A Weekly Journal of Gastroenterology and Hepatology

World Journal of Gastroenterology

Indexed and Abstracted in:Current Contents®/Clinical Medicine, Science Citation Index Expanded (also known as SciSearch®) and Journal Citation Reports/Science Edition, Index Medicus, MEDLINE and PubMed, Chemical Abstracts, EMBASE/Excerpta Medica, Abstracts Journals, Nature Clinical Practice Gastroenterology and Hepatology, CAB Abstracts and Global Health. ISI JCR 2003-2000 IF: 3.318, 2.532, 1.445 and 0.993.

Volume 13 Number 47December 21, 2007

World J Gastroenterol2007 December 21; 13(47): 6291-6454

Online Submissionswjg.wjgnet.com

www.wjgnet.com Printed on Acid-free Paper

ISSN 1007-9327 CN 14-1219/R Local Post Offices Code No. 82-261

World Journal of Gastroenterology ®

National Journal Award2005

Volume 13 Number 47December 21, 2007

World Journal of G

astroenterology ww

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jgnet.com Volum

e 13 Num

ber 47 Dec 21 2007

ISSN 1007-9327CN 14-1219/R

Editorial Department of World Journal of Gastroenterology77 Shuangta Xijie, Taiyuan 030001, Shanxi Province, China Telephone: +86-351-4078656 E-mail: wjg@wjgnet.com http://www.wjgnet.com

Baishideng

World Journal ofGastroenterology

Volume 13 Number 47December 21, 2007

Contents

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6291 What’s wrong with sentinel node mapping in colon cancer?Cahill RA

6295 A potpourri of pancreatic issuesByrne MF

6296 Review of idiopathic pancreatitisLee JK, Enns R

6314 Role of endoscopic retrograde cholangiopancreatography in acute pancreatitisCanlas KR, Branch MS

6321 Utility of endoscopic ultrasound in pancreatitis: A reviewRizk MK, Gerke H

6327 Autoimmune pancreatitis: A reviewZandieh I, Byrne MF

6333 Sphincter of Oddi dysfunction and pancreatitisMcLoughlin MT, Mitchell RMS

6344 Pancreatic endocrine and exocrine changes in celiac diseaseFreeman HJ

6347 Transplantation for the treatment of type 1 diabetesMeloche RM

6356 Evaluation of quantitative contrast harmonic imaging to assess malignancy of liver tumors: A prospective controlled two-center study

Jung EM, Clevert DA, Schreyer AG, Schmitt S, Rennert J, Kubale R, Feuerbach S, Jung F

6365 Analysis of the expression of coxsackievirus and adenovirus receptor in five colon cancer cell lines

Abdolazimi Y, Mojarrad M, Pedram M, Modarressi MH

6370 Probiotic bacteria change Escherichia coli -induced gene expression in cultured colonocytes: Implications in intestinal pathophysiology

Panigrahi P, Braileanu GT, Chen H, Stine OC

EDITORIAL

National Journal Award2005

Weekly Established in October 1995

Baishideng

TOPIC HIGHLIGHT

LIVER CANCER

COLORECTAL CANCER

BASIC RESEARCH

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6379 Inflammatory cytokines promote inducible nitric oxide synthase-mediated DNA damage in hamster gallbladder epithelial cells

Kitasato A, Tajima Y, Kuroki T, Tsutsumi R, Adachi T, Mishima T, Kanematsu T

6385 Intestinal endotoxemia plays a central role in development of hepatopulmonary syndrome in a cirrhotic rat model induced by multiple pathogenic factors

Zhang HY, Han DW, Su AR, Zhang LT, Zhao ZF, Ji JQ, Li BH, Ji C

6396 Two distinct pathways of p16 gene inactivation in gallbladder cancerTadokoro H, Shigihara T, Ikeda T, Takase M, Suyama M

6404 Left-sided gallbladder: Its clinical significance and imaging presentationsHsu SL, Chen TY, Huang TL, Sun CK, Concejero AM, Tsang LLC, Cheng YF

6410 New method for long-term monitoring of intragastric pHOno S, Kato M, Ono Y, Asaka M

6414 Personality factors and profiles in variants of irritable bowel syndrome Farnam A, Somi MH, Sarami F, Farhang S, Yasrebinia S

6419 Prevalence of fatty liver disease and its risk factors in the population of South China

Zhou YJ, Li YY, Nie YQ, Ma JX, Lu LG, Shi SL, Chen MH, Hu PJ

6425 Antitumor and antiangiogenic activities of anti-vascular endothelial growth factor hairpin ribozyme in human hepatocellular carcinoma cell cultures and xenografts

Li LH, Guo ZJ, Yan LL, Yang JC , Xie YF, Sheng WH, Huang ZH, Wang XH

6433 Complete pathological response following down-staging chemoradiation in locally advanced pancreatic cancer: Challenging the boundaries

Khan AZ, Pitsinis V, Mudan SS

6436 Metastatic hepatocellular carcinoma of the external auditory canalYasumatsu R, Okura K, Sakiyama Y, Nakamuta M, Matsumura T, Uehara S, Yamamoto T, Komune S

6439 Decompensated porto-pulmonary hypertension in a cirrhotic patient with thrombosis of portocaval shunt

Giannarelli C, De Giorgi A, De Negri F, Carmassi F

6441 Malignant fibrous histiocytoma presenting as hemoperitoneum mimicking hepatocellular carcinoma rupture

Chen HC, Chen CJ, Jeng CM, Yang CM

6444 Eosinophilic enteritis presenting as a rare cause for ileo-ileal intussusception Kshirsagar AY, Jagtap SV, Kanojiya RP, Langade YB, Shinde SL, Shekhar N

6446 Migrated endoclip and stone formation after cholecystectomy: A new danger of acute pancreatitis

Dolay K, Alis H, Soylu A, Altaca G, Aygun E

ContentsWorld Journal of Gastroenterology

Volume 13 Number 47 December 21, 2007

CASE REPORTS

RAPID COMMUNICATION

CLINICAL RESEARCH

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ContentsWorld Journal of Gastroenterology

Volume 13 Number 47 December 21, 2007

6449 Number of monthly hits to WJG articles linked to PubMed surpasses 70 000 Jiang Y

6450 Acknowledgments to Reviewers of World Journal of Gastroenterology

6451 Meetings

6452 Instructions to authors

I-V Editorial Board

Online Submissions

Online Submissions

FLYLEAF

INSIDE FRONT COVER

INSIDE BACK COVER

NAME OF JOURNAL World Journal of Gastroenterology

RESPONSIBLE INSTITUTIONDepartment of Science and Technology of Shanxi Province

SPONSOR Taiyuan Research and Treatment Center for Digestive Diseases, 77 Shuangta Xijie, Taiyuan 030001, Shanxi Province, China

EDITINGEditorial Board of World Journal of Gastroenterolog y, 77 Shuangta Xijie, Taiyuan 030001, Shanxi Province, ChinaTelephone: +86-351-4078656E-mail: wjg@wjgnet.com

PUBLISHINGEditorial Department of World Journal of Gastroenterology, 77 Shuangta Xijie, Taiyuan 030001, Shanxi Province, ChinaTelephone: +86-351-4078656E-mail: wjg@wjgnet.comhttp://www.wjgnet.com

PRINTINGBeijing Kexin Printing House

OVERSEAS DISTRIBUTORBeijing Bureau for Distribution of Newspapers and Journals (Code No. 82-261)China International Book Trading Corporation PO Box 399, Beijing, China (Code No. M4481)

PUBLICATION DATEDecember 21, 2007

EDITOR-IN-CHIEFLian-Sheng Ma, Taiyuan

SUBSCRIPTION RMB 50 Yuan for each issue, RMB 2400 Yuan for one year

CSSNISSN 1007-9327CN 14-1219/R

HONORARY EDITORS-IN-CHIEFKe-Ji Chen, BeijingLi-Fang Chou, TaipeiZhi-Qiang Huang, BeijingShinn-Jang Hwang, TaipeiMin-Liang Kuo, TaipeiNicholas F LaRusso, RochesterJie-Shou Li, NanjingGeng-Tao Liu, BeijingLein-Ray Mo, TainanBo-Rong Pan, Xi'anFa-Zu Qiu, WuhanEamonn M Quigley, CorkDavid S Rampton, LondonRudi Schmid, kentfieldNicholas J Talley, RochesterGuido NJ Tytgat, AmsterdamH-P Wang, TaipeiJaw-Ching Wu, TaipeiMeng-Chao Wu, ShanghaiMing-Shiang Wu, TaipeiJia-Yu Xu, ShanghaiTa-Sen Yeh, Taoyuan

ASSOCIATE EDITORS-IN-CHIEFGianfranco D Alpini, TempleBruno Annibale, RomaRoger William Chapman, OxfordChi-Hin Cho, Hong KongAlexander L Gerbes, MunichShou-Dong Lee, TaipeiWalter Edwin Longo, New HavenYou-Yong Lu, BeijingMasao Omata, TokyoHarry HX Xia, Hanover

EXECUTIVE VICE DIRECTORYe Liu, Beijing

DEPUTY DIRECTORJian-Zhong Zhang, Beijing

TECHNICAL DIRECTORMin Zhang, Beijing

LANGUAGE EDITORSDirector: Jing-Yun Ma, BeijingDeputy Director: Xian-Lin Wang, Beijing

MEMBERSGianfranco D Alpini, TempleBS Anand, HoustonRichard B Banati, LidcombeGiuseppe Chiarioni, ValeggioJohn Frank Di Mari, TexasShannon S Glaser, Temple Mario Guslandi, MilanoMartin Hennenberg, BonnAtif Iqbal, OmahaManoj Kumar, NepalPatricia F Lalor, BirminghamMing Li, New OrleansMargaret Lutze, ChicagoJing-Yun Ma, BeijingDaniel Markovich, BrisbaneSabine Mihm, GöttingenFrancesco Negro, GenèveBernardino Rampone, SienaRichard A Rippe, Chapel HillStephen E Roberts, Swansea Ross C Smith, SydneySeng-Lai Tan, SeattleXian-Lin Wang, BeijingEddie Wisse, KeerbergenDaniel Lindsay Worthley, Bedford

NEWS EDITORLixin Zhu, Berkeley

COPY EDITORSGianfranco D Alpini, TempleSujit Kumar Bhattacharya, Kolkata

Filip Braet, SydneyKirsteen N Browning, Baton RougeRadha K Dhiman, ChandigarhJohn Frank Di Mari, TexasShannon S Glaser, TempleMartin Hennenberg, BonnEberhard Hildt, BerlinPatricia F Lalor, BirminghamMing Li, New OrleansMargaret Lutze, ChicagoMI Torrs, JaénSri Prakash Misra, AllahabadGiovanni Monteleone, RomeGiovanni Musso, TorinoValerio Nobili, RomeOsman Cavit Ozdogan, IstanbulFrancesco Perri, San Giovanni RotondoThierry Piche, NiceBernardino Rampone, SienaRichard A Rippe, Chapel HillRoss C Smith, SydneyDaniel Lindsay Worthley, BedfordGeorge Y Wu, FarmingtonJian Wu, Sacramento

COPYRIGHT© 2007 Published by WJG. All rights reserved; no part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise without the prior permission of WJG. Authors are required to grant WJG an exclusive licence to publish.

SPECIAL STATEMENT All articles published in this journal represent the viewpoints of the authors except where indicated otherwise.

INSTRUCTIONS TO AUTHORSFull instructions are available online at http://www.wjgnet.com/wjg/help/instructions.jsp. If you do not have web access please contact the editorial office.

RESPONSIBLE EDITORSFOR THIS ISSUE

Assistant Editor: Yan Jiang Review Editor: You-De Chang Electronic Page Editor: Wen-Hua Ma

Technical Director: Min Zhang Editor-in-Charge: You-De Chang Copy Editor: Pamela Frederick

Associate Senior Editor: Ye Liu Proof Editor: Hai-Ning Zhang Layout Editor: Lian-Sheng Ma

ACKNOWLEDGMENTS

NEWS

APPENDIX

World Journal of Gastroenterology

Editorial Board 2007-2009

Baishidenghttp://www.wjgnet.com E-mail: wjg@wjgnet.com

HONORARY EDITORS-IN-CHIEFKe-Ji Chen, BeijingLi-Fang Chou, TaipeiZhi-Qiang Huang, BeijingShinn-Jang Hwang, TaipeiMin-Liang Kuo, TaipeiNicholas F LaRusso, RochesterJie-Shou Li, NanjingGeng-Tao Liu, BeijingLein-Ray Mo, TainanBo-Rong Pan, Xi'anFa-Zu Qiu, WuhanEamonn M Quigley, CorkDavid S Rampton, LondonRudi Schmid, Kentfi eldNicholas J Talley, RochesterGuido NJ Tytgat, AmsterdamH-P Wang, TaipeiJaw-Ching Wu, TaipeiMeng-Chao Wu, ShanghaiMing-Shiang Wu, TaipeiJia-Yu Xu, ShanghaiTa-Sen Yeh, Taoyuan

EDITOR-IN-CHIEFLian-Sheng Ma, Taiyuan

ASSOCIATE EDITORS-IN-CHIEFGianfranco D Alpini, TempleBruno Annibale, RomaRoger William Chapman, OxfordChi-Hin Cho, Hong KongAlexander L Gerbes, MunichShou-Dong Lee, TaipeiWalter Edwin Longo, New HavenYou-Yong Lu, BeijingMasao Omata, TokyoHarry HX Xia, Hanover

MEMBERS OF THE EDITORIAL BOARD

Albania Bashkim Resuli, Tirana

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ArgentinaJulio Horacio Carri, CórdobaAdriana M Torres, Rosario

AustraliaMinoti Vivek Apte, LiverpoolRichard B Banati, LidcombeMichael R Beard, AdelaidePatrick Bertolino, SydneyFilip Braet, SydneyAndrew D Clouston, SydneyDarrell HG Crawford, BrisbaneGuy D Eslick, SydneyMichael Anthony Fink, MelbourneRobert JL Fraser, Daw ParkMark D Gorrell, SydneyYik-Hong Ho, TownsvilleGerald J Holtmann, AdelaideMichael Horowitz, AdelaideJohn E Kellow, SydneyDaniel Markovich, BrisbanePhillip S Oates, PerthStephen M Riordan, SydneyIC Roberts-Thomson, AdelaideArthur Shulkes, MelbourneRoss C Smith, SydneyKevin John Spring, BrisbaneNathan Subramaniam, BrisbaneHerbert Tilg, InnsbruckMartin John Veysey, GosfordDL Worthley, Bedford

Austria Valentin Fuhrmann, ViennaAlfred Gangl, ViennaChristoph Gasche, ViennaKurt Lenz, LinzM Peck-Radosavljevic, ViennaRE Stauber, AuenbruggerplatzMichael Trauner, GrazHarald Vogelsang, ViennaGuenter Weiss, Innsbruck

Belarus Yury K Marakhouski, Minsk

Belgium Rudi Beyaert, GentBart Rik De Geest, LeuvenInge Irma Depoortere, LeuvenOlivier Detry, LiègeBY De Winter, AntwerpKarel Geboes, LeuvenThierry Gustot, BrusselsYves J Horsmans, BrusselsGeert G Leroux-Roels, GhentLouis Libbrecht, LeuvenEtienne M Sokal, BrusselsMarc Peeters, De PintelaanGert A Van Assche, LeuvenYvan Vandenplas, BrusselsEddie Wisse, Keerbergen

BrazilHeitor Rosa, Goiania

BulgariaZahariy Krastev, Sofi a

CanadaFernando Alvarez, QuébecDavid Armstrong, OntarioOlivier Barbier, QuébecNancy Baxter, TorontoMatthew Bjerknes, TorontoFrank J Burczynski, WinnipegMichael F Byrne, VancouverWang-Xue Chen, OttawaHugh J Freeman, VancouverChantal Guillemette, QuébecSamuel S Lee, CalgaryGary A Levy, TorontoAndrew Lawrence Mason, AlbertaJohn K Marshall, OntarioDonna-Marie McCafferty, CalgaryThomas I Michalak, St. John'sGerald Y Minuk, ManitobaPaul Moayyedi, HamiltonEldon Shaffer, CalgaryMorris Sherman, TorontoAlan BR Thomson, EdmontonEF Verdu, Ontario

I

John L Wallace, CalgaryEric M Yoshida, Vancouver

ChileSilvana Zanlungo, Santiago

ChinaHenry LY Chan, HongkongXiao-Ping Chen, WuhanZong-Jie Cui, BeijingDa-Jun Deng, BeijingEr-Dan Dong, BeijingSheung-Tat Fan, Hong Kong Jin Gu, BeijingDe-Wu Han, TaiyuanMing-Liang He, Hong KongWayne HC Hu, Hong KongChee-Kin Hui, Hong KongChing Lung Lai, Hong KongKam Chuen Lai, Hong KongJames YW Lau, Hong KongYuk Tong Lee, Hong KongSuet Yi Leung, Hong KongWai-Keung Leung, Hong KongChung-Mau Lo, Hong KongJing-Yun Ma, BeijingLun-Xiu Qin, ShanghaiYu-Gang Song, GuangzhouQin Su, BeijingWai-Man Wong, Hong KongHong Xiao, Beijing Dong-Liang Yang, WuhanWinnie Yeo, Hong KongYuan Yuan, ShenyangMan-Fung Yuen, Hong KongJian-Zhong Zhang, BeijingXin-Xin Zhang, ShanghaiShu Zheng, Hangzhou

CroatiaTamara Cacev, ZagrebMarko Duvnjak, Zagreb

CubaDamian Casadesus Rodriguez, Havana

CzechMilan Jirsa, Praha

DenmarkPeter Bytzer, CopenhagenHans Gregersen, AalborgJens H Henriksen, HvidovreClaus Peter Hovendal, OdenseFin Stolze Larsen, CopenhagenSØren MØller, Hvidovre

EgyptAbdel-Rahman El-Zayadi, GizaAmr Mohamed Helmy, CairoSanaa Moharram Kamal, CairoAyman Yosry, Cairo

FinlandIrma Elisabet Jarvela, HelsinkiKatri Maria Kaukinen, TampereMinna Nyström, HelsinkiPentti Sipponen, Espoo

FranceBettaieb Ali, DijonCorlu Anne, RennesDenis Ardid, Clermont-FerrandCharles Paul Balabaud, BordeauxSoumeya Bekri, RouenJacques Belghiti, Clichy

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Pierre Brissot, RennesPatrice Philippe Cacoub, ParisFranck Carbonnel, BesanconLaurent Castera, PessacBruno Clément, RennesJacques Cosnes, ParisThomas Decaens, CedexFrancoise Lunel Fabiani, AngersGérard Feldmann, ParisJean Fioramonti, ToulouseCatherine Guettier, VillejuifChantal Housset, ParisJuan Lucio Iovanna, MarseilleRene Lambert, LyonPhilippe Mathurin, LilleTamara Matysiak–Budnik, ParisFrancis Mégraud, BordeauxRichard Moreau, ClichyThierry Piche, NiceRaoul Poupon, ParisJean Rosenbaum, BordeauxJose Sahel, MarseilleJean-Philippe Salier, RouenJean-Yves Scoazec, LyonKhalid Ahnini Tazi, ClichyEmmanuel Tiret, ParisBaumert F Thomas, StrasbourgMC Vozenin-brotons, VillejuifJean-Pierre Henri Zarski, GrenobleJessica Zucman-Rossi, Paris

GermanyHD Allescher, Garmisch-PartenkirchenMartin Anlauf, KielRudolf Arnold, MarburgMax G Bachem, UlmThomas F Baumert, FreiburgDaniel C Baumgart, BerlinHubert Blum, FreiburgThomas Bock, TuebingenKatja Breitkopf, MannheimDunja Bruder, BraunschweigMarkus W Büchler, HeidelbergChrista Buechler, RegensburgReinhard Buettner, BonnElke Cario, EssenCF Dietrich, Bad MergentheimRainer Josef Duchmann, BerlinPaul Enck, TuebingenFred Fändrich, KielUlrich Robert Fölsch, KielHelmut Friess, HeidelbergPeter R Galle, MainzNikolaus Gassler, AachenAndreas Geier, AachenDieter Glebe, GiessenBurkhard Göke, MunichFlorian Graepler, TuebingenAxel M Gressner, AachenVeit Gülberg, MunichRainer Haas, MunichEckhart Georg Hahn, ErlangenStephan Hellmig, KielMartin Hennenberg, BonnJohannes Herkel, HamburgKlaus Herrlinger, StuttgartEberhard Hildt, BerlinJoerg C Hoffmann, BerlinFerdinand Hofstaedter, RegensburgWerner Hohenberger, ErlangenRG Jakobs, LudwigshafenJutta Keller, HamburgAndrej Khandoga, MunichSibylle Koletzko, MünchenStefan Kubicka, HannoverJoachim Labenz, SiegenFrank Lammert, BonnThomas Langmann, RegensburgChristian Liedtke, AachenMatthias Löhr, MannheimChristian Maaser, MuensterAhmed Madisch, Dresden

Michael Peter Manns, HannoverStephan Miehlke, DresdenSabine Mihm, GöttingenSilvio Nadalin, EssenMarkus F Neurath, MainzJohann Ockenga, BerlinFlorian Obermeier, RegensburgGustav Paumgartner, MunichUlrich Ks Peitz, MagdeburgMarkus Reiser, BochumSteffen Rickes, MagdeburgGerhard Rogler, RegensburgTilman Sauerbruch, BonnDieter Saur, MunichHans Scherubl, BerlinJoerg Schirra, MunichRoland M Schmid, MünchenVolker Schmitz, BonnAG Schreyer, RegensburgTobias Schroeder, EssenHans Seifert, OldenburgManfred V Singer, MannheimGisela Sparmann, RostockJurgen M Stein, FrankfurtUlrike Susanne Stein, BerlinManfred Stolte, BayreuthChristian P Strassburg, HannoverWR Stremmel, HeidelbergHarald F Teutsch, UlmRobert Thimme, FreiburgHL Tillmann, LeipzigTung-Yu Tsui, RegensburgAxel Ulsenheimer, MunichPatrick Veit, EssenClaudia Veltkamp, HeidelbergSiegfried Wagner, DeggendorfHenning Walczak, HeidelbergFritz von Weizsacker, BerlinJens Werner, HeidelbergBertram Wiedenmann, BerlinReiner Wiest, RegensburgStefan Wirth, WuppertalStefan JP Zeuzem, Homburg

GreeceElias A Kouroumalis, HeraklionIoannis E Koutroubakis, HeraklionSpiros Sgouros, Athens

HungaryPeter Laszlo Lakatos, BudapestZsuzsa Szondy, Debrecen

IcelandH Gudjonsson, Reykjavik

IndiaKA Balasubramanian, VelloreSujit K Bhattacharya, KolkataYogesh K Chawla, ChandigarhRadha K Dhiman, ChandigarhSri Prakash Misra, AllahabadND Reddy, Hyderabad

IranSeyed-Moayed Alavian, TehranReza Malekzadeh, TehranSeyed Alireza Taghavi, Shiraz

IrelandBilly Bourke, DublinRonan A Cahill, CorkAnthony P Moran, Galway

IsraelSimon Bar-Meir, HashomerAbraham Rami Eliakim, Haifa

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Yaron Ilan, JerusalemAvidan U Neumann, Ramat-GanYaron Niv, PardesiaRan Oren, Tel Aviv

ItalyGiovanni Addolorato, RomaLuigi E Adinolfi , NaplesDomenico Alvaro, RomeV Annese, San Giovanni RotondAdolfo Francesco Attili, RomaGiovanni Barbara, BolognaGabrio Bassotti, PerugiaPier Maria Battezzati, MilanStefano Bellentani, CarpiAntomio Benedetti, AnconaMauro Bernardi, BolognaLivia Biancone, RomeLuigi Bonavina, Milano Flavia Bortolotti, PadovaGiuseppe Brisinda, RomeGiovanni Cammarota, RomaAntonino Cavallari, BolognaGiuseppe Chiarioni, ValeggioMichele Cicala, RomeAmedeo Columbano, CagliariMassimo Conio, SanremoDario Conte, MilanoGino Roberto Corazza, PaviaFrancesco Costa, PisaAntonio Craxi, PalermoSilvio Danese, MilanRoberto De Giorgio, BolognaGiovanni D De Palma, NaplesFabio Farinati, PaduaGiammarco Fava, AnconaFrancesco Feo, SassariStefano Fiorucci, PerugiaAndrea Galli, FirenzeValeria Ghisett, TurinGianluigi Giannelli, BariEdoardo G Giannini, GenoaPaolo Gionchetti, BolognaMario Guslandi, MilanoPietro Invernizzi, MilanGiacomo Laffi , FirenzeGiovanni Maconi, MilanLucia Malaguarnera, CataniaED Mangoni, NapoliGiulio Marchesini, BolognaFabio Marra, FlorenceMarco Marzioni, AnconaGiuseppe Montalto, PalermoGiovanni Monteleone, RomeGiovanni Musso, TorinoGerardo Nardone, NapoliValerio Nobili, RomeLuisi Pagliaro, PalermoFrancesco Pallone, RomeFabrizio R Parente, MilanF Perri, San Giovanni RotondoRaffaele Pezzilli, BolognaA Pilotto, San Giovanni RotondoMario Pirisi, NovaraPaolo Del Poggio, TreviglioGabriele Bianchi Porro, MilanoPiero Portincasa, BariBernardino Rampone, SienaClaudio Romano, MessinaMarco Romano, NapoliGerardo Rosati, PotenzaEnrico Roda, BolognaDomenico Sansonno, BariVincenzo Savarino, GenovaMario Del Tacca, PisaGiovanni Tarantino, NaplesRoberto Testa, GenoaPier Alberto Testoni, Milan

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Dino Vaira, Bologna

JapanKyoichi Adachi, IzumoYasushi Adachi, SapporoTaiji Akamatsu, MatsumotoSk Md Fazle Akbar, EhimeTakafumi Ando, NagoyaAkira Andoh, OtsuTaku Aoki, TokyoMasahiro Arai, TokyoTetsuo Arakawa, OsakaYasuji Arase, TokyoMasahiro Asaka, SapporoHitoshi Asakura, TokyoTakeshi Azuma, Fukui Yoichi Chida, FukuokaTakahiro Fujimori, TochigiJiro Fujimoto, HyogoKazuma Fujimoto, SagaMitsuhiro Fujishiro, TokyoYoshihide Fujiyama, OtsuHirokazu Fukui, TochigiHiroyuki Hanai, HamamatsuKazuhiro Hanazaki, KochiNaohiko Harada, FukuokaMakoto Hashizume, FukuokaTetsuo Hayakawa, NagoyaKazuhide Higuchi, OsakaKeisuke Hino, UbeKeiji Hirata, KitakyushuYuji Iimuro, NishinomiyaKenji Ikeda, TokyoFumio Imazeki, ChibaYutaka Inagaki, KanagawaYasuhiro Inokuchi, YokohamaHaruhiro Inoue, YokohamaMasayasu Inoue, OsakaAkio Inui, KagoshimaHiromi Ishibashi, NagasakiShunji Ishihara, IzumoToru Ishikawa, NiigataKei Ito, SendaiMasayoshi Ito, TokyoHiroaki Itoh, AkitaRyuichi Iwakiri, SagaYoshiaki Iwasaki, OkayamaTerumi Kamisawa, TokyoHiroshi Kaneko, Aichi-GunShuichi Kaneko, KanazawaTakashi Kanematsu, NagasakiMitsuo Katano, FukuokaJunji Kato, SapporoMototsugu Kato, SapporoShinzo Kato, TokyoNorifumi Kawada, OsakaSunao Kawano, OsakaMitsuhiro Kida, KanagawaYoshikazu Kinoshita, IzumoTsuneo Kitamura, ChibaSeigo Kitano, OitaKazuhiko Koike, TokyoNorihiro Kokudo, TokyoSatoshi Kondo, SapporoShoji Kubo, OsakaMasato Kusunoki, Tsu MieKatsunori Iijima, SendaiShin Maeda, Tokyo Masatoshi Makuuchi, TokyoOsamu Matsui, KanazawaYasuhiro Matsumura, ChibaYasushi Matsuzaki, TsukubaKiyoshi Migita, OmuraTetsuya Mine, KanagawaHiroto Miwa, Hyogo Masashi Mizokami, NagoyaYoshiaki Mizuguchi, TokyoMotowo Mizuno, Hiroshima

Morito Monden, SuitaHisataka S Moriwaki, GifuYasuaki Motomura, IizukaYoshiharu Motoo, KanazawaKazunari Murakami, OitaKunihiko Murase, TusimaMasahito Nagaki, GifuMasaki Nagaya, KawasakiYuji Naito, KyotoHisato Nakajima, TokyoHiroki Nakamura, Yamaguchi Shotaro Nakamura, FukuokaMikio Nishioka, NiihamaShuji Nomoto, NagoyaSusumu Ohmada, MaebashiMasayuki Ohta, OitaTetsuo Ohta, KanazawaKazuichi Okazaki, OsakaKatsuhisa Omagari, Nagasaki Saburo Onishi, NankokuMorikazu Onji, EhimeSatoshi Osawa, HamamatsuMasanobu Oshima, KanazawaHiromitsu Saisho, Chiba Hidetsugu Saito, TokyoYutaka Saito, TokyoIsao Sakaida, Yamaguchi Michiie Sakamoto, TokyoYasushi Sano, ChibaHiroki Sasaki, TokyoIwao Sasaki, SendaiMotoko Sasaki, KanazawaChifumi Sato, TokyoShuichi Seki, OsakaHiroshi Shimada, YokohamaMitsuo Shimada, TokushimaTomohiko Shimatan, HiroshimaHiroaki Shimizu, ChibaIchiro Shimizu, TokushimaYukihiro Shimizu, KyotoShinji Shimoda, FukuokaTooru Shimosegawa, SendaiTadashi Shimoyama, HirosakiKen Shirabe, IizukaYoshio Shirai, NiigataKatsuya Shiraki, MieYasushi Shiratori, OkayamaMasayuki Sho, NaraYasuhiko Sugawara, TokyoHidekazu Suzuki, TokyoMinoru Tada, TokyoTadatoshi Takayama, TokyoTadashi Takeda, OsakaKoji Takeuchi, KyotoKiichi Tamada, Tochigi Akira Tanaka, KyotoEiji Tanaka, MatsumotoNoriaki Tanaka, Okayama Shinji Tanaka, Hiroshima Wei Tang, TokyoHideki Taniguchi, YokohamaKyuichi Tanikawa, KurumeAkira Terano, ShimotsugagunHitoshi Togash, YamagataKazunari Tominaga, OsakaTakuji Torimura, FukuokaMinoru Toyota, SapporoAkihito Tsubota, ChibaShingo Tsuji, OsakaTakato Ueno, KurumeShinichi Wada, TochigiHiroyuki Watanabe, KanazawaToshio Watanabe, OsakaYuji Watanabe, EhimeChun-Yang Wen, NagasakiKoji Yamaguchi, FukuokaTakayuki Yamamoto, YokkaichiTakashi Yao, Fukuoka

Ⅲ

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Masashi Yoneda, TochigiHiroshi Yoshida, TokyoMasashi Yoshida, TokyoNorimasa Yoshida, KyotoKentaro Yoshika, ToyoakeMasahide Yoshikawa, Kashihara

LebanonBassam N Abboud, BeirutAla I Sharara, BeirutJoseph Daoud Boujaoude, Beirut

LithuaniaLimas Kupcinskas, Kaunas

MacedoniaVladimir Cirko Serafi moski, Skopje

MalaysiaAndrew Seng Boon Chua, IpohKhean-Lee Goh, Kuala LumpurJayaram Menon, Sabah

MexicoGarcia-Compean Diego, MonterreyE R Marin-Lopez, Jesús GarcíaSaúl Villa-Treviño, MéxicoJK Yamamoto-Furusho, México

MonacoPatrick Rampal, Monaco

NetherlandsUlrich Beuers, AmsterdamGerd Bouma, AmsterdamLee Bouwman, LeidenJ Bart A Crusius, AmsterdamJanine K Kruit, GroningenErnst Johan Kuipers, RotterdamTon Lisman, UtrechtYi Liu, AmsterdamServaas Morré, AmsterdamChris JJ Mulder, AmsterdamMichael Müller, WageningenAmado Salvador Peña, AmsterdamRobert J Porte, GroningenIngrid B Renes, RotterdamAndreas Smout, UtrechtRW Stockbrugger, MaastrichtLuc JW van der Laan, RotterdamKarel van Erpecum, UtrechtGP VanBerge-Henegouwen,Utrecht

New ZealandIan David Wallace, Auckland

NigeriaSamuel Babafemi Olaleye, Ibadan

Norway Trond Berg, Oslo Tom Hemming Karlsen, OsloHelge Lyder Waldum, Trondheim

PakistanMuhammad S Khokhar, Lahore

PolandTomasz Brzozowski, Cracow Robert Flisiak, BialystokHanna Gregorek, WarsawDM Lebensztejn, BialystokWojciech G Polak, WroclawMarek Hartleb, Katowice

Portugal MP Cecília, LisbonMiguel Carneiro De Moura, Lisbon

RussiaVladimir T Ivashkin, Moscow Leonid Lazebnik, Moscow Vasiliy I Reshetnyak, Moscow

Saudi ArabiaIbrahim Abdulkarim Al Mofl eh, Riyadh

SerbiaDM Jovanovic, Sremska Kamenica

Singapore Bow Ho, Kent Ridge Khek-Yu Ho, SingaporeFrancis Seow-Choen, Singapore

SlovakiaAnton Vavrecka, Bratislava

SloveniaSasa Markovic, Ljubljana

South AfricaMichael C Kew, Parktown

South KoreaByung Ihn Choi, SeoulHo Soon Choi, SeoulM Yeo, SuwonSun Pyo Hong, Gyeonggi-doJae J Kim, SeoulJin-Hong Kim, Suwon Myung-Hwan Kim, Seoul Chang Hong Lee, SeoulJong Kyun Lee, SeoulEun-Yi Moon, SeoulJae-Gahb Park, Seoul Dong Wan Seo, Seoul Dong jin Suh, Seoul

SpainJuan G Abraldes, Barcelona Agustin Albillos, MadridRaul J Andrade, MálagaLuis Aparisi, ValenciaFernando Azpiroz, Barcelona Ramon Bataller, Barcelona Josep M Bordas, Barcelona Xavier Calvet, Sabadell Andres Cardenas, BarcelonaVicente Carreño, MadridJose Castellote, BarcelonaAntoni Castells, Barcelona Vicente Felipo, ValenciaJuan C Garcia-Pagán, Barcelona Jaime Bosch Genover, BarcelonaJaime Guardia, Barcelona Angel Lanas, Zaragoza María Isabel Torres López, JaénJosé M Mato, DerioJuan F Medina, PamplonaMA Muñoz-Navas, PamplonaJulian Panes, Barcelona Miguel Minguez Perez, ValenciaMiguel Perez-Mateo, Alicante Josep M Pique, BarcelonaJesús M Prieto, PamplonaSabino Riestra, Pola De SieroLuis Rodrigo, OviedoManuel Romero-Gómez, Sevilla

SwedenEinar Stefan Björnsson, GothenburgCurt Einarsson, Huddinge

Ulf Hindorf, LundHanns-Ulrich Marschall, StockholmLars Christer Olbe, Molndal Matti Sallberg, StockholmMagnus Simrén, GöteborgXiao-Feng Sun, Linköping Ervin Tóth, MalmöWeimin Ye, Stockholm

Switzerland Chrish Beglinger, Basel Pierre A Clavien, ZurichJean-Francois Dufour, BernFranco Fortunato, ZürichJean Louis Frossard, GenevaGerd A Kullak-Ublick, ZurichPierre Michetti, LausanneFrancesco Negro, GenèveBruno Stieger, Zurich Radu Tutuian, ZurichStephan Robert Vavricka, ZurichArthur Zimmermann, Berne

TurkeyYusuf Bayraktar, Ankara Figen Gurakan, Ankara Aydin Karabacakoglu, KonyaSerdar Karakose, KonyaHizir Kurtel, IstanbuOsman Cavit Ozdogan, IstanbulÖzlem Yilmaz, IzmirCihan Yurdaydin, Ankara

United Arab EmiratesSherif M Karam, Al-Ain

United KingdomDavid Adams, BirminghamNK Ahluwalia, StockportCG Antoniades, LondonAnthony TR Axon, Leeds Qasim Aziz, ManchesterNicholas M Barnes, BirminghamJim D Bell, LondonMairi Brittan, LondonAlastair David Burt, NewcastleSimon Scott Campbell, ManchesterSimon R Carding, LeedsPaul Jonathan Ciclitira, LondonEithne Costello, LiverpoolTatjana Crnogorac-Jurcevic, LondonAmar Paul Dhillon, LondonEmad M El-Omar, AberdeenAnnette Fristscher-Ravens, LondonElizabeth Furrie, DundeeDaniel Richard Gaya, EdinburghSubrata Ghosh, London William Greenhalf, LiverpoolIndra Neil Guha, SouthamptonPeter Clive Hayes, EdinburghGwo-Tzer Ho, EdinburghAnthony R Hobson, SalfordStefan G Hübscher, BirminghamRobin Hughes, LondonPali Hungin, StocktonDavid Paul Hurlstone, Sheffi eldRajiv Jalan, LondonJanusz AZ Jankowski, OxfordBrian T Johnston, BelfastDavid EJ Jones, NewcastleMichael A Kamm, HarrowPeter Karayiannis, LondonLaurens Kruidenier, HarlowPatricia F Lalor, BirminghamHong-Xiang Liu, Cambridge K E L McColl, GlasgowStuart AC McDonald, London

IV

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Dermot Patrick Mcgovern, OxfordGiorgina Mieli-Vergani, LondonNikolai V Naoumov, London John P Neoptolemos, Liverpool James Neuberger, BirminghamMark S Pearce, Newcastle Upon TyneStephen P Pereira, LondonD Mark Pritchard, LiverpoolStephen E Roberts, SwanseaMarco Senzolo, PadovaSoraya Shirazi-Beechey, LiverpoolRobert Sutton, LiverpoolSimon D Taylor-Robinson, LondonParis P Tekkis, LondonUlrich Thalheimer, LondonNick Paul Thompson, NewcastleDavid Tosh, BathFrank Ivor Tovey, London Chris Tselepis, BirminghamDiego Vergani, LondonGeoffrey Warhurst, SalfordPeter James Whorwell, ManchesterRoger Williams, LondonKaren Leslie Wright, BathMin Zhao, Foresterhill

United StatesGary A Abrams, BirminghamGolo Ahlenstiel, BethesdaBS Anand, HoustonFrank A Anania, AtlantaM Ananthanarayanan, New YorkGavin Edward Arteel, LouisvilleJasmohan Singh Bajaj, Milwaukee Jamie S Barkin, Miami Beach Kim Elaine Barrett, San DiegoMarc Basson, DetroitWallace F Berman, DurhamTimothy R Billiar, PittsburghEdmund J Bini, New YorkJennifer D Black, Buffalo Herbert L Bonkovsky, FarmingtonAndrea D Branch, New YorkRobert S Bresalier, HoustonAlan L Buchman, ChicagoAlan Cahill, PhiladelphiaJohn M Carethers, San DiegoDavid L Carr-Locke, BostonRavi S Chari, NashvilleJiande Chen, GalvestonXian-Ming Chen, OmahaRamsey Chi-man Cheung, Palo AltoWilliam D Chey, Ann ArborJohn Y Chiang, RootstownParimal Chowdhury, ArkansasRaymond T Chung, BostonJames M Church, ClevelandMark G Clemens, CharlotteVincent Coghlan, BeavertonDavid Cronin II, New HavenJohn Cuppoletti, CincinnatiMark James Czaja, New YorkPeter V Danenberg, Los Angeles Kiron Moy Das, New Brunswick Sharon DeMorrow, TempleDeborah L Diamond, SeattlePeter Draganov, FloridaBijan Eghtesad, ClevelandHala El-Zimaity, HoustonMichelle Embree-Ku, ProvidenceRonnie Fass, Tucson Mark A Feitelson, PhiladelphiaAriel E Feldstein, ClevelandAlessandro Fichera, ChicagoChris E Forsmark, GainesvilleChandrashekhar R Gandhi, PittsburghSusan L Gearhart, BaltimoreXupeng Ge, Boston

Ⅴ

John P Geibel, New HavenXin Geng, New BrunswickJean-Francois Geschwind, BaltimoreIgnacio Gil-Bazo, New YorkShannon S Glaser, TempleAjay Goel, DallasJulia Butler Greer, PittsburghJames Henry Grendell, New YorkDavid R Gretch, SeattleStefano Guandalini, ChicagoAnna S Gukovskaya, Los Angeles Sanjeev Gupta, BronxDavid J Hackam, PittsburghStephen B Hanauer, ChicagoGavin Harewood, Rochester Margaret McLean Heitkemper, SeattleAlan W Hemming, GainesvilleSamuel B Ho, San DiegoColin William Howden, ChicagoHongjin Huang, AlamedaJamal A Ibdah, ColumbiaAtif Iqbal, Omaha Hajime Isomoto, RochesterHartmut Jaeschke, TucsonDennis M Jensen, Los AngelesLeonard R Johnson, MemphisMichael P Jones, ChicagoPeter James Kahrilas, Chicago AN Kalloo, BaltimoreNeil Kaplowitz, Los AngelesRashmi Kaul, TulsaJonathan D Kaunitz, Los AngelesAli Keshavarzian, ChicagoMiran Kim, ProvidenceJoseph B Kirsner, Chicago Leonidas G Koniaris, MiamiBurton I Korelitz, New YorkRobert J Korst, New York Richard A Kozarek, Seattle Michael Kremer, Chapel HillShiu-Ming Kuo, Buffalo Daryl Tan Yeung Lau, GalvestoJoel E Lavine, San DiegoDirk J van Leeuwen, LebanonGlen A Lehman, IndianapolisAlex B Lentsch, CincinnatiAndreas Leodolter, La Jolla Gene LeSage, HoustonMing Li, New Orleans Zhiping Li, BaltimoreLM Lichtenberger, HoustonGR Lichtenstein, PhiladelphiaOtto Schiueh-Tzang Lin, SeattleMartin Lipkin, New York Edward V Loftus, RochesteRobin G Lorenz, BirminghamMichael Ronan Lucey, Madison JD Luketich, PittsburghHenry Thomson Lynch, OmahaPatrick M Lynch, HoustonPeter J Mannon, BethesdaCharles Milton Mansbach, MemphisJohn Frank Di Mari, TexasJohn M Mariadason, BronxWM Mars, PittsburghLaura E Matarese, PittsburghLynne V McFarland, WashingtonKevin McGrath, PittsburghHarihara Mehendale, MonroeStephan Menne, New YorkHoward Mertz, NashvilleGeorge W Meyer, SacramentoG Michalopoulos, PittsburghJames Michael Millis, ChicagoAlbert D Min, New YorkPramod Kumar Mistry, New Haven

Smruti Ranjan Mohanty, ChicagoSatdarshan Singh Monga, PittsburghTimothy H Moran, Baltimore Steven F Moss, ProvidenceMasaki Nagaya, BostonLaura Eleanor Nagy, ClevelandHiroshi Nakagawa, PhiladelphiaDouglas B Nelson, MinneaplisBrant K Oelschlager, WashingtonCurtis T Okamoto, Los AngelesStephen JD O’Keefe, PittsburghDimitry Oleynikov, OmahaNatalia A Osna, OmahaStephen J Pandol, Los AngelesPankaj Jay Pasricha, GavestonZhiheng Pei, New York Michael A Pezzone, PittsburghCS Pitchumoni, New BrunswiucJay Pravda, GainesvilleM Raimondo, JacksonvilleGS Raju, GalvestonMurray B Resnick, ProvidenceAdrian Reuben, Charleston Douglas K Rex, IndianapolisVictor E Reyes, GalvestonRichard A Rippe, Chapel HillMarcos Rojkind, WashingtonPhilip Rosenthal, San FranciscoHemant Kumar Roy, EvanstonShawn David Safford, NorfolkBruce E Sands, BostonNJ Shaheen, Chapel HillHarvey L Sharp, MinneapolisStuart Sherman, Indianapolis Shivendra Shukla, ColumbiaAlphonse E Sirica, VirginiaShanthi V Sitaraman, AtlantaShanthi Srinivasan, AtlantaMichael Steer, BostonGary D Stoner, Columbus Liping Su, ChicagoChristina Surawicz, SeattleGyongyi Szabo, WorcesterYvette Taché, Los AngelesSeng-Lai Tan, SeattleAndrzej Tarnawski, Long BeachAndrzej S Tarnawski, OrangeK-M Tchou-Wong, New YorkNeil D Theise, New YorkPJ Thuluvath, BaltimoreSwan Nio Thung, New YorkNatalie J Torok, SacramentoRA Travagli, Baton RougeG Triadafi lopoulos, Stanford James F Trotter, DenverChung-Jyi Tsai, LexingtonAndrew Ukleja, FloridaHugo E Vargas, ScottsdaleScott A Waldman, PhiladelphiaJian-Ying Wang, Baltimore Steven David Wexner, WestonKeith Tucker Wilson, BaltimoreJacqueline L Wolf, BostonJackie Wood, OhioGeorge Y Wu, FarmingtonJian Wu, SacramentoSamuel Wyllie, HoustonWen Xie, PittsburghYoshio Yamaoka, HoustonVincent W Yang, AtlantaFrancis Y Yao, San FranciscoMin You, TampaZobair M Younossi, VirginiaLiqing Yu, Winston-SalemDavid Yule, RochesterRuben Zamora, PittsburghMichael E Zenilman, New YorkZhi Zhong, Chapel Hill

UruguayHenry Cohen, Montevideo

Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6291-6294www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

What’s wrong with sentinel node mapping in colon cancer?

Ronan A Cahill

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EDITORIAL

Ronan A Cahill, Department of General Surgery, Cork University Hospital, Wilton, Cork, Ireland Correspondence to: Ronan A Cahill, IRCAD/EITS, 1 Place de l'Hopital, Strasbourg 67091, France. rcahill@rcsi.ieTelephone: +353-87-2886417 Received: September 17, 2007 Revised: October 10, 2007

AbstractDespite near-universal embrace of the concept and clinical relevance of lymphatic mapping for sentinel node identification and analysis for cancers of the breast and integument, the same technique has struggled to a find a role in gastrointestinal cancers in general and, perhaps, in colon cancer in particular. Despite many studies demonstrating its feasibility in malignancies of the large bowel, concern is continually aroused by the variable and often unacceptably low sensitivity rates. Additionally, many confess uncertainty as to what benefit it could ever confer to patients even if it were proven sufficiently accurate given that standard surgical resection incorporates mesenteric resection anyway. However, the huge impact sentinel node mapping has had on clinical practice in certain cancers means that each of these aspects merit careful reconsideration, from very first principles.

© 2007 WJG. All rights reserved.

Key words: Sentinel node; Lymphatic mapping; Colon cancer

Cahill RA. What’s wrong with sentinel node mapping in colon cancer? World J Gastroenterol 2007; 13(47): 6291-6294

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Despite being initially proposed many years before, the sentinel node concept has only recently impacted upon clinical practice. The major landmark work in validating the theory took place in melanoma patients[1], but the proof of the concept was quickly (and relatively painlessly) transferred to breast cancer[2]. However, perhaps the most salient aspect evident on reviewing these seminal publications now is their consistent focus on confining the technique to relatively early stage cancers. The rationale underlying this is that larger tumours involve a greater area of lymphatic channels and also that more advanced tumours may demonstrate aberrant lymphatic dynamics,

both within the primary and in the lymphatic channels in the immediate vicinity. Lymphatic mapping is, therefore, currently recommended only for intermediate thickness melanoma (1.2 mm to 3.5 mm deep)[3] and T1 and ‘early’ T2 breast tumours[4,5]. As discussants of the topic with regard to colon cancer largely tend to overlook this basic tenet, it is worth reviewing the literature for in vivo sentinel node detection in colon cancer from this perspective (ex vivo work has been excluded from this review as it primarily is of interest from the point of view of pathological evaluation rather than surgical approach).

To date, there have so far been 37 publications in the English language describing over 2500 patients (although some studies may have overlapped their patient groups). Explicit cognisance of tumour advancement and mural penetration has mostly only been peripherally addressed in these studies, however, and tumour size hardly at all. Only four studies excluded patients with evident macroscopic lymphadenopathy[6-9] and it is interesting to note that each of these despite being relatively small studies from groups outside of the main proponent centres have excellent results given the other characteristics of their patient cohorts (see below). The authors with the most consistently impressive results have also tended to include relatively high proportions of “early tumours” (approximately 50% or more of their cancers have been T1 or T2)[2,5,10]. However, when collaborating in a multicentre study resulting in patients with tumours of somewhat more advanced T-stage, sensitivity rates reduced. Further examination of these patients led to the conclusion that this was primarily due to lymphatic obstruction by tumour[11]. Furthermore, when a related group described a cohort of somewhat more advanced primary tumours (at least in terms of T-stage) for the purposes of a different study their lymphatic mapping results also appeared worse (82% detection rate with 12% false negative rate) than those previously published[12]. Other authors have also reported high sensitivity rates when dealing with earlier tumours although this aspect of the patient demographic was not specifically teased out[13,14].

