial pancreatic duct disruptions. Gastrointest Endosc Clin N Am 1998;8:39–53. 49 Bracher GA, Manocha AP, DeBanto JR, Gates LK Jr, Slivka A, Whitcomb DC, Bleau BL, Ulrich CD 2nd, Martin SP: Endoscopic pancreatic duct stenting to treat pancreatic ascites. Gastrointest Endosc 1999;49:710–715. 50 Bahsin DK, Poddar U: Endoscopic treatment of pancreatitis; in Bhutani MS, Tandon RK (eds): Advances in Gastrointestinal Endoscopy. New Delhi, Jaypee Brothers, 2001, vol 18, pp 284–302.

Mönkemüller/Kahl/Malfertheiner

51 Bahsin DK, Dharan S, Siriam PVJ, et al: Endoscopic management of pancreatic diseases. Indian J Gastroenterol 1997;16:30–31. 52 Sherman S, Lehman GA: Endoscopic pancreatic sphincterotomy: Techniques and complications. Gastrointest Endosc Clin N Am 1998; 8:115–125. 53 Ell C, Rabenstein T, Schneider HT, Ruppert T, Nicklas M, Bulling D: Safety and efﬁcacy of pancreatic sphincterotomy in chronic pancreatitis. Gastrointest Endosc 1998;48:244–249. 54 Esber E, Sherman S, Earle E, et al: Complications of major pancreatic duct sphincterotomy: A review of 106 patients. Gastrointest Endosc 1995;41:A422. 55 Elton E, Howell DA, Parsons WG, Qaseem T, Hanson BL: Endoscopic pancreatic sphincterotomy: Indications, outcome, and a safe stentless technique. Gastrointest Endosc 1998; 47: 240–249. 56 Song MH, Kim MH, Lee SK, Lee SS, Han J, Seo DW, Min YI, Lee DK: Endoscopic minor papilla interventions in patients without pancreas divisum. Gastrointest Endosc 2004; 59: 901–905. 57 Tarnasky P, Palesch YY, Cunningham JT, Maudldin PD, Cotton PB, Hawes RH: Pancreatic duct stenting prevents pancreatitis after biliary sphincterotomy in patients with sphincter of Oddi dysfunction. Gastroenterology 1998;115:1518–1524. 58 Wilcox CM, Mönkemüller KE: Wire-assisted minor papilla precut papillotomy. Gastrointest Endosc 2001;54:83–86. 59 Ikeneberry SO, Sherman S, Hawes RH, et al: The occlusion rate of pancreatic stents. Gastrointest Endosc 1994;40:611–613. 60 Sherman S, Alvarez C, Robert M, et al: Polyethylene pancreatic duct stent-induced changes in the normal dog pancreas. Gastrointest Endosc 1993;39:658–643. 61 Morgan DE, Smith JK, Hawkins K, Wilcox CM: Endoscopic stent therapy in advanced chronic pancreatitis: Relationships between ductal changes, clinical response, and stent patency. Am J Gastroenterol 2003;98:821–826. 62 Neuhaus H: Therapeutic pancreatic endoscopy. Endoscopy 2002;34:54–62.