Conversely, the studies with the highest false negative rates have also tended to have the greatest proportion of T3/T4 tumours in their study cohorts (with the T3-T4 proportion representing at least two thirds of the total study group)[15-21]. Better results in such patient populations is reported in those studies which specifically excluded those with evident lymph node disease intraoperatively[9]. Of the studies that have considered any potential impact of T-stage, Viehl et al[22] found identification rates most significantly affected by tumour size:dye instillate ratio, Saha et al[23] found 95 per cent of so called ‘skip metastases’

occurred in T3 and T4 tumours while Wood et al [24], Bilchik et al[25] and Kitagawa et al[26] all noted that their false negative cases occurred predominantly in T3/T4 patients. Furthermore, Ratanachaikanont et al[27] found significantly lower identification rates in these tumour categories and Yagci et al[28] documented lower sensitivities in those with advanced Dukes B cancers. The one exception to this very consistent trend has been the work of Paramo et al[29] who initially described a 0% false negative rate in their initial experience with predominantly T3 tumours. However, after further expanding their series (mostly by enlarging their numbers of T3 tumours), they found a 3% false negative rate[30] while, interestingly, neither report included any T4 cases.

Some other important studies have provided no meaningful details at all regarding T stage[31-39] and so do not lend themselves to be scrutinised in this fashion. However, given that they state no pre-selection criteria for their patients, it would seem likely that their patients were representative of typical presentation (which in the case of colon cancer is predominantly with transmural, node positive disease). Indeed, that the majority of studies include all comers in their validation studies is perhaps understandable given that most patients with potentially resectable tumours proceed to operation without particular consideration of tumour diameter or mural penetration. Nonetheless, inclusion of high proportions of more locally advanced tumours seems, both theoretically and empirically, to undermine the validation of the technique in this disease. While other factors such as operator experience and dye pharmacodynamics may, of course, also play a role, it would surely be of great interest to selectively, prospectively study sentinel node mapping in patients with early tumours. This would evidentially necessitate some means of preselection such as endoscopic ultrasound (proven efficacious in this tumour type as in other alimentary malignancies)[40] or by including for study only those with screen-detected cancers but would seem eminently feasible.

The second fundamental difference between SNM for cutaneous and breast tumours and that for colon cancer is in the overall intention of purpose. The fundamental principles ab initio diverge significantly, however, between the two as the intent of the technique when used in the former malignancies is to identify lymph node negative patients (in order to spare them from morbid lymph basin dissections) whereas in the latter it has focussed mainly on detecting lymph node positive patients (to identify those who have otherwise occult dissemination and who, therefore, could benefit from systemic therapy). Prognostic prediction (and, therefore, adjuvant therapy prescription) in colon cancer however is largely determined by lymph node involvement and recently much attention has focussed on the adequacy of nodal harvests (whether by surgeon resection or pathologist detection)[41]. It may well be, therefore, that the value of sentinel node identification and analysis is to confirm that lymphatic dissemination has not taken place in tumours likely to be of early stage in order to save the searching for sufficient numbers of nodes to prove this (in excess of 40 perhaps) that

would otherwise be necessary[42]. From this viewpoint, the upstaging of some cancers that are conventionally node negative becomes an added bonus rather than the sole outcome to justify the effort involved. While such a hypothesis can obviously only be purely speculative at present, some proof of concept could perhaps be advanced by the early adopters of lymphatic mapping in colon cancer in examining the survival of their sentinel node negative patients to date in comparison to those deemed node negative by conventional means but with low nodal counts.

Finally, it is intriguing to question the widely held assumption that sentinel node mapping in colon cancer has no value in minimizing the operative morbidity associated with resection of colonic tumours[43]. This basic tenet has become so dogmatic that a sentence to this effect nearly always forms part of the introduction, discussion and/or accompanying editorials of publications concerning the topic[44]. The intent behind the standard operations performed for colonic cancer is, however, to achieve full lymph node basin clearance concomitantly with resection of the primary in every case (“en bloc” or radical resection). The fact that lymphatic drainage closely follows the arterial (rather then venous) regional blood supply is what prompts the level of proximal vascular ligation (a “high-tie”) and it is this manoeuvre that then determines the extent of the segmental bowel resection required (in order to minimize the risk of ischemia of the residual bowel). In many cases, the magnitude of visceral resection provoked by radical lymphadenectomy is far in excess of what would be associated with curative surgery in terms of marginal clearance (colonic tumours rarely infiltrate more then 2 cm beyond the area of gross involvement and therefore a resection margin of 5-10 is considered appropriate)[45]. Although there is likely to be a therapeutic value in resecting nodes positive for metastatic disease in colon cancer, the main value of such clearance for truly lymph node negative patients can only be the gain of prognostic information for reassurance. If adoption of sentinel node mapping obviated the need for wide lymph basin clearance for intraperitoneal colon cancers, the potential benefits would include shortened operative times, reduced postoperative convalescence and, perhaps, improved bowel function[46] and diminished rates of anastomotic dehiscence[47]. Furthermore, the potential for ureteric, duodenal and (in the male) spermatic vessel injury would be greatly reduced if root mesenteric dissection became unnecessary as would the hazard of splenic laceration that occurs with mobilization of this flexure [often necessary after high ligation of the inferior mesenteric artery (IMA) to ensure tension-free anastomosis after radical left hemicolectomy for sigmoid tumours] and the risk of sexual impotence that may result if para-aortic nerve injury occurs during flush ligation of the IMA at its origin from the abdominal aorta. Lastly, if a localized resection of the bowel was all that was required, the facility by which excisional colonic cancer surgery is performed by minimally invasive means would be markedly enhanced (and the now arduous learning curve reduced). In short, the clinical significance of localized rather then

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radical colectomy is not at present known, but cannot be assumed to be negligible.

The reasons why the basic principles regarding the validation and clinical utility of lymphatic mapping for colon cancer have become so fundamentally divergent from that of breast and melanoma is not entirely clear and, to date, have rarely been discussed. The simplest reason to suspect why it has occurred may be simply that the operative terminology has led to a prevailing mindset. As breast cancer surgeons always considered two operations for their patients (mastectomy/wide local excision and axillary node clearance) and melanoma surgeons always advocated wide excision and lymph basin dissection, perhaps the basic similarities would be easier appreciated if operations such as left hemicolectomy were to have more standardised names such as ‘segmental colonic resection with mesenteric lymph node resection’.

REFERENCES1 Morton DL, Wen DR, Wong JH, Economou JS, Cagle LA,

Storm FK, Foshag LJ, Cochran AJ. Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg 1992; 127: 392-399

2 Giuliano AE , Kirgan DM, Guenther JM, Morton DL. Lymphatic mapping and sentinel lymphadenectomy for breast cancer. Ann Surg 1994; 220: 391-398; discussion 398-401

3 Balch CM, Cascinelli N. Sentinel-node biopsy in melanoma. N Engl J Med 2006; 355: 1370-1371

4 McMasters KM, Giuliano AE, Ross MI, Reintgen DS, Hunt KK, Byrd DR, Klimberg VS, Whitworth PW, Tafra LC, Edwards MJ. Sentinel-lymph-node biopsy for breast cancer--not yet the standard of care. N Engl J Med 1998; 339: 990-995

5 Lyman GH , Giuliano AE, Somerfield MR, Benson AB, Bodurka DC, Burstein HJ, Cochran AJ, Cody HS, Edge SB, Galper S, Hayman JA, Kim TY, Perkins CL, Podoloff DA, Sivasubramaniam VH, Turner RR, Wahl R, Weaver DL, Wolff AC, Winer EP. American Society of Clinical Oncology guideline recommendations for sentinel lymph node biopsy in early-stage breast cancer. J Clin Oncol 2005; 23: 7703-7720

6 Braat AE, Oosterhuis JW, Moll FC, de Vries JE. Successful sentinel node identification in colon carcinoma using Patent Blue V. Eur J Surg Oncol 2004; 30: 633-637

7 Dahl K, Westlin J, Kraaz W, Winqvist O, Bergkvist L, Thörn M. Identification of sentinel nodes in patients with colon cancer. Eur J Surg Oncol 2005; 31: 381-385

8 Kelder W, Braat AE, Karrenbeld A, Grond JA, De Vries JE, Oosterhuis JW, Baas PC, Plukker JT. The sentinel node procedure in colon carcinoma: a multi-centre study in The Netherlands. Int J Colorectal Dis 2007; 22: 1509-1514

9 Tuech JJ, Pessaux P, Di Fiore F, Nitu V, Lefebure B, Colson A, Michot F. Sentinel node mapping in colon carcinoma: in-vivo versus ex-vivo approach. Eur J Surg Oncol 2006; 32: 158-161

10 Bilchik AJ, Saha S, Wiese D, Stonecypher JA, Wood TF, Sostrin S, Turner RR, Wang HJ, Morton DL, Hoon DS. Molecular staging of early colon cancer on the basis of sentinel node analysis: a multicenter phase II trial. J Clin Oncol 2001; 19: 1128-1136

11 Bilchik AJ, DiNome M, Saha S, Turner RR, Wiese D, McCarter M, Hoon DS, Morton DL. Prospective multicenter trial of staging adequacy in colon cancer: preliminary results. Arch Surg 2006; 141: 527-533; discussion 533-534

12 Turner RR, Nora DT, Trocha SD, Bilchik AJ. Colorectal carcinoma nodal staging. Frequency and nature of cytokeratin-positive cells in sentinel and nonsentinel lymph nodes. Arch Pathol Lab Med 2003; 127: 673-679

13 Bianchi PP, Ceriani C, Rottoli M, Torzilli G, Roncalli M, Spinelli A, Montorsi M. Laparoscopic lymphatic mapping and sentinel lymph node detection in colon cancer: technical aspects and preliminary results. Surg Endosc 2007; 21: 1567-1571

14 Dan AG, Saha S, Monson KM, Wiese D, Schochet E, Barber KR, Ganatra B, Desai D, Kaushal S. 1% lymphazurin vs 10% fluorescein for sentinel node mapping in colorectal tumors. Arch Surg 2004; 139: 1180-1184

15 Bertagnolli M, Miedema B, Redston M, Dowell J, Niedzwiecki D, Fleshman J, Bem J, Mayer R, Zinner M, Compton C. Sentinel node staging of resectable colon cancer: results of a multicenter study. Ann Surg 2004; 240: 624-628; discussion 628-630

16 Codignola C , Zorzi F, Zaniboni A, Mutti S, Rizzi A, Padolecchia E, Morandi GB. Is there any role for sentinel node mapping in colorectal cancer staging? Personal experience and review of the literature. Jpn J Clin Oncol 2005; 35: 645-650

17 Cserni G, Vajda K, Tarján M, Bori R, Svébis M, Baltás B. Nodal staging of colorectal carcinomas from quantitative and qualitative aspects. Can lymphatic mapping help staging? Pathol Oncol Res 1999; 5: 291-296

18 Feig BW, Curley S, Lucci A, Hunt KK, Vauthey JN, Mansfield PF, Cleary K, Hamilton S, Ellis V, Brame M, Berger DH. A caution regarding lymphatic mapping in patients with colon cancer. Am J Surg 2001; 182: 707-712

19 Joosten JJ, Strobbe LJ, Wauters CA, Pruszczynski M, Wobbes T, Ruers TJ. Intraoperative lymphatic mapping and the sentinel node concept in colorectal carcinoma. Br J Surg 1999; 86: 482-486

20 Patten LC, Berger DH, Rodriguez-Bigas M, Mansfield P, Delpassand E, Cleary KR, Fagan SP, Curley SA, Hunt KK, Feig BW. A prospective evaluation of radiocolloid and immunohistochemical staining in colon carcinoma lymphatic mapping. Cancer 2004; 100: 2104-2109

21 Wiese DA, Saha S, Badin J, Ng PS, Gauthier J, Ahsan A, Yu L. Pathologic evaluation of sentinel lymph nodes in colorectal carcinoma. Arch Pathol Lab Med 2000; 124: 1759-1763

22 Viehl CT , Hamel CT, Marti WR, Guller U, Eisner L, Stammberger U, Terracciano L, Spichtin HP, Harder F, Zuber M. Identification of sentinel lymph nodes in colon cancer depends on the amount of dye injected relative to tumor size. World J Surg 2003; 27: 1285-1290

23 Saha S, Sehgal R, Patel M, Doan K, Dan A, Bilchik A, Beutler T, Wiese D, Bassily N, Yee C. A multicenter trial of sentinel lymph node mapping in colorectal cancer: prognostic implications for nodal staging and recurrence. Am J Surg 2006; 191: 305-310

24 Wood TF , Nora DT, Morton DL, Turner RR, Rangel D, Hutchinson W, Bilchik AJ. One hundred consecutive cases of sentinel lymph node mapping in early colorectal carcinoma: detection of missed micrometastases. J Gastrointest Surg 2002; 6: 322-329; discussion 329-330

25 Bilchik AJ, Nora D, Tollenaar RA, van de Velde CJ, Wood T, Turner R, Morton DL, Hoon DS. Ultrastaging of early colon cancer using lymphatic mapping and molecular analysis. Eur J Cancer 2002; 38: 977-985

26 Kitagawa Y, Watanabe M, Hasegawa H, Yamamoto S, Fujii H, Yamamoto K, Matsuda J, Mukai M, Kubo A, Kitajima M. Sentinel node mapping for colorectal cancer with radioactive tracer. Dis Colon Rectum 2002; 45: 1476-1480

27 Ratanachaikanont T, Karalak A. Sentinel node localization in breast cancer using intradermal dye injection: results, influencing factors and learning curve. J Med Assoc Thai 2005; 88: 386-390

28 Yagci G, Unlu A, Kurt B, Can MF, Kaymakcioglu N, Cetiner S, Tufan T, Sen D. Detection of micrometastases and skip metastases with ex vivo sentinel node mapping in carcinoma of the colon and rectum. Int J Colorectal Dis 2007; 22: 167-173

29 Paramo JC, Summerall J, Wilson C, Cabral A, Willis I, Wodnicki H, Poppiti R, Mesko TW. Intraoperative sentinel lymph node mapping in patients with colon cancer. Am J Surg 2001; 182: 40-43

30 Paramo JC, Summerall J, Poppiti R, Mesko TW. Validation of sentinel node mapping in patients with colon cancer. Ann Surg Oncol 2002; 9: 550-554

31 Bembenek AE, Rosenberg R, Wagler E, Gretschel S, Sendler A, Siewert JR, Nährig J, Witzigmann H, Hauss J, Knorr C, Dimmler A, Gröne J, Buhr HJ, Haier J, Herbst H, Tepel J, Siphos B, Kleespies A, Koenigsrainer A, Stoecklein NH, Horstmann O, Grützmann R, Imdahl A, Svoboda D, Wittekind C, Schneider

Cahill RA. Lymphatic mapping and colon cancer 6293

www.wjgnet.com

W, Wernecke KD, Schlag PM. Sentinel lymph node biopsy in colon cancer: a prospective multicenter trial. Ann Surg 2007; 245: 858-863

32 Bembenek A, Schneider U, Gretschel S, Fischer J, Schlag PM. Detection of lymph node micrometastases and isolated tumor cells in sentinel and nonsentinel lymph nodes of colon cancer patients. World J Surg 2005; 29: 1172-1175

33 Bendavid Y, Latulippe JF, Younan RJ, Leclerc YE, Dube S, Heyen F, Morin M, Girard R, Bastien E, Ferreira J, Cerino M, Dubé P. Phase I study on sentinel lymph node mapping in colon cancer: a preliminary report. J Surg Oncol 2002; 79: 81-84; discussion 85

34 Bertoglio S, Sandrucci S, Percivale P, Goss M, Gipponi M, Moresco L, Mussa B, Mussa A. Prognostic value of sentinel lymph node biopsy in the pathologic staging of colorectal cancer patients. J Surg Oncol 2004; 85: 166-170

35 Gandy CP, Biddlestone LR, Roe AM, O'Leary DP. Intra-operative injection of Patent Blue V dye to facilitate nodal staging in colorectal cancer. Colorectal Dis 2002; 4: 447-449

36 Levine EA, Shen P, Shiver SA, Waters G, Brant A, Geisenger KR. Intraoperative imprint cytology for evaluation of sentinel lymph nodes from visceral malignancies. J Gastrointest Surg 2003; 7: 687-691

37 Merrie AE, van Rij AM, Phillips LV, Rossaak JI, Yun K, Mccall JL. Diagnostic use of the sentinel node in colon cancer. Dis Colon Rectum 2001; 44: 410-417

38 Redston M , Compton CC, Miedema BW, Niedzwiecki D, Dowell JM, Jewell SD, Fleshman JM, Bem J, Mayer RJ,

Bertagnolli MM. Analysis of micrometastatic disease in sentinel lymph nodes from resectable colon cancer: results of Cancer and Leukemia Group B Trial 80001. J Clin Oncol 2006; 24: 878-883

39 Read TE, Fleshman JW, Caushaj PF. Sentinel lymph node mapping for adenocarcinoma of the colon does not improve staging accuracy. Dis Colon Rectum 2005; 48: 80-85

40 Hünerbein M, Totkas S, Ghadimi BM, Schlag PM. Preoperative evaluation of colorectal neoplasms by colonoscopic miniprobe ultrasonography. Ann Surg 2000; 232: 46-50

41 Bilchik AJ, Compton C. Close collaboration between surgeon and pathologist is essential for accurate staging of early colon cancer. Ann Surg 2007; 245: 864-866

42 Cahill RA. Alternative perspective on the rationale for lymphatic mapping for sentinel node identification in colon cancer. Ann Surg 2008;247:901-902; author reply 902-904

43 Cahill RA . A multicenter trial of sentinel lymph node mapping in colorectal cancer: prognostic implications for nodal staging and recurrence. Am J Surg 2007; 194: 139-140

44 Sticca RP. Is there clinical value to sentinel lymph node sampling in colon cancer? J Clin Oncol 2006; 24: 841-842

45 Yahanda AM, Chang AE. Colorectal Cancer. In Surgery, Scientific Principles and Practice. Greenfield LJ, Mulholland MW, Oldham KT, Zelenock KT, Lillemore KD Eds. Lippincott Willaims & Wilkins. 3rd edition. PA USA, Chpt 46, pg 1121

46 Ho YH, Low D, Goh HS. Bowel function survey after segmental colorectal resections. Dis Colon Rectum 1996; 39: 307-310

47 Dworkin MJ, Allen-Mersh TG. Effect of inferior mesenteric artery ligation on blood flow in the marginal artery-dependent sigmoid colon. J Am Coll Surg 1996; 183: 357-360

S- Editor Liu Y L- Editor Rippe RA E- Editor Liu Y

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A potpourri of pancreatic issues

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In this issue of World Journal of Gastroenterology, we present a series of articles relating to various aspects of pancreatic disease. Overall, our understanding of the etiology of pancreatic disease is improving, and there is much more of a move towards reserving endoscopic retrograde cholangiopancreatography (ERCP) for cases in which endotherapy is likely and utilising other less invasive modalities such as EUS. In this series, Branch et al discuss the utility of ERCP in the evaluation and management of acute pancreatitis, focusing on the need to move away from diagnostic ERCP. With this in mind, Gerke et al present an update on the role of EUS in pancreatic disease. What we do for the truly idiopathic pancreatitis population has been the subject of a large body of literature over the last few years and is highlighted here by the paper of Enns et al . The perpetually contentious disease process of sphincter of Oddi dysfunction is explored here by Mitchell et al and provides some food for thought as to who should be doing manometry and which patients should be exposed to this potentially curative but also risky procedure. Byrne et al focus on the emerging burden presented by autoimmune pancreatitis, a condition undoubtedly missed in the past and often confused for pancreatic cancer. Freeman tickles our fancy with some revelations in to the pancreatic endocrine and exocrine manifestations of pancreatic disease. Finally, Meloche presents a delightful review of the current state of play in relation to pancreatic transplantation for type 1 diabetes mellitus.

This is a somewhat mixed bag of topics but hopefully serves to remind us that there are a number of considerations in pancreatic disease, that we have more therapeutic options, safer imaging, and that we have a lot more to learn.

6296 Review of idiopathic pancreatitisLee JK, Enns R

6314 Role of endoscopic retrograde cholangiopancreatography in acute pancreatitisCanlas KR, Branch MS

6321 Utility of endoscopic ultrasound in pancreatitis: A reviewRizk MK, Gerke H

6327 Autoimmune pancreatitis: A reviewZandieh I, Byrne MF

6333 Sphincter of Oddi dysfunction and pancreatitisMcLoughlin MT, Mitchell RMS

6344 Pancreatic endocrine and exocrine changes in celiac diseaseFreeman HJ

6347 Transplantation for the treatment of type 1 diabetesMeloche RM

TOPIC HIGHLIGHT

Michael F Byrne, MD, Series Editor

Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6295www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

inflammation within the pancreas. Acute pancreatitis (AP) results in acute inflammation typically presenting as abdominal pain with elevated levels of pancreatic enzymes[1]. Chronic pancreatitis (CP) is defined as a clinical syndrome of progressive inflammatory changes in the pancreas leading to permanent structural damage with subsequent impairment of both exocrine and endocrine function[2]. Chronic pancreatitis is often preceded by recurrent bouts of acute pancreatitis, however, occasionally it can present in a ‘silent’ fashion.

The label of “idiopathic pancreatit is” (IP) was originally designated to cases of pancreatitis wherein a diagnosis could not be made through a thorough history, physical examination, laboratory studies, and noninvasive imaging modalities such as abdominal ultrasonography/computerized tomography. Previously, this nomenclature had accounted for 8%-44% of cases being termed “idiopathic”[3-9]. Recent new laboratory and technological advances have been able to shred the enigmatic veils of IP to a point wherein with extensive evaluation it is possible to reveal the etiology in 79%-80% of patients previously labeled as having “idiopathic pancreatitis”[10,11]. More modern imaging investigations include, but are not limited to fine cut computerized tomography, endoscopic retrog rade pancreatog raphy, magnet ic resonance pancreatography as well as endoscopic ultrasound.

EPIDEMIOLOGYLongitudinal data on incidence trends report an increase in AP[12] which may be attributable to increases in all causes of pancreatitis as well as improved detection methods. The fatality rate for pancreatitis has continued to range between 3%-10%[13-18] despite a marked decrease in pancreatitis case fatality presumably secondary to early recognition of severity and complications as well as improved intensive care management[14,15]. Thomson[6] and others[7,17] have noted that in AP mortality rates are greater when the etiology is IP (14.1%)[6], compared with gallstone pancreatitis (7.2%)[6]. In addition, the clinical impact of IP is further highlighted by the fact that 40 percent (the largest subgroup) of all AP fatalities are attributable to IP[19, 20]. The incidence of IP ranges from 4.21 per 100 000[21] to as high as 45.33 per 100 000[9] depending on the population studied and the time in which data was collected with a trend toward high incidence in populations studied more recently[12,13,22]. The incidence appears to be

TOPIC HIGHLIGHT

Review of idiopathic pancreatitis

Jason Kihyuk Lee, Robert Enns

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Jason Kihyuk Lee, University of British Columbia Department of Medicine, Vancouver, British Columbia, Canada Robert Enns, University of British Columbia Department of Medicine, Division of Gastroenterology, St. Paul’s Hospital, Vancouver, British Columbia, Canada Correspondence to: Robert Enns, MD, FRCP, University of British Columbia Department of Medicine, Division of Gastro-enterology, St. Paul’s Hospital, Vancouver, British Columbia, Canada. renns@interchange.ubc.caTelephone: +1-604-6886332 Fax: +1-604-6892004Received: June 15, 2007 Revised: July 16, 2007

AbstractRecent advances in understanding of pancreatitis and advances in technology have uncovered the veils of idiopathic pancreatitis to a point where a thorough history and judicious use of diagnostic techniques elucidate the cause in over 80% of cases. This review examines the multitude of etiologies of what were once labeled idiopathic pancreatitis and provides the current evidence on each. This review begins with a background review of the current epidemiology of idiopathic pancreatitis prior to discussion of various etiologies. Etiologies of medications, infections, toxins, autoimmune disorders, vascular causes, and anatomic and functional causes are explored in detail. We conclude with management of true idiopathic pancreatitis and a summary of the various etiologic agents. Throughout this review, areas of controversies are highlighted.

© 2007 WJG. All rights reserved.

Key words: Idiopathic pancreatitis; Recurrence; Etiology; Endoscopic retrograde cholangiopancreatography; Magnetic resonance cholangiopancreatography; Sphincter of Oddi dysfunction

Lee JK, Enns R. Review of idiopathic pancreatitis. World J Gastroenterol 2007; 13(47): 6296-6313

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INTRODUCTIONPancreatitis is a relatively common disorder with a myriad of etiologies all resulting in a common end result of

Michael F Byrne, MD, Series Editor

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equal between the two sexes and tends to increase with age in both before starting to plateau around 70 years[18].

Elucidating the etiology of pancreatitis, if possible is paramount as it guides therapy and may theoretically subsequently improve pat ient outcomes, thereby preventing relapses. Some studies have shown that over 50% of untreated patients with acute IP experience recurrent episodes[23-25]. This contrasts with other studies where only 1 of 31 patients with a first episode of unexplained AP suffered another attack during a median follow up of 36 mo[26]. These conflicting results may reflect different patient populations, perhaps even within the same category known as IP.

Aside from the initial objective to decrease acute patient mortality and morbidity, repeated insults to the pancreas may progress to chronic pancreatitis with irreversible morphologic and functional changes[2,24,27]. This concern typically results in aggressive investigation for those patients with more than one episode of AP. This approach is supported by a study by Kaw and Brodmerkel in a study that included 126 patients with two or more episodes of IP. They demonstrated that investigations including bile for microlithiasis, a secretin stimulation test, and sphincter of Oddi manometry (SOM) was able to clarify the etiologies in 79 percent of patients[11]. In this study they were able to offer 75% of these patients treatment that resulted in the absence of AP in over 60% of cases over a 30 mo follow-up period[11].

However there is ongoing debate against aggressive evaluation. The Kaw and Brodmerkel study confirmed previous morbidity data demonstrating that SOM is not a benign procedure and that complications occur[11]. Additionally, many aggressive techniques for diagnosing the etiology of pancreatitis, including SOM, may not be routinely available. Therefore the generalizability of this study has been brought into question.

This article will examine some of the etiologies that must be excluded prior to a diagnosis of IP. An attempt will be made to highlight the areas of controversy, and make suggestions based on the best available evidence. For the purpose of this IP article, gallstones and alcohol related pancreatic disease will not be discussed as their presentation is usually easily determined. For a more comprehensive list of all etiologies of pancreatitis please refer to Table 1.

Table 1 Etiologies of acute or acute recurrent pancreatitis

Category Agent/Diagnosis

Vascular AtheroembolismIntraoperative hypotensionHemorrhagic shockVasculitis (systemic lupus erythematosus and polyarteritis nodosa)

Infectious ViralMumpsCoxsackievirus type BHepatitis BCytomegalovirusHerpes simplexVaricella-zosterHIVRubella (probable)BacterialLegionellaLeptospiraSalmonellaMycoplasmaBrucellaMycoplasmaSalmonella typhiFungalAspergillusParasitesToxoplasmaCryptosporidiumAscaris lumbricoides

Trauma Blunt or penetrating abdominal injuryPost-ERCP pancreatitisERCP sphincterotomyManometry of sphincter of OddiIatrogenic operative complication

Metabolic Hypertriglyceridemia (typesⅠ, Ⅳ, Ⅴ)HypercalcemiaHyperparathyroidism

Toxins Ethyl alcoholScorpion venomMethyl alcoholOrganophosphorous insecticides

MedicationsThe following drugs were definitely associated with pancreatitis

Antimicrobial agentsMetronidazole, Stibogluconate, Sulfonamides, Tetracycline, Nitrofurantoin, Erythromycin, IsoniazidHIV TherapyDidanosine, PentamidineDiureticsFurosemide, ThiazidesCommonly used Gastroenterology Medications5-ASA, Sulphasalazine, Cimetidine, Ranitidine, Mercaptopurine, Proton pump inhibitorsCardiac AgentsProcainamideImmunosuppressives or ChemotherapeuticsL-asparaginase, Azathioprine, Cytosine arabinoside, DexamethasoneNeuropsychiatric Agents

Mechanical

Valproic Acid, αMethyl DopaOther Commonly UsedAcetaminophen, Salicylates, Sulindac, Calcium, Ethinylestradiol, Norethindrone

GallstonesMicrolithiasis and Biliary Sludge.Sphincter of Oddi dysfunctionPancreas divisumAnnular pancreasAutoimmune pancreatitis

Pancreatobiliary TumoursCholodochoceleDuodenal stricture or obstruction

Miscellaneous

Ascariasis

Post ERCPRenal transplant

Genetic

Hyper IgG4 disease

CFTRSerine protease inhibitor Kazal type 1 mutationCationic trypsinogen gene PRSS1 mutation

Autoimmune Sjogren’s syndromePrimary biliary cirrhosisRenal tubular acidosis

PANCREATITIS ETIOLOGIES OFTEN LABELLED AS IDIOPATHIC PANCREATITISMicrolithiasis and biliary sludgeBiliary sludge refers to the viscous suspension in gallbladder bile formed by modification of hepatic bile by the gallbladder mucosa that may contain small stones (< 5 mm in diameter)[28]. Microlithiasis refers to stones of < 3 mm in diameter and is often used interchangeably at times with “microcrystals” and sometimes with biliary sludge[28-30]. Microscopy of bile in patients with sludge often shows cholesterol monohydrate crystals, calcium carbonate microspheroliths, or calcium bilirubinate granules[31]. A known risk factor for the development of sludge typically includes prolonged fasting states as well as some antibiotics (ceftriaxone)[32]. Microlithiasis has been suggested to be the most common causes of IP[31,33] with a prevalence ranging from 6%-73%[11,25,31,33,34].

It has been demonstrated that treatment with chole-cystectomy, endoscopic sphincterotomy, or ursodiol significantly reduces further attacks of IP[31,33]. Therefore it was inferred that in the absence of other identifiable risk factors, the presence of microlithiasis was enough to cause IP[11,26,35]. The caveat to interpreting these studies is that combined, they involved only 74 patients who had IP and treatments were clearly not double blinded[31,33].

An Indian study published earlier this year evaluating 51 patients with recurrent IP found that microlithiasis accounted for a mere 13 percent of these patients (using duodenal bile samples)[36]. In the setting of suspected biliary pancreatitis up to 88% of patients will have microlithiasis demonstrating the difference between these two patient groups[37]. It is speculated that there may be some gender, age, racial, and procedural differences in bile sample collection and analysis as these are not well standardized.

The pathogenesis of AP via microlithiasis remains unclear however it is thought that the microlithiasis may transiently impact the papilla, cause pancreatic duct obstruction and thereby pancreatitis[29,37,38]. The diagnostic workup for microlithiasis includes routine abdominal ultrasound (US) which has limited sensitivity when looking for stones less than 3 mm in diameter[28,39]. Endoscopic US (EUS) may be considered as well as it carries a lower risk of complications than endoscopic retrograde cholangiopancreatography (ERCP)[34,40-45]. On average, EUS is able to identify gallbladder sludge in up to 75 percent of IP cases[40-45]. All patients with recurrent IP should have microlithiasis excluded and there is even some evidence that it should be excluded for patients who have their first IP[41-43].

It remains to be clarified which method of bile collection for microscopic analysis is clinically the best and there are no standardized methods or recommendations at this time[46-48].

Cholecystectomy is recommended for patients with biliary sludge/microlithiasis once they recover from their episode of pancreatitis as it reduces the relapse rates[31,33]. In patients who are poor surgical candidates, ERCP with biliary sphincterotomy or ursodeoxycholic acid may be alternative forms of treatment[31,33].

Sphincter of Oddi dysfunctionAt times referred to as hypertensive or fibrotic SOD, SOD causes diminished transphincteric flow of bile and/or pancreatic juice due to either organic obstruction (stenosis) or functional obstruction (dysmotility)[3,35,49]. SOD often causes recurrent pain with or without abnormalities of either hepatic/pancreatic profiles as well as duct dilation. It is thought that SOD causes pancreatitis as a result of bile reflux into the pancreatic duct or from pancreatic duct outflow obstruction[25,50]. SOD is considered to cause up to one third of all cases of IP[25,30,51,52].

In order to diagnose SOD, one often needs to perform sphincter of Oddi manometry (SOM)[53,54]. The diagnostic gold standard is a water-perfused catheter system that can be inserted into the common bile duct or pancreatic duct with the positive finding being a hypertensive sphincter of Oddi pressure greater than 40 mmHg[53]. Unfortunately whether ERCP in patients with suspected SOD is done for diagnostic SOM or therapeutic purposes it is undeniable that there have been high complication rates, particularly pancreatitis[51,55-59]. Additionally “severe” pancreatitis that is associated with death has even been quoted in 1-3% in SOM[15,25,51]. Despite this, ERCP with SOM has been advocated for the evaluation of recurrent IP[25,60]. In patients who have normal biliary manometry, pancreatic sphincter manometry may be reasonable to perform[61,62] which does carry a higher risk of pancreatitis[54] that may be reduced by aspirating through the middle port of the triple lumen manometry catheter[63].

Once the diagnosis of SOD is made, endoscopic sphincterotomy is the treatment of choice as it is believed to decrease the risk of recurrent pancreatitis[25,60,64-66].

The Geenen-Hogan (Milwaukee) criteria was devised to predict the overall probability of response to biliary sphincterotomy taking into account the presence of abnormal liver chemistries and dilated bile ducts[44]. On the basis of this, patients with pain and both abnormalities (typeⅠ) are definitely recommended to have sphincterotomy as they have high response rates ranging from 90%-100% regardless of SOM[67,68]. Along this stratification, type Ⅱ patients have either abnormal liver chemistry or dilated bile ducts. If SOM is abnormal in type Ⅱ patients they will have a response rate to sphincterotomy of 60%-91%[64,67,69] and thus this would be a reasonable course of action. On the contrary, type Ⅲ patients do not have objective biliary abnormalities and even if they have abnormal SOM they have poor response rates of 6-58 percent[69-71], making sphincterotomy of questionable benefit (Table 2) .

If a diagnosis of pancreatic sphincter dysfunction is made in patients who have had biliary sphincterotomy the answer to who best benefi ts from pancreat ic sphincterotomy is less clear. A study by Freeman et al[72] this year treated suspected SOD with biliary sphincterotomy with additional pancreatic sphincterotomy at initial or subsequent ERCP if there was abnormal pancreatic manometry in conjunction with pain refractory to biliary sphincterotomy, continuous pain, or a history of amylase elevation. In this study of 121 predominantly female (92%) and post cholecystectomy (87%) patients, all patients underwent biliary sphincterotomy while 40 percent also

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underwent pancreatic sphincterotomy regardless of the modified Milwaukee biliary classification[72]. The result from this study was that a positive response at final follow up was reported in 69 percent of patients and that this response was not significantly different between biliary typesⅠ, Ⅱ, and Ⅲ[72]. Freeman et al[72] concluded that patient characteristics of pancreatic manometry, delayed gastric emptying, daily opioid use, and age < 40 were significant as predictors of outcomes as opposed to the Milwaukee classification. Indeed it is becoming more accepted that a lack of improvement after biliary sphincterotomy may be representative of a failure to relieve pancreatic sphincter pressure[61,62,73-77].

Noninvasive strategies such as a low-fat diet, analgesics, anticholinergics, calcium-channel blockers, nitrates, and proton pump inhibitors are unfortunately seldom effective[78,79].

Pancreas divisumIn Pancreas divisum it is thought that 80%-95% of pancreatic juice volume flows via the dorsal duct through the smaller minor papillary orifice via the dorsal duct of Santorini as opposed to the more common route through the major papilla via the ventral duct of Wirsung[66]. It is postulated that the mechanism is that of relative minor papilla outflow obstruction leading to pancreatitis[23,80-82].

Pancreas divisum occurs as an anatomic variant in 5%-7.5% of patients[23,82]. There are those who are skeptical of the obstructive theory as fewer than 5% of patients with pancreas divisum develop pancreatitis[83-86]. It also appears that the incidence of pancreas divisum is the same in patients with and without pancreatitis[86]. Nonetheless there is consensus that while most individuals with pancreas divisum live normally, a few unfortunate patients are predisposed to develop recurrent AP[23-25,35] and that it accounts for 20 percent of the IP cases[23-25].

One explanation of why some patients with pancreas divisum are more likely to be affected was the smaller than usual minor papillary orifice causing a disproportionately high intrapancreatic dorsal ductal pressure especially during times of active secretion. In this situation a cascade of inadequate drainage leading to ductal distension and eventually pancreatitis could occur[23,87-89]. This theory is

supported by at least one surgical study that demonstrated relief of pain as well as diminished attacks of AP with sphincteroplasty[87].

Recently, Choudari et al[90] found that prevalence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations was similar in patients with pancreas divisum with recurrent acute pancreatitis and true idiopathic recurrent acute pancreatitis. Interesting, this study also found that the prevalence of CFTR gene mutations in patients with pancreas divisum but without recurrent AP was similar to controls without pancreatitis[90]. The significance of CFTR will be discussed in more detail under “Genetic Causes and Contributions” below, however, these genetic studies call into question the true role of pancreas divisum in IP.

At present, it is accepted that patients who present with severe pancreatobiliary type symptoms or recurrent IP should however be evaluated for pancreas divisum[35,48]. The diagnosis of divisum can be made either by ERCP or magnetic resonance cholangiopancreatography (MRCP)[91,92]. ERCP may be preferred to conventional MRCP as it has a higher sensitivity and specificity[91,92] and can offer therapy albeit with a complication rate that is higher than in MRCP. Secretin may be given intravenously to aid in finding the minor papillary orifice at ERCP[93], finding functional outflow obstructions[94-96], and assessing the relative obstruction of the minor papilla[94-96]. In settings where secretin is either contraindicated or does not produce a visible increase in pancreatic juice flow, a dilute (1:10) methylene blue solution can assist in the location of the minor papilla[97]. Confirmation is recommended to rule out pseudodivisum caused by a tumor.

An emerging area is the role of EUS as it is a minimally invasive tool for the general workup of IP and recurrent IP[48]. It appears that EUS is able to detect pancreas divisum but more research in comparing ERCP to EUS specific to divisum is needed before recommendations are made.

In the setting of recurrent AP, once divisum is diagnosed, ERCP therapy to relieve minor papillary obstruction is recommended to decrease the rate of recurrent pancreatitis[98-101], particularly in patients with a dilated pancreatic duct who may be the most likely to benefit from therapy[98]. The rate of decrease of recurrent pancreatitis is quoted to range from 70%-90%[99,100] with a 40%-60% response rate in patients with CP also demonstrated[102]. The ERCP therapy involves a minor papillotomy usually with temporary stenting[98-101,103]. In one study, more favorable long-term results were achieved with minor papilla sphincterotomy than with repeated stenting in terms of both recurrence and fewer complications (25% vs 44% respectively)[98]. Some endoscopists are particularly cautious about endoscopic stenting as the procedure may cause permanent damage to the pancreatic ducts, possibly promoting the development of chronic pancreatic disease[104,105].

Anomalous union of pancreaticobiliary ductAnomalous union of pancreaticobiliary duct (AUPBD) is defined as an anomalous junction of bile duct and

Table 2 Sphincter of Oddi dysfunction (Geenen and Hogan Classification)

Biliary type Pancreatic type

TypeⅠ TypeⅠBiliary-type pain Pancreatic-type painLFT elevation Amylase/lipase elevationCBD dilation PD dilationDelayed drainage Delayed drainage

Type Ⅱ Type ⅡBiliary-type pain Pancreatic-type painOne or two of above criteria One or two of above criteria

Type Ⅲ Type ⅢBiliary-type pain only Pancreatic-type pain only

CBD: Common bile duct; LFT: Liver function tests; PD: Pancreatic duct.

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pancreatic duct at an abnormal proximal site of these ducts[35]. The precise location of the junction is outside the duodenal musculature and independent of the sphincter of Oddi. This independence from the contractility of the sphincter of Oddi results in regurgitation of the pancreatic juice into the biliary tract and vice versa[106]. AUPBD is frequently associated with choledochal cysts which in itself has been linked to IP (see below)[107] and is more common in East Asia[35].

It is thought that in AUPBD reflux of bile into the pancreatic duct may cause recurrent AP[106,108] and that in cases with a dilated common channel the stasis of pancreatic secretion can occur in the common channel leading to pancreatitis[109,110]. Moreover it has also been found that SOD (see above) is sometimes associated with AUPBD[111].

The diagnosis of AUPBD is usually made during ERCP evaluation for IP when one notes a pancreatic duct and choledochus connecting with a long common channel of > 15 mm in adults[112]. Similarly AUPBD is also seen during MRCP[91]. The length of the common channel alone is not an absolute diagnostic criterion as other factors like the form of union, direction, and age and stature of patients need to be considered[108,113]. An alternate method of diagnosis, more importantly a ‘clue’ to the presence of AUPBD, may be the presence of high amylase levels in aspirated bile[114,115].

In AUPBD, endoscopic biliary sphincterotomy has been shown to prevent further attacks of AP[112] by a postulated mechanism of decreasing resistance at the major duodenal papilla[35]. Due to the fact that AUPBD has a propensity to increase gallbladder and biliary duct cancer[114,116-118], prophylactic cholecystectomy has been recommended in these patients[116]. As well, at this time there is some evidence that patients with AUPBD and choledochal cyst should have an extensive resection of the extrahepatic bile duct inclusive of the cyst in hopes of preventing development of biliary duct cancer[108] which may be relevant in 5% of patients[116]. It is reiterated that the understanding of AUPBD, its pathophysiology and therapy is very limited[35].

CholedochoceleCholedochocele is classified as a type Ⅲ choledochal cyst that represents a prolapse or herniation of the intramural segment of the distal common bile duct into the duodenal lumen which may be either congenital or acquired[35,119]. Pancreatitis and recurrent AP is an important complication occurring in choledochoceles occurring in 12%-30% of patients[120,121]. Choledochoceles do not represent a common cause of IP, especially in adults[25,35,120].

It is thought that the choledochocele may create an obstruction to the pancreatic duct intermittently when the choledochocele becomes distended and that this may lead to reflux of bile into the pancreatic duct[121-123].

A characteristic “bulging” appearance of the papilla at ERCP and a soft “pillow” sign with pressure applied at the catheter tip suggests a choledochocele[120,124]. A CT or US in isolation may miss this diagnosis[120]. The role of MRCP in adults to diagnose this is still undefined[91,125,126].

Trea tment can be in i t i a t ed w i th endoscop ic sphincterotomy combined with “unroofing” of the choledochocele with a papillotome with the goal of creating effective drainage of bile and pancreatic juice[120,127]. Patients that fail this treatment may be considered for a surgical sphincteroplasty[128]. Reports in the surgical literature about choledochal cysts in general recommend a single stage surgery usually comprising of complete cyst resection, cholecystectomy and Rou-en-Y hepatojejunostomy[129-131]. Malignancy is noted in 3-5 percent which may in some cases warrant prophylactic surgery[131,132].

Annular pancreasAnnular pancreas is a congenital condition that represents a band of pancreatic tissue partially or completely encircling the duodenum that is usually at the level of or immediately proximal to the major duodenal papilla[133]. It is thought that this defect occurs in utero due to the failure of the ventral bud to rotate with the duodenum[133]. This abnormality is rare and is detected in 1/7000-1/20 000 autopsies[133,134] and in 1/1500 ERCPs[135-137].

Clinically it usually manifests in childhood with intractable vomiting[136,137] and is thought to result from descending duodenal narrowing leading to duodenal obstruction or recurrent AP[138]. In adults annular pancreas may present with abdominal pain, acute recurrent IP, CP, peptic ulcer disease, postprandial fullness, vomiting, or biliary obstruction[137,139]. Associated congenital anomalies such as Down’s syndrome, cardiac defects, tracheoesophageal fistula, Meckel’s diverticulum, and imperforate anus may be present[133,136]. If the diagnosis is made in adults it usually occurs between the ages of 20 to 50[140].

From a diagnostic view, barium studies, abdominal CT, or MRCP may suggest the diagnosis but an ERCP is recommended for confirmation[135,136,141]. A typical ERCP is that of the duct of the pancreatic annulus encircling the duodenum[24] and in one third of cases a pancreas divisum is also present[139]. The annular duct may communicate with the central duct but rarely drains into the dorsal duct, common bile duct, or independently into the duodenum[135,139]. In cases where ERCP may not be feasible, an EUS may offer an alternative means of diagnosis[142]. Recently some data has also become available on the role of MRCP which appears encouraging as a non-invasive method of diagnosis[143-145].

Surgery is the procedure of choice in patients in whom symptoms can be attributed to annular pancreas with the goal to relieve duodenal or gastric outlet obstruction[137,146].

Pancreatobiliary tumorsAny mass that obstructs the main pancreatic or biliary ducts, benign or malignant can result in acute pancreatitis. It has been estimated that 5%-14% of patients with pancreatobiliary tumors, benign or malignant, present with apparent IP[147-150]. Pancreatic cancer should be suspected in any patient older than 40 years with IP especially with a prolonged or recurrent course[3,5]. Neoplasia should be suspected in a patient with weight loss, steatorrhea, ductal dilation, new onset diabetes, or evidence of a solid

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or cystic pancreatic mass[24,149,150]. In younger patients lesions such as curable islet cell tumors should be in the differential whereas in the elderly they may have potentially curable lesions such as cystic neoplasms[24].

It is well known that CT, MRI, ERCP, and EUS have a role in identifying pancreatobiliary neoplasms[151-154]. It is recommended that if there is any clinical suspicion of malignancy that aggressive investigation including ERCP be performed even on the first attack of IP in older patients. In patients who are less than 40 years old, a CT may be sufficient in first attacks of AP[24]. If a malignancy is suspected, EUS is a favorable technique that can be used for diagnostic and staging purposes[155,156] especially when combined with fine needle biopsy[157,158].

Intraductal papillary mucinous neoplasm of the pancreas (IPMN) is a distinct pathologic entity formed of papillary proliferations of mucin-producing epithelial cells with or without excessive mucus production and or cystic dilation of the pancreatic duct. IPMN represents a precancerous lesion with a well described adenoma and carcinoma sequence that causes recurrent acute IP with symptoms suggestive of chronic obstructive pancreatitis due to intermittent obstruction of the pancreatic duct with mucus plugs[159]. It is worth noting this entity has an insidious nature and lack of awareness often delays diagnosis[160-163]. On ERCP, in cases of main duct involvement, the papilla is patulous and resembles a “fish-eye” frequently with mucus extruding from the orifice[161,164,165]. In terms of management, since the ten-year actuarial risk of high grade dysplasia and invasive cancer is significant[166] surgery is usually recommended, particularly for main duct disease[159,167-169]. It should also be noted that patients with IPMN may be at increased risk for extrapancreatic malignancies and because of this gastricadenocarcinoma and colorectal cancer should be screened for using endoscopy[170,171]. Patients with branch type disease may be at a lower risk of malignancy and theoretically can be monitored in regards to mural wall thickening, size of branch cystic lesion and tumor markers.

Cystic pancreatic tumors including IPMN, serous cystadenomas, mucinous cystadenomas, and mucinous cystadenocarcinomas, can be premalignant or malignant and surgery is generally indicated[172-174]. Tumor markers such as CA 19-9, CA 15-3, CA 72-4, and carcinoembryonic antigen in aspirated cystic fluid along with fluid viscosity and amylase level may be used to increase the diagnostic yield of cyst fluid cytology[175-179].

Ampul lar y adenomas are premal ignant and in general, indicate the need for close monitoring and eventual removal[180,181]. In general, ampullary tumors have a more favorable prognosis than pancreatic tumors and pancreatoduodenectomy has historically been recommended[182-184]. An emerging development for ampullary tumors is the use of endoscopic techniques such as endoscopic ampullectomy for management of patients with small benign lesions or for carcinoma in situ[185-188] which appears to be safe and efficacious on long term follow up[188]. If endoscopic management is selected it has been recommended that surveillance and random biopsies be performed[24].

GENETIC ASSOCIATIONS AND CAUSESAn exciting development is the recognition of genetic mutations that are associated with pancreatitis.

CFTRThe cystic fibrosis transmembrane conductance regulator (CFTR) and its gene mutations cause cystic fibrosis in an autosomal recessive pattern. It is well established that CFTR mutations cause disease of the exocrine pancreas[189]. The CFTR gene encodes a chloride-channel protein that is regulated by cAMP. Bicarbonate ion is secreted into the duct lumen by the action of CFTR in the apical membranes and this sets up a gradient in which water follows[190]. In the absence of CFTR, the pancreatic duct cells cannot secrete fluid and bicarbonate and hence CF of the pancreas develops[191].

The actual mechanism that mutations in the CFTR gene causes recurrent AP or CP[90,192-194] is unknown. It may be that mutant CFTR channels are inefficient at flushing digestive enzymes out of the pancreatic duct and thereby limiting the major mechanism that prevents trypsin-associated injury within the pancreatic duct[195]. Along the same lines, it may be that mutant CFTR can limit bicarbonate secretion that can interfere with trypsin activation[195]. What is clear is that CFTR mutations have a demonstrated increased incidence in patients with CP, IP, and alcohol induced CP[194,196-201]. One recent study of 381 patients diagnosed with either CP or recurrent IP that used expansive CFTR genotyping showed mutant CFTR genes in 11% (43/381) of these patients[195]. This frequency is in keeping with previous reports of 28%-45% of IP patients being either heterozygous or homozygous for a CFTR gene mutation[202,203]. One study in Poland showed that the frequency of mutations in CFTR alleles was similar to controls (4.9% vs 5%, P = 0.587) but this study only looked at 3 CFTR defects[204].

Testing for this gene mutation may be considered in younger patients presenting with recurrent IP who have a positive family history of cystic fibrosis or IP[66,195,205]. If testing is to be done, a broad mutation panel to look for a multitude of mutations of CFTR is recommended[195].

SPINK1The N34S mutation of the serine protease inhibitor Kazal type 1 (SPINK1) has been repor ted to be strongly associated with IP and hereditary/familial pancreatitis[206-208]. SPINK1 is a specific trypsin and trypsin activated trypsin like inhibitor expressed within the pancreas that provides a defense against prematurely activated trypsinogen[209,210]. It has been suggested that SPINK1 mutations are disease modifiers in that they lower the threshold for pancreatitis from other genetic or environmental factors[207]. For example this mutation was found in one study to be associated with CP and recurrent IP in 7.7% and 10% respectively[204]. Likewise, a larger study showed that 15.7% of patients with CP or recurrent IP carried at least one SPINK1 mutation[195].

A genetic test for SPINK1 should be done in any younger patient presenting with recurrent AP or CP and a family history of IP or CP-particularly if they have had

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prior negative workup with ERCP. When genetic testing is done, it is advised to perform a concomitant CFTR and PRSS1 mutation screen[195].

Cationic trypsinogen gene PRSS1Two missense mutations R12211[211] and N291[212] in the human cationic trypsinogen gene [protease serine 1 (trypsin 1); PRSS1] were first detected in patients with hereditary pancreatitis and appear to manifest as autosomal dominant mutations[213]. Since the discovery of the first two genes many other PRSS1 mutations have been reported[214-219]. Pathophysiologically this mutation leads to impaired trypsin autolysis and degradation, impairment of SPINK1 defense mechanism, and promotion of auto-activation of trypsinogen all of which can cause AP and lead to CP[211,220].

Symptoms typically arise in childhood but may be delayed until the mid 30 s[221]. These usually include symptoms associated with CP[221]. The lifetime risk of pancreatic cancer is 40% and reportedly 75% with paternal inheritance[222]. Whereas in cases of CFTR and SPINK1 are associated with IP and CP, PRSS1 defects seem to be causative for AP[204].

As in CFTR and SPINK1, the diagnosis is usually suspected if younger patients with affected family members present with CP or recurrent IP. Complications of CP are usually dealt with endoscopically but given the high risk of malignancy surgical resection may be preferred[24].

Celiac diseaseThe frequent familial occurrence and the remarkably close association with the HLA-DQ2 and/or DQ8 gene locus suggests that celiac disease as an immune disorder that is triggered by an environmental agent, gliadin, in genetically predisposed individuals[223,224]. Intriguingly, recurrent pancreatitis can be caused by celiac disease[225,226]. The mechanism appears to be duodenal inflammation and associated papillary stenosis causing pancreatitis[225]. Endoscopic treatment is recommended at this time to relieve the obstruction.

There are consensus guidelines to the role of genetic testing in IP although they are evolving as our knowledge expands in the area[227].

MEDICATIONSThe list of medications that are associated with or that cause pancreatitis is increasing[228-230]. In one German study 1.4% of all acute pancreatitis was related to medications[231]. Another study reported an even lower association of 0.3%[232]. Drugs with the highest incidence of pancreatitis are azathioprine and mercaptopurine (incidence, 3 to 5%)[233] and didanosine (23%)[234].

Most medication related to pancreatitis is due to idiosyncratic response or a direct toxic effect. A high index of suspicion and astute drug history is crucial for making the diagnosis.

A few areas of medications causing pancreatitis will be highlighted. Firstly, physicians should be aware of a recent development showing an association of proton

pump inhibitors and pancreatitis[235-237]. Secondly, some medications, such as pentamidine, valproic acid, and didanosine, appear to cause injury weeks to months after exposure, possibly through the accumulation of a toxic metabolite[238]. Hypersensitivity reactions have been implicated in other drugs, such as azathioprine, mercaptopurine, metronidazole, aminosalicylates, and sulfonamides, and these drugs characteristically lead to pancreatitis within one month after exposure[238]. Lastly, some medications like acetaminophen may cause pancreatitis with a single dose[239].

A complete list of medications that are classified as being definitely associated with pancreatitis derived from multiple references is shown in Table 1[228-230,240-242].

TOXINSToxins that have been linked to pancreatitis include the most common alcohol. The more deadly methanol can also cause AP[243]. Although exceedingly rare scorpion bites from Trinidad have been known to cause AP[244,245]. Another rare cause of IP is organophosphorous poi-soning[246].

INFECTIOUS CAUSESA plethora of infectious agents have been associated with AP[247]. A full list of definite and probable etiologic agents is provided in Table 1 from data gathered from Parenti et al[247]. It is unclear how often an infectious etiology is responsible for IP but many appear to be associated with it[247].

A clinician must be suspicious of an infectious etiology in the characteristic syndrome caused by the particular infectious agent notwithstanding that this was evident in only 70% of patients in the prior review for definite cases[247]. A routine search for an infectious cause in IP is not recommended unless there is a strong clinical suspicion.

Ascariasis is an impor tant cause of infect ious obstructive AP in India where it is the second most common cause of AP[248]. At times this may also present with associated biliary tree infestation requiring endoscopic decompression[249].

An infectious group worth discussing in greater detail is HIV infections and their relation to AP[250]. One large series revealed that 4.7% of hospitalized patients with HIV had AP[251]. While primary HIV infection itself can be a cause of AP[252,253] it is more commonly attributable to a complication of medications taken as part of HIV treatment or medications for opportunistic infections such as Pneumocystis carinii and Mycobacterium avium-intracellulare[250].

METABOLICHypertriglyceridemiaSerum triglyceride concentrations above 11.3 mmol/L are capable of causing AP although admittedly the pathogenesis of inflammation is unclear[254]. It is thought that hypertriglyceridemia represents 1.3%-3.8% of AP

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cases[255]. The incidence has been best defined in children with inherited disorders of lipoprotein metabolism that is associated with severe hypertriglyceridemia which are 35%, 15%, and up to 40% in hyperlipidemia typesⅠ, Ⅱ, and Ⅴ respectively[256,257]. Other acquired causes of hypertriglyceridemia include obesity, diabetes mellitus, hypothyroidism, pregnancy, estrogen or tamoxifen therapy, glucocorticoid excess, nephritic syndrome, and beta blockers[258-260,255].

Drug-induced disease is more likely to occur in patients with underlying hypertriglyceridemia[256]. It is important to not neglect the lactescent serum as it is a vital clue to the diagnosis[255].

Controversy surrounds the contribution of hype-rlipidemia in causing AP in alcoholics[261] but it is thought that in most alcohol abusers the moderate elevations of triglyceride levels are transient and likely to be an epiphenomenon rather than a causative agent of pancreatitis[262].

HypercalcemiaOccurring in uncommon frequency, hypercalcemia of any cause is a known cause of AP[263,264]. Postulated mechanisms include calcium deposition in the pancreatic duct and calcium activation of trypsinogen within the pancreatic parenchyma[265,266].

There are questions however on hyperparathyroidism, a cause of hypercalcemia, and the link to pancreatitis rose in one large study of 1153 patients that found that AP occurred in only 1.5 percent of patients that was of a statistically non significant difference from that of the general population[267]. This finding was mirrored in two other studies[268,269]. This is still an open controversy however as other studies have supported at least an association and a significantly increased relative risk of AP in patients with hyperparathyroidism[270,271]. Until the debate is resolved it is recommended that the parathyroid be checked in recurrent IP if hypercalcemia is present.

VASCULAR DISEASEPancreatic ischemia is an uncommon but an established cause of pancreatitis reported in: (1) vasculitis (systemic lupus erythematosus, polyarteritis nodosa, and microscopic polyangi i t is ) [272-274], (2) atheroembol ism[275,276], (3) hypotension and shock[277-279].

While it is true that most patients have mild attacks of pancreatitis secondary to ischemia, fatal necrotizing pancreatitis is a rare occurrence[277].

TRAUMATIC CAUSESBlunt or penetrating abdominal injuries can cause pancreatitis although it is incredibly rare given the retroperitoneal location of the gland[280]. Types of trauma that cause pancreatitis can range from a mild contusion, severe crush injury, or transection of the gland[280].

Post ERCP pancreatitis remains the commonest severe complication of ERCP found to occur in 5%-7% of patients in a recent review[281]. It may be possible that

prophylactic stenting of the pancreatic duct in selective cases and minimally traumatic cannulation techniques may prevent some cases[281] and indeed this has been shown to be cost effective in high risk patients[282]. Octreotide infusion does not prevent ERCP-induced pancreatitis or affect serum amylase levels[283]. Likewise, transdermal glyceryl trinitrate did not improve the rate of success in ERCP cannulation or prevent post-ERCP pancreatitis in either average or high-risk patient groups[284]. Allopurinol at high doses has been shown in a prospective randomized trial to lower the risk of post-ERCP pancreatitis[285] but this study requires further verification as other studies have shown no benefit[285,286]. Risk factors for post-ERCP pancreatitis have been well described[287].

AUTOIMMUNE CAUSESAutoimmune pancreatitis is a more recently described type of chronic pancreat i t i s character ized by an autoimmune inflammatory process in which prominent lymphoplasmacytic infiltration with associated fibrosis of the pancreas causes organ dysfunction[288-290]. The reported prevalence of autoimmune pancreatitis is between 5 and 6% of all patients with chronic pancreatitis[291]. A series from the United States shows that 11% of patients (27 of 254) with chronic pancreatitis received a diagnosis of autoimmune pancreatitis based on histological findings[292].

Immunologic abnormalities including hypergamma-globulinemia, elevated serum IgG4 levels, and the presence of autoantibodies against carbonic anhydrase

and lactoferrin are important markers of the disease[288]. In particular, autoantibodies against lactoferrin and carbonic anhydrase Ⅱ have been identified as potential serologic markers of autoimmune pancreatitis[293,294]. The finding of increased serum IgG levels or the presence of autoantibodies is supportive of the diagnosis, whereas an

elevated serum IgG4 level is nearly diagnostic[288].CP has also been found to be in association with

Sjogren’s syndrome, primary biliary cirrhosis, and renal tubular acidosis[295,296]. The diagnosis is usually based on clinical suspicion and serum autoantibody to a pancreatic antigen previously discussed. Inf lammatory bowel disease is also occasionally associated with autoimmune pancreatitis[297].

Differentiating the focal form of autoimmune pancreatitis rather than pancreatic carcinoma can be very difficult on the basis of CT imaging only[288].

Diffuse pancreatic-ductal narrowing is highly diagnostic of autoimmune pancreatitis[288,289,291]. There are also nonspecific endoscopic findings in the stomach or colon in patients with autoimmune pancreatitis, foci of slightly pale, thickened mucosa with loss of visible vascular pattern were observed in some cases[298]. The role for MRCP is at this time undefined[288].

EUS is a key tool in the diagnosis of autoimmune pancreatitis and its differentiation from other pancreatic diseases. The most common finding on endoscopic

ultrasonography is diffuse or focal pancreatic enlargement along with a diffusely hypoechoic parenchyma, similar to findings on transabdominal ultrasonography[299,300].

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The use of corticosteroid therapy is not mandatory in autoimmune pancreatitis, as there are reports of the spontaneous resolution of a pancreatic mass, stricture, and jaundice[301,302]. When steroids are given the response is often dramatic and can be monitored via clinical, laboratory and radiological parameters[303-306].

MISCELLANEOUS CAUSESRecent reports of a new disease known as hyper IgG4 disease has been linked to IP[307] but more data is required before further comment. It is unclear if this is an entirely separate entity from autoimmune pancreatitis.

In renal transplant cases pancreatitis can occur as a result of the procedure itself[308], immunosuppressant medications (see above ‘Medications’), opportunistic infections[309] or through allograft pancreatitis[310].

CHRONIC PANCREATITISThe aforementioned etiologies of IP can all theoretically lead to CP. The diagnosis is best made after considering the results of ERCP, pancreatic function tests, and EUS[24].

It has been suggested that pancreatic function testing may help establish the diagnosis of CP at an earlier stage[311]. It appears based on recent evidence that EUS may be the most sensitive test[311,312] for CP diagnosis with the caveat of false positives[313].

Mortality rates for CP are 3-4 times greater than in controls[314,315]. Pancreatitis in CP accounts for 20 percent of mortality but mortality is usually from non-pancreatic causes[314,315]. CP carries with it many complications such as pancreatic duct strictures, stones, pseudocyst, fistulas, pseudoaneurysm, or ascites[316-320].

TRUE IDIOPATHIC PANCREATITISIn spite of extensive systematic investigations and exhaustive efforts, there will be patients with true IP (TIP). Recommendations are difficult to make given the heterogeneity of studies that have evaluated this problem. Generally there are 4 therapies that might be applicable in TIP taking into account that TIP most likely represents a group of heterogeneous disorders: antioxidants, ursodeoxycholic acid, pancreatic enzymes, and somatostatin or its analogue octreotide.

Antioxidants (e.g., vitamin C, and E, beta carotene) have been shown to reduce the pain involved in IP[321]. It is postulated that patients with AP or CP may have a deficiency in antioxidants either locally within the pancreatic parenchyma or systemically[322]. A cocktail of antioxidants including 600 μg of selenium, 9000 IU of β-carotene, 0.54 g of vitamin C, 270 IU of vitamin E, and 2 g of methionine daily[321] was shown to have a statistically significant benefit in reducing attacks of pancreatitis. It is again stressed that these trials have limited power since the above study evaluated only 28 patients[321].

In pa t i en ts who cont inue to have a t t acks of pancreatitis despite having cholecystectomy or endoscopic sphincterotomy, or patients with contraindications to surgical and endoscopic treatment, maintenance therapy

with ursodeoxycholic acid has also been suggested with some benefit[31,323].

Pancreatic enzyme therapy has recently been reviewed and the efficacy in IP or recurrent AP or CP is small[324]. However, given the low risk of enzyme therapy it has been suggested as a therapeutic trial in treating CP and IP[325,326].

Somatostatin or its analogue octreotide has been postulated to reduce pancreatic secretion and thereby be of benefit in the treatment of relapsing pancreatitis. This therapy entails either a continuous infusion or frequent injections in the past[327]. A 1 mo depot injection has recently become available and may make this therapy more attractive but data is limited with existing data on octreotide and somatostatin focused on altering outcomes during an index hospitalization of severe pancreatitis rather than preventing subsequent attacks[327].

APPROACH TO RECURRENT IPThe authors of this review propose the following systematic approach for patients who have had more than one attack of IP. The following is not validated and is meant to be only used as a guide. For specific details please refer to the aforementioned sections (Figure 1).

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Recurrent undiagnosed pancreatitis1

Rule out metabolic

Rule out medications

Rule out toxins

Rule out genetics

Rule out infection(s)

Rule out autoimmune

Possible sludge?

High resCT Scan

Anatomic abnormality suspected

ERCP with biliary analysis, EUS or MRCP

No answer

ERCP with biliary analysis

Microli-thiasis?

Yes

Cholecystectomy, ES, URSO

No

No Yes

+

-

Anatomic lesion found?

Treat accordingly

TIP, treat as TIP

No benefit

Sphincter of Oddi manometry

Figure 1 1To arrive at the label of recurrent undiagnosed pancreatitis, a history, physical exam, routine laboratory investigations, chest radiograph, abdominal ultrasound, and or computed tomography must fail to find the etiology of pancreatitis. Although sludge may be picked up on transabdominal ultrasound, the low sensitivity precludes exclusion of diagnosis of microlithiasis. ES: Endoscopic sphincterotomy; TIP: True idiopathic pancreatitis; EUS: Endoscopic ultrasound; CT: Computed tomography; URSO: Ursodeoxycholic acid.

CONCLUSIONTrue IP is declining as knowledge and technology advances. IP has been found now to represent a myriad of etiologies that have been elucidated with the advancement of laboratory and endoscopic studies. It is thought that a thorough workup of these cases should reveal an etiology in up to 80% of cases. If genetic screening is applied it is suspected the etiologies may be explained in a much higher percentage of cases. There are still many controversies surrounding some of the IP etiologies and full consensus agreement is lacking in many areas. This review comprehensively outlined the latest evidence in the etiology, pathogenesis, diagnosis, and treatment strategies. It is felt that establishing a diagnosis is key, for it has the potential to direct management. It is recognized, however, that for many of the disorders (particularly genetic abnormalities) there are limited therapies. Elucidating all the causes of IP is a challenge that needs to be faced so that patients can avoid unnecessary morbidity and mortality from subsequent invasive testing.

The treating physician should perform a detailed history to rule out medication, metabolic, and toxin related etiologies. Moreover a detailed history should raise the possibility of genetic associations and causes as well as infectious etiologies so that further testing can be best directed. Celiac disease, vascular disease, and autoimmune causes of pancreatitis should be considered in evaluating the optimal approach to IP.

The most common causes of IP include microlithiasis and biliary sludge, sphincter of Oddi Dysfunction, and anatomic abnormalities. When there is any risk or suggestion of malignancy either as a cause or as an association of the etiology, as in choledochoceles, appropriate management including the necessary diagnostic workup and consideration of surgical excision when the diagnosis is made needs to be considered. In the aforementioned causes it is clear that both imaging techniques such as MRCP, EUS, high resolution CT, and interventional techniques as ERCP play their respective roles.

Indeed with further research additional causes of IP and associations of IP will be revealed. Even with exhaustive efforts and considering all the etiologies in this article there will still be a few patients who will continue to have true IP. In these patients non specific therapy including pain control is often unfortunately the only option. Certainly further advances in pancreatitis will be welcome.

REFERENCES1 Sarles H. Revised classification of pancreatitis--Marseille 1984.

Dig Dis Sci 1985; 30: 573-5742 Steer ML, Waxman I, Freedman S. Chronic pancreatitis. N

Engl J Med 1995; 332: 1482-14903 Bank S, Indaram A. Causes of acute and recurrent pancreatitis.

Clinical considerations and clues to diagnosis. Gastroenterol Clin North Am 1999; 28: 571-589, viii

4 Banks PA. Epidemiology, natural history, and predictors of disease outcome in acute and chronic pancreatitis. Gastrointest Endosc 2002; 56: S226-S230

5 Grendell JH. Idiopathic acute pancreatitis. Gastroenterol Clin

North Am 1990; 19: 843-8486 Thomson HJ. Acute pancreatitis in north and north-east

Scotland. J R Coll Surg Edinb 1985; 30: 104-1117 Thomson SR, Hendry WS, McFarlane GA, Davidson AI.

Epidemiology and outcome of acute pancreatitis. Br J Surg 1987; 74: 398-401

8 Giggs JA, Bourke JB, Katschinski B. The epidemiology of primary acute pancreatitis in Greater Nottingham: 1969-1983. Soc Sci Med 1988; 26: 79-89

9 Svensson JO, Norbäck B, Bokey EL, Edlund Y. Changing pattern in aetiology of pancreatitis in an urban Swedish area. Br J Surg 1979; 66: 159-161

10 Coyle WJ, Pineau BC, Tarnasky PR, Knapple WL, Aabakken L, Hoffman BJ, Cunningham JT, Hawes RH, Cotton PB. Evaluation of unexplained acute and acute recurrent pancreatitis using endoscopic retrograde cholangiopancreatography, sphincter of Oddi manometry and endoscopic ultrasound. Endoscopy 2002; 34: 617-623

11 Kaw M, Brodmerkel GJ. ERCP, biliary crystal analysis, and sphincter of Oddi manometry in idiopathic recurrent pancreatitis. Gastrointest Endosc 2002; 55: 157-162

12 Yadav D, Lowenfels AB. Trends in the epidemiology of the first attack of acute pancreatitis: a systematic review. Pancreas 2006; 33: 323-330

13 Gislason H, Horn A, Hoem D, Andrén-Sandberg A, Imsland AK, Søreide O, Viste A. Acute pancreatitis in Bergen, Norway. A study on incidence, etiology and severity. Scand J Surg 2004; 93: 29-33

14 Goldacre MJ, Roberts SE. Hospital admission for acute pancreatitis in an English population, 1963-98: database study of incidence and mortality. BMJ 2004; 328: 1466-1469

15 Floyd A, Pedersen L, Nielsen GL, Thorladcius-Ussing O, Sorensen HT. Secular trends in incidence and 30-day case fatality of acute pancreatitis in North Jutland County, Denmark: a register-based study from 1981-2000. Scand J Gastroenterol 2002; 37: 1461-1465

16 Mann DV, Hershman MJ, Hittinger R, Glazer G. Multicentre audit of death from acute pancreatitis. Br J Surg 1994; 81: 890-893

17 Birgisson H, Möller PH, Birgisson S, Thoroddsen A, Asgeirsson KS, Sigurjónsson SV, Magnússon J. Acute pancreatitis: a prospective study of its incidence, aetiology, severity, and mortality in Iceland. Eur J Surg 2002; 168: 278-282

18 Lankisch PG, Assmus C, Maisonneuve P, Lowenfels AB. Epidemiology of pancreatic diseases in Lüneburg County. A study in a defined german population. Pancreatology 2002; 2: 469-477

19 Wilson C, Imrie CW, Carter DC. Fatal acute pancreatitis. Gut 1988; 29: 782-788

20 Banerjee AK , Kaul A, Bache E, Parberry AC, Doran J, Nicholson ML. An audit of fatal acute pancreatitis. Postgrad Med J 1995; 71: 472-475

21 Trapnell JE, Duncan EH. Patterns of incidence in acute pancreatitis. Br Med J 1975; 2: 179-183

22 Lindkvist B, Appelros S, Manjer J, Borgström A. Trends in incidence of acute pancreatitis in a Swedish population: is there really an increase? Clin Gastroenterol Hepatol 2004; 2: 831-837

23 Bernard JP, Sahel J, Giovannini M, Sarles H. Pancreas divisum is a probable cause of acute pancreatitis: a report of 137 cases. Pancreas 1990; 5: 248-254

24 Levy MJ, Geenen JE. Idiopathic acute recurrent pancreatitis. Am J Gastroenterol 2001; 96: 2540-2555

25 Venu RP, Geenen JE, Hogan W, Stone J, Johnson GK, Soergel K. Idiopathic recurrent pancreatitis. An approach to diagnosis and treatment. Dig Dis Sci 1989; 34: 56-60

26 Ballinger AB, Barnes E, Alstead EM, Fairclough PD. Is intervention necessary after a first episode of acute idiopathic pancreatitis? Gut 1996; 38: 293-295

27 Etemad B, Whitcomb DC. Chronic pancreatitis: diagnosis, classification, and new genetic developments. Gastroenterology 2001; 120: 682-707

28 Ko CW, Sekijima JH, Lee SP. Biliary sludge. Ann Intern Med

Lee JK et al. Review of idiopathic pancreatitis 6305

www.wjgnet.com

1999; 130: 301-31129 Farinon AM, Ricci GL, Sianesi M, Percudani M, Zanella

E. Physiopathologic role of microlithiasis in gallstone pancreatitis. Surg Gynecol Obstet 1987; 164: 252-256

30 Levy MJ. The hunt for microlithiasis in idiopathic acute recurrent pancreatitis: should we abandon the search or intensify our efforts? Gastrointest Endosc 2002; 55: 286-293

31 Ros E , Navarro S, Bru C, Garcia-Pugés A, Valderrama R. Occult microlithiasis in ‘idiopathic’ acute pancreatitis: prevention of relapses by cholecystectomy or ursodeoxycholic acid therapy. Gastroenterology 1991; 101: 1701-1709

32 Lopez AJ, O’Keefe P, Morrissey M, Pickleman J. Ceftriaxone-induced cholelithiasis. Ann Intern Med 1991; 115: 712-714

33 Lee SP, Nicholls JF, Park HZ. Biliary sludge as a cause of acute pancreatitis. N Engl J Med 1992; 326: 589-593

34 Tandon M, Topazian M. Endoscopic ultrasound in idiopathic acute pancreatitis. Am J Gastroenterol 2001; 96: 705-709

35 Kim HJ, Kim MH, Bae JS, Lee SS, Seo DW, Lee SK. Idiopathic acute pancreatitis. J Clin Gastroenterol 2003; 37: 238-250

36 Garg PK, Tandon RK, Madan K. Is biliary microlithiasis a significant cause of idiopathic recurrent acute pancreatitis? A long-term follow-up study. Clin Gastroenterol Hepatol 2007; 5: 75-79

37 Kohut M, Nowak A, Nowakowska-Duiawa E, Marek T. Presence and density of common bile duct microlithiasis in acute biliary pancreatitis. World J Gastroenterol 2002; 8: 558-561

38 Kohut M, Nowak A, Nowakowska-Duława E, Kaczor R, Marek T. The frequency of bile duct crystals in patients with presumed biliary pancreatitis. Gastrointest Endosc 2001; 54: 37-41

39 Hernández CA, Lerch MM. Sphincter stenosis and gallstone migration through the biliary tract. Lancet 1993; 341: 1371-1373

40 Grau F, Almela P, Aparisi L, Bautista D, Pascual I, Peña A, Rodrigo JM. Usefulness of alanine and aspartate aminotransferases in the diagnosis of microlithiasis in idiopathic acute pancreatitis. Int J Pancreatol 1999; 25: 107-111

41 Queneau PE, Zeeh S, Lapeyre V, Thibault P, Heyd B, Carayon P, Miguet JP. Feasibility of and interest in combined endoscopic ultrasonography and biliary drainage in unexplained acute biliopancreatic disorders. Dig Dis Sci 2002; 47: 2020-2024

42 Yusoff IF, Raymond G, Sahai AV. A prospective comparison of the yield of EUS in primary vs. recurrent idiopathic acute pancreatitis. Gastrointest Endosc 2004; 60: 673-678

43 Norton SA, Alderson D. Endoscopic ultrasonography in the evaluation of idiopathic acute pancreatitis. Br J Surg 2000; 87: 1650-1655

44 Frossard JL, Sosa-Valencia L, Amouyal G, Marty O, Hadengue A, Amouyal P. Usefulness of endoscopic ultrasonography in patients with “idiopathic” acute pancreatitis. Am J Med 2000; 109: 196-200

45 Kurol M, Forsberg L. Ultrasonography in the diagnosis of acute cholecystitis. Acta Radiol Diagn (Stockh) 1984; 25: 379-383

46 Marotta PJ, Gregor JC, Taves DH. Biliary sludge: a risk factor for ‘idiopathic’ pancreatitis? Can J Gastroenterol 1996; 10: 385-388

47 Goodman AJ, Neoptolemos JP, Carr-Locke DL, Finlay DB, Fossard DP. Detection of gall stones after acute pancreatitis. Gut 1985; 26: 125-132

48 Wilcox CM, Varadarajulu S, Eloubeidi M. Role of endoscopic evaluation in idiopathic pancreatitis: a systematic review. Gastrointest Endosc 2006; 63: 1037-1045

49 Lehman GA, Sherman S. Hypertensive pancreatic sphincter. Can J Gastroenterol 1998; 12: 333-337

50 Petersen BT. An evidence-based review of sphincter of Oddi dysfunction: part I, presentations with “objective” biliary findings (types I and II). Gastrointest Endosc 2004; 59: 525-534

51 S h e r m a n S , R u f f o l o T A , H a w e s R H , L e h m a n G A . Complications of endoscopic sphincterotomy. A prospective series with emphasis on the increased risk associated with sphincter of Oddi dysfunction and nondilated bile ducts. Gastroenterology 1991; 101: 1068-1075

52 Tarnasky P, Cunningham J, Cotton P, Hoffman B, Palesch

Y, Freeman J, Curry N, Hawes R. Pancreatic sphincter hypertension increases the risk of post-ERCP pancreatitis. Endoscopy 1997; 29: 252-257

53 Hogan WJ, Geenen JE, Dodds WJ. Dysmotility disturbances of the biliary tract: classification, diagnosis, and treatment. Semin Liver Dis 1987; 7: 302-310

54 Kuo WH, Pasricha PJ, Kalloo AN. The role of sphincter of Oddi manometry in the diagnosis and therapy of pancreatic disease. Gastrointest Endosc Clin N Am 1998; 8: 79-85

55 Freeman ML. Adverse outcomes of ERCP. Gastrointest Endosc 2002; 56: S273-S282

56 Cotton PB, Lehman G, Vennes J, Geenen JE, Russell RC, Meyers WC, Liguory C, Nickl N. Endoscopic sphincterotomy complications and their management: an attempt at consensus. Gastrointest Endosc 1991; 37: 383-393

57 Freeman ML, Nelson DB, Sherman S, Haber GB, Herman ME, Dorsher PJ, Moore JP, Fennerty MB, Ryan ME, Shaw MJ, Lande JD, Pheley AM. Complications of endoscopic biliary sphincterotomy. N Engl J Med 1996; 335: 909-918

58 Freeman ML , Guda NM. Prevent ion o f pos t -ERCP pancreatitis: a comprehensive review. Gastrointest Endosc 2004; 59: 845-864

59 Gottlieb K , Sherman S. ERCP and biliary endoscopic sphincterotomy-induced pancreatitis. Gastrointest Endosc Clin N Am 1998; 8: 87-114

60 Geenen JE, Nash JA. The role of sphincter of Oddi manometry and biliary microscopy in evaluating idiopathic recurrent pancreatitis. Endoscopy 1998; 30: A237-A241

61 Eversman D, Fogel EL, Rusche M, Sherman S, Lehman GA. Frequency of abnormal pancreatic and biliary sphincter manometry compared with clinical suspicion of sphincter of Oddi dysfunction. Gastrointest Endosc 1999; 50: 637-641

62 Aymerich RR, Prakash C, Aliperti G. Sphincter of oddi manometry: is it necessary to measure both biliary and pancreatic sphincter pressures? Gastrointest Endosc 2000; 52: 183-186

63 Sherman S, Troiano FP, Hawes RH, Lehman GA. Sphincter of Oddi manometry: decreased risk of clinical pancreatitis with use of a modified aspirating catheter. Gastrointest Endosc 1990; 36: 462-466

64 Geenen JE, Hogan WJ, Dodds WJ, Toouli J, Venu RP. The efficacy of endoscopic sphincterotomy after cholecystectomy in patients with sphincter-of-Oddi dysfunction. N Engl J Med 1989; 320: 82-87

65 Toouli J, Roberts-Thomson IC, Kellow J, Dowsett J, Saccone GT, Evans P, Jeans P, Cox M, Anderson P, Worthley C, Chan Y, Shanks N, Craig A. Manometry based randomised trial of endoscopic sphincterotomy for sphincter of Oddi dysfunction. Gut 2000; 46: 98-102

66 Somogyi L, Martin SP, Venkatesan T, Ulrich CD. Recurrent acute pancreatitis: an algorithmic approach to identification and elimination of inciting factors. Gastroenterology 2001; 120: 708-717

67 Rolny P, Geenen JE, Hogan WJ. Post-cholecystectomy patients with “objective signs” of partial bile outflow obstruction: clinical characteristics, sphincter of Oddi manometry findings, and results of therapy. Gastrointest Endosc 1993; 39: 778-781

68 Hogan WJ, Sherman S, Pasricha P, Carr-Locke D. Sphincter of Oddi manometry. Gastrointest Endosc 1997; 45: 342-348

69 Botoman VA, Kozarek RA, Novell LA, Patterson DJ, Ball TJ, Wechter DG, Neal LA. Long-term outcome after endoscopic sphincterotomy in patients with biliary colic and suspected sphincter of Oddi dysfunction. Gastrointest Endosc 1994; 40: 165-170

70 Wehrmann T, Wiemer K, Lembcke B, Caspary WF, Jung M. Do patients with sphincter of Oddi dysfunction benefit from endoscopic sphincterotomy? A 5-year prospective trial. Eur J Gastroenterol Hepatol 1996; 8: 251-256

71 Rosenblatt ML , Catalano MF, Alcocer E, Geenen JE. Comparison of sphincter of Oddi manometry, fatty meal sonography, and hepatobiliary scintigraphy in the diagnosis of sphincter of Oddi dysfunction. Gastrointest Endosc 2001; 54:

6306 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol December 21, 2007 Volume 13 Number 47

www.wjgnet.com

697-70472 Freeman ML , Gill M, Overby C, Cen YY. Predictors of

outcomes after biliary and pancreatic sphincterotomy for sphincter of oddi dysfunction. J Clin Gastroenterol 2007; 41: 94-102

73 Cohen S, Bacon BR, Berlin JA, Fleischer D, Hecht GA, Loehrer PJ, McNair AE, Mulholland M, Norton NJ, Rabeneck L, Ransohoff DF, Sonnenberg A, Vannier MW. National Institutes of Health State-of-the-Science Conference Statement: ERCP for diagnosis and therapy, January 14-16, 2002. Gastrointest Endosc 2002; 56: 803-809

74 Sherman S, Troiano FP, Hawes RH, O’Connor KW, Lehman GA. Frequency of abnormal sphincter of Oddi manometry compared with the clinical suspicion of sphincter of Oddi dysfunction. Am J Gastroenterol 1991; 86: 586-590

75 Lans JL, Parikh NP, Geenen JE. Application of sphincter of Oddi manometry in routine clinical investigations. Endoscopy 1991; 23: 139-143

76 Rolny P, Arlebäck A, Funch-Jensen P, Kruse A, Järnerot G. Clinical significance of manometric assessment of both pancreatic duct and bile duct sphincter in the same patient. Scand J Gastroenterol 1989; 24: 751-754

77 Silverman WB, Slivka A, Rabinovitz M, Wilson J. Hybrid classification of sphincter of Oddi dysfunction based on simplified Milwaukee criteria: effect of marginal serum liver and pancreas test elevations. Dig Dis Sci 2001; 46: 278-281

78 Khuroo MS, Zargar SA, Yattoo GN. Efficacy of nifedipine therapy in patients with sphincter of Oddi dysfunction: a prospective, double-blind, randomized, placebo-controlled, cross over trial. Br J Clin Pharmacol 1992; 33: 477-485

79 Kalloo AN , Pasricha PJ. Therapy of sphincter of Oddi dysfunction. Gastrointest Endosc Clin N Am 1996; 6: 117-125

80 Tarnasky PR, Hawes RH. Endoscopic diagnosis and therapy of unexplained (idiopathic) acute pancreatitis. Gastrointest Endosc Clin N Am 1998; 8: 13-37

81 Cotton PB. Pancreas divisum. Am J Gastroenterol 1995; 90: 189882 Cotton PB. Congenital anomaly of pancreas divisum as cause

of obstructive pain and pancreatitis. Gut 1980; 21: 105-11483 Delhaye M, Engelholm L, Cremer M. Pancreas divisum:

controversial clinical significance. Dig Dis 1988; 6: 30-3984 Burtin P, Person B, Charneau J, Boyer J. Pancreas divisum and

pancreatitis: a coincidental association? Endoscopy 1991; 23: 55-58

85 Carr-Locke DL. Pancreas divisum: the controversy goes on? Endoscopy 1991; 23: 88-90

86 Delhaye M, Engelholm L, Cremer M. Pancreas divisum: congenital anatomic variant or anomaly? Contribution of endoscopic retrograde dorsal pancreatography. Gastroenterology 1985; 89: 951-958

87 Lehman GA, Sherman S. Pancreas divisum. Diagnosis, clinical significance, and management alternatives. Gastrointest Endosc Clin N Am 1995; 5: 145-170

88 Krueger KJ, Wootton FT, Cunningham JT, Hoffman BJ. Unexpected anomalies of the common bile and pancreatic ducts. Am J Gastroenterol 1992; 87: 1492-1495

89 Cunningham JT. Pancreas divisum and acute pancreatitis: romancing the stone? Am J Gastroenterol 1992; 87: 802-803

90 Choudari CP, Imperiale TF, Sherman S, Fogel E, Lehman GA. Risk of pancreatitis with mutation of the cystic fibrosis gene. Am J Gastroenterol 2004; 99: 1358-1363

91 Kamisawa T, Tu Y, Egawa N, Tsuruta K, Okamoto A, Kamata N. MRCP of congenital pancreaticobiliary malformation. Abdom Imaging 2007; 32: 129-133

92 Bret PM, Reinhold C, Taourel P, Guibaud L, Atri M, Barkun AN. Pancreas divisum: evaluation with MR cholangiopancreatography. Radiology 1996; 199: 99-103

93 Devereaux BM, Fein S, Purich E, Trout JR, Lehman GA, Fogel EL, Phillips S, Etemad R, Jowell P, Toskes PP, Sherman S. A new synthetic porcine secretin for facilitation of cannulation of the dorsal pancreatic duct at ERCP in patients with pancreas divisum: a multicenter, randomized, double-blind comparative study. Gastrointest Endosc 2003; 57: 643-647

94 Warshaw AL, Simeone J, Schapiro RH, Hedberg SE, Mueller PE, Ferrucci JT. Objective evaluation of ampullary stenosis with ultrasonography and pancreatic stimulation. Am J Surg 1985; 149: 65-72

95 Lowes JR, Lees WR, Cotton PB. Pancreatic duct dilatation after secretin stimulation in patients with pancreas divisum. Pancreas 1989; 4: 371-374

96 Warshaw AL, Simeone JF, Schapiro RH, Flavin-Warshaw B. Evaluation and treatment of the dominant dorsal duct syndrome (pancreas divisum redefined). Am J Surg 1990; 159: 59-64; discussion 64-66

97 Park SH, de Bellis M, McHenry L, Fogel EL, Lazzell L, Bucksot L, Sherman S, Lehman GA. Use of methylene blue to identify the minor papilla or its orifice in patients with pancreas divisum. Gastrointest Endosc 2003; 57: 358-363

98 Heyries L, Barthet M, Delvasto C, Zamora C, Bernard JP, Sahel J. Long-term results of endoscopic management of pancreas divisum with recurrent acute pancreatitis. Gastrointest Endosc 2002; 55: 376-381

99 Lans JI, Geenen JE, Johanson JF, Hogan WJ. Endoscopic therapy in patients with pancreas divisum and acute pancreatitis: a prospective, randomized, controlled clinical trial. Gastrointest Endosc 1992; 38: 430-434

100 Lehman GA , Sherman S, Nisi R, Hawes RH. Pancreas divisum: results of minor papilla sphincterotomy. Gastrointest Endosc 1993; 39: 1-8

101 Ertan A. Long-term results after endoscopic pancreatic stent placement without pancreatic papillotomy in acute recurrent pancreatitis due to pancreas divisum. Gastrointest Endosc 2000; 52: 9-14

102 Kamisawa T. Clinical significance of the minor duodenal papilla and accessory pancreatic duct. J Gastroenterol 2004; 39: 605-615

103 Lehman GA, Sherman S. Diagnosis and therapy of pancreas divisum. Gastrointest Endosc Clin N Am 1998; 8: 55-77

104 Smith MT, Sherman S, Ikenberry SO, Hawes RH, Lehman GA. Alterations in pancreatic ductal morphology following polyethylene pancreatic stent therapy. Gastrointest Endosc 1996; 44: 268-275

105 Sherman S, Hawes RH, Savides TJ, Gress FG, Ikenberry SO, Smith MT, Zaidi S, Lehman GA. Stent-induced pancreatic ductal and parenchymal changes: correlation of endoscopic ultrasound with ERCP. Gastrointest Endosc 1996; 44: 276-282

106 Guelrud M, Morera C, Rodriguez M, Prados JG, Jaén D. Normal and anomalous pancreaticobiliary union in children and adolescents. Gastrointest Endosc 1999; 50: 189-193

107 Okada A , Nakamura T, Higaki J, Okumura K, Kamata S, Oguchi Y. Congenital dilatation of the bile duct in 100 instances and its relationship with anomalous junction. Surg Gynecol Obstet 1990; 171: 291-298

108 Matsumoto Y, Fujii H, Itakura J, Matsuda M, Nobukawa B, Suda K. Recent advances in pancreaticobiliary maljunction. J Hepatobiliary Pancreat Surg 2002; 9: 45-54

109 Komi N, Takehara H, Kunitomo K, Miyoshi Y, Yagi T. Does the type of anomalous arrangement of pancreaticobiliary ducts influence the surgery and prognosis of choledochal cyst? J Pediatr Surg 1992; 27: 728-731

110 Tashiro S, Imaizumi T, Ohkawa H, Okada A, Katoh T, Kawaharada Y, Shimada H, Takamatsu H, Miyake H, Todani T. Pancreaticobiliary maljunction: retrospective and nationwide survey in Japan. J Hepatobiliary Pancreat Surg 2003; 10: 345-351

111 Guelrud M , Morera C, Rodriguez M, Jaen D, Pierre R. Sphincter of Oddi dysfunction in children with recurrent pancreatitis and anomalous pancreaticobiliary union: an etiologic concept. Gastrointest Endosc 1999; 50: 194-199

112 Samavedy R, Sherman S, Lehman GA. Endoscopic therapy in anomalous pancreatobiliary duct junction. Gastrointest Endosc 1999; 50: 623-627

113 Nomura T, Shirai Y, Sandoh N, Nagakura S, Hatakeyama K. Cholangiographic criteria for anomalous union of the pancreatic and biliary ducts. Gastrointest Endosc 2002; 55: 204-208

Lee JK et al. Review of idiopathic pancreatitis 6307

www.wjgnet.com

114 Kinoshita H, Nagata E, Hirohashi K, Sakai K, Kobayashi Y. Carcinoma of the gallbladder with an anomalous connection between the choledochus and the pancreatic duct. Report of 10 cases and review of the literature in Japan. Cancer 1984; 54: 762-769

115 Davenport M, Stringer MD, Howard ER. Biliary amylase and congenital choledochal dilatation. J Pediatr Surg 1995; 30: 474-477

116 Chijiiwa K, Kimura H, Tanaka M. Malignant potential of the gallbladder in patients with anomalous pancreaticobiliary ductal junction. The difference in risk between patients with and without choledochal cyst. Int Surg 1995; 80: 61-64

117 Sandoh N, Shirai Y, Hatakeyama K. Incidence of anomalous union of the pancreaticobiliary ductal system in biliary cancer. Hepatogastroenterology 1997; 44: 1580-1583

118 Yamauchi S, Koga A, Matsumoto S, Tanaka M, Nakayama F. Anomalous junction of pancreaticobiliary duct without congenital choledochal cyst: a possible risk factor for gallbladder cancer. Am J Gastroenterol 1987; 82: 20-24

119 Todani T, Watanabe Y, Narusue M, Tabuchi K, Okajima K. Congenital bile duct cysts: Classification, operative procedures, and review of thirty-seven cases including cancer arising from choledochal cyst. Am J Surg 1977; 134: 263-269

120 Kim MH, Myung SJ, Lee SK, Yoo BM, Seo DW, Lee MH, Jung SA, Kim YS, Min YI. Ballooning of the papilla during contrast injection: the semaphore of a choledochocele. Gastrointest Endosc 1998; 48: 258-262

121 Sarris GE, Tsang D. Choledochocele: case report, literature review, and a proposed classification. Surgery 1989; 105: 408-414

122 Goldberg PB, Long WB, Oleaga JA, Mackie JA. Choledochocele as a cause of recurrent pancreatitis. Gastroenterology 1980; 78: 1041-1045

123 Ochiai K, Kaneko K, Kitagawa M, Ando H, Hayakawa T. Activated pancreatic enzyme and pancreatic stone protein (PSP/reg) in bile of patients with pancreaticobiliary maljunction/ choledochal cysts. Dig Dis Sci 2004; 49: 1953-1956

124 Venu RP, Geenen JE, Hogan WJ, Dodds WJ, Wilson SW, Stewart ET, Soergel KH. Role of endoscopic retrograde cholangiopancreatography in the diagnosis and treatment of choledochocele. Gastroenterology 1984; 87: 1144-1149

125 Shimizu T, Suzuki R, Yamashiro Y, Segawa O, Yamataka A, Kuwatsuru R. Magnetic resonance cholangiopancreatography in assessing the cause of acute pancreatitis in children. Pancreas 2001; 22: 196-199

126 Schaefer JF, Kirschner HJ, Lichy M, Schlemmer HP, Schick F, Claussen CD, Fuchs J. Highly resolved free-breathing magnetic resonance cholangiopancreatography in the diagnostic workup of pancreaticobiliary diseases in infants and young children--initial experiences. J Pediatr Surg 2006; 41: 1645-1651

127 Gerritsen JJ, Janssens AR, Kroon HM. Choledochocele: treatment by endoscopic sphincterotomy. Br J Surg 1988; 75: 495-496

128 Lopez RR, Pinson CW, Campbell JR, Harrison M, Katon RM. Variation in management based on type of choledochal cyst. Am J Surg 1991; 161: 612-615

129 Jordan PH , Goss JA, Rosenberg WR, Woods KL. Some considerations for management of choledochal cysts. Am J Surg 2004; 187: 790-795

130 Lipsett PA, Pitt HA. Surgical treatment of choledochal cysts. J Hepatobiliary Pancreat Surg 2003; 10: 352-359

131 Woon CY, Tan YM, Oei CL, Chung AY, Chow PK, Ooi LL. Adult choledochal cysts: an audit of surgical management. ANZ J Surg 2006; 76: 981-986

132 Ozawa K, Yamada T, Matumoto Y, Tobe R. Carcinoma arising in a choledochocele. Cancer 1980; 45: 195-197

133 Schulte S. Embryology, and congenital anomalies of the bile and pancreatic ducts. In: Silvis S, Rohrmann CA, Ansel HJ, editors. Text and Atlas of Endoscopic Retrograde Cholangiopancreatography. New York: Igaku-Shoin, 1995: 1-480

134 Ravitch MM. The pancreas in infants and children. Surg Clin

North Am 1975; 55: 377-385135 Itoh Y, Hada T, Terano A, Itai Y, Harada T. Pancreatitis in the

annulus of annular pancreas demonstrated by the combined use of computed tomography and endoscopic retrograde cholangiopancreatography. Am J Gastroenterol 1989; 84: 961-964

136 Kiernan PD, ReMine SG, Kiernan PC, ReMine WH. Annular pancreas: May Clinic experience from 1957 to 1976 with review of the literature. Arch Surg 1980; 115: 46-50

137 Lloyd-Jones W, Mountain JC, Warren KW. Annular pancreas in the adult. Ann Surg 1972; 176: 163-170

138 Rizzo RJ, Szucs RA, Turner MA. Congenital abnormalities of the pancreas and biliary tree in adults. Radiographics 1995; 15: 49-68; quiz 147-148

139 Dowsett JF, Rode J, Russell RC. Annular pancreas: a clinical, endoscopic, and immunohistochemical study. Gut 1989; 30: 130-135

140 Urayama S, Kozarek R, Ball T, Brandabur J, Traverso L, Ryan J, Wechter D. Presentation and treatment of annular pancreas in an adult population. Am J Gastroenterol 1995; 90: 995-999

141 Bret PM, Reinhold C. Magnetic resonance cholangiopancreatography. Endoscopy 1997; 29: 472-486

142 Gress F, Yiengpruksawan A, Sherman S, Ikenberry S, Kaster S, Ng RY, Cerulli MA, Lehman GA. Diagnosis of annular pancreas by endoscopic ultrasound. Gastrointest Endosc 1996; 44: 485-489

143 Choi JY, Kim MJ, Kim JH, Lim JS, Oh YT, Chung JJ, Song SY, Chung JB, Yoo HS, Lee JT, Kim KW. Annular pancreas: emphasis on magnetic resonance cholangiopancreatography findings. J Comput Assist Tomogr 2004; 28: 528-532

144 Chevallier P , Souci J, Buckley MJ, Oddo F, Hastier P, Diaine B. Annular pancreas: MR imaging including MR cholangiopancreatography (MRCP). Pancreas 1999; 18: 216-218

145 Hidaka T, Hirohashi S, Uchida H, Koh M, Itoh T, Matsuo Y, Matsuo N, Ohishi H. Annular pancreas diagnosed by single-shot MR cholangiopancreatography. Magn Reson Imaging 1998; 16: 441-444

146 Maker V, Gerzenshtein J, Lerner T. Annular pancreas in the adult: two case reports and review of more than a century of literature. Am Surg 2003; 69: 404-410

147 Simpson WF , Adams DB, Metcalf JS, Anderson MC. Nonfunctioning pancreatic neuroendocrine tumors presenting as pancreatitis: report of four cases. Pancreas 1988; 3: 223-231

148 K ö h l e r H , L a n k i s c h P G . A c u t e p a n c r e a t i t i s a n d hyperamylasaemia in pancreatic carcinoma. Pancreas 1987; 2: 117-119

149 Kahrilas PJ, Hogan WJ, Geenen JE, Stewart ET, Dodds WJ, Arndorfer RC. Chronic recurrent pancreatitis secondary to a submucosal ampullary tumor in a pat ient with neurofibromatosis. Dig Dis Sci 1987; 32: 102-107

150 Robertson JF, Imrie CW. Acute pancreatitis associated with carcinoma of the ampulla of Vater. Br J Surg 1987; 74: 395-397

151 Rösch T, Braig C, Gain T, Feuerbach S, Siewert JR, Schusdziarra V, Classen M. Staging of pancreatic and ampullary carcinoma by endoscopic ultrasonography. Comparison with conventional sonography, computed tomography, and angiography. Gastroenterology 1992; 102: 188-199

152 Legmann P, Vignaux O, Dousset B, Baraza AJ, Palazzo L, Dumontier I, Coste J, Louvel A, Roseau G, Couturier D, Bonnin A. Pancreatic tumors: comparison of dual-phase helical CT and endoscopic sonography. AJR Am J Roentgenol 1998; 170: 1315-1322

153 Müller MF , Meyenberger C, Bertschinger P, Schaer R, Marincek B. Pancreatic tumors: evaluation with endoscopic US, CT, and MR imaging. Radiology 1994; 190: 745-751

154 Rösch T. Staging of pancreatic cancer. Analysis of literature results. Gastrointest Endosc Clin N Am 1995; 5: 735-739