Endoscopic Therapy of Chronic Pancreatitis

63 Costamagna G, Gabbrielli A, Mutignani M, Perri V, Pandolﬁ M, Boscaini M, Crucitti F: Extracorporeal shock wave lithotripsy of pancreatic stones in chronic pancreatitis: Immediate and medium-term results. Gastrointest Endosc 1997;46:231–236. 64 Okolo PI 3rd, Pasricha PJ, Kalloo AN: What are the long-term results of endoscopic pancreatic sphincterotomy? Gastrointest Endosc 2000;52:15–19. 65 Rösch T, Daniel S, Scholz M, Huibregtse K, Smits M, Schneider T, Ell C, et al: Endoscopic treatment of chronic pancreatitis: A multicenter study of 1000 patients with long-term follow-up. Endoscopy 2002;34:765–771. 66 Ponchon T, Bory RM, Hedelius F, Roubein LD, Paliard P, Napoleon B, Chavaillon A: Endoscopic stenting for pain relief in chronic pancreatitis: Results of a standardized protocol. Gastrointest Endosc 1995;42:452–456. 67 Brand B, Kahl M, Sidhu S, Nam VC, et al: Prospective evaluation of morphology, function, and quality of life after extracorporeal shockwave lithotripsy and endoscopic treatment of chronic calciﬁc pancreatitis. Am J Gastroenterol 2000;95:3428–3438. 68 Tocchi A, Costa G, Lepre L, Liotta G, Mazzoni G, Sita A: The long-term outcome of hepaticojejunostomy in the treatment of benign bile duct strictures. Ann Surg 1996; 224: 162– 167. 69 Kahl S, Zimmermann S, Genz I, Schmidt U, Pross M, Schulz HU, Malfertheiner P: Biliary strictures are not the cause of pain in patients with chronic pancreatitis. Pancreas 2004; 28: 387–390. 70 Farnbacher MJ, Rabenstein T, Ell C, Hahn EG, Schneider HT: Is endoscopic drainage of common bile duct stenoses in chronic pancreatitis up-to-date? Am J Gastroenterol 2000;95: 1466–1471. 71 Smits ME, Rauws EA, van Gulik TM, Gouma DJ, Tytgat GN, Huibregtse K: Long-term results of endoscopic stenting and surgical drainage for biliary stricture due to chronic pancreatitis. Br J Surg 1996;83:764–768. 72 van Berkel AM, Cahen DL, van Westerloo DJ, Rauws EA, Huibregtse K, Bruno MJ: Self-expanding metal stents in benign biliary strictures due to chronic pancreatitis. Endoscopy 2004;36:381–384.

Dig Dis 2004;22:280–291

73 Eickhoff A, Jakobs R, Leonhardt A, Eickhoff JC, Riemann JF: Self-expandable metal mesh stents for common bile duct stenosis in chronic pancreatitis: Retrospective evaluation of long-term follow-up and clinical outcome pilot study. Z Gastroenterol 2003;41:649–654. 74 Eisendrath P, Deviere J: Expandable metal stents for benign pancreatic duct obstruction. Gastrointest Endosc Clin North Am 1999; 9: 547–554. 75 Kahl S, Zimmermann S, Glasbrenner B, Pross M, Schulz HU, McNamara D, Malfertheiner P: Treatment of benign biliary strictures in chronic pancreatitis by self-expandable metal stents. Dig Dis 2002;20:199–203. 76 Howell DA, Elton E, Parsons WG: Endoscopic management of pseudocysts of the pancreas. Gastrointest Endosc 1998;8:143–162. 77 Seifert H, Dietrich C, Schmitt T, Caspary W, Wehrmann T: Endoscopic ultrasound-guided one-step transmural drainage of cystic abdominal lesions with a large-channel echo endoscope. Endoscopy 2000;32:255–259. 78 Giovannini M, Pesenti C, Rolland AL, Moutardier V, Delpero JR: Endoscopic ultrasound-guided drainage of pancreatic pseudocysts or pancreatic abscesses using a therapeutic echo endoscope. Endoscopy 2001; 33: 473–477. 79 Seifert H, Faust D, Schmitt T, Dietrich C, Caspary W, Wehrmann T: Transmural drainage of cystic peripancreatic lesions with a new largechannel echo endoscope. Endoscopy 2001;33: 1022–1026. 80 Eisendrath P, Deviere J: Expandable metal stents for benign pancreatic duct obstruction. Gastrointest Endosc Clin N Am 1999; 9: 547– 554. 81 Barthet M, Sahel J, Bodiou-Bertei C, Bernard JP: Endoscopic transpapillary drainage of pancreatic pseudocysts. Gastrointest Endosc 1995;42:208–213. 82 Cunningham J, Tarnasky PR, Hawes RH, et al: ERCP in the management of pancreatic pseudocysts. Gastrointest Endosc 1999;49: AB189.