155 Mujica VR, Barkin JS, Go VL. Acute pancreatitis secondary to pancreatic carcinoma. Study Group Participants. Pancreas 2000; 21: 329-332

156 Rösch T, Lightdale CJ, Botet JF, Boyce GA, Sivak MV, Yasuda K, Heyder N, Palazzo L, Dancygier H, Schusdziarra V. Localization of pancreatic endocrine tumors by endoscopic

6308 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol December 21, 2007 Volume 13 Number 47

www.wjgnet.com

ultrasonography. N Engl J Med 1992; 326: 1721-1726157 Jhala NC, Jhala D, Eltoum I, Vickers SM, Wilcox CM, Chhieng

DC, Eloubeidi MA. Endoscopic ultrasound-guided fine-needle aspiration biopsy: a powerful tool to obtain samples from small lesions. Cancer 2004; 102: 239-246

158 Volmar KE, Vollmer RT, Jowell PS, Nelson RC, Xie HB. Pancreatic FNA in 1000 cases: a comparison of imaging modalities. Gastrointest Endosc 2005; 61: 854-861

159 Bassi C, Procacci C, Zamboni G, Scarpa A, Cavallini G, Pederzoli P. Intraductal papillary mucinous tumors of the pancreas. Verona University Pancreatic Team. Int J Pancreatol 2000; 27: 181-193

160 Bastid C, Bernard JP, Sarles H, Payan MJ, Sahel J. Mucinous ductal ectasia of the pancreas: a premalignant disease and a cause of obstructive pancreatitis. Pancreas 1991; 6: 15-22

161 Cellier C, Cuillerier E, Palazzo L, Rickaert F, Flejou JF, Napoleon B, Van Gansbeke D, Bely N, Ponsot P, Partensky C, Cugnenc PH, Barbier JP, Devière J, Cremer M. Intraductal papillary and mucinous tumors of the pancreas: accuracy of preoperative computed tomography, endoscopic retrograde pancreatography and endoscopic ultrasonography, and long-term outcome in a large surgical series. Gastrointest Endosc 1998; 47: 42-49

162 Obara T, Saitoh Y, Maguchi H, Ura H, Yokota K, Okamura K, Namiki M. Papillary adenoma of the pancreas with excessive mucin secretion. Pancreas 1992; 7: 114-117

163 Yamaguchi K, Chijiiwa K, Shimizu S, Yokohata K, Morisaki T, Yonemasu H, Tanaka M. Intraductal papillary neoplasm of the pancreas: a clinical review of 13 benign and four malignant tumours. Eur J Surg 1999; 165: 223-229

164 Nickl NJ, Lawson JM, Cotton PB. Mucinous pancreatic tumors: ERCP findings. Gastrointest Endosc 1991; 37: 133-138

165 Raijman I, Kortan P, Walden D, Kandel G, Marcon NE, Haber GB. Mucinous ductal ectasia: cholangiopancreatographic and endoscopic findings. Endoscopy 1994; 26: 303-307

166 Lévy P, Jouannaud V, O’Toole D, Couvelard A, Vullierme MP, Palazzo L, Aubert A, Ponsot P, Sauvanet A, Maire F, Hentic O, Hammel P, Ruszniewski P. Natural history of intraductal papillary mucinous tumors of the pancreas: actuarial risk of malignancy. Clin Gastroenterol Hepatol 2006; 4: 460-468

167 Traverso LW, Peralta EA, Ryan JA, Kozarek RA. Intraductal neoplasms of the pancreas. Am J Surg 1998; 175: 426-432

168 Sohn TA, Yeo CJ, Cameron JL, Iacobuzio-Donahue CA, Hruban RH, Lillemoe KD. Intraductal papillary mucinous neoplasms of the pancreas: an increasingly recognized clinicopathologic entity. Ann Surg 2001; 234: 313-321; discussion 321-322

169 Sohn TA, Yeo CJ, Cameron JL, Hruban RH, Fukushima N, Campbell KA, Lillemoe KD. Intraductal papillary mucinous neoplasms of the pancreas: an updated experience. Ann Surg 2004; 239: 788-797; discussion 797-799

170 Sugiyama M, Atomi Y. Extrapancreatic neoplasms occur with unusual frequency in patients with intraductal papillary mucinous tumors of the pancreas. Am J Gastroenterol 1999; 94: 470-473

171 Choi MG, Kim SW, Han SS, Jang JY, Park YH. High incidence of extrapancreatic neoplasms in patients with intraductal papillary mucinous neoplasms. Arch Surg 2006; 141: 51-56; discussion 56

172 Sarr MG, Carpenter HA, Prabhakar LP, Orchard TF, Hughes S, van Heerden JA, DiMagno EP. Clinical and pathologic correlation of 84 mucinous cystic neoplasms of the pancreas: can one reliably differentiate benign from malignant (or premalignant) neoplasms? Ann Surg 2000; 231: 205-212

173 Wilentz RE, Albores-Saavedra J, Zahurak M, Talamini MA, Yeo CJ, Cameron JL, Hruban RH. Pathologic examination accurately predicts prognosis in mucinous cystic neoplasms of the pancreas. Am J Surg Pathol 1999; 23: 1320-1327

174 Siech M, Tripp K, Schmidt-Rohlfing B, Mattfeldt T, Widmaier U, Gansauge F, Görich J, Beger HG. Cystic tumours of the pancreas: diagnostic accuracy, pathologic observations and surgical consequences. Langenbecks Arch Surg 1998; 383: 56-61

175 Anderson JH, Morran CG, Anderson JR, Carter DC. Acute

pancreatitis and non-Hodgkin’s lymphoma. Postgrad Med J 1987; 63: 137-139

176 McLatchie GR, Imrie CW. Acute pancreatitis associated with tumour metastases in the pancreas. Digestion 1981; 21: 13-17

177 Sand JA, Hyoty MK, Mattila J, Dagorn JC, Nordback IH. Clinical assessment compared with cyst fluid analysis in the differential diagnosis of cystic lesions in the pancreas. Surgery 1996; 119: 275-280

178 Hammel P, Voitot H, Vilgrain V, Lévy P, Ruszniewski P, Bernades P. Diagnostic value of CA 72-4 and carcinoembryonic antigen determination in the fluid of pancreatic cystic lesions. Eur J Gastroenterol Hepatol 1998; 10: 345-348

179 Kim HJ, Kim MH, Myung SJ, Lim BC, Park ET, Yoo KS, Seo DW, Lee SK, Min YI. A new strategy for the application of CA19-9 in the differentiation of pancreaticobiliary cancer: analysis using a receiver operating characteristic curve. Am J Gastroenterol 1999; 94: 1941-1946

180 Stolte M, Pscherer C. Adenoma-carcinoma sequence in the papilla of Vater. Scand J Gastroenterol 1996; 31: 376-382

181 Yamaguchi K, Enjoji M. Carcinoma of the ampulla of vater. A clinicopathologic study and pathologic staging of 109 cases of carcinoma and 5 cases of adenoma. Cancer 1987; 59: 506-515

182 Tarazi RY, Hermann RE, Vogt DP, Hoerr SO, Esselstyn CB, Cooperman AM, Steiger E, Grundfest S. Results of surgical treatment of periampullary tumors: a thirty-five-year experience. Surgery 1986; 100: 716-723

183 Sperti C, Pasquali C, Piccoli A, Sernagiotto C, Pedrazzoli S. Radical resection for ampullary carcinoma: long-term results. Br J Surg 1994; 81: 668-671

184 Chijiiwa K, Yamashita H, Kuroki S. Wide ampullectomy for patients with villous adenoma of duodenal papilla and follow-up results of pancreaticobiliary tract. Int Surg 1994; 79: 178-182

185 Binmoeller KF, Boaventura S, Ramsperger K, Soehendra N. Endoscopic snare excision of benign adenomas of the papilla of Vater. Gastrointest Endosc 1993; 39: 127-131

186 Ponchon T, Berger F, Chavaillon A, Bory R, Lambert R. Contribution of endoscopy to diagnosis and treatment of tumors of the ampulla of Vater. Cancer 1989; 64: 161-167

187 Dittrick GW, Mallat DB, Lamont JP. Management of ampullary lesions. Curr Treat Options Gastroenterol 2006; 9: 371-376

188 Katsinelos P , Paroutoglou G, Kountouras J, Beltsis A, Papaziogas B, Mimidis K, Zavos C, Dimiropoulos S. Safety and long-term follow-up of endoscopic snare excision of ampullary adenomas. Surg Endosc 2006; 20: 608-613

189 Davis PB, Drumm M, Konstan MW. Cystic fibrosis. Am J Respir Crit Care Med 1996; 154: 1229-1256

190 Steward MC , I shiguro H, Case RM. Mechanisms of bicarbonate secretion in the pancreatic duct. Annu Rev Physiol 2005; 67: 377-409

191 Naruse S, Fujiki K, Ishiguro H. Is genetic analysis helpful for diagnosing chronic pancreatitis in its early stage? J Gastroenterol 2007; 42 Suppl 17: 60-65

192 Shwachman H, Lebenthal E, Khaw KT. Recurrent acute pancreatitis in patients with cystic fibrosis with normal pancreatic enzymes. Pediatrics 1975; 55: 86-95

193 Atlas AB, Orenstein SR, Orenstein DM. Pancreatitis in young children with cystic fibrosis. J Pediatr 1992; 120: 756-759

194 Cohn JA, Friedman 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

195 Keiles S, Kammesheidt A. Identification of CFTR, PRSS1, and SPINK1 mutations in 381 patients with pancreatitis. Pancreas 2006; 33: 221-227

196 Audrézet MP, Chen JM, Le Maréchal C, Ruszniewski P, Robaszkiewicz M, Raguénès O, Quéré I, Scotet V, Férec C. Determination of the relative contribution of three genes-the cystic fibrosis transmembrane conductance regulator gene, the cationic trypsinogen gene, and the pancreatic secretory trypsin inhibitor gene-to the etiology of idiopathic chronic pancreatitis. Eur J Hum Genet 2002; 10: 100-106

197 Noone PG, Zhou Z, Silverman LM, Jowell PS, Knowles MR,

Lee JK et al. Review of idiopathic pancreatitis 6309

www.wjgnet.com

Cohn JA. Cystic fibrosis gene mutations and pancreatitis risk: relation to epithelial ion transport and trypsin inhibitor gene mutations. Gastroenterology 2001; 121: 1310-1319

198 Weiss FU, Simon P, Bogdanova N, Mayerle J, Dworniczak B, Horst J, Lerch MM. Complete cystic fibrosis transmembrane conductance regulator gene sequencing in patients with idiopathic chronic pancreatitis and controls. Gut 2005; 54: 1456-1460

199 Cohn JA. Reduced CFTR function and the pathobiology of idiopathic pancreatitis. J Clin Gastroenterol 2005; 39: S70-S77

200 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

201 Pezzilli R, Morselli-Labate AM, Mantovani V, Romboli E, Selva P, Migliori M, Corinaldesi R, Gullo L. Mutations of the CFTR gene in pancreatic disease. Pancreas 2003; 27: 332-336

202 Cohn JA, Neoptolemos JP, Feng J, Yan J, Jiang Z, Greenhalf W, McFaul C, Mountford R, Sommer SS. Increased risk of idiopathic chronic pancreatitis in cystic fibrosis carriers. Hum Mutat 2005; 26: 303-307

203 Bishop MD, Freedman SD, Zielenski J, Ahmed N, Dupuis A, Martin S, Ellis L, Shea J, Hopper I, Corey M, Kortan P, Haber G, Ross C, Tzountzouris J, Steele L, Ray PN, Tsui LC, Durie PR. The cystic fibrosis transmembrane conductance regulator gene and ion channel function in patients with idiopathic pancreatitis. Hum Genet 2005; 118: 372-381

204 Sobczyńska-Tomaszewska A, Bak D, Oralewska B, Oracz G, Norek A, Czerska K, Mazurczak T, Teisseyre M, Socha J, Zagulski M, Bal J. Analysis of CFTR, SPINK1, PRSS1 and AAT mutations in children with acute or chronic pancreatitis. J Pediatr Gastroenterol Nutr 2006; 43: 299-306

205 Teich N, Ockenga J, Keim V, Mössner J. Genetic risk factors in chronic pancreatitis. J Gastroenterol 2002; 37: 1-9

206 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

207 Pfützer RH, Barmada MM, Brunskill AP, Finch R, Hart PS, Neoptolemos J, Furey WF, Whitcomb DC. SPINK1/PSTI polymorphisms act as disease modifiers in familial and idiopathic chronic pancreatitis. Gastroenterology 2000; 119: 615-623

208 Threadgold J, Greenhalf W, Ellis I, Howes N, Lerch MM, Simon P, Jansen J, Charnley R, Laugier R, Frulloni L, Oláh A, Delhaye M, Ihse I, Schaffalitzky de Muckadell OB, Andrén-Sandberg A, Imrie CW, Martinek J, Gress TM, Mountford R, Whitcomb D, Neoptolemos JP. The N34S mutation of SPINK1 (PSTI) is associated with a familial pattern of idiopathic chronic pancreatitis but does not cause the disease. Gut 2002; 50: 675-681

209 Khalid A, Finkelstein S, Thompson B, Kelly L, Hanck C, Godfrey TE, Whitcomb DC. A 93 year old man with the PRSS1 R122H mutation, low SPINK1 expression, and no pancreatitis: insights into phenotypic non-penetrance. Gut 2006; 55: 728-731

210 Mitchell RM, Byrne MF, Baillie J. Pancreatitis. Lancet 2003; 361: 1447-1455

211 W h i t c o m b D C , G o r r y M C , P r e s t o n R A , F u r e y W , Sossenheimer MJ, Ulrich CD, Martin SP, Gates LK, Amann ST, Toskes PP, Liddle R, McGrath K, Uomo G, Post JC, Ehrlich GD. Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat Genet 1996; 14: 141-145

212 Gorry MC, Gabbaizedeh D, Furey W, Gates LK, Preston RA, Aston CE, Zhang Y, Ulrich C, Ehrlich GD, Whitcomb DC. Mutations in the cationic trypsinogen gene are associated with recurrent acute and chronic pancreatitis. Gastroenterology 1997; 113: 1063-1068

213 Whitcomb DC, Preston RA, Aston CE, Sossenheimer MJ, Barua PS, Zhang Y, Wong-Chong A, White GJ, Wood PG, Gates LK, Ulrich C, Martin SP, Post JC, Ehrlich GD. A gene for hereditary pancreatitis maps to chromosome 7q35. Gastroenterology 1996; 110: 1975-1980

214 Férec C, Raguénès O, Salomon R, Roche C, Bernard JP, Guillot

M, Quéré I, Faure C, Mercier B, Audrézet MP, Guillausseau PJ, Dupont C, Munnich A, Bignon JD, Le Bodic L. Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis. J Med Genet 1999; 36: 228-232

215 Teich N, Ockenga J, Hoffmeister A, Manns M, Mössner J, Keim V. Chronic pancreatitis associated with an activation peptide mutation that facilitates trypsin activation. Gastroenterology 2000; 119: 461-465

216 Chen JM , Piepoli Bis A, Le Bodic L, Ruszniewski P, Robaszkiewicz M, Deprez PH, Raguenes O, Quere I, Andriulli A, Ferec C. Mutational screening of the cationic trypsinogen gene in a large cohort of subjects with idiopathic chronic pancreatitis. Clin Genet 2001; 59: 189-193

217 Le Maréchal C, Chen JM, Quéré I, Raguénès O, Férec C, Auroux J. Discrimination of three mutational events that result in a disruption of the R122 primary autolysis site of the human cationic trypsinogen (PRSS1) by denaturing high performance liquid chromatography. BMC Genet 2001; 2: 19

218 Simon P, Weiss FU, Sahin-Toth M, Parry M, Nayler O, Lenfers B, Schnekenburger J, Mayerle J, Domschke W, Lerch MM. Hereditary pancreatitis caused by a novel PRSS1 mutation (Arg-122 --&gt; Cys) that alters autoactivation and autodegradation of cationic trypsinogen. J Biol Chem 2002; 277: 5404-5410

219 Pfützer R, Myers E, Applebaum-Shapiro S, Finch R, Ellis I, Neoptolemos J, Kant JA, Whitcomb DC. Novel cationic trypsinogen (PRSS1) N29T and R122C mutations cause autosomal dominant hereditary pancreatitis. Gut 2002; 50: 271-272

220 Sahin-Tóth M, Gráf L, Tóth M. Trypsinogen stabilization by mutation Arg117--&gt;His: a unifying pathomechanism for hereditary pancreatitis? Biochem Biophys Res Commun 1999; 264: 505-508

221 Perrault J. Hereditary pancreatitis. Gastroenterol Clin North Am 1994; 23: 743-752

222 Lowenfels AB, Maisonneuve P, DiMagno EP, Elitsur Y, Gates LK, Perrault J, Whitcomb DC. Hereditary pancreatitis and the risk of pancreatic cancer. International Hereditary Pancreatitis Study Group. J Natl Cancer Inst 1997; 89: 442-446

223 Kagnoff MF. Celiac disease. A gastrointestinal disease with environmental, genetic, and immunologic components. Gastroenterol Clin North Am 1992; 21: 405-425

224 Schuppan D. Current concepts of celiac disease pathogenesis. Gastroenterology 2000; 119: 234-242

225 Patel RS, Johlin FC, Murray JA. Celiac disease and recurrent pancreatitis. Gastrointest Endosc 1999; 50: 823-827

226 Thomson A . Celiac disease as a cause of pancreatitis. Gastroenterology 2005; 129: 1137

227 Ulrich CD. Pancreatic cancer in hereditary pancreatitis: consensus guidelines for prevention, screening and treatment. Pancreatology 2001; 1: 416-422

228 Rünzi M, Layer P. Drug-associated pancreatitis: facts and fiction. Pancreas 1996; 13: 100-109

229 Wilmink T, Frick TW. Drug-induced pancreatitis. Drug Saf 1996; 14: 406-423

230 McArthur KE. Review article: drug-induced pancreatitis. Aliment Pharmacol Ther 1996; 10: 23-38

231 Lankisch PG, Dröge M, Gottesleben F. Drug induced acute pancreatitis: incidence and severity. Gut 1995; 37: 565-567

232 Werth B, Kuhn M, Hartmann K, Reinhart WH. Drug-induced pancreatitis: experience of the Swiss Drug Adverse Effects Center (SANZ) 1981-1993. Schweiz Med Wochenschr 1995; 125: 731-734

233 Haber CJ, Meltzer SJ, Present DH, Korelitz BI. Nature and course of pancreatitis caused by 6-mercaptopurine in the treatment of inflammatory bowel disease. Gastroenterology 1986; 91: 982-986

234 Maxson CJ, Greenfield SM, Turner JL. Acute pancreatitis as a common complication of 2’,3’-dideoxyinosine therapy in the acquired immunodeficiency syndrome. Am J Gastroenterol 1992; 87: 708-713

6310 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol December 21, 2007 Volume 13 Number 47

www.wjgnet.com

235 Youssef SS , Iskandar SB, Scruggs J , Roy TM. Acute pancreatitis associated with omeprazole. Int J Clin Pharmacol Ther 2005; 43: 558-561

236 Eland IA, Alvarez CH, Stricker BH, Rodríguez LA. The risk of acute pancreatitis associated with acid-suppressing drugs. Br J Clin Pharmacol 2000; 49: 473-478

237 Sundström A, Blomgren K, Alfredsson L, Wiholm BE. Acid-suppressing drugs and gastroesophageal reflux disease as risk factors for acute pancreatitis--results from a Swedish Case-Control Study. Pharmacoepidemiol Drug Saf 2006; 15: 141-149

238 Steinberg W, Tenner S. Acute pancreatitis. N Engl J Med 1994; 330: 1198-1210

239 Mofenson HC , Caracc io TR, Nawaz H, S teck ler G. Acetaminophen induced pancreatitis. J Toxicol Clin Toxicol 1991; 29: 223-230

240 Mallory A, Kern F. Drug-induced pancreatitis: a critical review. Gastroenterology 1980; 78: 813-820

241 Banerjee AK, Patel KJ, Grainger SL. Drug-induced acute pancreatitis. A critical review. Med Toxicol Adverse Drug Exp 1989; 4: 186-198

242 Underwood TW, Frye CB. Drug-induced pancreatitis. Clin Pharm 1993; 12: 440-448

243 Bennett IL, Cary FH, Mitchell GL Jr, Cooper MN. Acute methyl alcohol poisoning: a review based on experiences in an outbreak of 323 cases. Medicine (Baltimore) 1953; 32: 431-463

244 Bartholomew C. Acute scorpion pancreatitis in Trinidad. Br Med J 1970; 1: 666-668

245 Bartholomew C, McGeeney KF, Murphy JJ, Fitzgerald O, Sankaran H. Experimental studies on the aetiology of acute scorpion pancreatitis. Br J Surg 1976; 63: 807-810

246 Lee HS. Acute pancreatitis and organophosphate poisoning--a case report and review. Singapore Med J 1989; 30: 599-601

247 Parenti DM, Steinberg W, Kang P. Infectious causes of acute pancreatitis. Pancreas 1996; 13: 356-371

248 Khuroo MS, Zargar SA, Mahajan R. Hepatobiliary and pancreatic ascariasis in India. Lancet 1990; 335: 1503-1506

249 Khuroo MS, Zargar SA, Yattoo GN, Javid G, Dar MY, Boda MI, Khan BA. Worm extraction and biliary drainage in hepatobiliary and pancreatic ascariasis. Gastrointest Endosc 1993; 39: 680-685

250 Dassopoulos T, Ehrenpreis ED. Acute pancreatitis in human immunodeficiency virus-infected patients: a review. Am J Med 1999; 107: 78-84

251 Cappell MS, Marks M. Acute pancreatitis in HIV-seropositive patients: a case control study of 44 patients. Am J Med 1995; 98: 243-248

252 Rizzardi GP, Tambussi G, Lazzarin A. Acute pancreatitis during primary HIV-1 infection. N Engl J Med 1997; 336: 1836-1837

253 Cuenca Carvajal C, Ortiz Vega M, Gómez Antúnez M, Pérez Tamayo I, Filgueira Rubio JS. Acute pancreatitis complicating primary HIV-1 infection. An Med Interna 1998; 15: 324-326

254 Toskes PP. Hyperlipidemic pancreatitis. Gastroenterol Clin North Am 1990; 19: 783-791

255 Fortson MR, Freedman SN, Webster PD. Clinical assessment of hyperlipidemic pancreatitis. Am J Gastroenterol 1995; 90: 2134-2139

256 Krauss RM, Levy AG. Subclinical chronic pancreatitis in type I hyperlipoproteinemia. Am J Med 1977; 62: 144-149

257 Salen S , Kessler JI, Janowitz HD. The development of pancreatic secretory insufficiency in a patient with recurrent pancreatitis and type V hyperlipoproteinemia. Mt Sinai J Med 1970; 37: 103-107

258 Nair S, Yadav D, Pitchumoni CS. Association of diabetic ketoacidosis and acute pancreatitis: observations in 100 consecutive episodes of DKA. Am J Gastroenterol 2000; 95: 2795-2800

259 G l u e c k C J , L a n g J , H a m e r T , T r a c y T . S e v e r e hypertriglyceridemia and pancreatitis when estrogen replacement therapy is given to hypertriglyceridemic women. J Lab Clin Med 1994; 123: 59-64

260 Hozumi Y, Kawano M, Saito T, Miyata M. Effect of tamoxifen

on serum lipid metabolism. J Clin Endocrinol Metab 1998; 83: 1633-1635

261 T o s k e s P P . I s t h e r e a r e l a t i o n s h i p b e t w e e n hypertriglyceridemia and development of alcohol- or gallstone-induced pancreatitis? Gastroenterology 1994; 106: 810-812

262 Haber PS, Wilson JS, Apte MV, Hall W, Goumas K, Pirola RC. Lipid intolerance does not account for susceptibility to alcoholic and gallstone pancreatitis. Gastroenterology 1994; 106: 742-748

263 Brandwein SL, Sigman KM. Case report: milk-alkali syndrome and pancreatitis. Am J Med Sci 1994; 308: 173-176

264 Ullian ME, Linas SL. The milk-alkali syndrome in pregnancy. Case report. Miner Electrolyte Metab 1988; 14: 208-210

265 M i t h ö f e r K , F e r n á n d e z - d e l C a s t i l l o C , F r i c k T W , Lewandrowski KB, Rattner DW, Warshaw AL. Acute hypercalcemia causes acute pancreatit is and ectopic trypsinogen activation in the rat. Gastroenterology 1995; 109: 239-246

266 Ward JB, Petersen OH, Jenkins SA, Sutton R. Is an elevated concentration of acinar cytosolic free ionised calcium the trigger for acute pancreatitis? Lancet 1995; 346: 1016-1019

267 Bess MA, Edis AJ, van Heerden JA. Hyperparathyroidism and pancreatitis. Chance or a causal association? JAMA 1980; 243: 246-247

268 Prinz RA , Aranha GV. The assoc ia t ion o f pr imary hyperparathyroidism and pancreatitis. Am Surg 1985; 51: 325-329

269 Shearer MG , Imrie CW. Parathyroid hormone levels, hyperparathyroidism and acute pancreatitis. Br J Surg 1986; 73: 282-284

270 Jacob JJ, John M, Thomas N, Chacko A, Cherian R, Selvan B, Nair A, Seshadri M. Does hyperparathyroidism cause pancreatitis? A South Indian experience and a review of published work. ANZ J Surg 2006; 76: 740-744

271 Abdullah M. Pancreatitis in primary hyperparathyroidism. Med J Malaysia 2003; 58: 600-603

272 Watts RA, Isenberg DA. Pancreatic disease in the autoimmune rheumatic disorders. Semin Arthritis Rheum 1989; 19: 158-165

273 Haraguchi K, Gunji K, Ito Y, Yokomori N, Kawaguchi A, Ohomori M, Inoue H, Shimura H, Saito T, Kobayashi T. Extensive pancreatic necrosis in microscopic polyangiitis. Clin Exp Nephrol 2005; 9: 326-331

274 Adsay NV, Basturk O, Thirabanjasak D. Diagnostic features and differential diagnosis of autoimmune pancreatitis. Semin Diagn Pathol 2005; 22: 309-317

275 Moolenaar W, Lamers CB. Cholesterol crystal embolization to liver, gallbladder, and pancreas. Dig Dis Sci 1996; 41: 1819-1822

276 Orvar K, Johlin FC. Atheromatous embolization resulting in acute pancreatitis after cardiac catheterization and angiographic studies. Arch Intern Med 1994; 154: 1755-1761

277 Fernández-del Castillo C, Harringer W, Warshaw AL, Vlahakes GJ, Koski G, Zaslavsky AM, Rattner DW. Risk factors for pancreatic cellular injury after cardiopulmonary bypass. N Engl J Med 1991; 325: 382-387

278 Sakorafas GH, Tsiotos GG, Sarr MG. Ischemia/Reperfusion-Induced pancreatitis. Dig Surg 2000; 17: 3-14

279 Warshaw AL, O’Hara PJ. Susceptibility of the pancreas to ischemic injury in shock. Ann Surg 1978; 188: 197-201

280 Wilson RH, Moorehead RJ. Current management of trauma to the pancreas. Br J Surg 1991; 78: 1196-1202

281 Frank CD , Adler DG. Post-ERCP pancreatitis and its prevention. Nat Clin Pract Gastroenterol Hepatol 2006; 3: 680-688

282 Das A, Singh P, Sivak MV, Chak A. Pancreatic-stent placement for prevention of post-ERCP pancreatitis: a cost-effectiveness analysis. Gastrointest Endosc 2007; 65: 960-968

283 Kisli E, Baser M, Aydin M, Guler O. The role of octreotide versus placebo in the prevention of post-ERCP pancreatitis. Hepatogastroenterology 2007; 54: 250-253

284 Kaffes AJ, Bourke MJ, Ding S, Alrubaie A, Kwan V, Williams SJ. A prospective, randomized, placebo-controlled trial of transdermal glyceryl trinitrate in ERCP: effects on technical

Lee JK et al. Review of idiopathic pancreatitis 6311

www.wjgnet.com

success and post-ERCP pancreatitis. Gastrointest Endosc 2006; 64: 351-357

285 Katsinelos P , Kountouras J, Chatzis J, Christodoulou K, Paroutoglou G, Mimidis K, Beltsis A, Zavos C. High-dose allopurinol for prevention of post-ERCP pancreatitis: a prospective randomized double-blind controlled trial. Gastrointest Endosc 2005; 61: 407-415

286 Mosler P, Sherman S, Marks J, Watkins JL, Geenen JE, Jamidar P, Fogel EL, Lazzell-Pannell L, Temkit M, Tarnasky P, Block KP, Frakes JT, Aziz AA, Malik P, Nickl N, Slivka A, Goff J, Lehman GA. Oral allopurinol does not prevent the frequency or the severity of post-ERCP pancreatitis. Gastrointest Endosc 2005; 62: 245-250

287 Cheng CL, Sherman S, Watkins JL, Barnett J, Freeman M, Geenen J, Ryan M, Parker H, Frakes JT, Fogel EL, Silverman WB, Dua KS, Aliperti G, Yakshe P, Uzer M, Jones W, Goff J, Lazzell-Pannell L, Rashdan A, Temkit M, Lehman GA. Risk factors for post-ERCP pancreatitis: a prospective multicenter study. Am J Gastroenterol 2006; 101: 139-147

288 Finkelberg DL , Sahani D, Deshpande V, Brugge WR. Autoimmune pancreatitis. N Engl J Med 2006; 355: 2670-2676

289 Sahani DV, Kalva SP, Farrell J, Maher MM, Saini S, Mueller PR, Lauwers GY, Fernandez CD, Warshaw AL, Simeone JF. Autoimmune pancreatitis: imaging features. Radiology 2004; 233: 345-352

290 van Buuren HR , Vleggaar FP, Willemien Erkelens G, Zondervan PE, Lesterhuis W, Van Eijck CH, Puylaert JB, Van Der Werf SD. Autoimmune pancreatocholangitis: a series of ten patients. Scand J Gastroenterol Suppl 2006: 70-78

291 Kim KP , Kim MH, Song MH, Lee SS, Seo DW, Lee SK. Autoimmune chronic pancreatitis. Am J Gastroenterol 2004; 99: 1605-1616

292 Pearson RK, Longnecker DS, Chari ST, Smyrk TC, Okazaki K, Frulloni L, Cavallini G. Controversies in clinical pancreatology: autoimmune pancreatitis: does it exist? Pancreas 2003; 27: 1-13

293 Okazaki K, Uchida K, Ohana M, Nakase H, Uose S, Inai M, Matsushima Y, Katamura K, Ohmori K, Chiba T. Autoimmune-related pancreatitis is associated with autoantibodies and a Th1/Th2-type cellular immune response. Gastroenterology 2000; 118: 573-581

294 Aparisi L, Farre A, Gomez-Cambronero L, Martinez J, De Las Heras G, Corts J, Navarro S, Mora J, Lopez-Hoyos M, Sabater L, Ferrandez A, Bautista D, Perez-Mateo M, Mery S, Sastre J. Antibodies to carbonic anhydrase and IgG4 levels in idiopathic chronic pancreatitis: relevance for diagnosis of autoimmune pancreatitis. Gut 2005; 54: 703-709

295 Epstein O, Chapman RW, Lake-Bakaar G, Foo AY, Rosalki SB, Sherlock S. The pancreas in primary biliary cirrhosis and primary sclerosing cholangitis. Gastroenterology 1982; 83: 1177-1182

296 Nishimori I, Yamamoto Y, Okazaki K, Morita M, Onodera M, Kino J, Tamura S, Yamamoto Y. Identification of autoantibodies to a pancreatic antigen in patients with idiopathic chronic pancreatitis and Sjögren's syndrome. Pancreas 1994; 9: 374-381

297 Zamboni G , Lüttges J, Capelli P, Frulloni L, Cavallini G, Pederzol i P , Leins A, Longnecker D, Klöppel G. Histopathological features of diagnostic and clinical relevance in autoimmune pancreatitis: a study on 53 resection specimens and 9 biopsy specimens. Virchows Arch 2004; 445: 552-563

298 Kamisawa T, Egawa N, Nakajima H, Tsuruta K, Okamoto A, Hayashi Y, Funata N. Gastrointestinal findings in patients with autoimmune pancreatitis. Endoscopy 2005; 37: 1127-1130

299 Farrell JJ, Garber J, Sahani D, Brugge WR. EUS findings in patients with autoimmune pancreatitis. Gastrointest Endosc 2004; 60: 927-936

300 Levy MJ, Reddy RP, Wiersema MJ, Smyrk TC, Clain JE, Harewood GC, Pearson RK, Rajan E, Topazian MD, Yusuf TE, Chari ST, Petersen BT. EUS-guided trucut biopsy in establishing autoimmune pancreatitis as the cause of obstructive jaundice. Gastrointest Endosc 2005; 61: 467-472

301 Wakabayashi T, Kawaura Y, Satomura Y, Fujii T, Motoo Y, Okai T, Sawabu N. Clinical study of chronic pancreatitis

with focal irregular narrowing of the main pancreatic duct and mass formation: comparison with chronic pancreatitis showing diffuse irregular narrowing of the main pancreatic duct. Pancreas 2002; 25: 283-289

302 Ozden I, Dizdaroğlu F, Poyanli A, Emre A. Spontaneous regression of a pancreatic head mass and biliary obstruction due to autoimmune pancreatitis. Pancreatology 2005; 5: 300-303

303 Ito T, Nakano I, Koyanagi S, Miyahara T, Migita Y, Ogoshi K, Sakai H, Matsunaga S, Yasuda O, Sumii T, Nawata H. Autoimmune pancreatitis as a new clinical entity. Three cases of autoimmune pancreatitis with effective steroid therapy. Dig Dis Sci 1997; 42: 1458-1468

304 Saito T, Tanaka S, Yoshida H, Imamura T, Ukegawa J, Seki T, Ikegami A, Yamamura F, Mikami T, Aoyagi Y, Niikawa J, Mitamura K. A case of autoimmune pancreatitis responding to steroid therapy. Evidence of histologic recovery. Pancreatology 2002; 2: 550-556

305 Kojima E, Kimura K, Noda Y, Kobayashi G, Itoh K, Fujita N. Autoimmune pancreatitis and multiple bile duct strictures treated effectively with steroid. J Gastroenterol 2003; 38: 603-607

306 Kamisawa T, Yoshiike M, Egawa N, Nakajima H, Tsuruta K, Okamoto A. Treating patients with autoimmune pancreatitis: results from a long-term follow-up study. Pancreatology 2005; 5: 234-238; discussion 238-240

307 Neild GH, Rodriguez-Justo M, Wall C, Connolly JO. Hyper-IgG4 disease: report and characterisation of a new disease. BMC Med 2006; 4: 23

308 Troppmann C, Gruessner AC, Dunn DL, Sutherland DE, Gruessner RW. Surgical complications requiring early relaparotomy after pancreas transplantation: a multivariate risk factor and economic impact analysis of the cyclosporine era. Ann Surg 1998; 227: 255-268

309 Sinha S, Jha R, Lakhtakia S, Narayan G. Acute pancreatitis following kidney transplantation - role of viral infections. Clin Transplant 2003; 17: 32-36

310 Pike JL, Rice JC, Sanchez RL, Kelly EB, Kelly BC. Pancreatic panniculitis associated with allograft pancreatitis and rejection in a simultaneous pancreas-kidney transplant recipient. Am J Transplant 2006; 6: 2502-2505

311 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

312 Sahai AV , Zimmerman M, Aabakken L, Tarnasky PR, Cunningham JT, van Velse A, Hawes RH, Hoffman BJ. Prospective assessment of the ability of endoscopic ultrasound to diagnose, exclude, or establish the severity of chronic pancreatitis found by endoscopic retrograde cholangiopancreatography. Gastrointest Endosc 1998; 48: 18-25

313 Wiersema MJ , Hawes RH, Lehman GA, Kochman ML, Sherman S , Kopecky KK. Prospect ive evaluat ion of endoscopic ultrasonography and endoscopic retrograde cholangiopancreatography in patients with chronic abdominal pain of suspected pancreatic origin. Endoscopy 1993; 25: 555-564

314 Levy P, Milan C, Pignon JP, Baetz A, Bernades P. Mortality factors associated with chronic pancreatitis. Unidimensional and multidimensional analysis of a medical-surgical series of 240 patients. Gastroenterology 1989; 96: 1165-1172

315 Ammann RW, Akovbiantz A, Largiader F, Schueler G. Course and outcome of chronic pancreatitis. Longitudinal study of a mixed medical-surgical series of 245 patients. Gastroenterology 1984; 86: 820-828

316 Dumonceau JM , Devière 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

317 DiMagno MJ, Dimagno EP. Chronic pancreatitis. Curr Opin Gastroenterol 2006; 22: 487-497

318 Adler DG, Lichtenstein D, Baron TH, Davila R, Egan JV, Gan SL, Qureshi WA, Rajan E, Shen B, Zuckerman MJ, Lee KK,

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www.wjgnet.com

VanGuilder T, Fanelli RD. The role of endoscopy in patients with chronic pancreatitis. Gastrointest Endosc 2006; 63: 933-937

319 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

320 Kozarek RA, Ball TJ, Patterson DJ. Endoscopic approach to pancreatic duct calculi and obstructive pancreatitis. Am J Gastroenterol 1992; 87: 600-603

321 Uden S, Bilton D, Nathan L, Hunt LP, Main C, Braganza JM. Antioxidant therapy for recurrent pancreatitis: placebo-controlled trial. Aliment Pharmacol Ther 1990; 4: 357-371

322 Uden S, Schofield D, Miller PF, Day JP, Bottiglier T, Braganza JM. Antioxidant therapy for recurrent pancreatitis: biochemical profiles in a placebo-controlled trial. Aliment Pharmacol Ther 1992; 6: 229-240

323 Testoni PA, Caporuscio S, Bagnolo F, Lella F. Idiopathic

recurrent pancreatitis: long-term results after ERCP, endoscopic sphincterotomy, or ursodeoxycholic acid treatment. Am J Gastroenterol 2000; 95: 1702-1707

324 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

325 Warshaw AL, Banks PA, Fernández-Del Castillo C. AGA technical review: treatment of pain in chronic pancreatitis. Gastroenterology 1998; 115: 765-776

326 Mössner J , Secknus R, Meyer J, Niederau C, Adler G. Treatment of pain with pancreatic extracts in chronic pancreatitis: results of a prospective placebo-controlled multicenter trial. Digestion 1992; 53: 54-66

327 Steinberg WM, Chari ST, Forsmark CE, Sherman S, Reber HA, Bradley EL, DiMagno E. Controversies in clinical pancreatology: management of acute idiopathic recurrent pancreatitis. Pancreas 2003; 27: 103-117

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Lee JK et al. Review of idiopathic pancreatitis 6313

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TOPIC HIGHLIGHT

Role of endoscopic retrograde cholangiopancreatography in acute pancreatitis

Karen R Canlas, Malcolm S Branch

Michael F Byrne, MD, Series Editor

Karen R Canlas, Malcolm S Branch, Division of Gastro-enterology and Hepatology, Duke University Medical Center, Durham, North Carolina 27710, United States Correspondence to: Malcolm S Branch, MD, Division of Gastroenterology and Hepatology, Duke University Medical Center, DUMC Box 3662, Durham, NC 27710, United States. branch003@mc.duke.eduTelephone: +1-919-6843787 Fax: +1-919-6818785 Received: July 12, 2007 Revised: September 13, 2007

AbstractEndoscopic retrograde cholangiopancreatography (ERCP) is a useful tool in the evaluation and management of acute pancreatitis. This review will focus on the role of ERCP in specific causes of acute pancreatitis, including microlithiasis and gallstone disease, pancreas divisum, Sphincter of Oddi dysfunction, tumors of the pancreaticobiliary tract, pancreatic pseudocysts, and pancreatic duct injury. Indications for endoscopic techniques such as biliary and pancreatic sphincterotomy, stenting, stricture dilation, treatment of duct leaks, drainage of fluid collections and stone extraction will also be discussed in this review. With the advent of less invasive and safer diagnostic modalities including endoscopic ultrasound (EUS) and magnetic retrograde cholangiopancreatography (MRCP), ERCP is appropriately becoming a therapeutic rather than diagnostic tool in the management of acute pancreatitis and its complications.

© 2007 WJG. All rights reserved.

Key words: Endoscopic retrograde cholangiopancreatography; Acute pancreatitis

Canlas KR, Branch MS. Role of endoscopic retrograde cholangiopancreatography in acute pancreatitis. World J Gastroenterol 2007; 13(47): 6314-6320

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INTRODUCTIONMost pancreatologists accept the 1992 Atlanta Symposium definition of acute pancreatitis as an acute inflammatory process of the pancreas with variable involvement of

other regional tissues or remote organ systems[1]. It is recognized that the underlying process is reversible and the gland returns to normal once the injury resolves. The annual incidence is estimated to be 17 per 100 000 in the United States[2]. Over 333 000 hospital admissions and 911 000 physician visits in the United States each year are due to acute pancreatitis[2]. The most common causes in US adults are gallstone disease and excessive alcohol use, although clinically detectable pancreatitis never develops in most persons with these risk factors[3].

The pathogenesis of acute pancreatitis involves the inappropriate activation of trypsinogen to trypsin in excessive quantities to overwhelm the mechanisms of elimination within the pancreas. Activation of these digestive enzymes causes pancreatic injury and an intense inflammatory response which results in microcirculatory injury, leukocyte chemoattraction, release of cytokines and oxidative stress. The release of pancreatic enzymes damages the vascular endothelium, the interstitium and acinar cells. Microcirculatory changes and progressive ischemia occur, which increase vascular permeability and leads to edema of the gland, commonly referred to as interstitial pancreatitis. Translocation of bacteria from the gut into the systemic circulation may occur as a consequence of gut ischemia secondary to hypovolemia and arteriovenous shunting. Severe pancreatitis may ensue, leading to life-threatening complications such as acute respiratory distress syndrome, renal failure, shock, metabolic derangements, and multi-organ failure. Approximately 20% of patients with acute pancreatitis will have a severe course with a 10% to 30% mortality rate[3]. Mortality related to acute pancreatitis decreased substantially from as high as 25%-30% in the 1970s to current rates since the early 1990s; however, since then, the mortality has remained relatively constant[3,4].Initial improvement appears to have been related to improvements in intensive care treatment rather than a better understanding of the natural history of disease or improved intervention.

Determining the etiology of acute pancreatitis can be challenging in those who do not give a significant history of alcohol use and in those who do not exhibit obvious gallstone disease. In about 20% to 40% of cases, no defined cause may be found and patients are subsequently labeled as having idiopathic acute pancreatitis[5]. Several causes for pancreatitis may be missed in the initial workup using the conventional imaging techniques of

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trans-abdominal ultrasound and/or CT scan and routine laboratory tests. Other modalities, including more advanced imaging techniques and endoscopic procedures, are often considered when working up the cause of unexplained acute pancreatitis. Although used primarily for diagnostic and therapeutic purposes in biliary disorders, endoscopic retrograde cholangiopancreatography (ERCP) has evolved as a diagnostic and therapeutic option in evaluating several pancreatic diseases[6].

CLINICAL USE OF ERCP IN ACUTE PANCREATITIS When clear clinical, laboratory and imaging evidence for persistent biliary obstruction is present, patients should directly proceed to ERCP. As with all invasive procedures, the risk of procedure-related complications must be weighed against the potential benefit of the procedure. ERCP has a reported complication rate of 5% to 7%, with the main complications including pancreatitis, hemorrhage, perforation, cholangitis, cardiopulmonary complications and, rarely, death. Patient-related factors, such as underlying co-morbidities, age, and need for invasive evaluation, are considerable determinants of complication risk in any endoscopic procedures, especially those that carry a higher possibility for complications such as ERCP. Because of the potential morbidity and mortality associated with ERCP and the improvements in other less-invasive imaging modalities, the role of ERCP has become more well-defined in the diagnosis and treatment acute pancreatitis.

In the 30% of patients who may have no identifiable cause for acute pancreatitis with traditional non-invasive methods, ERCP with empiric biliary sphincterotomy is often performed without a more thorough evaluation for cause[7]. Freeman et al evaluated this concern and determined the risk of post-ERCP pancreatitis to be as high as 20% with a 3%-4% risk for severe pancreatitis. Though tempting, empiric sphincterotomy appears to have about an equal chance of causing complications as treating the underlying cause of the acute pancreatitis and, therefore, is not advocated[6,8]. Determining the etiology of acute pancreatitis is important, as it helps direct therapy, limits further unnecessary evaluation, and may improve a patient’s long term prognosis. Advanced endoscopic procedures, including ERCP, are emerging as valuable tools in the evaluation of this challenging group. The role of ERCP in the diagnostic and therapeutic evaluation of acute pancreatitis will be discussed in this review.

MICROLITHIASISMicrolithiasis or biliary sludge as a causative etiology for acute pancreatitis remains controversial and not well understood. Several studies have demonstrated the presence of biliary sludge in as many as 75% of patients with unexplained acute pancreatitis[5]. Microlithiasis is a viscous precipitate containing mucin, cholesterol and calcium bilirubinate which can obstruct the pancreatic duct. Ultrasonography has a sensitivity of only about 55% in detecting microlithiasis and does not allow for analysis

of the chemical composition of bile [9]. Bile analysis with microscopic examination is considered the gold standard for diagnosis. Bile can be obtained directly while cannulating the bile duct during ERCP or following CCK stimulation on EGD. ERCP with bile aspiration from the common bile duct (CBD) has a reported sensitivity of 83% in detecting microlithiasis[7]. It is recognized, however, that gallbladder bile is preferred over ductal bile for examination, as transit through the hepatic and common ducts can be too rapid to allow formation of crystals large enough to detect on microscopy. Ko et al[9] propose the criteria of 2 or more crystals per 100X field or more than 4 crystals per sample as a positive result. It is also recommended to collect bile samples prior to contrast injection to avoid the formation of “pseudomicrolithiasis” from contrast precipitates. Using a guidewire under fluoroscopy with aspiration to clear the collection catheter prior to obtaining the bile sample can minimize contrast contamination and diminish this artifact. The specimen should be centrifuged immediately and examined under polarized microscopy to evaluate for crystals. If specimens are not examined immediately, crystals can precipitate and cause a false positive result. ERCP should be performed after complete recovery from acute pancreatitis, usually 4 to 6 wk after presentation. If microlithiasis is detected, patients should be considered for cholecystectomy or biliary sphincterotomy depending on surgical risk. In post- cholecystectomy patients, bile analysis need not be performed.

PANCREAS DIVISUMPancreas divisum (PD) is the most common congenital anomaly of the pancreas and occurs in approximately 7%-10% of the population; however, less than 5% of those with PD are symptomatic[7]. PD is a small or absent ventral pancreatic duct that fails to fuse with the dorsal duct during embryologic development, resulting in a lack of communication between the ducts (Figure 1A and B). This lack of communication leads to a prominent dorsal duct that drains entirely through the minor papilla. Incomplete PD is the partial communication between the ventral and dorsal ducts; however, incomplete PD functions similar to that of complete PD. The divided drainage routes may result in a relative obstruction of flow through the minor papilla, leading to acute pancreatitis. Some controversy exists about recognizing PD as a causative etiology for pancreatitis. Regardless of the debate, several studies have demonstrated that dorsal duct outflow obstruction can lead to both acute and chronic pancreatitis[10,11]. It has been reported that patients with divisum have an increased prevalence of pancreatitis. Parenchymal changes consistent with chronic pancreatitis isolated to the dorsal pancreas have been observed on autopsy studies[6].

ERCP plays a limited role in diagnosis of PD given the less invasive and reasonably accurate modalities of MRCP and EUS. ERCP can, however, offer a therapeutic option in those who experience recurrent acute pancreatitis. Several studies have shown that endoscopic intervention, including sphincterotomy with or without stenting of the minor

A

papilla, has resulted in decreased recurrence rates of acute pancreatitis and improved outcomes in patients with PD (Figure 2A-C)[12-17]. In the only randomized controlled trial evaluating endoscopic therapy in PD, Lans et al[18] observed an improvement in 90% of treated patients versus only 11% of controls over a mean of 12 mo follow-up. A retrospective study by Gerke et al[19] observed that 60% of patients who underwent minor papillotomy reported immediate improvement, but sustained results were seen in only half of those patients at a mean of 29 mo follow-up. Patients with well-defined bouts of pancreatitis appeared to benefit over those solely with chronic abdominal pain without objective evidence of pancreatitis. It is recognized that pain secondary to PD and chronic abdominal pain with incidentally found PD is difficult to differentiate. In order to distinguish between these two groups, objective criteria such as recurrent pain with ductal dilatation and elevations in pancreatic enzymes can be useful. Secretin stimulation imaging using either ultrasonography or MRI has proven promising as these modalities can give clues to the functionality of the pancreas. The study by Gerke et al underscores the importance of defining patients who may benefit from endoscopic therapy and reveals the difficulty in achieving long-term results in patients with PD.

The role of using pancreatic stents in PD is limited to the prevention of post-ERCP pancreatitis and has demonstrated little efficacy in treating PD in comparison to sphincterotomy. The long-term use of pancreatic stents in PD may induce duct strictures or irregularities. Most experts recommend only the short-term use of pancreatic stents (less than 2 wk) in patients without pre-existing duct strictures. Size 3 or 4 French, non-phlanged stents, without an internal flap are recommended to decrease the risk of post-ERCP pancreatitis (Figure 3). These stents can pass spontaneously, therefore, obviating the need for a repeat

procedure for stent removal. Note that the use of size 3 French stents requires a separate, smaller guidewire which may increase the overall cost of the procedure.