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Original Paper Dig Dis 2004;22:292–295 DOI: 10.1159/000082801

Chronic Parotitis: Not Another SPINKosis Felix Gundlinga, e Fabian Reitmeierb Andrea Tannapfelc Alexander Schützc Anette Weberd Jürgen Ussmüllerb Volker Keima Joachim Mössnera Niels Teicha a

Medizinische Klinik und Poliklinik II, Universitätsklinikum Leipzig, b Klinik für Hals-, Nasen- und Ohrenheilkunde, Universitätsklinikum Hamburg-Eppendorf, Hamburg, c Institut für Pathologie, Universitätsklinikum Leipzig, d Klinik für Hals-, Nasen- und Ohrenheilkunde, Universitätsklinikum Leipzig, und e II. Medizinische Abteilung für Gastroenterologie, Hepatologie und Gastroenterologische Onkologie, Städtisches Krankenhaus München-Bogenhausen, München, Deutschland

Key Words Parotitis SPINK1 N34S mutation

Abstract Introduction: Pancreatitis and parotitis share several etiological, pathohistological and functional similarities. It arose from recent pancreatitis research that some cases of chronic pancreatitis are associated with mutations of the serine protease inhibitor, Kazal type-1 (SPINK1). We tested the hypothesis that the pancreatitis-associated N34S mutation of SPINK1 is also a risk factor for chronic parotitis. Methods: Reverse-transcriptase polymerase chain reaction was used to investigate SPINK1 transcription in the parotid gland. Forty-five blocks of formalin-fixed, paraffin wax-embedded tissues with chronic parotitis of unknown cause were analyzed for the SPINK1-N34S mutation. Results: The SPINK1 gene is transcribed in the parotid gland. Two of the 45 patients (4.4%) with chronic parotitis carried the N34S mutation heterozygously. Of 82 healthy blood donors, 3 subjects (3.7%) were identified as carrying this mutation heterozygously (p = 0.83). Conclusion: The SPINK1-N34S mutation is not associated with chronic parotitis. Copyright © 2004 S. Karger AG, Basel

© 2004 S. Karger AG, Basel 0257–2753/04/0223–0292$21.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/ddi

Introduction

Pancreatitis and parotitis share various etiological, morphological and functional similarities. The best characterized association is acute pancreatitis and parotitis due to mumps virus infection [1]. Sagatelian et al. [2] found coexistent ductal abnormalities of the parotid gland in 78% of alcoholics with ductal lesions of the pancreas, and Frulloni et al. [3] investigated the sialographies of 49 patients with chronic pancreatitis of different origin. Fifteen patients and none of the 10 controls exhibited abnormalities of the glandular ducts compatible with chronic inﬂammation of the salivary ducts. The frequent ﬁnding of parotid ductal abnormalities in chronic pancreatitis suggests a common pathogenetic mechanism [2, 3]. An actual study demonstrated highly similar (immuno-)histochemical features of the pancreas and salivary glands in patients with autoimmune chronic pancreatitis. The authors suspected both lesions as parts of a systemic disease of multifocal ﬁbrosclerosis [4]. The same group investigated the function of the salivary glands by sialochemistry and salivary gland scintigraphy in patients with chronic pancreatitis, Sjogren’s syndrome and controls. Salivary gland function was frequently and signiﬁ-