SPHINCTER OF ODDI DYSFUNCTIONSphincter of Oddi dysfunction (SOD) as an etiology for acute pancreatitis has been questioned by many due to the lack of concrete pathologic findings[20]. SOD refers to an abnormality in sphincter of Oddi contractility resulting in intermittent biliary and pancreatic duct obstruction. It has been estimated that SOD accounts for approximately 1/3rd of those with recurrent unexplained pancreatitis.

Management depends upon the classification of SOD. A classification system similar to the Milwaukee Biliary Group Classification has been developed for pancreatic SOD (Table 1): type Ⅰ pancreatic SOD includes recurrent pancreatitis or pain suspected to be of pancreatic origin with elevated amylase and/or lipase up to 1.5 times upper limit of normal, a dilated pancreatic duct greater than 6 mm in the head and more than 5 mm in the body, and delayed emptying of greater than 9 min of the pancreatic duct. type Ⅱ SOD includes the presence of presumed pancreatic pain plus at least one additional factor defining type Ⅰ. type Ⅲ is defined by pain alone. Some experts have suggested that due to the high incidence of stone disease, patients suspected to have SOD with an intact gallbladder should undergo cholecystectomy as initial therapy before evaluation for sphincter dysfunction due to less risk involved in surgery compared with manometry. Our approach in patients with intact gallbladder, but no stone disease on standard imaging, is to perform endoscopic ultrasound (EUS) looking for undetected stones or biliary sludge. If EUS is unremarkable, then ERCP with bile analysis is

Figure 1 A: Pancreas divisum with filling of the small ventral duct; B: Pancreas divisum with filling of dorsal duct through the minor orifice.

Figure 2 A: Sphicterotome performing minor ampulla sphincter-otomy; B: Guidewire in place after minor sphincterotomy; C: Pancreatic stent placement post-sphincterotomy for pancreas divisum.

B

BA C

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performed to evaluate for microlithiasis. If microlithiasis is not detected, we will proceed with sphincter of Oddi manometry during the same exam. In those who have previously had a cholecystectomy, dual sphincterotomy becomes the recommended treatment modality. A study by Gelrud et al[21] demonstrated significantly better outcomes when dual sphincterotomy was performed over a biliary sphincterotomy alone.

Management of types Ⅱ and Ⅲ disease has posed more of a treatment challenge to the clinician. Patients who meet type Ⅱ classification criteria should undergo sphincter of Oddi manometry (SOM). A resting basal pressure of > 40 mm Hg is the best predictor of response to endoscopic sphincterotomy, with up to 90% demonstrating clinical benefit in 4 years follow-up[20]. Studies advocate the measurement of both biliary and pancreatic sphincter pressures during ERCP, as differences in pressure may be detected if either duct is measured alone[22-25]. There is no role for ERCP without manometry in the evaluation of type Ⅱ SOD patients.

Traditional teaching has been that performing SOM increases the risk for pancreatitis [26]. However, more recently, it appears that performing any ERCP (with or without manometry) in the subset of patients at highest risk for SOD (typically young women with unexplained recurrent abdominal pain, normal anatomy, and normal serum bilirubin) increases the risk for pancreatitis[8,27,28]. Several randomized trials have proven a decreased risk of post-ERCP pancreatitis in this group of patients by performing prophylactic stent placement[29,30]. The NIH has recommended that diagnostic and therapeutic procedures in this select group of patients should be performed ONLY by endoscopist possessing expertise in this particular area because of the high rate of severe complications in this young, otherwise healthy population[20]. Because of this thought, the diagnosis and management of type Ⅲ disease is the most difficult. Invasive procedures should be delayed or avoided if possible. Trials of anticholinergics, antidepressants, nonspecific pain relievers and/or calcium channel blockers should precede invasive approaches. Diagnostic ERCP without manometry has no role in the assessment of type Ⅲ pancreatic SOD patients[20].OCCULT TUMORS OF THE PANCREAS

AND BILIARY TRACTApproximately 30 000 new cases of pancreatic cancer and 7000 biliary tract cancers are diagnosed annually in the United States. Prognosis remains dismal with less than 20% survival at 1 year and a 5 years mortality rate of > 95%[31]. Infrequently, tumors of the pancreas and biliary tree may present as acute pancreatitis. ERCP has proven to be valuable in the diagnosis and treatment of ampullary tumors and intraductal papillary mucinous tumors (IPMT) of the pancreas, both conditions which may present with acute pancreatitis due to obstruction from the pancreatic duct. ERCP may be the best means for direct visualization of the ampullary and periampullary region and offers the capability of sampling through biopsy. In non-operative patients with obstructing ampullary tumors, palliation or possibly cure can be achieved with endoscopic snare ampullectomy and ablative thermal therapy. Post-procedure stent placement in this clinical scenario has been demonstrated to reduce the risk of post-procedure pancreatitis[6]. IPMT can be diagnosed on ERCP with the classic “fish eye” appearance of the dilated ampulla and mucin extruding from the orifice (Figure 4). In non-operative patients with IPMT, ERCP with stent placement and/or sphincterotomy may offer palliation by decreasing the risk of acute recurrent pancreatitis by minimizing mucin impaction in the pancreatic duct.

Peroral pancreatoscopy (PPS) is useful in cases of suspected IPMT by not only diagnosing but also localizing the tumor for planning surgical resection. PPS may also provide tissue sampling for histologic diagnosis[32].

GALLSTONESGallstones account for nearly half of the cases of acute pancreatitis in the Western world. More than 70% of patients will spontaneously pass the culprit stone into the duodenum, however, approximately 3% to 7% may go on to develop acute pancreatitis[33]. Because differentiating patients who may have an uncomplicated course due to a transiently impacted gallstone from those who may

Figure 3 Various designs of pancreatic stents (with permission from Cook Endoscopy).

Table 1 Sphincter of Oddi Dysfunction

Type Ⅰ1 Typical biliary-pancreatic pain2 Liver chemistries (total bilirubin, alkaline phosphatase, transaminases) ≥ 1.5-2X ULN and/or pancreatic chemistries (amylase and/or lipase) ≥ 1.5-2X ULN3 Dilated common bile duct (≥ 12 mm) or pancreatic duct (head ≥ 6 mm, body ≥ 5 mm) diameter4 Prolonged biliary drainage (> 45 min) with patient in supine position or pancreatic drainage (> 9 min) with patient in the prone position

Type Ⅱ1 Typical biliary-pancreatic pain2 Positive findings for one or two items (2, 3, or 4) from type Ⅰ

Type Ⅲ1 Typical biliary-pancreatic pain and no other abnormalities

Modified from Sherman et al[25].

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progress on to severe acute pancreatitis with necrosis and sepsis is difficult, several studies have evaluated scoring systems and variables to predict severity[34-36]. Urgent endoscopy is generally reserved for patients who fail to demonstrate liver enzyme improvement within 24 to 48 h of admission, especially in total bilirubin by hospital d 2[34]; those demonstrating persistent choledocholithiasis on imaging; and those with clinical cholangitis. Fan et al[37] evaluated the role of early ERCP in patients with acute biliary pancreatitis prior to the onset of complications, regardless of mild or severe presentation. Within 24 h of presentation, patients were randomized to early ERCP versus conservative treatment with selective ERCP. A significant reduction in progression to biliary sepsis was seen in the early ERCP patients. Interestingly, however, the incidence of local and systemic complications was not significantly different, which suggests that removal of the impacted stone may not reverse the damage already occurring in the pancreas during the first hours or days of the illness. Other studies have advocated early intervention within 72 h after admission if persistent CBD stones were suspected[38,39].

Patients who recover from gallstone pancreatitis carry a 29% to 67% risk of recurrent pancreatitis if subsequent cholecystectomy and/or sphincterotomy are not performed[6,40]. In mild to moderate gallstone pancreatitis, ERCP is rarely required before cholecyste-ctomy unless cholangitis or clear evidence of persistent choledocholithiasis by imaging and laboratory data is observed. Chang et al[34] evaluated patients with acute gallstone pancreatitis who were suspected of persistent choledocholithiasis. Patients were randomized to either pre-operative ERCP or selective post-operative ERCP if choledocholithiasis was found intraoperatively. Hospital stay was significantly longer in the routine pre-operative ERCP group (11.7 d vs 9.0 d). In the post-operative group, ERCP was necessary in only 24% of patients, suggesting that the diagnostic and therapeutic yield of pre-operative ERCP is low. These findings are consistent with the NIH consensus statement recommending that patients suspected of having choledocholithiasis should undergo an operative cholangiogram at the time of cholecystectomy. Operative cholangiogram is efficient and preferable when surgical proficiency in this technique is available. Otherwise, post-operative ERCP is indicated for patients who demonstrate retained stones. In patients who have had a prior cholecystectomy and have a low probability of common bile duct stones, diagnostic evaluation for choledocholithiasis should be performed with less-invasive

modalities including MRCP or EUS. In the clinical scenario where the potential for retained common bile duct stones is substantial, ERCP and, when indicated, sphincterotomy with stone removal is the preferred diagnostic and therapeutic option[20]. ERCP with sphincterotomy is the preferred therapeutic modality if cholangitis from retained common bile duct stones is present. Patients should receive close medical care and treatment with Ⅳ fluid resuscitation, hemodynamic monitoring, and intravenous antibiotic therapy. Patients who fail to improve should undergo ERCP with sphincterotomy as soon as possible. Those who do improve still require urgent ERCP with sphincterotomy, usually within 24 h, to relieve the obstruction[20].

Recurrence of pancreatitis after ERCP with sphinctero-tomy for gallstone pancreatitis is rare. Cholecystectomy versus endoscopic sphincterotomy for the treatment of recurrent gallstone pancreatitis remains a controversial topic. Several experts advocate that cholecystectomy should only be considered if there are overt manifestations of gallbladder disease (e.g., biliary pain, cholecystitis, cystic duct obstruction), but not for prevention of recurrent gallstone pancreatitis. Studies have demonstrated that ERCP can be an effective therapeutic option for prevention of recurrent gallstone pancreatitis[41]. Siegel et al [42] demonstrate that ERCP with sphincterotomy can be performed safely in both the elective and urgent setting in patients who are otherwise not ideal operative candidates, such as the aged or younger patients at risk for surgical complications. Our approach is to proceed with cholecystectomy if the patient is a good surgical candidate.

PANCREATIC PSEUDOCYSTS AND FLUID COLLECTIONSPancreatic pseudocysts occur mainly as a result of acute pancreatitis, pancreatic trauma or chronic pancreatitis. Fluid usually contains a high concentration of pancreatic enzymes and variable amount of tissue debris. Most pseudocysts are sterile. ERCP has a reported success rate of 65%-95% in treatment of pancreatic pseudocysts, with a complication rate of 10%-35%. Drainage of fluid collections is generally reserved for a later date, usually 4-6 wk after the acute pancreatitis episode resolves.

PANCREATIC DUCT INJURY Pancreatic duct disruptions may result from acute and chronic pancreatitis, or they may be the primary cause for pancreatitis in cases of trauma or surgical injury. ERCP can be successful in detecting the presence of contrast extravasation from the duct, localizing the suspected site of injury, and treating the leak or fistula with stent or drain placement.

Approximately 37%-67% of patients with acute pancreatitis have pancreatic duct injury, suggesting that acute duct injury can be a relatively common finding[43,44]. Lau et al [43] observed that the presence of a leak was associated with a higher incidence of necrosis and prolonged length of stay. ERCP in this patient population was determined to be safe and not associated with

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Figure 4 Classic “fish eye” appearance of IPMT with mucin draining from minor ampulla.

increased mortality, prolonged hospital stay or need for necrosectomy provided that pancreatic duct leaks were detected and immediately treated.

ERCP to evaluate for pancreatic duct disruption in acute pancreatitis is controversial and should be reserved for investigational studies. A multidisciplinary approach is advocated when considering pancreatic stenting in the setting of acute necrosis, as the procedure carries a risk of introducing infection into an otherwise sterile environment. In patients with evidence of pancreatic duct injury or leak who are not responding to conservative treatment, ERCP should be considered.

T he use o f ERCP in the t r ea tment o f acu te pancreatitis from traumatic pancreatic duct injury has also been evaluated in both the adult and pediatric patient population. Several studies have demonstrated that ERCP with transpapillary stent placement is an effective technique in closing pancreatic duct disruption[45-47]. Successful therapy, however, appears to be associated with positioning of the stent to bridge the disruption and leak, not simply across the papilla as in biliary leaks[48,49]. Several studies in children have also reported successful results in treating traumatic duct injury, however the authors call attention to the risk of iatrogenic ductitis when stenting smaller pancreatic ducts, especially those in children[50,51].

UNUSUAL CAUSES OF ACUTE PANCREATITISType Ⅲ choledochal cysts are dilations of the joined portion of the pancreaticobiliary ducts. These cysts can be large enough to obstruct the pancreatic duct, which may result in recurrent acute pancreatitis [52,53]. Biliary sphincterotomy has been suggested as treatment; however, some patients may require a dual sphincterotomy for long term benefit.

Annular pancreas, anomalous pancreaticobiliary junction, and pancreatic intraductal parasites have all been reported as causes for acute and recurrent acute pancreatitis. ERCP can occasionally offer benefit in treatment of these rare conditions[6].

CONCLUSIONERCP is a useful tool in the evaluation and management of acute pancreatitis. The main role of ERCP in acute pancreatitis is the diagnosis and treatment of biliary tract stone disease and other potential causes of pancreatic duct obstruction including sphincter dysfunction or anomalies such as pancreas divisum. With the advent of less-invasive and safer diagnostic modalities, ERCP is appropriately becoming a therapeutic tool in the management of acute pancreatitis and its complications.

REFERENCES1 Bradley EL. A clinically based classification system for acute

pancreatitis. Summary of the International Symposium on Acute Pancreatitis, Atlanta, Ga, September 11 through 13, 1992. Arch Surg 1993; 128: 586-590

2 National Digestive Disease Information Clearinghouse.

National Institute of Health 1976-20023 Whitcomb DC. Clinical practice. Acute pancreatitis. N Engl J

Med 2006; 354: 2142-21504 Neoptolemos JP , Raraty M, Finch M, Sutton R. Acute

pancreatitis: the substantial human and financial costs. Gut 1998; 42: 886-891

5 Lee SP, Nicholls JF, Park HZ. Biliary sludge as a cause of acute pancreatitis. N Engl J Med 1992; 326: 589-593

6 Kinney TP, Lai R, Freeman ML. Endoscopic approach to acute pancreatitis. Rev Gastroenterol Disord 2006; 6: 119-135

7 Hernandez L, Catalano M. Endoscopic Techniques (ERCP, EUS) for the Evaluation of Unexplained Acute Pancreatitis. Techn in Gastrointestinal Endoscopy 2004; 6: 84-90

8 Freeman ML, Nelson DB, Sherman S, Haber GB, Herman ME, Dorsher PJ, Moore JP, Fennerty MB, Ryan ME, Shaw MJ, Lande JD, Pheley AM. Complications of endoscopic biliary sphincterotomy. N Engl J Med 1996; 335: 909-918

9 Ko CW, Sekijima JH, Lee SP. Biliary sludge. Ann Intern Med 1999; 130: 301-311

10 Bank S, Indaram A. Causes of acute and recurrent pancreatitis. Clinical considerations and clues to diagnosis. Gastroenterol Clin North Am 1999; 28: 571-589, viii

11 Bernard JP, Sahel J, Giovannini M, Sarles H. Pancreas divisum is a probable cause of acute pancreatitis: a report of 137 cases. Pancreas 1990; 5: 248-254

12 Bradley EL, Stephan RN. Accessory duct sphincteroplasty is preferred for long-term prevention of recurrent acute pancreatitis in patients with pancreas divisum. J Am Coll Surg 1996; 183: 65-70

13 Keith RG, Shapero TF, Saibil FG, Moore TL. Dorsal duct sphincterotomy is effective long-term treatment of acute pancreatitis associated with pancreas divisum. Surgery 1989; 106: 660-666; discussion 666-667

14 Coleman SD , E isen GM, Troughton AB, Cotton PB. Endoscopic treatment in pancreas divisum. Am J Gastroenterol 1994; 89: 1152-1155

15 Ertan A. Long-term results after endoscopic pancreatic stent placement without pancreatic papillotomy in acute recurrent pancreatitis due to pancreas divisum. Gastrointest Endosc 2000; 52: 9-14

16 Lehman GA , Sherman S, Nisi R, Hawes RH. Pancreas divisum: results of minor papilla sphincterotomy. Gastrointest Endosc 1993; 39: 1-8

17 Soehendra N, Kempeneers I, Nam VC, Grimm H. Endoscopic dilatation and papillotomy of the accessory papilla and internal drainage in pancreas divisum. Endoscopy 1986; 18: 129-132

18 Lans JI, Geenen JE, Johanson JF, Hogan WJ. Endoscopic therapy in patients with pancreas divisum and acute pancreatitis: a prospective, randomized, controlled clinical trial. Gastrointest Endosc 1992; 38: 430-434

19 Gerke H, Byrne MF, Stiffler HL, Obando JV, Mitchell RM, Jowell PS, Branch MS, Baillie J. Outcome of endoscopic minor papillotomy in patients with symptomatic pancreas divisum. JOP 2004; 5: 122-131

20 Cohen S, Bacon BR, Berlin JA, Fleischer D, Hecht GA, Loehrer PJ, McNair AE, Mulholland M, Norton NJ, Rabeneck L, Ransohoff DF, Sonnenberg A, Vannier MW. National Institutes of Health State-of-the-Science Conference Statement: ERCP for diagnosis and therapy, January 14-16, 2002. Gastrointest Endosc 2002; 56: 803-809

21 Gelrud M, Plaz J, Mendoz S. Endoscopic treatment in type II pancreatic sphincter dysfunction. Gastrointest Endosc 1995; 41: 398A

22 Eversman D, Fogel EL, Rusche M, Sherman S, Lehman GA. Frequency of abnormal pancreatic and biliary sphincter manometry compared with clinical suspicion of sphincter of Oddi dysfunction. Gastrointest Endosc 1999; 50: 637-641

23 Silverman WB, Ruffolo TA, Sherman S, Hawes RH, Lehman GA. Correlation of basal sphincter pressures measured from the bile duct and the pancreatic duct in patients with suspected sphincter of Oddi dysfunction. Gastrointest Endosc 1992; 38: 440-443

Canlas KR et al . Role of ERCP in acute pancreatitis 6319

www.wjgnet.com

24 Funch-Jensen P , Kruse A. Manometric activity of the pancreatic duct sphincter in patients with total bile duct sphincterotomy for sphincter of Oddi dyskinesia. Scand J Gastroenterol 1987; 22: 1067-1070

25 Sherman S, Troiano FP, Hawes RH, O'Connor KW, Lehman GA. Frequency of abnormal sphincter of Oddi manometry compared with the clinical suspicion of sphincter of Oddi dysfunction. Am J Gastroenterol 1991; 86: 586-590

26 Cotton PB, Lehman G, Vennes J, Geenen JE, Russell RC, Meyers WC, Liguory C, Nickl N. Endoscopic sphincterotomy complications and their management: an attempt at consensus. Gastrointest Endosc 1991; 37: 383-393

27 Sherman S, Hawes RH, Troiano FP, Lehman GA. Pancreatitis following bile duct sphincter of Oddi manometry: utility of the aspirating catheter. Gastrointest Endosc 1992; 38: 347-350

28 Freeman ML, DiSario JA, Nelson DB, Fennerty MB, Lee JG, Bjorkman DJ, Overby CS, Aas J, Ryan ME, Bochna GS, Shaw MJ, Snady HW, Erickson RV, Moore JP, Roel JP. Risk factors for post-ERCP pancreatitis: a prospective, multicenter study. Gastrointest Endosc 2001; 54: 425-434

29 Tarnasky PR, Palesch YY, Cunningham JT, Mauldin PD, Cotton PB, Hawes RH. Pancreatic stenting prevents pancreatitis after biliary sphincterotomy in patients with sphincter of Oddi dysfunction. Gastroenterology 1998; 115: 1518-1524

30 Fogel EL, Eversman D, Jamidar P, Sherman S, Lehman GA. Sphincter of Oddi dysfunction: pancreaticobiliary sphincterotomy with pancreatic stent placement has a lower rate of pancreatitis than biliary sphincterotomy alone. Endoscopy 2002; 34: 280-285

31 Ries L, Eisner M, Kosary C. Seer Cancer Statistics Review 1973-1996. National Cancer Institute 2000

32 Yasuda K, Sakata M, Ueda M, Uno K, Nakajima M. The use of pancreatoscopy in the diagnosis of intraductal papillary mucinous tumor lesions of the pancreas. Clin Gastroenterol Hepatol 2005; 3: S53-S57

33 Moreau JA , Zinsmeister AR, Melton LJ, DiMagno EP. Gallstone pancreatitis and the effect of cholecystectomy: a population-based cohort study. Mayo Clin Proc 1988; 63: 466-473

34 Chang L, Lo SK, Stabile BE, Lewis RJ, de Virgilio C. Gallstone pancreatitis: a prospective study on the incidence of cholangitis and clinical predictors of retained common bile duct stones. Am J Gastroenterol 1998; 93: 527-531

35 Chatzicostas C, Roussomoustakaki M, Vlachonikolis IG, Notas G, Mouzas I, Samonakis D, Kouroumalis EA. Comparison of Ranson, APACHE II and APACHE III scoring systems in acute pancreatitis. Pancreas 2002; 25: 331-335

36 Leese T, Shaw D. Comparison of three Glasgow multifactor prognostic scoring systems in acute pancreatitis. Br J Surg 1988; 75: 460-462

37 Fan ST, Lai EC, Mok FP, Lo CM, Zheng SS, Wong J. Early treatment of acute biliary pancreatitis by endoscopic papillotomy. N Engl J Med 1993; 328: 228-232

38 Neoptolemos JP, Carr-Locke DL, London NJ, Bailey IA, James D, Fossard DP. Controlled trial of urgent endoscopic

retrograde cholangiopancreatography and endoscopic sphincterotomy versus conservative treatment for acute pancreatitis due to gallstones. Lancet 1988; 2: 979-983

39 Fölsch UR, Nitsche R, Lüdtke R, Hilgers RA, Creutzfeldt W. Early ERCP and papillotomy compared with conservative treatment for acute biliary pancreatitis. The German Study Group on Acute Biliary Pancreatitis. N Engl J Med 1997; 336: 237-242

40 Frakes JT. Biliary pancreatitis: a review. Emphasizing appropriate endoscopic intervention. J Clin Gastroenterol 1999; 28: 97-109

41 Kaw M, Al-Antably Y, Kaw P. Management of gallstone pancreatitis: cholecystectomy or ERCP and endoscopic sphincterotomy. Gastrointest Endosc 2002; 56: 61-65

42 Siegel JH, Veerappan A, Cohen SA, Kasmin FE. Endoscopic sphincterotomy for biliary pancreatitis: an alternative to cholecystectomy in high-risk patients. Gastrointest Endosc 1994; 40: 573-575

43 Lau ST, Simchuk EJ, Kozarek RA, Traverso LW. A pancreatic ductal leak should be sought to direct treatment in patients with acute pancreatitis. Am J Surg 2001; 181: 411-415

44 Kozarek R , Traverso L, Bail T. Pancreatic duct leak in necrotizing pancreatitis: role of diagnostic and therapeutic ERCP as part of a multidisciplinary approach. Gastrointest Endosc 2000; 51: AB138

45 Kim HS, Lee DK, Kim IW, Baik SK, Kwon SO, Park JW, Cho NC, Rhoe BS. The role of endoscopic retrograde pancreatography in the treatment of traumatic pancreatic duct injury. Gastrointest Endosc 2001; 54: 49-55

46 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

47 Kozarek RA. Endoscopic therapy of complete and partial pancreatic duct disruptions. Gastrointest Endosc Clin N Am 1998; 8: 39-53

48 Varadarajulu S, Noone TC, Tutuian R, Hawes RH, Cotton PB. Predictors of outcome in pancreatic duct disruption managed by endoscopic transpapillary stent placement. Gastrointest Endosc 2005; 61: 568-575

49 Telford JJ, Farrell JJ, Saltzman JR, Shields SJ, Banks PA, Lichtenstein DR, Johannes RS, Kelsey PB, Carr-Locke DL. Pancreatic stent placement for duct disruption. Gastrointest Endosc 2002; 56: 18-24

50 Canty TG , Weinman D. Treatment of pancreatic duct disruption in children by an endoscopically placed stent. J Pediatr Surg 2001; 36: 345-348

51 Canty TG, Weinman D. Management of major pancreatic duct injuries in children. J Trauma 2001; 50: 1001-1007

52 Goldberg PB, Long WB, Oleaga JA, Mackie JA. Choledochocele as a cause of recurrent pancreatitis. Gastroenterology 1980; 78: 1041-1045

53 Greene FL , Brown J J , Rubinste in P , Anderson MC. Choledochocele and recurrent pancreatitis. Diagnosis and surgical management. Am J Surg 1985; 149: 306-309

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ultrasound. This problem has been overcome by integrating the ultrasound probe into an endoscope in order to place it directly into the gastric and duodenal lumen. The close proximity of the endoscopic ultrasound probe to the pancreas results in high spatial resolution that is superior to that of Computer Tomography (CT) and magnetic resonance imaging (MRI). In addition, endoscopic ultrasound (EUS) is a minimally invasive procedure that does not share the relatively high complication rate of endoscopic retrograde cholangiopancreatography (ERCP). Due to these advantages, EUS has evolved into an important technique to assess pancreatobiliary disease.

This review will discuss the role of EUS in patients with pancreatitis. The indications can be divided into acute pancreatitis and chronic pancreatitis. In acute pancreatitis, EUS is used to determine the etiology; in suspected chronic pancreatitis it is helpful to establish the diagnosis. Another indication that will be discussed is biliary pancreatitis with suspicion for persistent choledocholithiasis.

ACUTE IDIOPHATHIC PANCREATITISThe diagnosis of acute idiopathic pancreatitis (AIP) is applied when an etiology cannot be determined after the initial evaluation that includes a thorough history and physical exam, laboratory evaluation and abdominal ultrasound or CT[1-4]. In-depth evaluation of AIP using EUS often yields the diagnosis of microlithiasis, pancreatic divisum, chronic pancreatitis[1,5-7] or even neoplasm[1,4].

Occult gallstones and microlithiasis A substantial number of patients with AIP and unexplained biliary pain turn out to have biliary sludge or small gallstones that have gone undetected by abdominal ultrasound (US) or CT. The term ‘biliary microlithiasis’ was coined to describe gallstones of < 3 mm in diameter[8-10]. Although sonographic characteristics of cholelithiasis do not differ between EUS and trans-abdominal ultrasound, EUS is more sensitive in detecting gallstones[11] due to the proximity of the endoscope tip to the gallbladder. Small gallbladder stones present as bright floating foci. Larger stones have posterior shadowing. Sludge presents as hyperechoic content within the gallbladder or bile duct (Figures 1 and 2).

The reported incidence of occult gallstones in patients with AIP varies widely. It ranges from 10%-73%[12-15]. Gallstones remain the most common cause of pancreatitis in patients with intact gallbladder. Therefore, it is

TOPIC HIGHLIGHT

Utility of endoscopic ultrasound in pancreatitis: A review

Maged K Rizk, Henning Gerke

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Maged K Rizk, Henning Gerke, University of Iowa Hospitals and Clinics, Division of Gastroenterology and Hepatology, Department of Internal Medicine, 200 Hawkins Drive, Iowa City, IA 52242, United States Correspondence to: Henning Gerke, MD, Assistant Professor, Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of Iowa Hospitals and Clinics, 200 Hawkins Drive, Iowa City, IA 52242, United States. henning-gerke@uiowa.eduTelephone: +1-319-3562132 Fax: +1-319-3536399Received: June 15, 2007 Revised: August 30, 2007

AbstractThe close proximity of the endoscopic ultrasound probe to the pancreas results in superior spatial resolution compared to CT scan and MRI. In addition, endoscopic ultrasound (EUS) is a minimally invasive procedure that does not share the relatively high complication rate of ERCP. Due to these advantages, EUS has evolved into an important technique to assess pancreatobiliary disease. This review will discuss the role of EUS in patients with pancreatitis. The indications can be divided into acute pancreatitis and chronic pancreatitis. In acute pancreatitis, EUS is used to determine the etiology; in suspected chronic pancreatitis it is helpful to establish the diagnosis. Lastly, this review will discuss biliary pancreatitis with suspicion for persistent choledocholithiasis.

© 2007 WJG. All rights reserved.

Key words: Idiopathic pancreatitis; Acute pancreatitis; Chronic pancreatitis; Endoscopic ultrasound; Endosono-graphy; Pancreas divisum; Cholelithiasis; Microlithiasis; Choledocholithiasis; Biliary pancreatitis

Rizk MK, Gerke H. Utility of endoscopic ultrasound in pancreatitis: A review. World J Gastroenterol 2007; 13(47): 6321-6326

http://www.wjgnet.com/1007-9327/13/6321.asp

INTRODUCTIONOverlying intestinal gas and the retroperitoneal location of the pancreas distant from the abdominal wall can impair the visualization of this organ with trans-abdominal

Michael F Byrne, MD, Series Editor

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commonly believed that the finding of microlithiasis explains the etiology of the pancreatitis. This has recently been challenged. In a study by Garg et al. Seventy-five patients with AIP were studied with duodenal bile microscopy and EUS. Initially, the cause of the recurrent pancreatitis was attributed to biliary microlithiasis in 10 of 75 patients. Eight of these 10 patients underwent cholecystectomy or endoscopic sphincterotomy yet continued to have recurrent pancreatitis flares[16] which implies that gallstones or biliary crystals were innocent bystanders in these patients. In contrast, other studies have demonstrated response to cholecystectomy[5,17], sphincterotomy[11] or ursodeoxycholic acid (UDCA)[10] in patients with microlithiasis suggesting a causal relationship.

Liu et al prospectively evaluated 89 consecutive patients who presented with symptoms of acute pancreatitis with trans-abdominal ultrasound, CT, or both. ERCP was performed in all patients with confirmed or suspected biliary pancreatitis. EUS was performed in patients suspected of having idiopathic pancreatitis. Of the 18 patients classified as idiopathic pancreatitis who underwent EUS, 14 had stones that were between 1 and 9 mm in size which was confirmed by cholecystectomy. Three had concomitant choledocholithiasis confirmed by ERCP[17].

Another study retrospectively evaluated 31 patients with AIP who underwent EUS 2-3 wk after resolution of symptoms[5]. Five of 31 patients had microlithiasis diagnosed by EUS (n = 3), or by bile microscopy after EUS (n = 2). All 5 patients underwent cholecystectomy and remained asymptomatic during the follow-up period. Sludge was found on pathology examination in all 5 gallbladders. Gallstones or sludge were not diagnosed in any of the other 26 subjects during the follow-up period.

In summary, EUS is an effective modality in diagnosing microlithiasis and may strengthen the indication for a subsequent intervention. Treatment with cholecystectomy, endoscopic sphincterotomy or ursodeoxycholic acid may reduce recurrent attacks of pancreatitis[10,11]. However, it remains debatable how intensively we have to search for occult gallstones. Statistically, gallstones remain by far the likeliest cause of unexplained recurrent pancreatitis in patients with intact gallbladders. The morbidity of laparoscopic cholecystectomy is very low, and one could argue that this procedure is justified regardless of the findings of cross sectional imaging.

Pancreas divisumPancreas divisum is a common congenital malformation. The prevalence is estimated at 5%-10% in a Western population[18]. This abnormality is characterized by lack of connection between the dorsal and ventral pancreatic ducts due to incomplete fusion of the pancreatic buds during embryologic development. As a result, the ventral duct drains only a small portion of the pancreas via the major papilla, whereas the dorsal pancreatic duct drains the majority of the pancreas via the minor papilla. The small size of the minor papilla in relation to the drainage volume may lead to relative outflow obstruction. Since only a minority of patients with pancreas divisum becomes symptomatic, it has been suggested that symptomatic disease requires additional factors leading to minor papilla stenosis. Symptomatic patients present with recurrent acute pancreatitis, chronic pancreatitis, or chronic abdominal pain without evidence of pancreatitis. Pancreas divisum has been implicated in as much as 20% of patients with AIP[12]. Patients with discrete episodes of acute pancreatitis commonly improve after ERCP with minor papillotomy, whereas the results are less favorable for those with chronic pancreatitis or chronic abdominal pain[19] .

ERCP is the gold standard for the diagnosis of pancreas divisum but poses a risk of post procedure pancreatitis. Small series suggest that EUS enables a fairly reliable diagnosis of pancreas divisum and may therefore present an alternative to ERCP with minimal complication rate[5,7,20,21]. Different EUS-criteria have been used: Bhutani et al suggest that the absence of a "stack sign" may be useful in determining the diagnosis. The stack sign is obtained by positioning a radial echoendoscope in the long position with the transducer in the duodenal bulb. The balloon is then inflated and advanced snugly into the apex of the bulb. From this position, the bile duct and the pancreatic duct can be seen running parallel through the pancreatic head. In six patients with known pancreas divisum that underwent EUS, the stack sign was found in only two patients. Of the two patients with presence of a stack sign, one had a ventral duct that was markedly dilated, and the other patient had an unusually large ventral pancreas[20]. Tandon et al used different sonographic criteria. The authors required direct visualization of the dorsal duct coursing to the duodenal wall, and excluded patients with a sonographically visible ventral pancreatic duct. The authors feel that their criteria will exclude some

Figure 1 Linear EUS of gallbladder w i t h s h a d o w i n g stone.

Figure 2 Linear EUS of gallbladder with sludge, and bright shadowing foci representing small stones.

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cases of pancreas divisum and many cases of "incomplete pancreas divisum," but may be more specific as compared to the absence of a stack sign[5]. Lai et al suggests that evaluation using a linear-array echoendoscope is possible. The main pancreatic duct can be followed continuously from the major papilla into the pancreatic body. The duct can be seen crossing a sonographic border between the ventral and dorsal pancreas. Absence of this feature suggests pancreas divisum. In the retrospective study, of the 78% who had adequate visualization of the pancreatic duct, sensitivity, specificity, positive and negative predictive values for EUS were 95%, 97%, 86%, and 99%, respectively[21].

Occult neoplasmIt has been estimated that pancreatic neoplasms cause pancreatitis at some point in the disease course in up to 7 percent of patients[22]; however, they are a rare differential diagnosis in patients with AIP.

Mujica et al surveyed 19 physicians regarding 45 patients who presented with acute pancreatitis prior to a diagnosis of a neoplasm. The patients had a mean number of 2 episodes of acute pancreatitis prior to the diagnosis of neoplasm. The mean time to diagnosis of the neoplasm after the initial episode was 34 wk. The majority of patients were diagnosed using conventional cross-sectional imaging, whereas only 3 patients in the series were diagnosed using EUS[22].

Albeit rare, it has been suggested that pancreatic malignancy should be suspected in patients with unexplained pancreatitis who are older than 40 years of age[23]. EUS is superior to CT in detecting small pancreatic neoplasms[24,25], however, inflammatory changes during a pancreatitis flare may decrease the image quality. Therefore, cross-sectional imaging and/or EUS should be repeated after the resolution of the acute attack.

Single episode of idiopathic pancreatitisThe utility of an evaluation with EUS after a single episode of unexplained pancreatitis is not well studied and remains unclear[4]. In a small series by Tandon et al, EUS found an etiology in 7 of 14 patients with a single episode of idiopathic pancreatitis (3 microlithiasis, 1 pancreas divisum, 3 alcoholic chronic pancreatitis). The diagnosis changed in only 1 patient[23] during the follow-up period. A series reported by Yusoff et al included 201 patients with a single episode of acute pancreatitis. A presumptive diagnosis was made after EUS in 31%; chronic pancreatitis and sludge were the most common diagnoses in those with a gallbladder, whereas chronic pancreatitis and pancreatic divisum were the most prevalent diagnoses in patients who had a prior cholecystectomy[6].

Although these studies suggest a high yield of EUS in patients with a single episode of unexplained pancreatitis, some skepticism remains. Only 20%-50% of patients will have recurrent symptoms[26] following the initial attack. Furthermore, it is difficult to be sure about the causal relationship of an abnormal EUS finding after a single episode of pancreatitis. Pancreas divisum, for example, is common in the general population, and

may be a coincidental finding rather than the cause of the pancreatitis. Even microlithiasis may be a harmless bystander[16]. As discussed in detail in a later paragraph, the diagnosis of chronic pancreatitis with EUS is problematic due to lack of specificity in early stages. In our opinion, further studies are necessary before advocating EUS for every patient after a single episode of idiopathic pancreatitis.

CHRONIC PANCREATITISThe diagnosis of chronic pancreatitis (CP) can be challenging. The normal pancreas has a homogeneous fine granular echo-pattern (salt and pepper appearance), with a thin and regular main pancreatic duct. Certain sonographic changes can be observed in patients with CP. In an attempt to develop diagnostic scores for the EUS-diagnosis of chronic pancreatitis, "EUS criteria" have been developed. These were first described by Jones et al[27], and later refined by Wiersema et al[28]. The criteria can be divided into pancreatic duct findings and parenchymal findings. Parenchymal findings include hyperechoic foci, hyperechoic strands, lobularity, heterogeneity, shadowing calcifications, and cysts. Pancreatic duct findings include dilation (> 4 mm in the head, > 3 mm in the body, > 2 mm in the tail), irregularity, hyperechoic duct margins, and visible side-branches (Table 1, Figures 3-6). Multiple studies have evaluated the ability of EUS to diagnose CP using the above criteria. In a prospective, blinded study by Sahai et al, 126 patients who were admitted for abdominal pain underwent ERCP followed by EUS performed by a blinded operator. ERCP diagnosis of CP was based on Cambridge Criteria. EUS sensitivity was uniformly greater than 85% when the diagnosis of CP was based on the

Table 1 EUS criteria of chronic pancreatitis

Parenchymal criteria Pancreatic ductal criteria

Hyperechoic foci Dilation (4 mm in head, 3 mm in body, 2 mm in tail)

Hyperechoic strands IrregularityLobularity Hyperechoic duct marginsHeterogeneity Visible branch ductsShadowing calcifications Intraductal stonesCysts

Figure 3 Linear E U S s h o w i n g a shadowing stone w i t h i n t h e p a n -creat ic duct (PD STONE).

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presence of fewer than three criteria, but the specificity was less than 60%. Specificity increased as the number of criteria increased and was greater than 85% when more than five criteria were used. "Moderate to severe chronic pancreatitis" was unlikely (NPV > 85%) when fewer than three criteria were present[29]. When criteria that can easily be detected by other imaging methods (ductal dilation, calcification, and cysts) were excluded, the number of parenchymal EUS criteria remained an independent predictor of CP[29].

There are nuances that need to be considered when using the above score. Firstly, the role of ERCP as a diagnostic "gold-standard" is debatable[30]. Thus, it is difficult to determine whether EUS is over-diagnosing pancreatic disease based on minimal changes or whether ERCP is a false negative in those with abnormal EUS findings but normal ERCP. In a study by Kahl et al[31], 32 patients with abnormal EUS but normal initial pancreatogram developed findings of CP on repeat ERCP after a median follow-up of 18 mo suggesting that EUS findings may precede ERCP findings. The sensitivity to diagnose chronic pancreatitis was 100% for EUS, but only 81% for ERCP.

Another concern when using an EUS scoring system to diagnose CP is that not all criteria may be equally important. For example, the presence of intraductal calcifications or parenchymal calcifications alone may be diagnostic of CP even in the absence of other criteria[30]. Age related changes in the pancreas may also affect the diagnostic threshold. The pancreatic duct becomes progressively wider with a hyperechoic wall with increased age. Another aspect to consider is interobserver

variability of different criteria. Wiersema et al[28] found excellent interobserver agreement among 3 experienced endosonographers reading individual criteria of CP. There was 88% interobserver agreement on presence of echogenic foci, 94% agreement on focally reduced echogenicity, 94% agreement on lobular gland pattern, 83% agreement on the main pancreatic duct echogenicity, and 94% agreement on main pancreatic duct irregularity. On the contrary, Wallace et al [32] could not confirm these optimistic results. EUS-exams on 33 patients with suspected CP and 12 controls without suspected CP were videotaped by 3 experienced endosonographers. Eleven expert endosonographers, who were blinded to clinical information, independently evaluated the examinations for the presence of CP and were asked to rank the importance of individual EUS features. There was moderately good interobserver agreement in the final diagnosis of CP (Kappa = 0.45).

Interobserver agreement was good for the individual criteria "ductal dilation" and "lobularity" but was poor for the other 7 criteria. The presence of stones was regarded as the most predictive feature of CP by all endosonographers, followed by visible side branches, cysts, lobularity, irregular main pancreatic duct, hyperechoic strands, main pancreatic duct dilation and main duct hyperechoic margins[32].

In our opinion, the early diagnosis of CP remains problematic due to lack of specificity and the presence of interobserver variability. The overall interpretation of the experienced endosonographer may be more valuable than a diagnosis based on a scoring system.

Only a few studies have evaluated the utility of biopsy in addition to EUS for the diagnosis of CP. One small study suggested that fine needle aspiration may improve the negative predictive value but not the specificity of EUS[33], however this study was limited by the small number of patients without chronic pancreatitis. Out of 37 patients, 31 had chronic pancreatitis. Only 4 patients had normal EUS findings, 3 without and one with chronic pancreatitis (negative predictive value of 75%). The negative predictive value was improved to 100% by FNA-cytology. In our opinion, it is difficult to draw conclusions based on such small numbers. Another study found that EUS-guided core biopsies with a Trucut needle was poor at diagnosing CP[34].

In conclusion, current data do not support a role of EUS-guided biopsies in the diagnosis of CP. In addition

Figure 5 Linear trans-gastric EUS showing a smal l p a n c r e a t i c c y s t (labeled with mea-surement markers).

Figure 6 Linear EUS of pancreatic body with echoge-n i c s t rands and lobularity.

Figure 4 Linear trans-gastric EUS o f the pancreas showing parench-ymal calcifications (CALC) caus ing d i la ta t ion o f the upstream pancreatic duct (PD).

PD

CALC

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to their questionable diagnostic value, pancreatic biopsies carry a potential risk of post-procedure pancreatitis.

CP makes the detection of pancreatic cancer more difficult. In a series of 282 patients with pancreatic mass (210 with adenocarcinoma), a lower sensitivity for EUS-FNA was observed in patients with CP (more than 4 EUS-criteria) than in those without CP (73.9% vs 91.3%). Patients with CP required more EUS-FNA passes to establish a diagnosis versus those without CP (5 vs 2)[35].

In summary, the diagnosis of CP remains challenging. EUS criteria have been established. Although these criteria are highly sensitive, they lack specificity in early stages. EUS is accurate in ruling out CP if no pancreatic abnormalities are found and in diagnosing CP if multiple criteria are present. However, a wide grey zone remains for patients with minimal to moderate findings.

CP decreases the sensitivity of EUS-FNA in the evaluation of pancreatic masses.

BILIARY PANCREATITIS AND CHOLEDOCHOLITHIASISIn most patients with biliary pancreatitis, the causal gallstone has already passed. This makes it difficult to iden t i f y those pa t i en t s in whom ERCP wi th sphincterotomy may be beneficial. In this context, EUS may provide a minimally invasive modality to diagnose or exclude choledocholithiasis. A review of five studies by Verma et al evaluating the efficacy of different modalities in diagnosing choledocholithiasis found an aggregated sensitivity of EUS of 0.93, a specificity of 0.96, a positive predictive value of 0.93, and a negative predictive value of 0.96. There was no statistical difference between MRCP and EUS[36]. In a study by Lui et al[37], 100 patients admitted for acute pancreatitis were evaluated with trans-abdominal ultrasound, EUS, and ERCP. EUS was found to be as sensitive as ERCP in the detection of choledocholithiasis, but with a lower complication rate.

Arguedas et al proposed a decision analysis model in evaluating biliary pancreatitis. Cost-effectiveness of strategies involving observation, intraoperative cholangiography, EUS, MRCP, and ERCP was evaluated. The results demonstrated that the choice of strategy is strongly influenced by the pretest probability of choledocholithiasis. If cost-minimization is the goal, observation with intraoperative cholangiography at the time of cholecystectomy is preferred in patients considered at "low risk" for choledocholithiasis. EUS is cost effective in patients at "intermediate risk" and ERCP is the preferable strategy in patients at "high-risk". There was no utility for MRCP in this model, as EUS was less costly[38]. Scheiman et al[39], also suggested that there is no role for MRCP for biliary pancreatitis in centers where EUS is available.

Sugiyama et al prospectively evaluated 35 patients with suspected acute biliary pancreatitis. All patients underwent trans-abdominal ultrasound, CT, EUS, and ERCP. The severity of pancreatitis was graded using APACHE Ⅱ scores. EUS and ERCP were significantly more sensitive in the detection of CBD stones than trans-abdominal

ultrasound and CT. ERCP and EUS were equivalent in CBD stone detection. Based on the severity of the pancreatitis, 20 of 35 ERCP were determined to be potentially avoidable[38].

In summary, EUS is both sensitive and specific in the detection of common bile duct stones and has a considerably lower complication rate than ERCP. While patients with high likelihood of cholelithiasis should undergo ERCP directly, EUS may enable selective use of ERCP in those with intermediate likelihood[1,3,7,10,38,39].

CONCLUSIONEUS is helpful in the evaluating patients with AIP and in diagnosing CP. In patients with AIP, EUS enables the diagnosis of occult cholelithiasis, pancreas divisum, chronic pancreatitis or an occult neoplasm. While EUS may be more sensitive than ERCP in diagnosing CP, the specificity is limited in early stages. In biliary pancreatitis, EUS allows accurate detection of common bile duct stones and can be used to select patients who will benefit from ERCP.

REFERENCES1 Norton SA, Alderson D. Endoscopic ultrasonography in the

evaluation of idiopathic acute pancreatitis. Br J Surg 2000; 87: 1650-1655

2 Frossard JL, Sosa-Valencia L, Amouyal G, Marty O, Hadengue A, Amouyal P. Usefulness of endoscopic ultrasonography in patients with "idiopathic" acute pancreatitis. Am J Med 2000; 109: 196-200

3 Draganov P , Forsmark CE. "Idiopathic" pancreatitis. Gastroenterology 2005; 128: 756-763

4 Levy MJ, Geenen JE. Idiopathic acute recurrent pancreatitis. Am J Gastroenterol 2001; 96: 2540-2555

5 Tandon M, Topazian M. Endoscopic ultrasound in idiopathic acute pancreatitis. Am J Gastroenterol 2001; 96: 705-709

6 Yusoff IF, Raymond G, Sahai AV. A prospective comparison of the yield of EUS in primary vs. recurrent idiopathic acute pancreatitis. Gastrointest Endosc 2004; 60: 673-678

7 Coyle WJ, Pineau BC, Tarnasky PR, Knapple WL, Aabakken L, Hoffman BJ, Cunningham JT, Hawes RH, Cotton PB. Evaluation of unexplained acute and acute recurrent pancreatitis using endoscopic retrograde cholangiopancreatography, sphincter of Oddi manometry and endoscopic ultrasound. Endoscopy 2002; 34: 617-623

8 Levy MJ. The hunt for microlithiasis in idiopathic acute recurrent pancreatitis: should we abandon the search or intensify our efforts? Gastrointest Endosc 2002; 55: 286-293

9 Rashdan A, Fogel E, McHenry L, Lehman G, Sherman S. Frequency of biliary crystals in patients with suspected sphincter of Oddi dysfunction. Gastrointest Endosc 2003; 58: 875-878

10 Saraswat VA, Sharma BC, Agarwal DK, Kumar R, Negi TS, Tandon RK. Biliary microlithiasis in patients with idiopathic acute pancreatitis and unexplained biliary pain: response to therapy. J Gastroenterol Hepatol 2004; 19: 1206-1211

11 Mirbagheri SA, Mohamadnejad M, Nasiri J, Vahid AA, Ghadimi R, Malekzadeh R. Prospective evaluation of endoscopic ultrasonography in the diagnosis of biliary microlithiasis in patients with normal transabdominal ultrasonography. J Gastrointest Surg 2005; 9: 961-964

12 Kaw M, Brodmerkel GJ. ERCP, biliary crystal analysis, and sphincter of Oddi manometry in idiopathic recurrent pancreatitis. Gastrointest Endosc 2002; 55: 157-162

13 Lee SP, Hayashi A, Kim YS. Biliary sludge: curiosity or

Rizk MK et al. EUS and pancreatitis 6325

www.wjgnet.com

culprit? Hepatology 1994; 20: 523-52514 Ros E , Navarro S, Bru C, Garcia-Pugés A, Valderrama

R. Occult microlithiasis in 'idiopathic' acute pancreatitis: prevention of relapses by cholecystectomy or ursodeoxycholic acid therapy. Gastroenterology 1991; 101: 1701-1709

15 Venu RP, Geenen JE, Hogan W, Stone J, Johnson GK, Soergel K. Idiopathic recurrent pancreatitis. An approach to diagnosis and treatment. Dig Dis Sci 1989; 34: 56-60

16 Garg PK, Tandon RK, Madan K. Is biliary microlithiasis a significant cause of idiopathic recurrent acute pancreatitis? A long-term follow-up study. Clin Gastroenterol Hepatol 2007; 5: 75-79

17 Liu CL, Lo CM, Chan JK, Poon RT, Fan ST. EUS for detection of occult cholelithiasis in patients with idiopathic pancreatitis. Gastrointest Endosc 2000; 51: 28-32

18 Dhar A, Goenka MK, Kochhar R, Nagi B, Bhasin DK, Singh K. Pancrease divisum: five years' experience in a teaching hospital. Indian J Gastroenterol 1996; 15: 7-9

19 Gerke H, Byrne MF, Stiffler HL, Obando JV, Mitchell RM, Jowell PS, Branch MS, Baillie J. Outcome of endoscopic minor papillotomy in patients with symptomatic pancreas divisum. JOP 2004; 5: 122-131

20 Bhutani MS, Hoffman BJ, Hawes RH. Diagnosis of pancreas divisum by endoscopic ultrasonography. Endoscopy 1999; 31: 167-169

21 Lai R, Freeman ML, Cass OW, Mallery S. Accurate diagnosis of pancreas divisum by linear-array endoscopic ultrasonography. Endoscopy 2004; 36: 705-709

22 Mujica VR, Barkin JS, Go VL. Acute pancreatitis secondary to pancreatic carcinoma. Study Group Participants. Pancreas 2000; 21: 329-332

23 Wilcox CM, Varadarajulu S, Eloubeidi M. Role of endoscopic evaluation in idiopathic pancreatitis: a systematic review. Gastrointest Endosc 2006; 63: 1037-1045

24 Rösch T , Lorenz R, Braig C, Feuerbach S, Siewert JR, Schusdziarra V, Classen M. Endoscopic ultrasound in pancreatic tumor diagnosis. Gastrointest Endosc 1991; 37: 347-352

25 Hunt GC, Faigel DO. Assessment of EUS for diagnosing, staging, and determining resectability of pancreatic cancer: a review. Gastrointest Endosc 2002; 55: 232-237

26 Gullo L, Migliori M, Pezzilli R, Oláh A, Farkas G, Levy P, Arvanitakis C, Lankisch P, Beger H. An update on recurrent acute pancreatitis: data from five European countries. Am J Gastroenterol 2002; 97: 1959-1962

27 Jones SN, Lees WR, Frost RA. Diagnosis and grading of chronic pancreatitis by morphological criteria derived by ultrasound and pancreatography. Clin Radiol 1988; 39: 43-48

28 Wiersema MJ , Hawes RH, Lehman GA, Kochman ML, Sherman S , Kopecky KK. Prospect ive evaluat ion of

endoscopic ultrasonography and endoscopic retrograde cholangiopancreatography in patients with chronic abdominal pain of suspected pancreatic origin. Endoscopy 1993; 25: 555-564

29 Sahai AV , Zimmerman M, Aabakken L, Tarnasky PR, Cunningham JT, van Velse A, Hawes RH, Hoffman BJ. Prospective assessment of the ability of endoscopic ultrasound to diagnose, exclude, or establish the severity of chronic pancreatitis found by endoscopic retrograde cholangiopancreatography. Gastrointest Endosc 1998; 48: 18-25

30 Raimondo M , Wallace MB. Diagnosis of early chronic pancreatitis by endoscopic ultrasound. Are we there yet? JOP 2004; 5: 1-7

31 Kahl S, Glasbrenner B, Zimmermann S, Malfertheiner P. Endoscopic ultrasound in pancreatic diseases. Dig Dis 2002; 20: 120-126

32 Wallace MB, Hawes RH, Durkalski V, Chak A, Mallery S, Catalano MF, Wiersema MJ, Bhutani MS, Ciaccia D, Kochman ML, Gress FG, Van Velse A, Hoffman BJ. The reliability of EUS for the diagnosis of chronic pancreatitis: interobserver agreement among experienced endosonographers. Gastrointest Endosc 2001; 53: 294-299

33 Hollerbach S, Klamann A, Topalidis T, Schmiegel WH. Endoscopic ultrasonography (EUS) and fine-needle aspiration (FNA) cytology for diagnosis of chronic pancreatitis. Endoscopy 2001; 33: 824-831

34 DeWitt J, McGreevy K, LeBlanc J, McHenry L, Cummings O, Sherman S. EUS-guided Trucut biopsy of suspected nonfocal chronic pancreatitis. Gastrointest Endosc 2005; 62: 76-84

35 Varadarajulu S, Tamhane A, Eloubeidi MA. Yield of EUS-guided FNA of pancreatic masses in the presence or the absence of chronic pancreatitis. Gastrointest Endosc 2005; 62: 728-736; quiz 751, 753

36 Verma D, Kapadia A, Eisen GM, Adler DG. EUS vs MRCP for detection of choledocholithiasis. Gastrointest Endosc 2006; 64: 248-254

37 Liu CL, Lo CM, Chan JK, Poon RT, Lam CM, Fan ST, Wong J. Detection of choledocholithiasis by EUS in acute pancreatitis: a prospective evaluation in 100 consecutive patients. Gastrointest Endosc 2001; 54: 325-330

38 Arguedas MR, Dupont AW, Wilcox CM. Where do ERCP, endoscopic ultrasound, magnetic resonance cholangiopancreatography, and intraoperative cholangiography fit in the management of acute biliary pancreatitis? A decision analysis model. Am J Gastroenterol 2001; 96: 2892-2899

39 Scheiman JM, Carlos RC, Barnett JL, Elta GH, Nostrant TT, Chey WD, Francis IR, Nandi PS. Can endoscopic ultrasound or magnetic resonance cholangiopancreatography replace ERCP in patients with suspected biliary disease? A prospective trial and cost analysis. Am J Gastroenterol 2001; 96: 2900-2904

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have profound treatment and prognostic implications, distinguished by the rapid reversal of its characteristic lesions with corticosteroid therapy.

DEFINITION AND NOMENCLATUREWith mounting evidence suggesting an underlying au to immune mechan i sm, the te r m auto immune pancreatitis was originally introduced by Yoshida et al[2] in 1995. Characterized histologically by a predominant lymphoplasmacytic infiltrate and fibrosis that can lead to both endocrine and exocrine dysfunction, autoimmune pancreatitis likely accounts for a significant proportion of cases previously classified as idiopathic pancreatitis. Various disease descriptors have been previously proposed such as chronic sclerosing pancreatitis[3], lymphoplasmacytic sclerosing pancreatitis with cholangitis[4], sclerosing pancreatocholangitis[5], non-alcoholic duct-destructive chronic pancreat i t i s [6], chronic pancreat i t i s wi th irregular narrowing of the main pancreatic duct[7], and pseudotumorous pancreatitis[8]. Retrospectively, these various descriptors likely describe the entity that we now refer to as autoimmune pancreatitis, however focused on its specific radiologic or histologic findings. As will be discussed further, although autoimmune pancreatitis is now the preferred term, its clinical, biochemical, radiologic, and pathologic findings are heterogeneous.

DEMOGRAPHICS AND EPIDEMIOLOGYDespite the increasing cases of autoimmune pancreatitis being reported around the world, its true prevalence and incidence have yet to be determined. Three case series have reported prevalence rates of 4% to 6% of all patients diagnosed with chronic pancreatitis[9,10]. The mean age of diagnosis is 55, this however can vary with cases presenting from 30 to 70 years of age[3,6,11,12]. A male predilection of 1.7:1-2:1 has been reported in three surgical series[12-14]. Considering its postulated autoimmune pathogenesis, it has been associated with Sjogren's syndrome[15,16], rheumatoid arthritis[14], primary sclerosing cholangitis[16,17], retroperitoneal fibrosis[18] and inflammatory bowel disease[19]. Although the prevalence of a concurrent autoimmune diagnosis has been reported in various series, they are likely underestimates as some diagnoses of autoimmune pancreatitis may precede the diagnosis of the concurrent autoimmune condition[9,10,20].

PATHOGENESIS

TOPIC HIGHLIGHT

Autoimmune pancreatitis: A review

Iman Zandieh, Michael F Byrne

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Iman Zandieh, Michael F Byrne, Division of Gastroenterology, Department of Medicine, University of British Columbia, Vancouver, British Columbia, CanadaCorrespondence to: Dr. Michael F Byrne, Vancouver General Hospital, Division of Gastroenterology, 5th Floor-2775 Laurel Street, Vancouver, British Columbia, Canada, V5Z 1M9, Canada. michael.byrne@vch.caTelephone: +1-604-8755640 Fax: +1-604-8755447Received: June 14, 2007 Revised: August 30, 2007

AbstractAutoimmune pancreatitis has emerged over the last 40 years from a proposed concept to a well established and recognized entity. As an efficient mimicker of pancreatic carcinoma, its early and appropriate recognition are crucial. With mounting understanding of its pathogenesis and natural history, significant advances have been made in the diagnosis of autoimmune pancreatitis. The characteristic laboratory features and imaging seen in autoimmune pancreatitis are reviewed along with some of the proposed diagnostic criteria and treatment algorithms.

© 2007 WJG. All rights reserved.

Key words: Autoimmune pancreatitis; Chronic pancreatitis; Idiopathic pancreatitis; Sclerosing pancreatitis

Zandieh I, Byrne MF. Autoimmune pancreatitis: A review. World J Gastroenterol 2007; 13(47): 6327-6332

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INTRODUCTIONIn a fashion similar to other solid organs, the pancreas has been linked with autoimmune disease in the form of "autoimmune pancreat i t i s" . The concept was originally postulated by Sarles et al[1] over 40 years ago, when non-alcoholic pancreatitis was associated with hypergammaglobulinemia. However, only in the last 10 years has this clinical entity been firmly established in the list of diseases affecting the pancreas and thus appropriately gained significant attention. An efficient mimicker of pancreatic carcinoma on both clinical and radiologic grounds, its early and accurate diagnosis can

Michael F Byrne, MD, Series Editor

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Although the precise etiology and pathogenesis of autoimmune pancreatitis remains unknown, mounting evidence supports an autoimmune cause. Similar to other autoimmune diseases, an association of the HLA haplotype DRB1*0405-DQB1*0401 with autoimmune pancreatitis in the Japanese population has been discovered[21]. Significant autoimmune markers of the disease include its association with other autoimmune diseases, a predominant lymphoplasmacytic infiltrate on histology, hypergammaglobulinemia, elevated IgG4 levels and the presence of autoantibodies. A number of autoantibodies including antinuclear antibody (ANA), antismooth muscle antibody (ASMA), rheumatoid factor, antilactoferrin antibody (ALF) and anticarbonic anhydrase Ⅱ antibody (CA-Ⅱ) have been frequently detected in patients with autoimmune pancreatitis[22]. Both CA-Ⅱ and ALF are found in the normal pancreas, with CA-Ⅱ located in duct cells and ALF being found in the acinar cells. However, they are also distributed in the cells of several other organs including the lactating breast, biliary ducts, distal renal tubules, and salivary, bronchial and gastric glands. Thus potentially explaining some of the extrapancreatic sequelae of autoimmune pancreatitis.

As in Sjogren's syndrome or primary sclerosing cholangitis, CD4+ T-cells inducing a Th1 type of immune response are predominantly involved in the development of autoimmune pancreatitis as effector cells over Th2 type CD4+ T-cells [22,23]. Further strengthening this discovery, an animal model of autoimmune pancreatitis, using neonatally thymectomized mice immunized with CA-Ⅱ or ALF found that CD4+ Th1 cells are mainly involved in the early development of murine autoimmune pancreatitis[24]. Pancreatic specimens from this mouse model revealed histologic features consistent with those seen in autoimmune pancreatitis in humans. Therefore, an autoimmune reaction against CA-Ⅱ or ALF via CD4+ Th1 T-cells has a role in the early development of autoimmune pancreatitis. However many questions remain regarding the underlying triggering event and precise mechanisms leading to autoimmune pancreatitis. CLINICAL CHARACTERISTICSThe present ing symptoms are var iable and most commonly include painless jaundice, weight loss and abdominal pain. Although common, abdominal pain tends to be mild and variable in duration, usually lasting weeks to months. Patients rarely present with acute attacks of pain, more typical of acute pancreatitis. Jaundice has been reported in up to 70%-80% of patients in some series and is usually due to an accompanying stricture in the distal common bile duct[25]. Considering the patient demographics and common presenting symptoms, it is clear why many patients are initially diagnosed with pancreatobiliary malignancies and prior to the recognition of autoimmune pancreatitis would undergo unnecessary invasive procedures.

In some series, up to 60% of patients with autoimmune pancreatitis have diabetes mellitus[26,27]. The precise pathogenesis is unclear however different theories have

been proposed. The majority are felt to manifest as type 2 diabetics with a degree of impaired glucose tolerance, however a proportion have been found to have antibodies to islet cells and glutamic decarboxylase and thus can manifest as Type 1 diabetics. It has been reported that a proportion of patients with autoimmune pancreatitis associated diabetes mellitus improve following steroid therapy[28].

The presence of potential extrapancreatic target antigens in the lungs, breasts and kidneys can also lead to clinical manifestations. Breast, renal and pulmonary inflammatory masses have been detected in patients with autoimmune pancreatitis and found to be composed of a lymphoplasmacytic infiltrate containing numerous IgG4 positive plasma cells[29,30]. Cases of autoimmune pancreatitis associated with interstitial pneumonia and mild renal failure due to interstitial nephritis have also been reported[31-33].

DIAGNOSIS-DIAGNOSTIC IMAGINGDiagnostic imaging plays a critical role in the diagnosis of autoimmune pancreatitis. It is often the first investigative modality that raises the possibility of autoimmune pancreatitis, based on characteristic imaging features. As a result, both radiologists and non-radiologists need to be familiar with its unique features on ultrasound, computed tomography and endoscopic retrograde cholangio-pancreatography.

UltrasoundAlthough abdominal ultrasonography is a commonly ordered initial radiologic investigation for painless jaundice, overlying bowel gas and obesity greatly limit its ability to accurately visualize the pancreas. Furthermore, ultrasound findings in patients with autoimmune pancreatitis are non-specific and include many features commonly seen in other types of acute and chronic pancreatitis. The role of contrast-enhanced ultrasonography is evolving and may prove to be an important modality in the differentiation of pancreatic lesions[34].

Computed tomography (CT)In patients with diffuse pancreatic involvement, the findings on CT usually consist of a diffusely enlarged pancreas, commonly referred to as 'sausage-like' or 'bulky', which correlates with the pathologic finding of marked stromal edema on gross examination (Figure 1). Focal involvement typically appears as a mass, most commonly involving the head of the pancreas, with low attenuation or isoattenuation. Peri-pancreatic fat infiltration is uncommon. Pancreatic parenchymal calcification and intraductal stones, commonly seen in chronic pancreatitis, are also rarely observed in autoimmune pancreatitis. Mild peri-pancreatic lymphadenopathy and loss of parenchymal lobularity are commonly seen.

Another distinguishing feature of autoimmune pancreatitis is delayed enhancement of the pancreatic parenchyma. The pancreas tends to appear hypodense in comparison to the spleen on the arterial enhanced

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phase with increasing attenuation in the delayed phase images. It has been proposed that delayed enhancement is a reflection of the heterogeneous lymphoplasmacytic infiltrate and fibrosis observed on histologic examination of the pancreas[35]. Finally, a capsule-like low density rim can also be seen surrounding the pancreas on both arterial and delayed images. This may represent clear demarcation of the inflammatory process in autoimmune pancreatitis from the surrounding peripancreatic fat, due to the absence of peripancreatic fat infiltration.

Magnetic resonance cholangio-pancreatography (MRCP)The role of MRCP continues to evolve, especially as a non-invasive modality in assessing pancreatobiliary ductal anatomy. Its role however in autoimmune pancreatitis remains limited, primarily because the lack of exogenous contrast and lower resolution compared to ERCP, greatly limit its ability to detect the characteristic pancreatic duct changes.

Endoscopic retrograde cholangio-pancreatography (ERCP)The hallmark findings on direct pancreatography are focal, segmental, or diffuse narrowing of the main pancreatic duct (Figure 2)[36]. The pattern and degree of ductal narrowing is a direct result of ductal compression due to the heterogenous lymphoplasmacytic infiltrate and fibrosis affecting the gland. Although uncommon, the focal variant of autoimmune pancreatitis represents a diagnostic challenge because its findings are quite similar to those expected in pancreatic cancer. Usually associated with a mass in the head of the pancreas on CT, the focal type of autoimmune pancreatitis results in localized stenosis of the main pancreatic duct and upstream dilatation.

In the 'segmental' variant, multiple strictures affecting different sections of the pancreatic dust are visualized with intervening duct that appears non-dilated. In the 'diffuse' form, the entire pancreatic duct is narrowed. Rather than representing distinct variants of autoimmune pancreatitis, some reports have documented progression of the segmental form to a more diffuse appearance on ERCP without therapy[12]. Thus suggesting that the different patterns of ductal narrowing are primarily a result of the timing of ERCP and represent the spectrum of the disease

rather than distinct entities[20,36].Other potential ERCP findings in autoimmune

pancreatitis include narrowing of the intrapancreatic portion of the common bile duct and irregular narrowing of extrahepatic bile ducts. Although uncommon, dilatation of the intrahepatic bile ducts has also been reported[36].

Endoscopic ultrasonography (EUS)Due to its ability to accurately visualize the pancreas and perform fine needle aspiration, EUS is a crucial modality in the diagnosis of autoimmune pancreatitis. The absence of exogenous contrast makes it an important diagnostic test, even prior to CT, especially in women of child-bearing age. The findings seen in autoimmune pancreatitis closely resemble those found on CT, including a hypoechoic gland with focal or diffuse parenchymal swelling. Above all, the ability to perform tissue acquisition either by fine needle aspiration or core biopsy further strengthens the role of EUS in the diagnosis of this elusive entity.

DIAGNOSIS-PATHOLOGYThe hallmark histologic finding of autoimmune pancreatitis includes a dense lymphoplasmacytic infiltrate and fibrosis (Figure 3). The infiltrate is often heterogeneous in its distribution throughout the gland and its degree of cellularity. Although consisting predominantly of lymphocytes, the cellular infiltrate also contains neutrophils and eosinophils. Studies using immunohistochemistry have further defined that the lymphocyte population consists predominantly of CD4+ T lymphocytes with a smaller but detectable population of CD8+T cells and B cells[24].

Another important histologic finding is the presence of periductal inflammation, usually involving medium-sized and large interlobular ducts (Figure 4)[37]. Sometimes referred to as a 'collar' of inflammation, it consists predominantly of plasma cells and lymphocytes, sometimes forming germinal centers. The infiltrate is primarily subepithelial, with the epithelium only rarely being infiltrated by lymphocytes. It completely encompasses the duct leading to luminal narrowing by way of epithelial infolding and gives the lumen a star-like appearance[24]. Periphlebitis and perineural inflammation are also often

Figure 1 Contrast enhanced CT showing sausage-like swelling of the pancreatic tail along with a surrounding low attenuation rim (arrows).

Figure 2 Direct pancreatography delineating a pancreatic duct with multiple segments of narrowing (arrows).

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observed (Figure 5)[12,30].Finally, an important histologic observation in

the pathologic diagnosis of autoimmune pancreatitis is the absence of ductal dilatation, calcifications and proteinaceous plugs, commonly found in chronic pancreatitis.

DIAGNOSTIC CRITERIAConsidering the difficulty in accurately differentiating autoimmune pancreatitis from other forms of chronic pancreatitis and pancreatic carcinoma, a number of diagnostic criteria have been proposed. The earliest was proposed by the Japanese Pancreas Society and is outlined in Table 1[38]. It is based on three criteria encompassing imaging, laboratory and histopathologic findings. The mandatory imaging findings include diffuse narrowing of the pancreatic duct involving at least one third of the entire length along with pancreatic enlargement. One of the two 'minor' laboratory or pathologic criteria also needs to be present for the diagnosis. Aparisi et al[39] proposed a different scoring system based on clinical presentation, laboratory parameters and morphologic findings. More recently, investigators have specifically included an elevation in the IgG4 level and response to steroids as supporting criteria for the diagnosis of autoimmune

pancreatitis[11,40]. Kim et al [11] retrospectively applied the original

diagnostic criteria of the Japanese Pancreas Society to a series of 28 patients diagnosed with autoimmune pancreatitis who had responded to steroid therapy. The original diagnosis required the presence of diffuse enlargement of the pancreas and diffuse or segmental ir regular nar rowing of the main pancreat ic duct . Supporting criteria included elevated levels of IgG and/or IgG4, the presence of autoantibodies and histopathologic findings. Despite the absence of laboratory abnormalities or histopathologic findings in some patients, steroid therapy was initiated if the imaging criteria were met. The authors concluded that had they strictly applied the diagnostic criteria of the Japanese Pancreas Society, 9 of 28 patients would have not been diagnosed with autoimmune pancreatitis and appropriately treated. Seven of the 9 patients would have been missed because the extent of ductal narrowing was less than one third of the entire length of the main pancreatic duct. Another two patients had normal IgG levels, absence of autoantibodies and non-diagnostic pancreatic histopathology. As a result, the authors revised the diagnostic criteria by including the response to steroids and association with other autoimmune diseases as supporting criteria. They also abolished the need for more than one third of the pancreatic duct to be affected.

Although none of the proposed diagnostic criteria have been validated, they underscore the highly characteristic

Figure 3 Diffuse pancreatic lymphoplasmacytic infiltrate with early fibrosis and atrophy.

Figure 4 Small pancreatic duct surrounded by a cuff of lymphocytes and plasma cells that is extending into the atrophic pancreatic parenchyma.

Figure 5 Medium sized artery with infiltration of the vessel wall by lymphocytes and plasma cells.

Table 1 Diagnostic criteria for autoimmune pancreatitis proposed by the Japanese Pancreas Society

A Imaging criterion: Diffuse narrowing of the main pancreatic duct with an irregular wall (more than 1/3 the length of the entire pancreatic duct) and enlargement of the pancreas

B Laboratory criterion: Abnormally elevated levels of serum gamma-globulin and/or IgG, or the presence of autoantibodies

C Histopathologic criterion: Marked lymphoplasmacytic infiltration and dense fibrosis

For the diagnosis, criterion A must be present together with criterion B and/or C

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imaging findings in autoimmune pancreatitis. Finally, the histopathologic findings which have previously been felt to represent the gold standard for the diagnosis can be absent on specimens due to the patchy distribution of the disease and have also been seen in patients with alcohol induced chronic pancreatitis[30].

TREATMENT AND PROGNOSISCorticosteroids are the first line therapy in the treatment of autoimmune pancreatitis, often providing dramatic and rapid results. The role of other immunosuppressive agents in this patient population remains largely undefined.

Prednisone is usually initiated at a dose of 0.4-0.6 mg/kg per day for a period of months. Although a detailed steroid schedule has not yet been fully defined, most patients are usually treated for a period of 2-3 mo, with a tapering schedule of 5 mg every 1-2 wk.

From a laboratory perspective, hypergammaglo-bulinemia and elevated IgG4 levels can resolve and previously identified autoantibodies can become undete-ctable[41]. Marked changes are also observed in the pre-treatment findings of CT and ERCP[20]. The diffuse enlargement of the gland, characteristic pattern of enhancement and capsule-like low density rim seen on CT revert to normal. ERCP findings of focal, segmental or diffuse pancreatic duct narrowing also disappear. Narrowing of the distal common bile duct normalizes. Since almost all patients undergo ERCP prior to the initiation of steroid therapy, the finding of distal common bile duct narrowing is often treated with stent placement. The rapid reversal of biliary narrowing with steroid therapy usually allows for stent removal within 1-2 mo.

These radiologic changes closely parallel the clinical improvement that patients experience, specifically regarding the most common presenting symptoms of abdominal pain or painless jaundice. They are often observed within 2-4 wk of corticosteroid initiation and serve to further confirm the diagnosis of autoimmune pancreatitis.

In terms of prognosis, although the majority of patients achieve a sustained clinical remission with a tapering course of corticosteroid therapy, a subset may go on to require chronic maintenance dosing of 5-10 mg/d. The long term prognosis of autoimmune pancreatitis remains as yet undefined considering its recent discovery. Two different cohorts involving 23 patients followed for a mean of 56 mo and 17 patients followed for 16 mo, each had 1 patient relapse[9,35]. In both cases the patient was successfully treated with a second course of corticosteroids and maintained on low dose steroid therapy.

CONCLUSIONIn the last 40 years autoimmune pancreatitis has gone from a proposed concept to a well-recognized clinical entity. Despite the fact that our understanding regarding its pathogenesis, presentation, diagnosis and treatment have evolved, many questions remain unanswered. Its relationship to other autoimmune diseases, precise

pathogenesis, accurate diagnosis and long-term prognosis require further clarification.

Above all, as an efficient mimicker of pancreatic carc inoma, the accurate and t imely d iagnosis of autoimmune pancreatitis can have drastic consequences on therapy and prognosis. Thereby underlining the critical importance of its awareness among internists, gastroenterologists, radiologists, pathologists and surgeons.

ACKNOWLEDGMENTSWe wish to acknowledge Dr. David Owen, Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada for providing histology images.

REFERENCES1 Sarles H, Sarles JC, Muratore R, Guien C. Chronic infla-

mmatory sclerosis of the pancreas--an autonomous pancreatic disease? Am J Dig Dis 1961; 6: 688-698

2 Yoshida K, Toki F, Takeuchi T, Watanabe S, Shiratori K, Hayashi N. Chronic pancreatitis caused by an autoimmune abnormality. Proposal of the concept of autoimmune pancreatitis. Dig Dis Sci 1995; 40: 1561-1568

3 Sood S , Fossard DP, Shorrock K. Chronic sclerosing pancreatitis in Sjögren's syndrome: a case report. Pancreas 1995; 10: 419-421

4 Kawaguchi K, Koike M, Tsuruta K, Okamoto A, Tabata I, Fujita N. Lymphoplasmacytic sclerosing pancreatitis with cholangitis: a variant of primary sclerosing cholangitis extensively involving pancreas. Hum Pathol 1991; 22: 387-395

5 Wakabayashi T, Kawaura Y, Satomura Y, Watanabe H, Motoo Y, Sawabu N. Long-term prognosis of duct-narrowing chronic pancreatitis: strategy for steroid treatment. Pancreas 2005; 30: 31-39

6 Ectors N, Maillet B, Aerts R, Geboes K, Donner A, Borchard F, Lankisch P, Stolte M, Lüttges J, Kremer B, Klöppel G. Non-alcoholic duct destructive chronic pancreatitis. Gut 1997; 41: 263-268

7 Toki F, Kozu T, Oi I. An unusual type of chronic pancreatitis showing diffuse irregular narrowing of the entire main pancreatic duct on ERCP – A report of four cases. Endoscopy 1992; 24: A640

8 Kodama T, Abe M, Sato H, Imamura Y, Koshitani T, Kato K, Uehira H, Yamane Y, Horii Y, Yamagishi M, Yamagishi H. A case of pseudotumorous pancreatitis that presented unique pancreatoscopic findings with the peroral electronic pancreatoscope. J Gastroenterol Hepatol 2003; 18: 108-111

9 Kim KP , Kim MH, Song MH, Lee SS, Seo DW, Lee SK. Autoimmune chronic pancreatitis. Am J Gastroenterol 2004; 99: 1605-1616

10 Pearson RK, Longnecker DS, Chari ST, Smyrk TC, Okazaki K, Frulloni L, Cavallini G. Controversies in clinical pancreatology: autoimmune pancreatitis: does it exist? Pancreas 2003; 27: 1-13

11 Kim KP , Kim MH, Kim JC, Lee SS, Seo DW, Lee SK. Diagnostic criteria for autoimmune chronic pancreatitis revisited. World J Gastroenterol 2006; 12: 2487-2496

12 Zamboni G , Lüttges J, Capelli P, Frulloni L, Cavallini G, Pederzol i P , Leins A, Longnecker D, Klöppel G. Histopathological features of diagnostic and clinical relevance in autoimmune pancreatitis: a study on 53 resection specimens and 9 biopsy specimens. Virchows Arch 2004; 445: 552-563

13 Yadav D, Notahara K, Smyrk TC, Clain JE, Pearson RK, Farnell MB, Chari ST. Idiopathic tumefactive chronic pancreatitis: clinical profile, histology, and natural history after resection. Clin Gastroenterol Hepatol 2003; 1: 129-135

14 Weber SM, Cubukcu-Dimopulo O, Palesty JA, Suriawinata A, Klimstra D, Brennan MF, Conlon K. Lymphoplasmacytic sclerosing pancreatitis: inflammatory mimic of pancreatic

Zandieh I et al. Autoimmune pancreatitis 6331

www.wjgnet.com

carcinoma. J Gastrointest Surg 2003; 7: 129-137; discussion 137-139

15 Kamisawa T, Tu Y, Egawa N, Sakaki N, Inokuma S, Kamata N. Salivary gland involvement in chronic pancreatitis of various etiologies. Am J Gastroenterol 2003; 98: 323-326

16 Külling D, Tresch S, Renner E. Triad of sclerosing cholangitis, chronic pancreatitis, and Sjögren's syndrome: Case report and review. Gastrointest Endosc 2003; 57: 118-120

17 Epstein O, Chapman RW, Lake-Bakaar G, Foo AY, Rosalki SB, Sherlock S. The pancreas in primary biliary cirrhosis and primary sclerosing cholangitis. Gastroenterology 1982; 83: 1177-1182

18 Uchida K, Okazaki K, Asada M, Yazumi S, Ohana M, Chiba T, Inoue T. Case of chronic pancreatitis involving an autoimmune mechanism that extended to retroperitoneal fibrosis. Pancreas 2003; 26: 92-94

19 Niemelä S, Lehtola J, Karttunen T, Lähde S. Pancreatitis in patients with chronic inflammatory bowel disease. Hepatogastroenterology 1989; 36: 175-177

20 Horiuchi A , Kawa S, Hamano H, Hayama M, Ota H, Kiyosawa K. ERCP features in 27 patients with autoimmune pancreatitis. Gastrointest Endosc 2002; 55: 494-499

21 Kawa S, Ota M, Yoshizawa K, Horiuchi A, Hamano H, Ochi Y, Nakayama K, Tokutake Y, Katsuyama Y, Saito S, Hasebe O, Kiyosawa K. HLA DRB10405-DQB10401 haplotype is associated with autoimmune pancreatitis in the Japanese population. Gastroenterology 2002; 122: 1264-1269

22 Okazaki K, Uchida K, Ohana M, Nakase H, Uose S, Inai M, Matsushima Y, Katamura K, Ohmori K, Chiba T. Autoimmune-related pancreatitis is associated with autoantibodies and a Th1/Th2-type cellular immune response. Gastroenterology 2000; 118: 573-581

23 Uchida K, Okazaki K, Nishi T, Uose S, Nakase H, Ohana M, Matsushima Y, Omori K, Chiba T. Experimental immune-mediated pancreatitis in neonatally thymectomized mice immunized with carbonic anhydrase II and lactoferrin. Lab Invest 2002; 82: 411-424

24 Klöppel G, Lüttges J, Löhr M, Zamboni G, Longnecker D. Autoimmune pancreatitis: pathological, clinical, and immunological features. Pancreas 2003; 27: 14-19

25 Okazaki K, Uchida K, Chiba T. Recent concept of autoimmune-related pancreatitis. J Gastroenterol 2001; 36: 293-302

26 Taniguchi T, Seko S, Okamoto M, Hamasaki A, Ueno H, Inoue F, Nishida O, Miyake N, Mizumoto T. Association of autoimmune pancreatitis and type 1 diabetes: autoimmune exocrinopathy and endocrinopathy of the pancreas. Diabetes Care 2000; 23: 1592-1594

27 Tanaka S, Kobayashi T, Nakanishi K, Okubo M, Murase T, Hashimoto M, Takeuchi K. Corticosteroid-responsive diabetes mellitus associated with autoimmune pancreatitis. Lancet 2000; 356: 910-911

28 Zen Y, Kasahara Y, Horita K, Miyayama S, Miura S, Kitagawa S, Nakanuma Y. Inflammatory pseudotumor of the breast in a patient with a high serum IgG4 level: histologic similarity to sclerosing pancreatitis. Am J Surg Pathol 2005; 29: 275-278

29 Taniguchi T, Ko M, Seko S, Nishida O, Inoue F, Kobayashi H, Saiga T, Okamoto M, Fukuse T. Interstitial pneumonia associated with autoimmune pancreatitis. Gut 2004; 53: 770; author reply 770-771

30 Deshpande V, Mino-Kenudson M, Brugge W, Lauwers GY. Autoimmune pancreatitis: more than just a pancreatic disease? A contemporary review of its pathology. Arch Pathol Lab Med 2005; 129: 1148-1154

31 Takeda S, Haratake J, Kasai T, Takaeda C, Takazakura E. IgG4-associated idiopathic tubulointerstitial nephritis complicating autoimmune pancreatitis. Nephrol Dial Transplant 2004; 19: 474-476

32 Uchiyama-Tanaka Y , Mori Y, Kimura T, Sonomura K, Umemura S, Kishimoto N, Nose A, Tokoro T, Kijima Y, Yamahara H, Nagata T, Masaki H, Umeda Y, Okazaki K, Iwasaka T. Acute tubulointerstitial nephritis associated with autoimmune-related pancreatitis. Am J Kidney Dis 2004; 43: e18-e25

33 Kitano M, Kudo M, Maekawa K, Suetomi Y, Sakamoto H, Fukuta N, Nakaoka R, Kawasaki T. Dynamic imaging of pancreatic diseases by contrast enhanced coded phase inversion harmonic ultrasonography. Gut 2004; 53: 854-859

34 Yoshikawa J , Matsui O, Kadoya M, Gabata T, Arai K, Takashima T. Delayed enhancement of fibrotic areas in hepatic masses: CT-pathologic correlation. J Comput Assist Tomogr 1992; 16: 206-211

35 Koga Y, Yamaguchi K, Sugitani A, Chijiiwa K, Tanaka M. Autoimmune pancreatitis starting as a localized form. J Gastroenterol 2002; 37: 133-137

36 Sahani DV, Kalva SP, Farrell J, Maher MM, Saini S, Mueller PR, Lauwers GY, Fernandez CD, Warshaw AL, Simeone JF. Autoimmune pancreatitis: imaging features. Radiology 2004; 233: 345-352

37 Members of the Criteria Committee for Autoimmune Pancreatitis of the Japan Pancreas Society. Diagnostic criteria for autoimmune pancreatitis by the Japan Pancreas Society. J Jpn Panc Soc 2002; 17: 585-587

38 Aparisi L, Farre A, Gomez-Cambronero L, Martinez J, De Las Heras G, Corts J, Navarro S, Mora J, Lopez-Hoyos M, Sabater L, Ferrandez A, Bautista D, Perez-Mateo M, Mery S, Sastre J. Antibodies to carbonic anhydrase and IgG4 levels in idiopathic chronic pancreatitis: relevance for diagnosis of autoimmune pancreatitis. Gut 2005; 54: 703-709

39 Chari ST, Smyrk TC, Levy MJ, Topazian MD, Takahashi N, Zhang L, Clain JE, Pearson RK, Petersen BT, Vege SS, Farnell MB. Diagnosis of autoimmune pancreatitis: the Mayo Clinic experience. Clin Gastroenterol Hepatol 2006; 4: 1010-1016; quiz 934

40 Hamano H , Kawa S, Horiuchi A, Unno H, Furuya N, Akamatsu T, Fukushima M, Nikaido T, Nakayama K, Usuda N, Kiyosawa K. High serum IgG4 concentrations in patients with sclerosing pancreatitis. N Engl J Med 2001; 344: 732-738

41 Irie H, Honda H, Baba S, Kuroiwa T, Yoshimitsu K, Tajima T, Jimi M, Sumii T, Masuda K. Autoimmune pancreatitis: CT and MR characteristics. AJR Am J Roentgenol 1998; 170: 1323-1327

S- Editor Liu Y L- Editor Alpini GD E- Editor Ma WH

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INTRODUCTIONSphincter of Oddi dysfunction (SOD) is the term used to describe a heterogenous group of clinical pain syndromes caused by abnormalities in sphincter contractility. The sphincter of Oddi (SO), a fibromuscular sheath encircling the distal common bile duct (CBD), pancreatic duct (PD) and common channel, controls the flow of bile and pancreatic secretions into the duodenum and prevents reflux of duodenal contents into the pancreaticobiliary system.

SOD describes SO dysmotility or stenosis leading to reduced transphincteric flow of bile or pancreatic juice[1]. SO stenosis is a structural abnormality where there is a physical alteration of the sphincter due to inflammation and fibrosis. SO dyskinesia results in a hypo- or hypertonic sphincter with altered motility causing an intermittent functional blockade of the sphincter[2]. As it is often difficult to distinguish SO stenosis from dyskinesia, the term Sphincter of Oddi dysfunction is used to cover both conditions.

Because of the anatomical position of the SO patients with SOD typically present with recurrent biliary or pancreatic type pain. The Rome Ⅱ diagnostic criteria for biliary pain are episodes of severe steady pain in the epigastrium and right upper quadrant, associated with all of the following: (1) Symptom episodes lasting at least 30 min with pain free episodes in between; (2) At least one attack of pain in the last 12 mo; (3) Pain that is steady and interrupts daily activities or requires consultation with a doctor; (4) No evidence of structural abnormalities to explain the symptoms.

Pancreatic pain is described as post-prandial, episodic, prolonged pain in the upper abdomen and/or back[3]. It is often presumed in the setting of acute recurrent pancreatitis in the absence of biliary stone disease or anatomical abnormalities. The true prevalence of SOD is not known but ongoing biliary type pain occurs in 10%-20% of patients who have had a cholecystectomy[4]. Sphincter ablation, usually by endoscopic sphincterotomy, is at the forefront in the management of SOD and one of the challenges of this condition is to identify which patients will benefit from it.

TOPIC HIGHLIGHT

Sphincter of Oddi dysfunction and pancreatitis

MT McLoughlin, RMS Mitchell

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MT McLoughlin, RMS Mitchell, Department of Gastroenterology, Belfast City Hospital, Northern IrelandCorrespondence to: Dr. RMS Mitchell, Consultant Gastro-enterologist, Belfast City Hospital Trust, Lisburn Road, Belfast, BT9 7AB, Northern Ireland. michael.mitchell@bch.n-i.nhs.uk Telephone: +44-28-90263573 Fax: +44-28-90263973Received: July 15, 2007 Revised: October 17, 2007

AbstractSphincter of Oddi dysfunction (SOD) is a term used to describe a group of heterogenous pain syndromes caused by abnormalities in sphincter contractility. Biliary and pancreatic SOD are each sub-classified as typeⅠ, Ⅱ or Ⅲ, according to the Milwaukee classification. SOD appears to carry an increased risk of acute pancreatitis as well as rates of post ERCP pancreatitis of over 30%. Various mechanisms have been postulated but the exact role of SOD in the pathophysiology of acute pancreatitis is unknown. There is also an association between SOD and chronic pancreatitis but it is still unclear if this is a cause or effect relationship. Management of SOD is aimed at sphincter ablation, usually by endoscopic sphincterotomy (ES). Patients with typeⅠSOD will benefit from ES in 55%-95% of cases. Sphincter of Oddi manometry is not necessary before ES in typeⅠSOD. For patients with types Ⅱ and Ⅲ the benefit of ES is lower. These patients should be more thoroughly evaluated before performing ES. Some researchers have found that manometry and ablation of both the biliary and pancreatic sphincters is required to adequately assess and treat SOD. In pancreatic SOD up to 88% of patients will benefit from sphincterotomy. Therefore, there have been calls from some quarters for the current classification system to be scrapped in favour of an overall system encompassing both biliary and pancreatic types. Future work should be aimed at understanding the mechanisms underlying the relationship between SOD and pancreatitis and identifying patient factors that will help predict benefit from endoscopic therapy.

© 2007 WJG. All rights reserved.

Key words: Sphincter of Oddi dysfunction; Pancreatitis, Post-ERCP pancreatitis; Sphincter of Oddi manometry; Endoscopic sphincterotomy

McLoughlin MT, Mitchell RMS. Sphincter of Oddi dysfunction and pancreatitis. World J Gastroenterol 2007; 13(47):

Michael F Byrne, MD, Series Editor

Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6333-6343www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

CLASSIFICATION OF SODThe Milwaukee Classification, proposed by Hogan and Geenen[5,6], sub-classifies biliary and pancreatic SOD into three types on the basis of symptoms, laboratory tests and radiological imaging (Table 1). Abnormally high basal sphincter pressure identified during sphincter of Oddi manometry (SOM) confirms the presumed diagnosis. As biliary drainage time is difficult and somewhat impractical to measure and may increase the risk of an ERCP it is rarely performed in clinical practice. In any case, there may be little or no correlation between basal sphincter of Oddi pressures and drainage times[7]. Therefore, a contemporary modified version of the Milwaukee classification, which does not include duct drainage times, is generally used in practice[3]. Sub classification of SOD into typesⅠ, Ⅱ and Ⅲ helps predict the underlying pathology and the likelihood of symptom relief after treatment. TypeⅠdisease is thought to result from a fixed stenosis caused by chronic inflammation and fibrosis and has the highest response rate to therapy. An episodic dysmotility is the presumed underlying abnormality in the other types and often does not respond as well to treatment[8,9].

There are some potential problems with the Milwaukee classification. For example, the description of typical biliary or pancreatic pain may be interpreted differently between individuals and this may lead to inappropriate referral for SOM, particularly for patients with presumptive type Ⅲ SOD. Also, according to the Milwaukee criteria, LFTs should normalize between attacks but patients are often labeled with type Ⅱ SOD on the basis of pain and abnormal LFTs which do not normalize[10]. CBD diameter of at least 12 mm is one of the criteria in the diagnosis of SOD. Most patients being investigated for SOD have had their gallbladder removed and in the past it was accepted that it was normal for a post-cholecystectomy CBD to be 2-3 mm dilated. However, in a cohort of 59 patients, Majeed et al[11] found no difference between pre- and post-cholecystectomy CBD diameter. As the upper limit of normal for CBD diameter is 7 mm, a cut off of 12 mm potentially leaves a large number of patients misdiagnosed. Also, variations in basal pressure and response to sphincterotomy between the biliary and pancreatic portions of the SO have led to calls for this dual classification system to be scrapped in favor of a single, overall system.

SPHINCTER OF ODDI MANOMETRY SOM remains the gold standard for the diagnosis of SOD. It is usually combined with a diagnostic ERCP examination and involves cannulating the ampulla with the manometry catheter. A triple lumen catheter allows continuous aspiration of PD fluid that may reduce the risk of post- procedural pancreatitis[12]. To determine which duct has been cannulated a small amount of contrast is injected or some fluid aspirated to determine its color. A catheter “pull-through” of the sphincter is performed to assess the pressure profile and to localize the point of peak basal pressure. Normal basal sphincter pressure is approximately 15 mmHg but ranges from 3 to 35 mmHg.

It is generally accepted that a basal pressure greater than 40 mmHg (based on a threshold of 3 standard deviations above the median) is abnormal[13]. In patients with SO stenosis this recording is reproducible and does not respond to muscle relaxants[1]. In contrast, SO dyskinesia is characterized by a response to smooth muscle relaxants[5], an excess of retrograde contractions (> 50%), tachyoddia (rapid contraction frequency > 7/min) and a paradoxical contraction response of the SO following an intravenous dose of CCK[1,14].

In typeⅠSOD SOM will be abnormal in 75%-95%[15]. However, the frequency of abnormal biliary manometry varies from 28% to 60% for type Ⅱ patients and from 7 to 55% in type Ⅲ patients[16]. Various factors may explain the differences in frequencies of SOD in published reports. For example, selection of patients with a typical biliary or pancreatic type pain rather than a non-specific pain will increase the yield of basal pressure abnormality. SOM measures a “snap shot” of sphincter pressure during the study period that may not always be reproducible. A study of 12 patients with previously normal SOM showed evidence of elevated SO pressures in 5 (42%) when re-tested after a median of 337 d[17]. Also, the pressure in the pancreatic and biliary portions of the SO can vary so assessment of only one sphincter component, rather than both, will reduce the frequency of SOD detection. Current data suggests a discordance rate of between 35% and 65%[16,18-22]. Therefore, both portions of the SO should be measured separately for a full assessment. This necessitates classifying each patient with respect to the pancreatic and biliary components of the SO and is one of the reasons some experts have called for a single overall classification system. When both sides of the sphincter are evaluated there is little difference between them in predicting

Table 1 Milwaukee classification of sphincter of Oddi dysfunction

1 Biliary type:TypeⅠ:Typical biliary type painLiver enzymes (AST, ALT or ALP) > 2 times normal limit documented on at least 2 occasions during episodes of painDilated CBD > 12 mm in diameterProlonged biliary drainage time (> 45 min)

Type Ⅱ:Biliary type pain andOne or two of the above criteria

Type Ⅲ:Biliary type pain only

2 Pancreatic type SOD TypeⅠ:Pancreatic type painAmylase and/or lipase > 2 times upper normal limit on at least 2 occasions during episodes of painDilated pancreatic duct (head > 6 mm, body > 5 mm)Prolonged pancreatic drainage time (> 9 min)

Type Ⅱ:Pancreatic type pain, andOne or two of the above criteria

Type Ⅲ:Pancreatic type pain only

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abnormal basal pressure[16]. Because SOM is technically difficult, invasive, has a

variable diagnostic yield and has recognized complications, other indirect methods of evaluating SO function have been developed. These include the Morphine-Prostigmin provocative test (Nardi test; now obsolete), the ultrasound- or MRCP-secretin test, and quantitative hepatobiliary scintigraphy. However, current data suggests that non-invasive tests have a relatively low specificity and sensitivity[15], although there is some evidence that secretin stimulated MRCP may be useful in selecting patients with suspected type Ⅱ SOD who are most likely to benefit from sphincterotomy [23]. Therefore, despite the risk, and assuming careful patient selection, SOM remains the diagnostic tool of choice for most clinicians.

PANCREATITIS POST SOMAcute pancreatitis is the main complication of SOM. Increased intraductal pressure, overfilling of the ductal system, difficult and repeated cannulation of the PD causing spasm and trauma have all been postulated as etiological factors, possibly by affecting pancreatic duct drainage [24]. This hypothesis is indirectly supported by the obser vat ion that PD stenting after bi l iar y sphincterotomy[24] and needle knife sphincterotomy over a PD stent[25] have been found to reduce the incidence of pancreatitis in patients with SOD.

The rate of post-SOM pancreatit is in patients suspected of having SOD has been found to be as high as 31%[27-30]. Sherman et al[27] found a much lower rate of pancreatitis when an aspirating catheter was used (1 of 33 patients; 4%) compared with an infusion catheter (8 of 34 patients; 31%). Walters et al[31], however, found no difference in the incidence of pancreatitis when comparing the two types of manometry catheter (8% vs 13%). In a case series of 146 patients (207 SOM measurements), Rolny et al[28] reported a 6% incidence of pancreatitis when using the standard catheter. In addition, acute pancreatitis developed in 10 of 95 (11%) patients who had undergone pancreatic manometry alone, compared with 1 of 93 (1%) who had biliary manometry alone. Recommended methods of reducing the rate of pancreatitis from SOM include evaluating biliary SO alone in patients with suspected biliary disease[32], limiting SO perfusion to 1-2 min[33] and careful patient selection. For example, Scicchitano et al[29] found a significantly higher rate of pancreatitis when the indication for SOM was idiopathic acute recurrent pancreatitis (IARP) compared to unexplained abdominal pain (29% vs 6%). The incidence of pancreatitis was 50% in the patients with IARP and high SO basal pressure. Temporary prophylactic pancreatic duct stenting has been shown to reduce the incidence of pancreatitis in a variety of patient groups, including those undergoing SOM[25,26,34,35].

A retrospective review of 100 patients who had undergone SOM found an overall incidence of pancreatitis of 17%[30]. The incidence was significantly higher in patients who had undergone SOM and ERCP, compared to those who had only undergone SOM (26.1% vs 9.3%). Multiple regression analysis showed that sphincterotomy

added no additional risk beyond that associated with ERCP. These results imply that other factors during ERCP, and not the manometry itself, predispose to pancreatitis. The authors recommended that ERCP should be performed at another session, possibly 24 h after SOM.

Results from other studies suggest that the risks of pancreatitis are intrinsic to the patient group undergoing the procedure and the therapy provided, rather than the SOM itself. Freeman et al[36] recorded complication rates for sphincterotomy in patients with suspected SOD and those in whom it was already confirmed. The complication rate was 21% for patients who underwent SOM and 25% when sphincterotomy was not preceded by SOM. Another study compared the pancreatitis rate from ERCP between patients with suspected SOD, some of whom also underwent SOM, and a control group of patients with biliary stones[37]. 27% of patients with suspected SOM developed post-procedural pancreatitis, compared with 3.2% of the control group (P < 0.001). However, there was no significant difference in the rate of acute pancreatitis in the first group between those who had SOM and those who did not (OR 0.72: 95% CI 0.08-9.2). Similarly, in a large trial of over 1000 patients who underwent ERCP with or without SOM, Cheng et al[38] found that SOM was not a risk factor for post-ERCP pancreatitis.

The variability in complication rates between studies is probably multifactorial and related to the timing and duration of the procedure, the number of passes with the manometry catheter and technique and skill of the operator. However, it is probable that, in skilled hands, SOM does not significantly increase the risks of post-ERCP pancreatitis and remains a useful tool in the diagnosis of SOD, particularly for types Ⅱ and Ⅲ.