Niels Teich, MD University of 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]

cantly impaired in the course of chronic pancreatitis of alcoholic, autoimmune and idiopathic etiologies [5]. Even less common etiologies may lead to acute inﬂammation of both glands. In recent reports, acute parotitis due to organophosphate intoxication has been reported, which is a rare cause of acute pancreatitis [6, 7], and recurrent simultaneous acute pancreatitis and parotitis induced by methimazole was reported in 1 patient with Graves’ disease [8]. In 1998, a family was presented in which 4 members had juvenile recurrent parotitis and 2 other family members may have also had chronic parotitis. The segregation pattern in the family suggested an autosomal dominant inheritance with incomplete penetration. This suggested that genetic factors may be implicated in chronic parotitis [9]. The parallel to autosomal dominant hereditary pancreatitis is obvious. During the last years, the latter disease has been intensively studied from genetic, clinical and biochemical points of view. In brief, hereditary pancreatitis is mainly associated with two distinct mutations of the cationic trypsinogen gene, which increase trypsinogen autoactivation of cationic trypsinogen in vitro [10– 12]. Further, less common mutations may induce reduced autoactivation due to trypsinogen misfolding or, on the contrary, an increased transactivation of anionic trypsinogen [13, 14]. Due to intensive research, hundreds of kindreds with autosomal dominant chronic pancreatitis have been detected worldwide. In contrast, chronic parotitis is a predominantly sporadically occurring disease. Recent pancreatitis research has shown that some cases of sporadically occurring chronic pancreatitis are associated with mutations of the serine protease inhibitor, Kazal type-1 (SPINK1). The N34S mutation of this gene was found in 20% of patients with ‘idiopathic’ chronic pancreatitis [15].Tropical and alcoholic chronic pancreatitis are also associated with this mutation in 50 and 6%, respectively [16, 17]. Hassan et al. [18] found an association between the SPINK1-N34S mutation and ﬁbrocalculous pancreatic diabetes in 33% of 180 patients on the Indian subcontinent. The close association of the SPINK1-N34S mutation to variable etiologies of chronic pancreatitis and even ﬁbrocalculous pancreatic diabetes led us to the hypothesis that chronic inﬂammatory diseases of other organs may also be associated with this mutation. Due to the parallels reported above, chronic parotitis seems to be the most likely candidate. The aim of this study was to determine whether SPINK1 is expressed in the parotid gland and whether the SPINK1-N34S mutation is associated with chronic idiopathic parotitis.

Chronic Parotitis: Not Another SPINKosis

Table 1. Primer sequences

SPINKrtfw SPINKfw SPINKrtrev GAPDHfw GAPDHrev

CAG TGC CTT GGC CCT GTT GAG GTG TTT AAT TCC ATT TTT AGG C GGT CAT ATA TCT TGG TGC ATC GGC ACC GTC AAG GCT GAG AA AGA GGC AGG GAT GAT GTT CT

Materials and Methods rtPCR of the SPINK1 Gene in Parotid Tissue 15 mg each of two different human parotid tissue samples were homogenized with a mortar and pestle in liquid nitrogen. Total RNA was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany, cat. No. 74104) according to the manufacturer’s protocol. Reverse transcriptase reaction was carried out with 5 g total pancreatic RNA using the SuperScript II RNAse H-Reverse Transcriptase according to the manufacturer’s instructions (Invitrogen, Karlsruhe, Germany, cat. No. 18064-022). 5 l of the resulting cDNA solution was used for subsequent polymerase chain reaction (PCR). The PCR was performed with the GeneAmp XL PCR Kit (Applied Biosystems, Foster City, Calif., USA, cat. No. N808-0192) using the primer pair SPINKrtfw (in exon 1) and SPINKrtrev (in exon 3). The resulting PCR product has a length of 106 bp. As control, the housekeeping gene GAPDH was ampliﬁed with the primer pair GAPDHfw and GAPDHrev, which led to a 459-bp PCR product. All primer sequences are shown in table 1. Chronic Parotitis Patients Forty-ﬁve patients with chronic parotitis of unknown etiology from the German Salivary Gland Registry, Hamburg, and the ENT Hospital of Leipzig University took part in this investigation. All patients gave their informed consent for the scientiﬁc use of the resected specimens. Germline DNA Extraction Patients’ parotid glands were investigated from formalin-ﬁxed, parafﬁn wax-embedded tissue specimens. The diagnosis of chronic parotitis was histological conﬁrmed in all cases. From each block, 4 slices of 5 m thickness were cut. Germline DNA extraction was performed by microwave extraction [19]. In brief, microcentrifuge tubes containing tissue slices and 200 l of digestion buffer (50 mM Tris/HCl, pH 8.5; 1 mM EDTA; 0.5% Tween 20) were tightly capped and subjected to high-power microwave irradiation for 2 min, with the irradiation time split into 15-second segments to prevent over boiling. After centrifugation for 2 min at room temperature, the solid parafﬁn wax ring above the buffer was removed, and the tissue pellet was digested overnight in a buffer containing 200 l/ml proteinase K at 56 ° C. After full-speed centrifugation at room temperature for 30 min, the supernatant containing the DNA was removed and boiled for 10 min at 95 ° C to denature residual proteinase. 10 l of these preparations were used for subsequent PCR. PCR Ampliﬁcation and Restriction Digestion The ampliﬁcation of the SPINK1 gene was carried out with the primer pair SPINKfw and SPINKrtrev (table 1) to obtain a 66-bp