SOD AND ACUTE PANCREATITISSOD may contribute to the risk of acute pancreatitis by causing abnormal biliary or pancreatic juice flow. In the Australian Bush opossum, which has a similar biliary and pancreatic anatomy to humans, the combination of pancreatic duct ligation and stimulation of pancreatic exocrine secretion with cholecystokinin/secretin uniformly causes acute pancreatitis[39]. In another group, reduced transphincteric flow was achieved by applying topical carbachol to the SO, causing PD pressures comparable with those opossums in which the PD was ligated. However, acute pancreatitis only occurred when carbachol application was combined with pancreatic secretory stimulation. Decompression of the PD negated the effects. Therefore, the combination of PD obstruction with increased exocrine secretion was needed to produce acute pancreatitis. Although it is a recognized complication of SOD, this study demonstrated that SOD might be a causative factor in the production of acute pancreatitis.

Kruszyna et al[40] carried out ERCP with pre- and post- sphincterotomy SOM in a group of 30 patients with mild acute biliary pancreatitis and compared results with a control group of 30 patients with no evidence of CBD stones or pancreatitis. The patients with pancreatitis had a significantly elevated CBD pressure, SO basal pressure and wave amplitude compared to controls.

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There was a significant reduction in all parameters after sphincterotomy. They concluded that SO dysfunction, either primary or secondary to spasm caused by a gallstone migrating through the ampulla, may have a role in acute biliary pancreatitis.

Although there is very little direct evidence supporting the role of the SO in causing pancreatitis in humans, there is plenty of circumstantial evidence. Fazel et al[41] measured intrapancreatic ductal pressure blindly in 263 patients presenting with either recurrent abdominal pain, acute recurrent pancreatitis or chronic pancreatitis. Complete SOM was then performed and patients with SOD were found to have a significantly higher ductal pressure compared to those with normal SO motility. This difference was seen across all three groups (P < 0.01) and patients with acute and chronic pancreatitis did not have a significant elevation in intraductal pressure compared to individuals with abdominal pain only. The authors concluded that SOD leads to an increase in intrapancreatic ductal pressure but this rise in pressure is not the sole cause of pancreatitis.

Warshaw et al [42] showed that infusion of secretin caused PD dilatation of > 1 mm in 83% of patients with SO stenosis and 72% with accessory papilla stenosis, compared with controls. This dilatation response was abolished after surgical sphincteroplasty. A positive secretin test was associated with a good surgical outcome in 90% of cases. It has been shown that in patients undergoing surgery for idiopathic acute recurrent pancreatitis (IARP) the SO narrows at the opening of the PD, suggesting that this narrowing may play a role in its development[43].

An abnor mal i ty of SO function has a lso been implicated in the pathogenesis of acute pancreatitis attributed to other causes. An organophosphate insecticide is a recognized cause of acute pancreatitis in humans. It acts by irreversibly inhibiting cholinesterase resulting in delayed breakdown of synaptic acetylcholine[44], and has been shown to cause pancreatitis in animals[45], probably due to the combination of obstruction at the level of the SO and cholinergic stimulation of pancreatic secretions. Scorpion venom causes acetylcholine release, stimulating the pancreas and SO, and causes pancreatitis in a similar way to organophosphate poisoning[46].

Other rare causes of acute pancreatitis including hypercalcemia and hyperlipidemia may involve abnormalities of SO function. High extra-cellular calcium stimulates smooth muscle and stimulates pancreatic secretion in animal models and it is thought that abnormal calcium regulation of the SO may be an underlying factor in the pathophysiology[47]. A study of hypercholesterolemic rabbits showed a failure of SO relaxation again indirectly suggesting that SO dysfunction may contribute to the risk of pancreatitis[48]. Therefore, although its exact role is not known, the evidence, taken together, suggests that the SO at some level is an important factor in the development of acute pancreatitis, including pancreatitis that may be attributed to another aetiology. SOD IN RECURRENT ACUTE PANCREATITISClinical evaluation, blood testing and imaging will yield a

cause of acute recurrent pancreatitis in 70%-90% of cases. In the remaining “idiopathic” acute recurrent pancreatitis (IARP) cases more extensive evaluation may be required, including assessment for SOD. Abnormal SOM in IARP ranges from 15%-72% with a mean of 30.5% (Table 2)[16,49-54]. The high incidence of abnormal SOM in IARP reflects the fact that a substantial proportion of these patients are likely to have SOD .

With the exception of a study by Eversman et al[16], the published studies measured sphincter pressure in only one duct, i.e., either pancreatic OR biliary, although in some cases it is not clear which duct was actually measured. Eversman et al, however, performed SOM of the biliary and pancreatic ducts in 593 patients, of whom 360 had intact sphincters. Of the 47 patients with idiopathic acute pancreatitis, 12 had increased pressure in the pancreatic portion of the SO, 3 had increased pressure in the biliary portion and 19 had it in both. The measurement of sphincter pressure in both ducts accounts for the much higher frequency of SOD in IARP that was found in this study. Choudari et al[55] also reported a higher frequency of basal sphincter abnormality of at least one duct in patients with chronic pancreatitis.

Of the 360 pat ients measured in the study by Eversman et al[16], 68 (18.9%) had abnormal pancreatic sphincter basal pressure alone, 41 (11.4%) had abnormal biliary basal sphincter pressure alone and in 113 (31.4%) the basal pressure was abnormal for both sphincters. Therefore, 219 (60.1%) of the patients had sphincter dysfunction. The authors concluded that assessment of both the pancreatic and biliary portions of the SO is necessary to accurately detect SOD. The frequency of SOD did not differ whether typed by biliary or pancreatic criteria (65% type Ⅱ and 59% type Ⅲ). As there was so little difference in the frequency of SOD according to the modified Geenen-Hogan criteria, the authors argued for an overall classification for SOD encompassing biliary and pancreatic types.

Guelrud et al[56] retrospectively reviewed ERCP studies from 64 children (> 1 year old) and adolescents with recurrent pancreatitis. SOM and sphincterotomy were performed in 9 patients, all of whom had SOD. Seven of these patients had a choledochal cyst and 2 had anomalous pancreaticobiliary union (APBU). After a mean follow up of 26.4 mo (range 18-38), 8 of these patients were symptom free and one had occasional pain but no further episodes of pancreatitis. They concluded that recurrent

Table 2 Frequency of abnormal sphincter of Oddi manometry in idiopathic acute recurrent pancreatitis

Author Year Patient number, n Abnormal SOM Frequency (%)

Gregg et al[49] 1984 125 28 22Toouli et al[50] 1985 28 14 50Venu et al[51] 1989 116 17 15Sherman et al[52] 1992 49 15 31Eversman et al[16] 1999 47 34 72Coyle et al[53] 2002 90 28 31Kaw et al[54] 2002 126 41 33Total 581 177 30.5

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pancreatitis and ABPU are associated with SOD in children and adolescents and that sphincterotomy was beneficial to these patients.

SOD AND CHRONIC PANCREATITISEarly studies investigating the association of SOD and chronic pancreatit is were inconclusive. Some studies showed no difference in pancreatic sphincter pressures between patients with chronic pancreatitis and controls[57-61]. However, these studies involved patients with chronic pancreatitis due to alcohol and in two of the studies the controls were patients with unexplained abdominal pain[58] or suspected biliary dyskinesia [60]. Also, although one of these studies found no significant difference between SO basal pressure in patients with chronic pancreatitis and controls, the pancreatic duct pressure was significantly higher in the early stages of chronic pancreatitis than normal subjects[57]. Other trials have shown a correlation between elevated pancreatic sphincter pressures and chronic pancreatitis[19,62-64]. Many of these also used patients with chronic pancreatitis secondary to alcohol. However, in the only one of these studies that excluded alcoholic patients, basal pancreatic sphincter pressures were significantly higher in the early stages of chronic pancreatitis than controls[62]. Laugier[64] performed manometry of the SOD and main pancreatic duct before and after intravenous injection of secretin in chronic pancreatitis patients and controls. Secretin transiently increased pancreatic duct pressure in controls, but chronic pancreatitis patients had a persistently elevated pancreatic duct pressure and a manometric pattern of SOD. The secretin-induced elevation in ductal pressure was greater and more sustained in patients with chronic pancreatitis, particularly of recent onset (less than 4 years).

It has been shown that local installation of alcohol on the SO results in elevated SO pressures, suggesting a role in the pathogenesis of alcoholic pancreatitis[65]. Tarnasky et al[66] looked for evidence of chronic pancreatitis in patients undergoing manometry for investigation of unexplained upper abdominal pain (n = 104). Pancreatic ductography, EUS and pancreatic fluid bicarbonate concentration measurements were carried out. Patients with SOD were 4 times more likely to have evidence of chronic pancreatitis than those with normal sphincter pressure (P = 0.01). Of 68 patients with SOD, 20 (29%) had structural evidence of chronic pancreatitis and 20 of 23 patients (87%) with chronic pancreatitis had SOD. The authors concluded that SOD is associated with structural evidence of chronic pancreatitis in patients with unexplained pancreaticobiliary pain. Patients with chronic pancreatitis and SOD were significantly older than those with SOD but no chronic pancreatitis. This raises the possibility that SOD precedes the development of pancreatitis.

The available evidence certainly suggests a link between SOD and chronic pancreatitis. However, it is still not clear if this is a cause or effect relationship, i.e., does the generalized scarring associated with chronic pancreatitis also involve the sphincter or does the hypertensive sphincter cause elevated pressure and, hence,

morphological changes? Further work is required to clarify this issue.

SPHINCTEROTOMY FOR SODBiliary type SODManagement of SOD has traditionally been aimed at sphincter ablation by endoscopic sphincterotomy. Most data on sphincterotomy relates to biliary sphincter ablation alone and clinical improvement has been reported to occur in 55%-95% of patients[15] with the grade of SOD having a significant effect on outcome. Outcomes are generally measured using pain scores or quality of life measures[2], although a lack of standardization in characterizing the patients and assessing response make comparisons between trials problematic.

There are no randomized or controlled trials of therapy for typeⅠSOD and the available evidence is derived from small retrospective trials. Rolny et al[67] carried out ERCP and SOM on 17 post-cholecystectomy patients with suspected typeⅠSOD. All patients had a dilated CBD at ERCP and delayed contrast drainage and 11 had elevated SO pressure. Sphincterotomy resulted in symptom relief in all patients after a mean follow up of 28 mo. It was concluded that, in symptomatic post-cholecystectomy patients, the triad of abnormal LFTs, dilated CBD and delayed contrast drainage was sufficient to make a diagnosis of definitive SO abnormality and, as these patients invariably benefit from sphincterotomy, SOM was unnecessary.

Other studies have reported the effect of sphincte-rotomy for both typeⅠand type Ⅱ patients. Thatcher et al[68] retrospectively reviewed 46 patients (31 with typeⅠand 15 with type Ⅱ) who had undergone sphincterotomy for SOD. In the patients with typeⅠSOD 87% had improved pain scores at 3 mo and 77% after a mean follow up of 12.5 mo. When evaluated along with the patients with type Ⅱ SOD, patients with a dilated bile duct and delayed contrast drainage at ERCP had a better response to therapy (P = 0.01) and reduced complication rate (P = 0.03) compared to those with normal ducts at ERCP. 29 patients underwent SOM but a favorable treatment outcome did not correlate with manometric assessment, particularly in patients with abnormal ducts. Therefore, patients suspected of having typeⅠSOD benefited from sphincterotomy, irrespective of SOM results.

Lin et al[69] performed sphincterotomy on 24 patients based on clinical findings of post-cholecystectomy pain, biochemical abnormalities and/or dilated bile ducts. Enzyme abnormalities were a significant predictor of response to therapy (P = 0.018) whereas duct dilatation was not (P = 1.0).

These smal l s tud ies sug ges t tha t endoscopic sphincterotomy without SOM is effective in suspected typeⅠbiliary SOD. However, patients with presumptive type Ⅱ SOD have, by definition, less concrete evidence for obstruction at the level of the sphincter so more extensive evaluation is necessary to predict those who would benefit from sphincterotomy.

Three randomized trials of endoscopic therapy for types Ⅱ and Ⅲ SOD have been reported. In one of these

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47 patients with presumed type Ⅱ SOD were randomly assigned to endoscopic sphincterotomy (n = 23) or a sham procedure (n = 24) in a prospective double-blind study[6]. All patients had biliary type pain, clinical characteristics in keeping with biliary obstruction and had a previous cholecystectomy. Eleven patients in the treatment group had manometric evidence of elevated sphincter pressure and 10/11 described improved pain scores at 1 year. In contrast, only 3 out of 12 patients in the control group who had elevated pressure had an improved pain score over the same time period. Pain scores were unchanged in patients with normal sphincter pressures, irrespective of treatment. After one year sphincterotomy was performed in 12 symptomatic patients who had initially undergone the sham procedure, 7/12 with elevated sphincter pressure and 5/12 with normal pressure. A total of 40 patients were followed for 4 years and after that time 17 of the 18 patients (95%) with SOD verified by manometry had benefited from sphincterotomy. However, only 30%-40% of patients with an elevated sphincter pressure treated with sham sphincterotomy or with a normal pressure treated by sphincterotomy or sham benefited from therapy. The authors concluded that SOM predicted outcome from sphincterotomy and that sphincterotomy offers long-term pain relief in patients with verified SOD.

An Australian study of SOM in 81 post-cholecystectomy patients with biliary-type pain compared outcomes among a mixed group of patients with types Ⅰ(n = 9),Ⅰ-Ⅱ (n = 27), Ⅱ (n = 27) and Ⅲ (n = 18)[70]. The manometric records were categorized as SO stenosis, SO dyskinesia or normal, after which patients were randomized in each category to sphincterotomy or a sham procedure in a prospective double blind study. In the SO stenosis group symptoms improved in 11/13 patients treated with sphincterotomy compared to 5/13 who had a sham procedure (P = 0.041). Results from each treatment group did not differ for patients with SO dyskinesia and normal SOM. This trial provided further evidence that patients with presumed SO dysfunction, with subsequent manometrically diagnosed SOD, benefit from endoscopic sphincterotomy. The authors hypothesized a generalized motility disorder to account for the lack of benefit in patients with normotensive but dyskinetic sphincter function.

Sherman et al[71] reported results of a randomized t r ia l compar ing sphincterotomy, surg ica l b i l i a r y sphincteroplasty with pancreatic septoplasty (with or without cholecystectomy) to sham sphincterotomy for types Ⅱ and Ⅲ biliary patients with manometrically documented SOD (n = 52). After 3 years, 69% of patients undergoing endoscopic or surgical sphincterotomy had symptomatic improvement compared to 24% in the sham sphincterotomy group (P = 0.009). Type Ⅱ patients had an 81% response to sphincter ablation compared to 58% for type Ⅲ patients; double that of the sham sphincterotomy group.

These trials suggest that SOM is a useful guide in predicting benefit from sphincterotomy in type Ⅱ SOD. However, other (non-randomized) trials have suggested that manometric findings do not correlate with clinical outcome. For example, Botoman et al[72] included types Ⅱ (n = 35) and Ⅲ (n = 38) patients to assess response

to sphincterotomy. There was no difference between the two groups with respect to sphincter hypertension (60% vs 55% respectively), symptomatic improvement at 3 years (60% vs 56%) or post-procedure pancreatitis rates (15% vs 16%). The authors suggested that current classifications are inadequate to define either incidence of SOD or response to sphincterotomy. In another trial SOM was performed in all but 3 patients from a total of 35 patients with suspected type Ⅱ SOD and 29 with type Ⅲ[73]. Sphincterotomy was performed in all patients with SO pressure greater than 40 mmHg, which included 62.5% of the type Ⅱ patients and 50% of the type Ⅲ patients. After 6 wk 70% of the patients with type Ⅱ SOD and 39% of the type Ⅲ SOD who had sphincterotomy reported benefit (P = 0.13, type Ⅱ vs type Ⅲ). None of the patients with normal manometry had symptomatic improvement. After long-term follow up (median 2.5 years) sustained improvement occurred in 60% of the type Ⅱ patients but only 8% of those with type Ⅲ (P < 0.01). The investigators felt that the current classification helps predict outcome after sphincterotomy but again acknowledged a lack of difference in the incidence of abnormal SO baseline pressure between type Ⅱ and type Ⅲ SOD.

Cicala et al [74] performed SOM and quantitative scintigraphy in 30 patients with suspected typeⅠor type Ⅱ SOD. Fourteen (6 typeⅠand 8 type Ⅱ) of the 22 patients were offered and underwent sphincterotomy. At long term follow up, all 14 patients were asymptomatic, biochemical abnormalities had resolved and hepatic hilum-duodenum transit time (HHDT) at scintigraphy had significantly decreased. The patients who had refused sphincterotomy had no change in symptoms or HHDT. Scintigraphy predicted favorable outcomes in 93% of cases compared to 57% for SOM. Two other studies found no correlation between response to sphincterotomy and sphincter pressure for either typeⅠor type Ⅱ patients[68,75].

The frequency of hypertension in either sphincter among patients with presumptive type Ⅲ SOD ranges from 25%-70%[76]. The previously cited trial by Sherman et al[71], which was published as an abstract, is the only randomized controlled trial that has dealt with outcomes post-sphincterotomy for patients with type Ⅲ SOD. 29 patients with presumed type Ⅲ SOD were randomized and after a 3-year follow up period symptoms had improved in 8/13 (62%) who had undergone endoscopic sphincterotomy, 3/10 (30%) who has sham sphincterotomy and 3/6 (50%) after surgery. A follow up study after dual sphincterotomy for biliary and pancreatic SOD, which included 166 patients with type Ⅲ SOD, found no significant difference in re-intervention rates between different classes of SOD (i.e., biliary vs pancreatic, type Ⅱ vs type Ⅲ)[77]. After a mean follow up of 44 mo, persistent symptoms prompted re-intervention in 28.3% of patients with type Ⅲ SOD, compared to 20.4% for combined typeⅠand Ⅱ (P = 0.105). Other studies report response rates between 8%-65% for type Ⅲ SOD[73,76].

It has been postulated that type Ⅲ SOD is part of a spectrum of functional GI disorders and many patients labeled with it may in fact have a diffuse gastrointestinal motility disturbance. Desautels et al [78], for example,

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showed that patients with type Ⅲ SOD exhibit duodenal-specific visceral hyperalgesia and their symptoms are re-produced by duodenal distension. The challenge remains to identify which patients will most likely to benefit from a particular therapy. Varadarajulu et al [2] suggest that patients who present with discrete, self-limiting episodes of typical biliary or pancreatic type pain are the ones most likely to benefit from SOM and sphincterotomy. With the current evidence available it is reasonable to consider medical therapy as the first line of treatment for patients with suspected type Ⅲ SOD. ERCP with SOM should be considered in the event of failure of medical therapy with sphincterotomy if manometry is abnormal.

Pancreatic type SODEvidence that SOD may be a cause of IARP is supported by the resolution of pancreatitis after sphincterotomy, with up to 80% improvement in patients with IARP after biliary sphincterotomy[79]. Tarnasky et al[80] showed that biliary sphincterotomy reduced pancreatic basal pressure to within the normal range in 30% of patients immediately after the procedure and 20% after longer term follow up, presumably by ablation of the common channel sphincter, and hence a reduction in the length of the residual pancreatic portion. In a proportion of patients therefore, biliary sphincterotomy alone may resolve pancreatitis or pancreatic pain.

In the one controlled trial addressing response to therapy in patients with acute recurrent pancreatitis presumed to be secondary to SOD, Jacob et al[81] compared response to ERCP with or without stent insertion in patients with negative investigations including SOM. Stent insertion reduced the rate of recurrence of pancreatitis from 53% to 11% over a 3-year study period.

Kaw et al[54] assessed the relationship between micro-lithiasis and sphincter hypertension in 67 patients with IARP. After endoscopic biliary sphincterotomy, 88% of patients with typeⅠSOD and 73% with type Ⅱ were asymptomatic, irrespective of microlithiasis or gallbladder status. In a study in which ERCP, SOM and endoscopic ultrasound (EUS) were carried out on 90 patients with acute recurrent pancreatitis, SOD was found to be the most common cause found (n = 28) [53]. Of the 22 of these patients who underwent biliary sphincterotomy 21 had reduced episodes of acute pancreatitis after 6 mo.

It has been suggested that inadequate pain relief after biliary sphincterotomy may be due to inadequate biliary sphincterotomy, recurrent biliary stenosis, chronic pancreatitis, other residual pancreaticobiliary disease or a non-pancreaticobiliary cause, e.g., irritable bowel syndrome or a persistent abnormality in pancreatic sphincter pressure[28,82,83]. In the latter case, dual biliary and pancreatic sphincterotomy may improve outcome. Eversman et al[84] reported long term outcome of biliary sphincterotomy alone in patients with SOD. Patients with SOD and an abnormal pancreatic sphincter pressure needed re-intervention more often than those with abnormal biliary sphincter pressure alone (39.4% vs 16.2%, P < 0.05) or dual sphincter hypertension (29%, P < 0.05). These results support the theory that an untreated pancreatic SOD

may cause recurrent pain in patients who have undergone biliary sphincterotomy alone. A previously cited study by the same authors[16] showed that manometry of both pancreatic and biliary portions of the SO is necessary for complete evaluation for SOD. Other studies have drawn the same conclusions[22,85].

Guelrud et al[86] reported the response to four different therapeutic options in patients with normal pancreatography and elevated sphincter pressures (pancreatic type Ⅱ SOD). Symptomatic improvement occurred in 28% of patients treated by biliary sphincterotomy alone, in 54% who had biliary sphincterotomy combined with pancreatic orifice dilatation, in 77% who underwent dual sphincterotomies at two separate sessions and in 86% of patients who had dual sphincterotomies performed during a single session. Compared to biliary sphincterotomy alone, dual sphincterotomy had significantly better outcomes (P < 0.0005), irrespective of whether they were performed at a single or at separate sessions. The authors suggested that pancreatic sphincter ablation should be considered for patients with type Ⅱ SOD and an abnormal pancreatic basal sphincter pressure. Other studies have shown similar results. Soffer and Johlin[87] found symptomatic improvement following pancreatic sphincterotomy in 16 out of 25 (64%) patients unresponsive to biliary sphincterotomy. In a further trial, 43 patients who had not responded to biliary sphincterotomy were followed up for a median of 14 mo after pancreatic sphincterotomy. 39/43 patients (91%) showed clinical improvement with 31/43 having a complete response[88].

Another group of investigators followed-up 313 patients who had undergone endoscopic dual sphincterotomy for manometry documented SOD of at least one sphincter for a mean of 43.1 mo[77]. Hypertension was demonstrated in both sphincters in 57%, in the pancreatic sphincter alone in 35% and in the biliary sphincter alone in 26%. Immediate complications occurred in 15% of patients and re-intervention was required in 24.6% of patients at a median follow-up of 8 mo. Re-intervention rates were similar irrespective of ducts with abnormal basal sphincter pressure or previous cholecystectomy. Compared to biliary sphincterotomy alone in historical controls, dual sphincterotomy had a lower re-intervention rate in patients with pancreatic SOD alone (21.3% vs 39.4%, P = 0.034) and a comparable outcome in those with SOD of both ducts (26.6% vs 29%, P = 0.412) or isolated biliary SOD (25% vs 16.2%, P = 0.285). Immediate complication rates occurred in 47/313 patients (15%) with pancreatitis in 45/313 (14.4%). Severe pancreatitis occurred in 0.9% of patients. These complication rates are lower than those reported for biliary sphincterotomy in the prospective study by Freeman et al[36] when 21.7% of patients developed pancreatitis, of which 3.7% were severe. This may relate to differences in the quality of pancreatic drainage between the two trials. Fogel et al[26] also noted that biliary, as opposed to dual, sphincterotomy was more likely to induce pancreatitis in patients with suspected SOD. Therefore, dual sphincterotomy seems to be beneficial for patients with pancreatic SOD, but not in those with biliary SOD alone. It remains unclear

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whether dual sphincterotomy should be performed at the initial procedure. Further randomized trials comparing single versus dual sphincterotomy in patients with SOD are necessary to determine the most appropriate sphincter therapy based on SOM findings. However, other factors should also be taken into account. In a recent trial which included patients with biliary typesⅠ, Ⅱ and Ⅲ SOD, all 121 patients underwent biliary sphincterotomy[89] and 49 patients had pancreatic sphincterotomy at initial or subsequent ERCP if there was a history of abnormal pancreatic manometry in the setting of continuous pain, persistent pain after biliary sphincterotomy or a history of amylase elevation. There was no significant difference in patient response according to Milwaukee classification. (However, this may reflect the numbers of patients involved, with only 18 meeting the criteria for typeⅠSOD). Significant predictors of poor response were normal pancreatic manometry, delayed gastric emptying, daily opioid use and age < 40. Abnormal liver function tests and a dilated bile duct were not significant predictors of outcome. These findings support the argument that we cannot rely on the Milwaukee classification alone to predict response to treatment. The authors suggested that patient factors and pancreatic manometry may be more important predictors of outcome of dual sphincterotomy for SOD. These issues should be taken into account before embarking on therapy.

POST-ERCP PANCREATITIS IN SODOverall pancreatitis rates post- ERCP are usually quoted to be between 5%-15%[36,90]. Prospective studies have consistently shown that SOD confers increased risk of post-ERCP pancreatitis (PEP). Cheng et al[38] evaluated risk factors for ERCP-induced pancreatitis in 1115 patients who had undergone ERCP. Suspected SOD was a significant risk factor with an OR of 2.6. In a prospective study of 1223 ERCP procedures, Vandervoort et al[91] found that patients with manometrically proven SOD had a threefold risk of PEP (21.7% vs 7.2%). Freeman et al[92] found an overall pancreatitis rate of 6.7% in 1963 ERCP procedures with an odds ratio of 2.6 for suspected SOD. A meta-analysis of 15 prospective clinical trials found that patients with suspected SOD had a relative risk of developing pancreatitis of 4.09 (95% CI 3.37-4.96, P < 0.001)[93]. SOD is therefore an independent risk factor for post-ERCP acute pancreatitis and the decision to proceed to ERCP, with or without SOM and/or sphincterotomy, should be made with care.

Sphincterotomy for SOD increases the risk of PEP. One randomized control trial, albeit small (n = 36), found a post-sphincterotomy pancreatitis rate of 33% in patients with SOD in whom a PD stent was not placed[94]. Five prospective randomized trials have compared PEP rates between high risk patients with or without PD stent placement. Four of these included patients with SOD (Table 3)[25,35,94,95]. Of these four studies all showed a trend to reduction of PEP with PD stent placement, and two reached statistical significance. A meta-analysis of five prospective studies showed a 3-fold increased risk of post-ERCP pancreatitis if a pancreatic stent was not used (15.5%

vs 5.8%, OR 3.2, 95% CI 1.6-6.4)[96].At least three case control studies have also included

patients with SOD. In two of these there was a significant reduction in PEP with a pancreatic stent[26,88] and in the other the reduction of pancreatitis rate from 66.7% to 14.4% did not quite reach significance (P = 0.06)[97]. Therefore, there is substantial evidence that pancreatic stent placement reduces the incidence of post-ERCP pancreatitis in high-risk groups such as SOD. However, failure to deploy the stent successfully may occur in up to 10% of patients[98], and failed pancreatic stent placement can increase the rate of PEP sixteen fold[97]. Therefore, pancreatic stent placement should not be attempted unless the likelihood of success is very high.

CONCLUSIONThe relationship between SOD and pancreatitis is a complex one. An association between SOD and acute pancreatitis appears to be beyond doubt, not least because of the high frequency of abnormal SOM in IARP. SOD also carries a significantly increased risk of post-ERCP pancreatitis with rates of over 30%, although correct placement of a pancreatic stent at the time of the procedure appears to reduce this risk. However, although various mechanisms have been postulated, the exact role of SOD in the pathophysiology of pancreatitis is not known and it is unclear if SO dysfunction as a primary event or secondary to other factors is the principal mechanism. There is also evidence linking SOD with chronic pancreatitis but whether this is a cause or effect relationship is still unknown.

Sphincterotomy remains the management of choice for SOD. All patients with typeⅠSOD should have their sphincter ablated and, by general consensus, this group does not require manometry prior to the procedure. The question whether dual sphincterotomies should be carried out remains unanswered and further randomized trials are required to clarify this. For patients with type Ⅱ SOD grade A studies have found that SOM is a useful guide in predicting response to sphincterotomy, although some smaller studies showed that manometric findings do not correlate with clinical outcome. However, most experts agree that patients with suspected type Ⅱ SOD should have SOM before considering sphincterotomy.

The management of patients with type Ⅲ SOD is more difficult still with response rates to sphincterotomy ranging from 8% to 65%. In general, sphincter ablation is probably warranted if SOM is abnormal but medical

Table 3 Role of pancreatic stent insertion in prevention of post- ERCP pancreatitis; results of randomized controlled trials that included patients with SOD

Author Year No. of patients

Pancreatitis rate (%)

Stent No stent

Smithline et al[95] 1993 93 14 18 P = 0.299Tarnasky et al[25] 1998 80 7 26 P = 0.03Patel et al[94] 1999 36 11 33 P > 0.05Fazel et al[35] 2003 76 5 28 P < 0.05

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therapy should be tried before proceeding to manometry. Detailed history taking is paramount for these patients. The more the pain pattern differs from that set out in the Rome Ⅱ criteria, the less likely is the patient to benefit from treatment. Until there is a more adequate method of characterizing patients with type Ⅲ SOD it will not be possible to carry out a randomized trial of sphincterotomy against placebo. Ultimately, this will be the only way of proving the benefit or otherwise of sphincterotomy for patients with presumptive type Ⅲ SOD.

Recent evidence supports the need to measure both portions of the SO to maximize the detection rate of SOD. This dual classification has prompted a call for a single overall classification system from some quarters. A recently published trial[89] has also shown that other patient factors such as age, opioid use, delayed gastric emptying and pancreatic manometry are more important predictors of response to dual sphincterotomy than abnormal liver function tests and a dilated ductal system, on which the traditional classification system is heavily based. Further large prospective trials are required to identify other potential patient factors that may help predict response to therapy; such factors should be taken into account in any future overhaul of the current classification system.

REFERENCES1 Toouli J, Roberts-Thomson IC, Dent J, Lee J. Sphincter of

Oddi motility disorders in patients with idiopathic recurrent pancreatitis. Br J Surg 1985; 72: 859-863

2 Varadarajulu S, Hawes R. Key issues in sphincter of Oddi dysfunction. Gastrointest Endosc Clin N Am 2003; 13: 671-694

3 Petersen BT. An evidence-based review of sphincter of Oddi dysfunction: part I, presentations with "objective" biliary findings (types I and II). Gastrointest Endosc 2004; 59: 525-534

4 Black NA, Thompson E, Sanderson CF. Symptoms and health status before and six weeks after open cholecystectomy: a European cohort study.ECHSS Group. European Collaborative Health Services Study Group. Gut 1994; 35: 1301-1305

5 Hogan WJ, Geenen JE. Biliary dyskinesia. Endoscopy 1988; 20 Suppl 1: 179-183

6 Geenen JE, Hogan WJ, Dodds WJ, Toouli J, Venu RP. The efficacy of endoscopic sphincterotomy after cholecystectomy in patients with sphincter-of-Oddi dysfunction. N Engl J Med 1989; 320: 82-87

7 Khusro Q, Lehman GA. Delayed biliary drainage: help or hype? Am J Gastroenterol 1993; 88: 962-963

8 Hogan WJ, Geenen JE, Dodds WJ. Dysmotility disturbances of the biliary tract: classification, diagnosis, and treatment. Semin Liver Dis 1987; 7: 302-310

9 Geenen JE, Hogan WJ, Dodds WJ, Stewart ET, Arndorfer RC. Intraluminal pressure recording from the human sphincter of Oddi. Gastroenterology 1980; 78: 317-324

10 Baillie J. Sphincter of Oddi dysfunction: overdue for an overhaul. Am J Gastroenterol 2005; 100: 1217-1220

11 Majeed AW, Ross B, Johnson AG. The preoperatively normal bile duct does not dilate after cholecystectomy: results of a five year study. Gut 1999; 45: 741-743

12 Sherman S, Troiano FP, Hawes RH, Lehman GA. Does continuous aspiration from an end and side port in a sphincter of Oddi manometry catheter alter recorded pressures? Gastrointest Endosc 1990; 36: 500-503

13 Guelrud M, Mendoza S, Rossiter G, Villegas MI. Sphincter of Oddi manometry in healthy volunteers. Dig Dis Sci 1990; 35: 38-46

14 Toouli J, Di Francesco V, Saccone G, Kollias J, Schloithe A, Shanks N. Division of the sphincter of Oddi for treatment of dysfunction associated with recurrent pancreatitis. Br J Surg

1996; 83: 1205-121015 Sherman S, Lehman GA. Sphincter of Oddi dysfunction:

diagnosis and treatment. JOP 2001; 2: 382-40016 Eversman D, Fogel EL, Rusche M, Sherman S, Lehman GA.

Frequency of abnormal pancreatic and biliary sphincter manometry compared with clinical suspicion of sphincter of Oddi dysfunction. Gastrointest Endosc 1999; 50: 637-641

17 Varadarajulu S, Hawes RH, Cotton PB. Determination of sphincter of Oddi dysfunction in patients with prior normal manometry. Gastrointest Endosc 2003; 58: 341-344

18 Raddawi HM, Geenen JE, Hogan WJ, Dodds WJ, Venu RP, Johnson GK. Pressure measurements from biliary and pancreatic segments of sphincter of Oddi. Comparison between patients with functional abdominal pain, biliary, or pancreatic disease. Dig Dis Sci 1991; 36: 71-74

19 Rolny P, Arlebäck A, Funch-Jensen P, Kruse A, Järnerot G. Clinical significance of manometric assessment of both pancreatic duct and bile duct sphincter in the same patient. Scand J Gastroenterol 1989; 24: 751-754

20 Silverman WB, Ruffolo TA, Sherman S, Hawes RH, Lehman GA. Correlation of basal sphincter pressures measured from the bile duct and the pancreatic duct in patients with suspected sphincter of Oddi dysfunction. Gastrointest Endosc 1992; 38: 440-443

21 Chan YK , Evans PR, Dowsett JF, Kellow JE, Badcock CA. Discordance of pressure recordings from biliary and pancreatic duct segments in patients with suspected sphincter of Oddi dysfunction. Dig Dis Sci 1997; 42: 1501-1506

22 Aymerich RR, Prakash C, Aliperti G. Sphincter of oddi manometry: is it necessary to measure both biliary and pancreatic sphincter pressures? Gastrointest Endosc 2000; 52: 183-186

23 Pereira SP, Gillams A, Sgouros SN, Webster GJ, Hatfield AR. Prospective comparison of secretin-stimulated magnetic resonance cholangiopancreatography with manometry in the diagnosis of sphincter of Oddi dysfunction types II and III. Gut 2007; 56: 809-813

24 Hamilton I , Lintott DJ, Rothwell J , Axon AT. Acute pancreatitis following endoscopic retrograde cholangiopancreatography. Clin Radiol 1983; 34: 543-546

25 Tarnasky PR, Palesch YY, Cunningham JT, Mauldin PD, Cotton PB, Hawes RH. Pancreatic stenting prevents pancreatitis after biliary sphincterotomy in patients with sphincter of Oddi dysfunction. Gastroenterology 1998; 115: 1518-1524

26 Fogel EL, Eversman D, Jamidar P, Sherman S, Lehman GA. Sphincter of Oddi dysfunction: pancreaticobiliary sphincterotomy with pancreatic stent placement has a lower rate of pancreatitis than biliary sphincterotomy alone. Endoscopy 2002; 34: 280-285

27 Sherman S, Troiano FP, Hawes RH, Lehman GA. Sphincter of Oddi manometry: decreased risk of clinical pancreatitis with use of a modified aspirating catheter. Gastrointest Endosc 1990; 36: 462-466

28 Rolny P, Anderberg B, Ihse I, Lindström E, Olaison G, Arvill A. Pancreatitis after sphincter of Oddi manometry. Gut 1990; 31: 821-824

29 Scicchitano J, Saccone GT, Baker RA, Roberts-Thomson IC, Toouli J. How safe is endoscopic sphincter of Oddi manometry? J Gastroenterol Hepatol 1995; 10: 334-336

30 Maldonado ME, Brady PG, Mamel JJ, Robinson B. Incidence of pancreatitis in patients undergoing sphincter of Oddi manometry (SOM). Am J Gastroenterol 1999; 94: 387-390

31 Walters DA , Geenen JE, Catalano MF. A randomized controlled trial comparing the use of an aspirating catheter vs a standard perfused catheter on the incidence of pancreatitis following sphincter of Oddi manometry (SOM). Gastrointest Endosc 1997; 45: AB152 (Abstract)

32 Sherman S, Hawes RH, Madura JA, Lehman GA. Comparison of intraoperative and endoscopic manometry of the sphincter of Oddi. Surg Gynecol Obstet 1992; 175: 410-418

33 Lehman GA. Endoscopic sphincter of Oddi manometry: a clinical practice and research tool. Gastrointest Endosc 1991; 37: 490-492

McLoughlin MT et al. SOD and pancreatitis 6341

www.wjgnet.com

34 Aizawa T, Ueno N. Stent placement in the pancreatic duct prevents pancreatitis after endoscopic sphincter dilation for removal of bile duct stones. Gastrointest Endosc 2001; 54: 209-213

35 Fazel A, Quadri A, Catalano MF, Meyerson SM, Geenen JE. Does a pancreatic duct stent prevent post-ERCP pancreatitis? A prospective randomized study. Gastrointest Endosc 2003; 57: 291-294

36 Freeman ML, Nelson DB, Sherman S, Haber GB, Herman ME, Dorsher PJ, Moore JP, Fennerty MB, Ryan ME, Shaw MJ, Lande JD, Pheley AM. Complications of endoscopic biliary sphincterotomy. N Engl J Med 1996; 335: 909-918

37 Singh P, Gurudu SR, Davidoff S, Sivak MV, Indaram A, Kasmin FE, Nozdak V, Wong RC, Isenberg G, Stark B, Bank S, Chak A. Sphincter of Oddi manometry does not predispose to post-ERCP acute pancreatitis. Gastrointest Endosc 2004; 59: 499-505

38 Cheng CL, Sherman S, Watkins JL, Barnett J, Freeman M, Geenen J, Ryan M, Parker H, Frakes JT, Fogel EL, Silverman WB, Dua KS, Aliperti G, Yakshe P, Uzer M, Jones W, Goff J, Lazzell-Pannell L, Rashdan A, Temkit M, Lehman GA. Risk factors for post-ERCP pancreatitis: a prospective multicenter study. Am J Gastroenterol 2006; 101: 139-147

39 Chen JW, Thomas A, Woods CM, Schloithe AC, Toouli J, Saccone GT. Sphincter of Oddi dysfunction produces acute pancreatitis in the possum. Gut 2000; 47: 539-545

40 Kruszyna T, Zajac A, Karcz D. Sphincter of Oddi manometry in patients with acute biliary pancreatitis: evidence for sphincter of Oddi dysfunction in acute biliary pancreatitis. Scand J Gastroenterol 2004; 39: 696-697

41 Fazel A , Geenen JE , MoezArdalan K, Catalano MF. Intrapancreatic ductal pressure in sphincter of Oddi dysfunction. Pancreas 2005; 30: 359-362

42 Warshaw AL, Simeone J, Schapiro RH, Hedberg SE, Mueller PE, Ferrucci JT. Objective evaluation of ampullary stenosis with ultrasonography and pancreatic stimulation. Am J Surg 1985; 149: 65-72

43 Moody FG, Vecchio R, Calabuig R, Runkel N. Transduodenal sphincteroplasty with transampullary septectomy for stenosing papillitis. Am J Surg 1991; 161: 213-218

44 Dressel TD, Goodale RL, Hunninghake DB, Borner JW. Sensitivity of the canine pancreatic intraductal pressure to subclinical reduction in cholinesterase acitivity. Ann Surg 1979; 190: 6-12

45 Dressel TD , Goodale RL, Arneson MA, Borner JW. Pancreatitis as a complication of anticholinesterase insecticide intoxication. Ann Surg 1979; 189: 199-204

46 Bartholomew C, McGeeney KF, Murphy JJ, Fitzgerald O, Sankaran H. Experimental studies on the aetiology of acute scorpion pancreatitis. Br J Surg 1976; 63: 807-810

47 Frick TW, Spycher MA, Heitz PU, Largiadèr F, Goodale RL. Hypercalcaemia and pancreatic ultrastructure in cats. Eur J Surg 1992; 158: 289-294

48 Szilvassy Z, Nagy I, Szilvassy J, Jakab I, Csati S, Lonovics J. Impaired nitrergic relaxation of the sphincter of Oddi of hyperlipidaemic rabbits. Eur J Pharmacol 1996; 301: R17-R18

49 Gregg JA , Carr-Locke DL. Endoscopic pancreatic and biliary manometry in pancreatic, biliary, and papillary disease, and after endoscopic sphincterotomy and surgical sphincteroplasty. Gut 1984; 25: 1247-1254

50 Toouli J, Roberts-Thomson IC, Dent J, Lee J. Sphincter of Oddi motility disorders in patients with idiopathic recurrent pancreatitis. Br J Surg 1985; 72: 859-863

51 Venu RP, Geenen JE, Hogan W, Stone J, Johnson GK, Soergel K. Idiopathic recurrent pancreatitis. An approach to diagnosis and treatment. Dig Dis Sci 1989; 34: 56-60

52 Sherman S. Idiopathic acute recurrent pancreatitis: endoscopic approach to diagnosis and therapy. Gastrointest Endosc 1992; 38: 261A (Abstract)

53 Coyle WJ, Pineau BC, Tarnasky PR, Knapple WL, Aabakken L, Hoffman BJ, Cunningham JT, Hawes RH, Cotton PB. Evaluation of unexplained acute and acute recurrent pancreatitis using endoscopic retrograde cholangiopancre

atography, sphincter of Oddi manometry and endoscopic ultrasound. Endoscopy 2002; 34: 617-623

54 Kaw M, Brodmerkel GJ. ERCP, biliary crystal analysis, and sphincter of Oddi manometry in idiopathic recurrent pancreatitis. Gastrointest Endosc 2002; 55: 157-162

55 Choudari CP, Fogel EL, Sherman S. Frequency of abnormal sphincter of Oddi manometry (SOM) in alcoholic pancreatitis. Gastrointest Endosc 1999; 49: 78A (Abstract)

56 Guelrud M , Morera C, Rodriguez M, Jaen D, Pierre R. Sphincter of Oddi dysfunction in children with recurrent pancreatitis and anomalous pancreaticobiliary union: an etiologic concept. Gastrointest Endosc 1999; 50: 194-199

57 Okazaki K, Yamamoto Y, Kagiyama S, Tamura S, Sakamoto Y, Morita M, Yamamoto Y. Pressure of papillary sphincter zone and pancreatic main duct in patients with alcoholic and idiopathic chronic pancreatitis. Int J Pancreatol 1988; 3: 457-468

58 Novis BH , Bornman PC, Girdwood AW, Marks IN. Endoscopic manometry of the pancreatic duct and sphincter zone in patients with chronic pancreatitis. Dig Dis Sci 1985; 30: 225-228

59 Okazaki K, Yamamoto Y, Ito K. Endoscopic measurement of papillary sphincter zone and pancreatic main ductal pressure in patients with chronic pancreatitis. Gastroenterology 1986; 91: 409-418

60 Rolny P, Arlebäck A, Järnerot G, Andersson T. Endoscopic manometry of the sphincter of Oddi and pancreatic duct in chronic pancreatitis. Scand J Gastroenterol 1986; 21: 415-420

61 Ugljesić M, Bulajić M, Milosavljević T, Stimec B. Endoscopic manometry of the sphincter of Oddi and pancreatic duct in patients with chronic pancreatitis. Int J Pancreatol 1996; 19: 191-195

62 Vestergaard H, Kruse A, Rokkjaer M, Frøbert O, Thommesen P, Funch-Jensen P. Endoscopic manometry of the sphincter of Oddi and the pancreatic and biliary ducts in patients with chronic pancreatitis. Scand J Gastroenterol 1994; 29: 188-192

63 Ochi T, Nakazawa S, Naito Y, Tsukamoto Y. Endoscopic manometry of the sphincter of Oddi and pancreatic duct in patients with papillary stenosis. Endoscopy 1991; 23: 255-258

64 Laugier R. Dynamic endoscopic manometry of the response to secretin in patients with chronic pancreatitis. Endoscopy 1994; 26: 222-227

65 Guelrud M, Mendoza S, Rossiter G, Gelrud D, Rossiter A, Souney PF. Effect of local instillation of alcohol on sphincter of Oddi motor activity: combined ERCP and manometry study. Gastrointest Endosc 1991; 37: 428-432

66 Tarnasky PR, Hoffman B, Aabakken L, Knapple WL, Coyle W, Pineau B, Cunningham JT, Cotton PB, Hawes RH. Sphincter of Oddi dysfunction is associated with chronic pancreatitis. Am J Gastroenterol 1997; 92: 1125-1129

67 Rolny P, Geenen JE, Hogan WJ. Post-cholecystectomy patients with "objective signs" of partial bile outflow obstruction: clinical characteristics, sphincter of Oddi manometry findings, and results of therapy. Gastrointest Endosc 1993; 39: 778-781

68 Thatcher BS, Sivak MV, Tedesco FJ, Vennes JA, Hutton SW, Achkar EA. Endoscopic sphincterotomy for suspected dysfunction of the sphincter of Oddi. Gastrointest Endosc 1987; 33: 91-95

69 Lin OS, Soetikno RM, Young HS. The utility of liver function test abnormalities concomitant with biliary symptoms in predicting a favorable response to endoscopic sphincterotomy in patients with presumed sphincter of Oddi dysfunction. Am J Gastroenterol 1998; 93: 1833-1836

70 Toouli J, Roberts-Thomson IC, Kellow J, Dowsett J, Saccone GT, Evans P, Jeans P, Cox M, Anderson P, Worthley C, Chan Y, Shanks N, Craig A. Manometry based randomised trial of endoscopic sphincterotomy for sphincter of Oddi dysfunction. Gut 2000; 46: 98-102

71 Sherman S, Lehman G, Jamidar P, Hawes R, Silverman W, Madura J. Efficacy of endoscopic sphincterotomy and surgical sphincteroplasty for patients with sphincter of Oddi dysfunction (SOD): randomized, controlled study. Gastrointest Endosc 1994; 40: 125 (Abstract)

72 Botoman VA, Kozarek RA, Novell LA, Patterson DJ, Ball TJ,

6342 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol December 21, 2007 Volume 13 Number 47

www.wjgnet.com

Wechter DG, Neal LA. Long-term outcome after endoscopic sphincterotomy in patients with biliary colic and suspected sphincter of Oddi dysfunction. Gastrointest Endosc 1994; 40: 165-170

73 Wehrmann T, Wiemer K, Lembcke B, Caspary WF, Jung M. Do patients with sphincter of Oddi dysfunction benefit from endoscopic sphincterotomy? A 5-year prospective trial. Eur J Gastroenterol Hepatol 1996; 8: 251-256

74 Cicala M, Habib FI, Vavassori P, Pallotta N, Schillaci O, Costamagna G, Guarino MP, Scopinaro F, Fiocca F, Torsoli A, Corazziari E. Outcome of endoscopic sphincterotomy in post cholecystectomy patients with sphincter of Oddi dysfunction as predicted by manometry and quantitative choledochoscintigraphy. Gut 2002; 50: 665-668

75 Viceconte G, Micheletti A. Endoscopic manometry of the sphincter of Oddi: its usefulness for the diagnosis and treatment of benign papillary stenosis. Scand J Gastroenterol 1995; 30: 797-803

76 Sherman S . What is the role of ERCP in the setting of abdominal pain of pancreatic or biliary origin (suspected sphincter of Oddi dysfunction)? Gastrointest Endosc 2002; 56: S258-S266

77 Park SH, Watkins JL, Fogel EL, Sherman S, Lazzell L, Bucksot L, Lehman GA. Long-term outcome of endoscopic dual pancreatobiliary sphincterotomy in patients with manometry-documented sphincter of Oddi dysfunction and normal pancreatogram. Gastrointest Endosc 2003; 57: 483-491

78 Desautels SG, Slivka A, Hutson WR, Chun A, Mitrani C, DiLorenzo C, Wald A. Postcholecystectomy pain syndrome: pathophysiology of abdominal pain in sphincter of Oddi type III. Gastroenterology 1999; 116: 900-905

79 Devereaux BM, Sherman S, Lehman GA. Sphincter of Oddi (pancreatic) hypertension and recurrent pancreatitis. Curr Gastroenterol Rep 2002; 4: 153-159

80 Tarnasky PR , Cunningham JT, Knapple WL. Repeat pancreatic sphincter manometry after biliary sphincterotomy in patients with sphincter of Oddi dysfunction. Gastrointest Endosc 1997; 45: 151A (Abstract)

81 Jacob L, Geenen JE, Catalano MF, Geenen DJ. Prevention of pancreatitis in patients with idiopathic recurrent pancreatitis: a prospective nonblinded randomized study using endoscopic stents. Endoscopy 2001; 33: 559-562

82 Hogan WJ, Geenen JE, Kruidenier J, Venu R, Helm J, Dodds WJ. Ineffectiveness of conventional sphincteroplasty in relieving pancreatic duct sphincter pressure in patients with idiopathic recurrent pancreatitis. Gastroenterology 1983; 84: 1189 (Abstract)

83 Tarnasky PR, Cunningham JT, Cotton PB, Hoffman B, Hawes R. Repeat pancreatic sphincter of Oddi manometry immediately after biliary sphincterotomy. Am J Gastroenterol 1996; 91: 1943 (Abstract)

84 Eversman D, Fogel EL, Phillips S, Sherman S, Lehman GA. Sphincter of Oddi dysfunction (SOD): long-term outcome of biliary sphincterotomy (BES) correlated with abnormal biliary

and pancreatic sphincters. Gastrointest Endosc 1999; 49: 78 (Abstract)

85 Kaw M, Verma R, Brodmerkel GJJ. Biliary and/or pancreatic sphincter of Oddi dysfunction (SOD): response to endoscopic sphincterotomy (ES). Gastrointest Endosc 1996; 43: 384 (Abstract)

86 Guelrud M, Plaz J, Mendoza S, Beker B, Rojas O, Rossiter G. Endoscopic treatment in type II pancreatic sphincter dysfunction. Gastrointest Endosc 1995; 41: 398 (Abstract)

87 Soffer EE, Johlin FC. Intestinal dysmotility in patients with sphincter of Oddi dysfunction. A reason for failed response to sphincterotomy. Dig Dis Sci 1994; 39: 1942-1946

88 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

89 Freeman ML , Gill M, Overby C, Cen YY. Predictors of outcomes after biliary and pancreatic sphincterotomy for sphincter of oddi dysfunction. J Clin Gastroenterol 2007; 41: 94-102

90 Tarnasky PR . Mechanical prevention of post-ERCP pancreatitis by pancreatic stents: results, techniques, and indications. JOP 2003; 4: 58-67

91 Vandervoort J, Soetikno RM, Tham TC, Wong RC, Ferrari AP, Montes H, Roston AD, Slivka A, Lichtenstein DR, Ruymann FW, Van Dam J, Hughes M, Carr-Locke DL. Risk factors for complications after performance of ERCP. Gastrointest Endosc 2002; 56: 652-656

92 Freeman ML, DiSario JA, Nelson DB, Fennerty MB, Lee JG, Bjorkman DJ, Overby CS, Aas J, Ryan ME, Bochna GS, Shaw MJ, Snady HW, Erickson RV, Moore JP, Roel JP. Risk factors for post-ERCP pancreatitis: a prospective, multicenter study. Gastrointest Endosc 2001; 54: 425-434

93 Masci E, Mariani A, Curioni S, Testoni PA. Risk factors for pancreatitis following endoscopic retrograde cholangiopancreatography: a meta-analysis. Endoscopy 2003; 35: 830-834

94 Patel R, Tarnasky PR, Hennessy WS, Hawes R, Payne KM, Nelles SE, et al. Does stenting after pancreatic sphincterotomy reduce post-ERCP pancreatitis in patients with prior biliary sphincterotomy? Preliminary results of a prospective randomized trial. Gastrointest Endosc 1999; 49: AB80 (Abstract)

95 Smithline A, Silverman W, Rogers D, Nisi R, Wiersema M, Jamidar P, Hawes R, Lehman G. Effect of prophylactic main pancreatic duct stenting on the incidence of biliary endoscopic sphincterotomy-induced pancreatitis in high-risk patients. Gastrointest Endosc 1993; 39: 652-657

96 Singh P, Das A, Isenberg G, Wong RC, Sivak MV, Agrawal D, Chak A. Does prophylactic pancreatic stent placement reduce the risk of post-ERCP acute pancreatitis? A meta-analysis of controlled trials. Gastrointest Endosc 2004; 60: 544-550

97 Freeman ML, Overby C, Qi D. Pancreatic stent insertion: consequences of failure and results of a modified technique to maximize success. Gastrointest Endosc 2004; 59: 8-14

98 Freeman ML. Role of pancreatic stents in prevention of post-ERCP pancreatitis. JOP 2004; 5: 322-327

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McLoughlin MT et al. SOD and pancreatitis 6343

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Pancreatic endocrine and exocrine changes in celiac disease

Hugh J Freeman

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Michael F Byrne, MD, Series Editor

Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6344-6346www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

Hugh J Freeman, Department of Medicine (Gastroenterology), University of British Columbia, Vancouver, BC, CanadaCorrespondence to: Dr. Hugh J Freeman, MD, FRCPC, FACP, Gastroenterology, UBC Hospital, 2211 Wesbrook Mall, Vancouver, BC, V6T 1W8, Canada. hugfree@shaw.caTelephone: +1-604-8227216 Fax: +1-604-8227236Received: June 15, 2007 Revised: July 16, 2007

AbstractAlthough there is a great deal of information on celiac disease and associated involvement of other non-intestinal sites, data on concomitant changes in the structure and function of the pancreas is limited. The present review critically examines pancreatic endocrine changes that have been well documented in the literature, including insulin-dependent diabetes mellitus. Pancreatic exocrine alterations may also occur, and if severe, marked malnutrition with pancreatic failure and ductal calcification have been observed. Finally, other pancreatic disorders have been recorded with celiac disease.