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1

2

3

4

GAPDH SPINK1

Fig. 1. Ethidium bromide-stained 1.4% agarose gel. Lane 1 = Mo-

lecular weight marker; lane 2 = PCR containing no cDNA (negative control); lanes 3 and 4 = two bands representing the GAPDH- and SPINK1-PCR products in two different cDNA samples of normal parotid gland.

PCR product spanning the N34 site of the SPINK1 gene. Other primer combinations which would result in longer PCR products were not successful in all samples or only successful in just a few samples (data not shown). The hot start PCR program was as follows: 94 ° C for 2 min, 35 cycles (94 ° C for of 15 s, 52 ° C for 30 s, and at 72 ° C for 30 s), and a ﬁnal extension step at 72 ° C for 4 min. 10 l of the resulting PCR product were subsequently digested with 1 unit of the restriction enzyme HpyCh4III (New England Biolabs cat. No. R0618S), which speciﬁcally detects the SPINK1-N34S mutation. In this case, the 66-bp PCR product will be digested into two 33-bp fragments. After 1-hour incubation at 37 ° C, the resulting fragments were electrophoretically separated on a 12% polyacrylamide gel. This approach has been adapted to our previously reported protocol [20]. As controls, DNA preparations from each 10 homozygous and heterozygous carriers of the N34S mutation and 10 wild-type carriers were used. All controls are participants of the German Registry of Hereditary Pancreatitis and the SPINK1-N34 status has previously been double-strand sequenced.

Results

Despite its initial designation as ‘pancreatic secretory trypsin inhibitor’, the powerful protease inhibitor SPINK1 is expressed in various gastrointestinal epithelia [21], and even extragastrointestinal tissues and tumors. The latter led to the third synonym ‘tumor-associated trypsin inhibitor’ [22, 23]. Due to its expression in this variety of organs, a potential inﬂuence of pancreatitis-associated mutations on chronic inﬂammatory diseases of these organs is conceivable. In parallel to the cystic ﬁbrosis syndrome [24] with various associated diseases (lung disease, chronic pancreatitis, liver cirrhosis, congenital bilateral absence of the vasa deferentia, chronic sialadenitis, etc.), and the already proven association of the SPINK1-N34S mutation to multiple etiologies of chronic pancreatitis, we hypothesized a SPINK syndrome with various ‘SPINKoses’. However, in this ﬁrst attempt to investigate the genetic basis of chronic parotitis, we failed to demonstrate an association of chronic parotitis with the SPINK1N34S mutation. A drawback of our experimental set-up is the restriction of our analysis to the N34S mutation, but none of the 30 other SPINK1 variants which have been identiﬁed so far (www.uni-leipzig.de/pancreasmutation). This restriction had to be made due to the bad DNA quality from some parafﬁn blocks that were more than 10 years old. Ampliﬁcation attempts with longer SPINK1 PCR products [15, 20, 21] failed due to the PCR product length of some hundred basepairs. The technology used here, however, led to the reproducible analysis of the SPINK1-N34S mutation in even bad DNA quality. Although N34S is the only proven SPINK1 mutation in chronic pancreatitis, the impact of other genetic variants of the SPINK1 gene for chronic parotitis may be overlooked by our approach. Whether rare mutations of the SPINK1 gene can have an impact in the pathogenesis of chronic parotitis should be evaluated in a prospective study.

The SPINK1 gene is transcribed in the parotid gland as shown in ﬁgure 1. Two of the 45 patients (4.4%) with chronic parotitis carried the N34S mutation heterozygously. In 82 healthy blood donors, 3 subjects (3.7%) were identiﬁed to carry this mutation heterozygously. This difference is not signiﬁcant (p = 0.83).

Discussion

We demonstrated the transcription of the SPINK1 gene in the parotid gland. Its highly frequent N34S mutation in various forms of chronic pancreatitis is not associated with chronic parotitis.

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References 1 Parenti DM, Steinberg W, Kang P: Infectious causes of acute pancreatitis. Pancreas 1996;13: 356–371. 2 Sagatelian MA, Fravel J, Gallo SH, et al: Do parotid duct abnormalities occur in patients with chronic alcoholic pancreatitis? Am J Gastroenterol 1998;93:197–200. 3 Frulloni L, Morana G, Bovo P, et al: Salivary gland involvement in patients with chronic pancreatitis. Pancreas 1999;19:33–38. 4 Kamisawa T, Funata N, Hayashi Y, et al: Close relationship between autoimmune pancreatitis and multifocal ﬁbrosclerosis. Gut 2003; 52: 683–687. 5 Kamisawa T, Tu Y, Egawa N, et al: Salivary gland involvement in chronic pancreatitis of various etiologies. Am J Gastroenterol 2003; 98:323–326. 6 Gokel Y, Gulalp B, Acikalin A: Parotitis due to organophosphate intoxication. J Toxicol Clin Toxicol 2002;40:563–565. 7 Sahin I, Onbasi K, Sahin H, et al: The prevalence of pancreatitis in organophosphate poisonings. Hum Exp Toxicol 2002;21:175–177. 8 Taguchi M, Yokota M, Koyano H, et al: Acute pancreatitis and parotitis induced by methimazole in a patient with Graves’ disease. Clin Endocrinol 1999;51:667–670. 9 Reid E, Douglas F, Crow Y, et al: Autosomal dominant juvenile recurrent parotitis. J Med Genet 1998;35:417–419. 10 Whitcomb DC, Gorry MC, Preston RA, et al: Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat Genet 1996;14:141–145.

Chronic Parotitis: Not Another SPINKosis

11 Gorry MC, Gabbaizedeh D, Furey W, et al: Mutations in the cationic trypsinogen gene are associated with recurrent acute and chronic pancreatitis. Gastroenterology 1997; 113: 1063–1068. 12 Teich N, Mössner J, Keim V: Mutations of the cationic trypsinogen in hereditary pancreatitis. Hum Mutat 1998;12:39–43. 13 Simon P, Weiss UF, Sahin-Tóth M, et al: Hereditary pancreatitis caused by a novel PRSS1 mutation (Arg-122rCys) that alters autoactivation and autodegradation of cationic trypsinogen. J Biol Chem 2002;277:5404–5410. 14 Teich N, Le Marechal C, Kukor Z, et al: Interaction between trypsinogen isoforms in genetically determined pancreatitis: Mutation E79K in cationic trypsin (PRSS1) causes increased transactivation of anionic trypsinogen (PRSS2). Hum Mutat 2004;23:22–31. 15 Witt H, Luck W, Hennies HC, et al: Mutations in the gene encoding the serine protease inhibitor, Kazal type 1 are associated with chronic pancreatitis. Nat Genet 2000;25:213–216. 16 Chandak GR, Idris MM, Reddy DN, et al: Mutations in the pancreatic secretory trypsin inhibitor gene (PSTI/SPINK1) rather than the cationic trypsinogen gene (PRSS1) are signiﬁcantly associated with tropical calciﬁc pancreatitis. J Med Genet 2002;39:347–351. 17 Witt H, Luck W, Becker M, et al: Mutation in the SPINK1 trypsin inhibitor gene, alcohol use, and chronic pancreatitis. JAMA 2001; 285:2716–2717.

Dig Dis 2004;22:292–295

18 Hassan Z, Mohan V, Ali L, et al: SPINK1 is a susceptibility gene for ﬁbrocalculous pancreatic diabetes in subjects from the Indian subcontinent. Am J Hum Genet 2002; 71: 964– 968. 19 Banerjee SK, Makdisi WF, Weston AP, et al: Microwave-based DNA extraction from parafﬁn-embedded tissue for PCR ampliﬁcation. Biotechniques 1995;18:768–773. 20 Teich N, Bauer N, Mössner J, et al: Mutational screening of patients with nonalcoholic chronic pancreatitis: Identiﬁcation of further trypsinogen variants. Am J Gastroenterol 2002;97:341–346. 21 Bohe M, Lindstrom C, Ohlsson K: Immunoreactive pancreatic secretory trypsin inhibitor in gastrointestinal mucosa. Adv Exp Med Biol 1988;240:101–105. 22 Fukayama M, Hayashi Y, Koike M, et al: Immunohistochemical localization of pancreatic secretory trypsin inhibitor in fetal and adult pancreatic and extrapancreatic tissues. J Histochem Cytochem 1986;34:227–235. 23 Stenman UH: Tumor-associated trypsin inhibitor. Clin Chem 2002;48:1206–1209. 24 Durie PR, Kent G, Phillips MJ, et al: Characteristic multiorgan pathology of cystic ﬁbrosis in a long-living cystic ﬁbrosis transmembrane regulator knockout murine model. Am J Pathol 2004;164:1481–1493. 25 Chen JM, Mercier B, Audrezet MP, et al: Mutational analysis of the human pancreatic secretory trypsin inhibitor (PSTI) gene in hereditary and sporadic chronic pancreatitis. J Med Genet 2000;37:67–69.

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Author Index Vol. 22, No. 3, 2004

Apte, M.V. 273 Beger, H.G. 247 Bödeker, H. 239 Büchler, M.W. 267 Di Sebastiano, P. 267 Friess, H. 267 Gaiser, S. 239 Garg, P.K. 258 Gundling, F. 292 Iovanna, J.L. 239 Kahl, S. 280 Keim, V. 292 Malfertheiner, P. 280 Mola, F.F. di 267

Mönkemüller, K. 280 Mössner, J. 233, 235, 292 Rau, B. 247 Reitmeier, F. 292 Savkovic, V. 239 Schilling, M.K. 247 Schütz, A. 292 Tandon, R.K. 258 Tannapfel, A. 292 Teich, N. 235, 292 Ussmüller, J. 292 Weber, A. 292 Wilson, J.S. 273

Subject Index Vol. 22, No. 3, 2004

Chronic pancreatitis 235, 258, 273 Diabetes, pancreatic 258 Endotherapy, pancreatic 258 Exocrine pancreas 239 Gene expression 239 Inflammation, chronic pancreatic 280 Interleukins 247 Interstitial hypertension 267 Intraductal pressure 267 Ischemia 267 Molecular analysis, pancreatic juice 235 Nerve entrapment, pancreatic 280 Pancreatic calcification 258 – cancer 233 – duct 280 – fibrosis 267, 273 – juice, molecular analysis 235

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Pancreatitis, acute 239, 247 –, chronic 267 –, management 280 Parotitis 292 Peripancreatic fibrosis 280 Procalcitonin 247 Procarboxypeptidase B activation peptide 247 Serum amyloid A 247 SPINK1-N34S mutation 292 Stellate cells, pancreatic 273 Stress response 239 Tropical pancreatitis, SPINK1 gene mutation 258 Trypsinogen 247