© 2007 WJG. All rights reserved.

Key words: Celiac disease; Gluten-sensitive enteropathy; Diabetes mellitus; Chronic pancreatitis; Pancreatic function

Freeman HJ. Pancreatic endocrine and exocrine changes in celiac disease. World J Gastroenterol 2007; 13(47): 6344-6346

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INTRODUCTIONConsiderable information has already been published on both the intestinal and the extra-intestinal manifestations of celiac disease. Pancreatic changes may be caused by celiac disease or co-exist with celiac disease. Both endocrine and exocrine function of the pancreas may be substantially changed in celiac disease. As a result, superimposed or more severe clinical changes may appear and marked nutritional disturbances may result. Although data is very limited, there is increasing evidence that impaired endocrine and exocrine pancreatic function in

celiac disease may be favorably influenced by gluten-free diet treatment.

PANCREATIC ENDOCRINE CHANGES Over the past decade or so, a number of studies from Europe[1,2] and North America[3,4] have demonstrated that the prevalence of celiac disease in patients with type 1 diabetes is increased. This association is due, at least in part, to sharing of the human leukocyte antigen allele, DR3, and, by linkage disequilibrium, DQ2[5]. In addition to a common “autoimmune” basis, it is conceivable that some celiacs have developed diabetic changes secondary to severe pancreatic insufficiency with exocrine dysfunction. Earlier serological studies employed IgA antibody studies for celiac screening, particularly using IgA endomysial antibody (EMA) testing. This eventually became the “gold standard” for serological studies, until subsequent identification of tissue transglutaminase (TTG) as the tissue antigen for endomysial antibodies[6]. As a result, IgA TTG antibodies (using ELISA methodology) became a very attractive alternative for first line screening and also permitted development of a quantitative assay.

A recent TTG prospective study[7] in children and adolescents with type 1 diabetes evaluated TTG antibody titres in 125 males and 108 females followed in a pediatric diabetes center. Altogether, 26 patients, including 15 males and 11 females, had positive TTG titres and, of these, 19 were also positive for EMA. In those positive for both TTG and EMA, small intestinal biopsies were done. Histopathological abnormalities described in celiac disease were detected ranging from increased numbers of intraepithelial lymphocytes to severe crypt hyperplastic villous atrophy (Marsh 3 lesion)[7]. These studies also suggested that serial TTG serological measurements in insulin-dependent diabetics might play a role in monitoring their serological responses as well as the compliance to the gluten-free diet. In children and adolescents, close monitoring is critical as compliance in these age groups may be especially difficult to assess. While over 40% of the diabetics in this study were asymptomatic[7], prospective serological screening appeared to facilitate selection for biopsy evaluation.

Earlier detection of celiac disease has been urged in type 1 diabetes, even in children, because of the long-term risks of undiagnosed celiac disease. It has been suggested that the longer the duration of untreated celiac disease (also in dermatitis herpetiformis), the higher the risk of

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enteropathy-associated T cell lymphoma[8]. Moreover, adherence to a gluten-free diet appears to reduce the risk of enteropathy-associated T cell lymphomas in celiac disease. Finally, the association of lymphoma, celiac disease and type 1 diabetes has also been documented in 4 cases[9].

Other long-term complications may occur including iron deficiency anemia, osteoporosis, infertility and growth retardation. These appear to be most significant when patients are poorly compliant with a gluten-free diet, or if diagnosis is delayed until later in life[10]. Finally, improved glucose control with a gluten-free diet has also been shown in type 1 diabetes with concomitant celiac disease[11]. In these studies with type 1 diabetes in celiac disease, pancreatic exocrine function was not evaluated.

PANCREATIC EXOCRINE CHANGESPancreatic exocrine function may also be substantially altered in cel iac disease. Even though the precise prevalence of altered pancreatic function in celiac disease is not known, impaired pancreatic function may be a cause of impaired digestion and absorption resulting in malnutrition[12]. It has been estimated that over 20% of patients with celiac disease have defective exocrine pancreatic function[13]. This may be related to several factors. First, impaired secretion and/or release of pancreatic stimulating hormones from the diseased proximal small intestine may be important[14]. Immunohistochemical studies on small intestinal biopsies from untreated celiac disease have demonstrated significant alterations in enteric endocrine cells, including an absence of secretin cells[15].

Moreover, studies with test meals in celiacs have suggested impaired secretion of cholecystokinin-pancreozymin, resulting in reduced pancreatic exocrine cell stimulation[16]. Second, deficiencies of amino acids may result from impaired small intestinal amino acid uptake, leading to reduction in precursors of pancreatic enzyme synthesis[12,17]. Third, protein malnutrition may lead to structural changes in the pancreas, including atrophy of acinar cells and pancreatic fibrosis, resulting in impaired pancreatic exocrine function[12]. Pancreatic enzyme measurements were also found to be reduced with mucosal atrophy and could be inversely correlated with the degree of intestinal damage[18]. Earlier studies measuring xylose absorption were limited in ability to separate pancreatic and intestinal causes of steatorrhea, especially if the degree of impairment was mild[19]. Subsequent attempts to define altered intestinal permeability using large molecules (e.g., cellulobiose, mannitol)[20] or separate pancreatic insufficiency from intestinal dysfunction with noninvasive stable isotopes[21] have not become widely accepted.

Severe structural changes have also been documented. Pancreatic calcification, most often associated with chronic or persisting pancreatic inflammation, has been traditionally associated with excessive alcohol use.

Atrophy, fibrosis and altered pancreatic function have also been detected in experimental animals treated with diets deficient in protein, in adults with protein energy malnutrition, in children with kwashiorkor and in some

early autopsy studies of patients with celiac disease[16,17,22-24]. In addition, pancreatic calcification has been reported with chronic protein malnutrition in the Indian subcontinent and in some African countries[25]. Finally, an adult patient with celiac disease and non-alcohol related pancreatitis with calcification has been described in North America[26]. Possible effects on pancreatic exocrine function in celiac disease poorly compliant to a gluten-free diet has not been evaluated.

MISCELLANEOUS DISORDERSA number of other miscellaneous pancreatic disorders have been associated with celiac disease. Pancreatic mucinous adenomas have been associated with celiac disease in a patient with polycystic kidney disease[27]. In addition, pancreatic and ampullary carcinomas have been recorded in celiac disease[28,29], although the celiac disease has often only been recognized after pancreatoduodenectomy suggesting that subclinical celiac disease may be “unmasked” by major upper gastrointestinal surgery and should be considered in the differential diagnosis after pancreatoduodenectomy[22,29].

Celiac disease has been associated with chronic calcific pancreatitis in patients with pancreas divisum[30]. Diabetes-related autoantibodies may appear in children with celiac disease, including antibodies to insulin, immunoglobulin, islet cells and glucagon[31]. Early-onset vitamin B12 malabsorption in children with celiac disease has also been documented and related to impaired pancreatic exocrine secretion[32]. Finally, macroamylasemia has also been attributed to gluten-related amylase autoantibodies (particularly in childhood)[33].

Because of the possible deleterious effects of biliary tract lithiasis on pancreatic structure and function, gal lbladder function in celiac disease may also be important and carefully examined in celiac disease. In some, slow emptying of the gallbladder has been documented, associated with impaired contraction response to fat[34,35]. Studies of enteric endocrine cells have demonstrated significant quantitative and qualitative changes in celiacs[15]. In addition, studies with test meals have suggested impaired secretion of cholecystokinin in patients with celiac disease, and, possibly, impaired gallbladder responsiveness to cholecystokinin[15,34]. In spite of these physiological alterations, there does not appear to be a significant predisposition to gallstones in celiac disease and consequently, secondary pancreatic damage. Only 9 of 350 celiac patients had a cholecystectomy for gallstone disease[14]; however, in a survey of elderly celiacs initially diagnosed after the age of 60 years, approximately 20% had developed gallstone disease[36,37].

CONCLUSIONIn summary the endocrine and exocrine function of the pancreas may be impaired in celiac disease and their pathogenesis may be closely linked. Further studies are needed to explore the importance of detection of endocrine and exocrine pancreatic disorders in celiac disease, along with potential treatment options.

REFERENCES 1 Saukkonen T, Savilahti E, Reijonen H, Ilonen J, Tuomilehto-

Wolf E, Akerblom HK. Coeliac disease: frequent occurrence after clinical onset of insulin-dependent diabetes mellitus. Childhood Diabetes in Finland Study Group. Diabet Med 1996; 13: 464-470

2 Mäki M, Huupponen T, Holm K, Hällström O. Seroconversion of reticulin autoantibodies predicts coeliac disease in insulin dependent diabetes mellitus. Gut 1995; 36: 239-242

3 Fraser-Reynolds KA, Butzner JD, Stephure DK, Trussell RA, Scott RB. Use of immunoglobulin A-antiendomysial antibody to screen for celiac disease in North American children with type 1 diabetes. Diabetes Care 1998; 21: 1985-1989

4 Hill I, Fasano A, Schwartz R, Counts D, Glock M, Horvath K. The prevalence of celiac disease in at-risk groups of children in the United States. J Pediatr 2000; 136: 86-90

5 Lie BA, Sollid LM, Ascher H, Ek J, Akselsen HE, Rønningen KS, Thorsby E, Undlien DE. A gene telomeric of the HLA class I region is involved in predisposition to both type 1 diabetes and coeliac disease. Tissue Antigens 1999; 54: 162-168

6 Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, Schuppan D. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997; 3: 797-801

7 Gillett PM, Gillett HR, Israel DM, Metzger DL, Stewart L, Chanoine JP, Freeman HJ. High prevalence of celiac disease in patients with type 1 diabetes detected by antibodies to endomysium and tissue transglutaminase. Can J Gastroenterol 2001; 15: 297-301

8 Holmes GK, Prior P, Lane MR, Pope D, Allan RN. Malignancy in coeliac disease--effect of a gluten free diet. Gut 1989; 30: 333-338

9 O'Connor TM, Cronin CC, Loane JF, O'Meara NM, Firth RG, Shanahan F, O'Halloran DJ. Type 1 diabetes mellitus, coeliac disease, and lymphoma: a report of four cases. Diabet Med 1999; 16: 614-617

10 Holmes GK. Non-malignant complications of coeliac disease. Acta Paediatr Suppl 1996; 412: 68-75

11 Acerini CL, Ahmed ML, Ross KM, Sullivan PB, Bird G, Dunger DB. Coeliac disease in children and adolescents with IDDM: clinical characteristics and response to gluten-free diet. Diabet Med 1998; 15: 38-44

12 Freeman HJ, Kim YS, Sleisenger MH. Protein digestion and absorption in man. Normal mechanisms and protein-energy malnutrition. Am J Med 1979; 67: 1030-1036

13 Carroccio A, Iacono G, Montalto G, Cavataio F, Di Marco C, Balsamo V, Notarbartolo A. Exocrine pancreatic function in children with coeliac disease before and after a gluten free diet. Gut 1991; 32: 796-799

14 D i M a g n o E P , G o W L , S u m m e r s k i l l W H . I m p a i r e d cholecystokinin-pancreozymin secretion, intraluminal dilution, and maldigestion of fat in sprue. Gastroenterology 1972; 63: 25-32

15 Buchan AM, Grant S, Brown JC, Freeman HJ. A quantitative study of enteric endocrine cells in celiac sprue. J Pediatr Gastroenterol Nutr 1984; 3: 665-671

16 Regan PT, DiMagno EP. Exocrine pancreatic insufficiency in celiac sprue: a cause of treatment failure. Gastroenterology 1980; 78: 484-487

17 Weinstein LD, Herskovic T. Rectal seepage of oil in a patient with celiac disease and secondary pancreatic insufficiency. Am J Dig Dis 1968; 13: 762-765

18 Nousia-Arvanitakis S , Karagiozoglou-Lamboudes T, Aggouridaki C, Malaka-Lambrellis E, Galli-Tsinopoulou A, Xefteri M. Influence of jejunal morphology changes on exocrine pancreatic function in celiac disease. J Pediatr Gastroenterol Nutr 1999; 29: 81-85

19 Hindmarsh JT. Xylose absorption and its clinical significance. Clin Biochem 1976; 9: 141-143

20 Cobden I, Rothwell J, Axon AT. Intestinal permeability and screening tests for coeliac disease. Gut 1980; 21: 512-518

21 Deutsch JC, Santhosh-Kumar CR, Kolli VR. A noninvasive stable-isotope method to simultaneously assess pancreatic exocrine function and small bowel absorption. Am J Gastroenterol 1995; 90: 2182-2185

22 Benson GD, Kowlessar OD, Sleisenger MH. Adult celiac dissease with emphasis upon response to tth gluten-free Diet. Medicine (Baltimore) 1964; 43: 1-40

23 Pink IJ, Creamer B. Response to a gluten-free diet of patients with the coeliac syndrome. Lancet 1967; 1: 300-304

24 Pitchumoni CS, Thomas E, Balthazar E, Sherling B. Chronic calcific pancreatitis in association with celiac disease. Am J Gastroenterol 1977; 68: 358-361

25 Pitchumoni CS. Pancreas in primary malnutrition disorders. Am J Clin Nutr 1973; 26: 374-379

26 Freeman HJ. Hepatobiliary tract and pancreatic disorders in celiac disease. Can J Gastroenterol 1997; 11: 77-81

27 Takeh H, Phillipart P, Brandelet B, Nemec E, Bidgoli SJ, da Costa PM. Pancreatic mucinous cystadenoma associated with celiac disease and polycystic kidneys. Case report and short review of the literature. Hepatogastroenterology 2002; 49: 944-946

28 C h e d i d A D , K r u e l C R , C h e d i d M F , T o r r e s i n i R J , Geyer GR. Development of clinical celiac disease after pancreatoduodenectomy: a potential complication of major upper abdominal surgery. Langenbecks Arch Surg 2005; 390: 39-41

29 Maple JT, Pearson RK, Murray JA, Kelly DG, Lara LF, Fan AC. Silent celiac disease activated by pancreaticoduodenectomy. Dig Dis Sci 2007; 52: 2140-2144

30 Arya S, Rana SS, Sinha SK, Nagi B, Bhasin DK. Celiac disease and chronic calcific pancreatitis with pancreas divisum. Gastrointest Endosc 2006; 63: 1080-1081

31 Rapoport MJ, Bistritzer T, Vardi O, Broide E, Azizi A, Vardi P. Increased prevalence of diabetes-related autoantibodies in celiac disease. J Pediatr Gastroenterol Nutr 1996; 23: 524-527

32 Hjelt K. The role of the exocrine pancreas in early-onset vitamin B12 malabsorption in gluten-challenged celiac children. J Pediatr Gastroenterol Nutr 1991; 13: 27-31

33 Barera G, Bazzigaluppi E, Viscardi M, Renzetti F, Bianchi C, Chiumello G, Bosi E. Macroamylasemia attributable to gluten-related amylase autoantibodies: a case report. Pediatrics 2001; 107: E93

34 Low-Beer TS, Schneider RE, Dobbins WO. Morphological changes of the small-intestinal mucosa of guinea pig and hamster following incubation in vitro and perfusion in vivo with unconjugated bile salts. Gut 1970; 11: 486-492

35 Low-Beer TS, Heaton KW, Heaton ST, Read AE. Gallbladder inertia and sluggish enterohepatic circulation of bile-salts in coeliac disease. Lancet 1971; 1: 991-994

36 Freeman HJ. Clinical spectrum of biopsy-defined celiac disease in the elderly. Can J Gastroenterol 1995; 9: 42-46

37 Freeman H, Lemoyne M, Pare P. Coeliac disease. Best Pract Res Clin Gastroenterol 2002; 16: 37-49

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TOPIC HIGHLIGHT

Transplantation for the treatment of type 1 diabetes

R Mark Meloche

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Michael F Byrne, MD, Series Editor

Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6347-6355www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

R Mark Meloche, Department of Surgery, University of British Columbia, Vancouver, British Columbia, CanadaCorrespondence to: R Mark Meloche, MD, FRCSC, Department of Surgery, University of British Columbia, 5th Floor Diamond Centre, 2775 Laurel Street, Vancouver, BC, V5Z 1M9,Canada. meloche@interchange.ubc.ca Telephone: +1-604-8755287Received: June 15, 2007 Revised: August 8, 2007

AbstractTransplantation of pancreatic tissue, as either the intact whole pancreas or isolated pancreatic islets has become a clinical option to be considered in the treatment of patients with type 1 insulin-dependant diabetes mellitus. A successful whole pancreas or islet transplant offers the advantages of attaining normal or near normal blood glucose control and normal hemoglobin A1c levels without the risks of severe hypoglycemia associate with intensive insulin therapy. Both forms of transplants are also effective at eliminating the occurrence of significant hypoglycemic events (even with only partial islet function evident). Whereas whole pancreas transplantation has also been shown to be very effective at maintaining a euglycemic state over a sustained period of time, thus providing an opportunity for a recipient to benefit from improvement of their blood glucose control, it is associated with a significant risk of surgical and post-operative complications. Islet transplantation is attractive as a less invasive alternative to whole pancreas transplant and offers the future promise of immunosuppression-free transplantation through pre-transplant culture. Islet transplantation however, may not always achieve the sustained level of tight glucose control necessary for reducing the risk of secondary diabetic complications and exposes the patient to the adverse effects of immunosuppression. Although recent advances have led to an increased rate of obtaining insulin-independence following islet transplantation, further developments are needed to improve the long-term viability and function of the graft to maintain improved glucose control over time.

© 2007 WJG. All rights reserved.

Key words: Type 1 diabetes; Insulin-dependant diabetes mellitus; Pancreas transplantation; Pancreatic islet transplantation; Immunosuppression; Glucose control

Meloche RM. Transplantation for the treatment of type 1 diabetes. World J Gastroenterol 2007; 13(47): 6347-6355

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TRANSPLANTATION FOR THETREATMENT OF TYPE 1 DIABETESIt has been clearly shown that patients with type 1 insulin-dependent diabetes mellitus (IDDM) benefit from improved blood glucose control. In 1993, the Diabetes Control and Complications Trial Research Group reported that patients with IDDM treated with intensive insulin therapy showed a reduced risk of developing retinopathy, albuminuria or microalbuminuria and clinical neuropathy when compared to patients who received conventional insulin therapy[1]. In this trial the intensive therapy group was shown to have achieved sustained lowered blood glucose concentrations over time as reflected by significantly lower glycosylated hemoglobin values compared to those of the conventional insulin therapy group. Although the intensive therapy group benefited from reduced long-term complications, the risk of severe hypoglycemia associated with tight glycemic control was three times greater than in the conventional therapy group.

Transplantation of pancreatic tissue, as either the intact whole pancreas or isolated pancreatic islets has become a clinical option to be considered in the treatment of patients with IDDM. Successful pancreatic transplantation offers the advantages of attaining normal or near normal blood glucose control without the risks of severe hypoglycemia associated with intensive insulin therapy. Pancreatic transplantation however, may not always achieve the sustained level of tight glucose control necessary for reducing the risk of secondary diabetic complications and exposes the patient to peri-operative or procedural risks, the adverse effects of immunosuppression and the risk of an eventual significant loss of graft function necessitating a return to exogenous insulin.

When either transplantation of a whole pancreas or pancreatic islets is being offered for the treatment of IDDM, the goals of the program should include: (1) A transplant procedure, which can be performed with overall low morbidity and mortality and subject the recipient to a minimal degree of side effects and complications of the post-procedural care; (2) Elimination of the need for insulin administration and close blood glucose monitoring; (3) Eliminate the occurrence of significant hypoglycemic events; (4) Creation of a euglycemic state, with preprandial and postprandial blood glucose concentrations and

hemoglobin A1c levels comparable to those in the non-diabetic population; (5) Achieving sustained effect of the transplant as to maintain normal glucose homeostasis over time.

WHOLE PANCREAS TRANSPLANTATIONWhole pancreas transplantation, first performed in 1966 in combination with a kidney in IDDM patients suffering from end-stage renal failure, demonstrated that a euglycemic state could be obtained without the need for exogenous insulin[2]. The early procedures however, were complicated by a high morbidity rate; early graft failure and poor patient survival and few transplants were performed[3]. Improvements in transplant techniques, immunosuppression therapies and post-transplant monitoring of graft function and rejection has resulted in a dramatic improvement in patient morbidity and graft survival. With the improved outcomes and demonstrated efficaciousness in controlling the diabetic glycemic state, whole pancreas transplantation is now recognized by the American Diabetes Association as an acceptable therapeutic alternative to continued insulin therapy in diabetic patients with imminent or established end-stage renal disease who had or plan to have a kidney transplant[4].

Pancreas transplantation may be considered as a group of three separate, clinical entities: simultaneous pancreas and kidney transplant (SPK), pancreas after kidney transplant (PAK) and pancreas transplant alone (PTA). Each form of transplant is characterized by its own indications, risks and outcomes.

Simultaneous pancreas kidney transplantationDiabetes is a major cause of renal disease and is associated with approximately 40% of new cases of end-stage renal failure (ESRF) in the US, who will subsequently require renal dialysis or kidney transplantation[5]. When an IDDM patient develops ESRF and requires a kidney transplant; consideration is now commonly given to whether the patient would also benefit from receiving a pancreas transplant. SPK transplantation is the most common form of pancreas transplantation performed accounting for 60% of the total number of pancreas transplants performed each year in the US (approximately 900/year)[6-8]. The annual number of SPK transplants has remained stable over the last 10 years, however this may reflect the increasing interest in the option of PAK transplantation (living donor kidney transplantation) by the patient in ESRF with IDDM.

When SPK transplantat ion i s perfor med, the patient undergoes only one operation and following the surgery may be managed on the same (or very similar) immunosuppressive drugs they would have received for a renal transplant alone. The combined procedure offers excellent patient and pancreatic graft survival, producing a sustained euglycemic state off of exogenous insulin or oral hyperglycemic agents[7,9]. The renal transplant provides an effective method of surveillance of both grafts for acute rejection (creatinine clearance, biopsy) and may in part explain the improved one-year and long-term pancreatic

graft survival rates when compared to a pancreas transplant without a donor-matched kidney transplant. There also has been no evidence that the pancreas transplant may have a deleterious effect on the simultaneously transplanted kidney.

Pancreas after kidney transplantationAs the number of patients with ESRF increased in the past decade, the demand for renal transplantation outpaced the supply of cadaveric kidneys available. By 2000, the number of living donor kidney transplants performed in the US exceeded the number of cadaveric donor transplants. Whereas SPK transplantation is usually restricted to the use of cadaveric kidneys only, PAK transplantation offers the option of using a living donor kidney, and in doing so both expands the number of kidneys available for transplant and allows the diabetic patient the opportunity to benefit from early living donor kidney transplant (better long term outcome, avoidance of dialysis). With improved surgical technique, better immunosuppressive drugs and rejection monitoring, outcomes for solitary pancreas transplants have improved such that PAK transplantation is now routinely considered[6,7]. The kidney transplant recipient is required to demonstrate stable renal function and minimal post procedure complications to be acceptable for subsequent pancreas transplantation. The PAK transplant option does require the patient to undergo two major operations, however the post transplant immunosuppression and care are similar to kidney transplant alone.

Pancreas transplantation alonePTA is offered in some transplant centers to IDDM patients who have difficult to manage diabetes and suffer from severe hypoglycemic episodes (often with hypoglycemic unawareness) with little or no evidence of renal disease. As with PAK transplantation, improvements in PTA graft survival has significantly improved in recent years, although the question of whether or not the procedure may have an adverse long term affect on the recipients renal function (calcineurin antagonist based immunosuppression) has not been resolved. The normalizing of blood glucose levels with the pancreas transplant may offer this patient group the possibility of long-term improvement in renal function. The American Diabetes Association’s 2006 Position Statement on pancreas and islet transplantation recommends “In the absence of indications for kidney transplantation, pancreas-alone transplantation should only be considered a therapy in patients who exhibit these three criteria: (1) a history of frequent, acute and severe metabolic complications (hypoglycemia, marked hyperglycemia, ketoacidosis) requiring medical attention; (2) clinical and emotional problems with exogenous insulin therapy that are so severe as to be incapacitating; and (3) consistent failure of insulin-based management to prevent acute complications”[4]. Since 2001, PTA has accounted for approximately 12% of pancreas transplants performed annually in the US[6].

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WHOLE PANCREAS TRANSPLANTATION SURGERYBladder verses enteric pancreatic drainageIn the early development of pancreas transplantation, the surgical procedure involved drainage of the exocrine pancreas secretions in to the bladder. This was best accomplished by transplanting (in continuity) a short segment of donor duodenum with the pancreas, which provides a conduit from the pancreas to the bladder through which the secretions could be drained. Use of the duodenum-to-bladder drainage reduced the rate of transplant exocrine secretion leaks and allowed monitoring for evidence of early graft rejection by the frequent measurement of amylase activity in the urine. Direct biopsy of the transplanted pancreas and duodenum by cystoscopic technique could also be performed[3]. However, the loss of bicarbonate-rich pancreatic secretions into the bladder is associated with a number of problems. Patients who have undergone bladder drainage of their pancreas transplant require daily oral bicarbonate and fluid replacement to offset the severe metabolic acidosis and at times significant fluid loss associated with the procedure. Other problems associated with bladder drainage include: reflux graft pancreatitis (bladder dysfunction associated with diabetic neurogenic bladder); cystitis; urethritis and stricture formation; recurrent bladder infections. For some patients (approximately 15% at three years from the time of transplantation) the complications were severe enough that surgical reversal of the bladder drained pancreas transplant, with re-direction of the secretions into the small bowel was necessary[3,9].

In the late nineties transplant centers began to perform primary enteric drainage of pancreatic secretions in order to avoid the complications of bladder drainage. Improvements in immunosuppression had led to a reduced number of episodes of acute graft rejection and increasing experience with direct percutaneous needle biopsy reduced the need for urinary amylase monitoring. Enteric drainage of pancreatic secretions prevented the obligatory loss of sodium bicarbonate and fluid and the subsequent hypovolemia, metabolic acidosis and the other complications associated with bladder drainage. Initially the procedure often involved anastomosis of the transplant duodenal segment to a Roux-en-Y limb of the recipient’s mid-small bowel to reduce the likelihood of an anastomotic leak, however the surgery is now routinely performed directly to the small bowel. Enteric drainage of the pancreatic transplant secretions is now the more commonly performed procedure, used in 81% of SPK, 67% of PAK and 56% of PTA transplants performed in the US during 2000-2004 (when a SPK was performed, the donor-matched kidney may be followed for evidence of acute graft rejection which may be a better early marker than urinary amylase output)[6-10].

Systemic verses portal venous drainageVascular drainage of the pancreas transplant may be performed either into the systemic venous system (most commonly used is the external iliac vein) or into the portal

venous system (using a vein of the small bowel mesentery). With systemic venous drainage the insulin secreted by the pancreas transplant avoids early hepatic extraction and results in an elevation of both basal and stimulated serum insulin concentrations. Portal venous drainage directs the insulin released by the pancreas transplant initially to the liver in a fashion similar to the release of insulin in a non-diabetic person. Although it is felt to be “more physiological”, portal venous drainage of the pancreas transplant has not been shown to offer any advantage over systemic venous drainage in maintaining normal glucose homeostasis or lipid metabolism. It has been suggested that direct presentation of the pancreas transplant alloantigen to the liver may provide an immunologic benefit. However, registry data has not found evidence for improved graft survival when portal venous drainage has been performed. Systemic venous drainage remains the more commonly performed procedure, used in 77% of SPK, 73% of PAK and 56% of PTA transplants performed in the US during 2000-2004[6-9].

Surgical complicationsThe complexity of the whole pancreas transplant procedure, along with the likelihood of pre-existing disease secondary to their IDDM, exposes the recipient to a variety of significant operative and post-operative risks. The extent of the post-operative problems likely limited the widespread acceptance of pancreas transplantation in the early era of its development. Serious surgical complications following the procedure include: thrombosis of graft vessels, intra-abdominal hemorrhage, anastomotic leak (enteric or bladder), graft pancreatitis, pancreatic fistula formation and intra-abdominal sepsis, all of which may require re-laparotomy and the possibility of graft loss. In recent years, with improvements in donor and recipient selection criteria, surgical technique, immunosuppression protocols (reduced incidence of early, acute rejection) and prophylaxis regimes (anti-viral, anti-bacterial and antithrombosis), there has been a significant decrease in the overall incidence of serious complications and the rate of re-laparotomy[11,12].

ImmunosuppressionSince 2000, in the US the most common primary protocol for maintenance immunosuppression for whole pancreas transplantation is Tacrolimus and Mycophenolate Mofetil (MMF), although other agents such as Cyclosporine, Sirolimus and Azathioprin in varying combinations are also being used in a small number of centers[7,8]. In addition, the majority of transplant centers continue to use corticosteroids, although there is an movement towards “steroid-free” immunosuppression protocols in an attempt to reduce their adverse effects (glucose control, dyslipidemia, bone loss) in the diabetic patient transplant population. The initial success with pancreatic islet transplantation in 2000 was obtained using an immunosuppression protocol of Sirolimus and Tacrolimus in combination and avoiding steroid entirely[13]. Sirolimus has proven to be difficult to use in some patients (mouth ulcers, hyperlipidemia)[14] and recent studies

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have demonstrated poorer graft survival and inferior renal function when Sirolimus is used as a primary agent in combination with Tacrolimus when compared with the use of Tacrolimus and MMF in kidney and heart transplantation[15-17]. Induction immunosuppression is routinely used in whole pancreas transplantation, with > 75% of recipients receiving either: (1) a T-cell depleting polyclonal antibody (Thymoglobulin, ATGAM) or monoclonal antibody (OKT3, Campath), (2) a non-depleting monoclonal anti-CD25 antibody (Zenapax, Simulect), or (3) both[7,8].

Long-term use of immunosuppression is associated with a number of significant side effects and complications. The side effects most commonly seen with standard maintenance immunosuppression include: nephrotoxicity, hypertension, hyperlipidemia, microvascular disease, glucose intolerance, gastrointestinal problems, weight gain, skin changes/alopecia/hirsutism[18]. Immunosuppression-relate complications include: infections (viral, bacterial, fungal, parasitic) and malignancy (skin, lymphoproliferative, genitourinary). The risk of infection depends upon the degree of immune compromise created by the immu-nosuppressive regiment and the exposure to possible pathogens, either through re-activation of pre-existing infection (e.g., viral, tuberculosis) or introduction by the transplant process (e.g., surgical technique, transfer from donor)[19]. The risk of skin cancer for a patient on immunosuppression following renal transplantation is cumulative, ranging from 10% to 40% at 10 and 20 years respectively (ratio of squamous cell to basal cell carcinoma 2:1)[20]. The overall prevalence of post-transplant lymphoproliferative disease and leukemia varies from 1% to 2%[21].

OUTCOMES OF WHOLE PANCREASTRANSPLANTATIONPatient and graft survivalWhole pancreas transplantation has proven to be a safe procedure with a 1 year and 3 year patient survival rates for all forms of pancreas transplant (SPK, PAK, PTA) in the US since 1998 at about 95% and 89% respectively (unadjusted patient survival rates, 2005 OPTN/SRTR Annual Report)[6] (Table 1). Transplantation of a pancreas simultaneously with a kidney may also increase long-term IDDM patient survival compared to kidney transplantation alone. Whereas the mortality risk is increased over the first 18 months for the SPK recipient (associated with an increased rate of complications such as infection when compared to kidney transplant alone), when the pancreas continues to function post transplant, recipient survival is superior to SPK recipients who have had their pancreas graft fail or diabetic patients who received only a cadaveric kidney transplant[22,23].

One and 3 year graft survival rates for SPK transplant in the US since 1998 for kidney are ≥ 91% and ≥ 83% and pancreas are ≥ 82% and ≥ 75% respectively. Pancreas graft survival rates for PAK and PTA over a similar period were slightly less that for SPK: for 1 year 72% to 81% and 74% to 83% respectively, and at 3 years

63% to 71% and 60% to 69% respectively (unadjusted graft survival rates, 2005 OPTN/SRTR Annual Report). Although the incidence of acute kidney graft rejection has been shown to be greater following SPK than for kidney transplant alone (15% versus 9%), patients following SPK transplantation as a group general demonstrate better kidney graft function. This advantage of SPK on renal function disappears however when the analyses are adjusted for donor and recipient variables[9].

CONSEQUENCES OF WHOLE PANCREAS TRANSPLANTATIONBlood glucose controlSuccessful whole pancreas transplantation produces a normoglycemic state in the majority of recipients, usually within minutes of completion of the procedure without the need for exogenous insulin. Transient hypoglycemia may occur over the first 24 h requiring I.V. glucose support. Patients demonstrate normal fasting and post-prandial blood glucose concentrations and a lowering of hemoglobin A1c to normal levels. Where systemic venous drainage of the pancreas has been performed, fasting and meal-stimulated insulin concentrations are elevate, the likely result of the elimination of first-pass hepatic extraction. Portal venous drainage typically results in a more normal pattern of fasting and meal-stimulated insulin concentrations, with similar glucose control. Although insulin levels are elevated by systemic venous drainage, blood glucose homeostasis appears to be unaffected, demonstrating normal glucose utilization and hepatic glucose production. Whole pancreatic transplantation is also an effective treatment for patients who had a long history of severe, symptomatic hypoglycemia. The normal glucagon response to hypoglycemia is restored and hypoglycemic episodes are uncommon. Whole pancreas transplantation has been shown to be effective in providing recipients with long-term normal glycemic control off insulin (10 years or more). Reduced hemoglobin A1c levels are maintained and patients demonstrate fasting

Source: 2005 OPTN/SRTR annual report.

Year of transplantSurvival (%) 1998 1999 2000 2001 2002 2003

SPK Patient One year 93.9 94.8 95.1 94.1 94.8 95.5Three years 89.6 90.6 90.2 89.6 + +

PAK Patient One year 94.5 94.4 96.0 95.2 95.7 95.5Three years 90.8 88.1 91.2 89.1 + +

PTA Patient One year 96.8 97.3 99.1 97.5 97.6 94.5Three years 90.5 90.9 95.5 90.8 + +

SPK Pancreas One year 82.7 83.1 84.0 85.0 85.4 85.8Three years 75.8 76.0 77.0 78.9 + +

Kidney One year 91.2 91.5 92.5 91.5 91.2 91.7Three years 83.9 83.0 83.5 83.7 + +

PAK Pancreas One year 72.0 80.3 74.0 81.8 77.3 77.9Three years 63.4 66.8 62.3 71.5 + +

PTA Pancreas One year 79.2 83.3 75.3 78.3 79.3 74.4Three years 60.3 69.3 60.5 64.4 + +

Table 1 Unadjusted patient and graft survival following SPK, PAK or PTA by year of transplant at 1 and 3 years

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blood glucose and glycemic control in response to a meal or glucose challenge similar to those of the non-diabetic population[9,24].

Secondary complications of IDDMThe microvascular, neurologic and macrovascular diseases associated with IDDM has been attributed to long-term poor glycemic control. Whereas the Diabetes Control and Complications Research Group reported that improved glucose control through intensive insulin therapy effectively delayed the onset, or slowed the progression of diabetic retinopathy, nephropathy and neuropathy, the risk of severe hypoglycemia was significant and only a small percentage of patients could sustain the required improvement in metabolic control. Whole pancreas transplantation has now been performed over a long enough period of time to allow study of the effect of sustained normal glycemic control in patients with IDDM.

Diabetic nephropathyWhole pancreas transplantation does prevent de-novo diabetic changes, which would otherwise occur in a diabetic recipient of a kidney transplant[25]. There is also evidence that long-term successful pancreas transplantation may improve pre-existing histological changes secondary to diabetes in the native kidneys, although the effect is only observed after 5 or more years[26]. Whether native renal function benefits from PTA is uncertain, as the nephrotoxic effect of calcineurin inhibitor based immunosuppression therapy must be considered. Registry data has identified that from 2% to 8% of PTA recipients develop ESRF and require a kidney transplant by one year[9,27]. A recent report of case matched PTA with diabetic controls found however that although native renal function decreased significantly after PTA in patients with decreased creatinine clearance (CrCl ≤ 70 mL/min) at the time of transplantation, it was well tolerated among patients with a CrCl ≥ 70 mL/min[28]. Another study also found evidence for improvement of renal function after pancreas transplantation, documented by reduction of urinary excretion of protein with stable creatinine concentration and CrCl[29].

Diabetic retinopathyThe diabetic population undergoing pancreas transplantation typically has already developed some degree of retinal pathology and most have received laser therapy. Advanced retinal change does not seem to benefit from pancreatic transplantation as the damage has already occurred. Initial studies that examined the short-term effect of pancreas transplantation on diabetic retinopathy were unable to demonstrate any positive effect of corrected blood glucose control when compared to diabetic recipients of a kidney alone or SPK with a failed pancreas graft[30]. Studies which followed successful pancreas transplants for 5 or more years however, do show a benefit to the recipient with mild to moderate disease, with stabilization of established retinopathy, delay in the progression of new disease, improvement in visual acuity and a reduction in the use of laser therapy[9,31].

Diabetic neuropathyPolyneuropathy is a common complication of both IDDM and ESRF and advanced motor, sensory and autonomic neuropathies are frequently seen in patients undergoing whole pancreas transplantation. Improvement of both motor and sensory neuropathies symptoms will occur following kidney transplantation alone, however correction of uremia secondary to diabetic nephropathy by kidney transplantation does not halt the progression of the underlying diabetic neuropathic process. Diabetic patients studied following pancreas transplantation with return to a normoglycemic state do show a significant early improvement in sensory and motor nerve conduction studies that continue to improve over time. However clinical neurological examination and testing for autonomic nerve dysfunction demonstrated little improvement, even when followed over a longer period of time[32-35].

Micro- and macrovascular diseaseDiabet ic microangiopathy, the resul t of chronic hyperglycemia and subsequent metabolic disturbances seen in IDDM, is the principle cause of many of the severe late complications of diabetes[36]. The progression of microvascular disease-related problems (e.g., nephropathy, retinopathy neuropathy) is reduced when tight glucose control has been obtained, either through intensive insulin therapy or following successful whole pancreas transplantation. Direct evidence for improvement of the microvasculature following pancreas transplantation can also be demonstrated. Skin blood flow characteristics (as measured by laser Doppler), a measure of the degree of microcirculation impairment, have been shown to improve (but not normalize) following pancreas transplantation when compared to non-diabet ic controls [37]. The calcineurin-inhibitors, Cyclosporine and Tacrolimus, in addition to being nephrotoxic, have been shown to produce microangiopathy following transplantation and their use may negate any benefit a diabetic recipient might obtain from tight glucose control[38,39].

There is evidence that whole pancreas transplantation may reduce the risk of macrovascular disease. Carotid intima media thickness (determined by carotid ultrasound), a measure shown to cor relate with the l ikel ihood of cardiovascular events in IDDM (coronary artery disease, atherosclerotic vascular disease, mortality), is reduced following whole pancreas transplantation. The reduction of the carotid intima media thickness occurs early following transplantation and is independent of other causative factors such as smoking, age, serum lipid concentration and failure of a kidney transplant[40]. However, the typical recipient of a pancreas transplant has had long-standing IDDM and frequently has established vascular intimal disease and plaque formation and clinically significant vascular disease that may progress following transplantation[41].

Quality of lifePatients who have received a whole pancreas transplant consistently report an improvement in their quality of life (QOL), although pancreas transplantation is a complex

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surgical procedure, requires life-long immunosuppression and careful follow-up, and is associated with a significant incidence of complications, including re-hospitalization. The improvement in QOL is maintained unless the pancreas recipient experienced graft loss. For patients with IDDM who are in ESRF, a significant improvement in their health may be expected following either correction of uremia or elimination of their diabetic state. In a prospective, longitudinal study of patients undergoing either SPK or kidney transplant alone (KTA), Gross et al found that the addition of a pancreas transplant increased the measure of QOL beyond the improvement seen with KTA and the pancreas recipients reported greater improvement in areas that are diabetes-specific[9,42-44].

Pancreatic islet transplantationAs d i s cus sed above , the D iabe t e s Cont ro l and Complications Trial Research Group reported that improved glycemic control through intensive insulin therapy delays the onset and slows the progression of diabetic complications. Improved glycemic control however, was hard to sustain and associated with intense insulin therapy was a significant increase in severe hypoglycemic episodes. Whole pancreas transplantation is capable of producing a sustained, euglycemic state, reducing the incidences of hypoglycemia and offering the possible benefit of reducing microvascular, macrovascular and neurologic complications. Pancreas transplantation however, is a major, complex surgical procedure associated with significant risk and cost that may limit its general acceptability, especially when a potential diabetic recipient has little evidence of renal impairment and does not need a kidney transplant.

Within the past 20 years, pancreatic islet transplantation has become a clinical reality and an option in the treatment of IDDM. Islet transplantation has a distinct advantage over whole pancreas transplantation in regards to reduced peri-procedure morbidity. The procedure avoids major surgery and the risk of associated post-operative complications, re-laparotomy and acute (vascularized) graft loss. Islet transplantation, with its ability to be cultured for a period of time prior to transplantation, also offers the future possibility of reducing the immunogenicity (both allo and auto) of the tissue such that little or no immunosuppression will be required.

Early efforts to treat IDDM patients with pancreatic islet transplantation were mostly unsuccessful. Although the first human pancreatic islet allografts were performed in 1974[45], it wasn’t until 1991 that a pancreatic islet transplant recipient achieved sustained euglycemia off insulin (for 1 year)[46]. In 2000, the Edmonton group repor ted 7 consecutive is let transplant recipients achieving insulin independence. All recipients received islets from 2 pancreas donors (one received islets from 4 donors), and were maintained on a glucocorticoid-free immunosuppression protocol using Sirolimus and low-dose Tacrolimus[13]. The success of the Edmonton program has led to a general acceptance that islet transplantation is a clinically feasible therapy, which may be considered for the treatment of patients with IDDM, especially when

accompanied by severe hypoglycemia. Since the report of success from Edmonton interest has grown in islet transplantation and now more than 40 centers in North America and many more worldwide are performing this procedure[47].

Patient selectionPancreatic islet transplant, in general, has been restricted to patients with IDDM who suffer from hypoglycemic unawareness or metabolic instability, or have early evidence of secondary complications due to their diabetes. The patients require long-term, calcineurin-based immunosuppression and thus are subjected to the risks of these agents, such as nephrotoxicity, infection and malignancy. The patient may also become sensitized due to their alloimmune response to the transplant, potentially interfering with subsequent transplantation. Patients with evidence of significant diabetic renal impairment are excluded from islet transplantation until they either require or have undergone a kidney transplant. To improve the likelihood of attaining euglycemia off insulin, most transplant programs and clinical trials will restrict islet transplantation therapy to patients weighting less than 70 kg, a body mass index (BMI) < 27 and not requiring an excessive amount of daily insulin for glycemic control[47-51].

PANCREATIC ISLET TRANSPLANTPROCEDUREIslet isolation and cultureOne of the key elements attributed to the success of the Edmonton program (Edmonton Protocol) was the need to transplant high-quality, purified islets in sufficient numbers. This usually requires isolation of islets from two or more whole pancreata. Refinement of the islet isolation process, with the standardizing of pancreas digestion (the universal use of controlled pancreatic duct perfusion with collagenase and the Ricordi digestion chamber) and use of the COBE cell processor (continuous gradient purification system) for islet purification from exocrine tissue now allows many transplant centers a source of high-quality islets[52-54]. Although some centers transplant the isolated, purified pancreatic islets immediately, other centers maintained the islets in culture for a short period (up to 48 h) prior to transplantation. Holding islets in culture for a short period does not seem to have a detrimental affect on their viability and function and allows the transplant center the option of pre-conditioning the islet tissue (to reduce immunogenicity or improve post-transplant viability), or initiate immunosuppression treatment of the recipient prior to the transplant[55-57].

In the islet recipient, access to the portal vein is obtained by either percutaneous transhepatic portal venous catheterization or mini-laparotomy. The pancreatic islets are suspended in an albumin solution and infused by gravity, along with heparin through the portal vein to embolize in the liver. Prophylactic anticoagulation is continued for several days to reduce the likelihood of an instant blood-mediated inflammatory reaction (IBMIR) with subsequent clot formation and inf lammatory

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response that has been shown to lead to islet damage[58-60]. Islet recipients are routinely given exogenous insulin to maintain blood glucose levels in a physiologic range as hyperglycemia in the early post-transplant period has been shown to be detrimental to islet function and may interfere with islet engraftment[55,56,61].

Procedural complicationsPancreatic islet transplantation is a less invasive alternative to whole pancreas transplantation and has been shown to be associated with a much lower risk of serious complications. The majority of serious adverse events reported in 2006 by the Collaborative Islet Transplant Registry (CITR) were related to the infusion procedure: bleeding, hematoma and portal vein thrombosis (41.9%)[47]. Newer catheters and radiologic techniques for sealing the intra-hepatic tract used for portal vein infusion of the islets has resulted in a reduction in the incidence of post procedural bleeding[62]. Increased purity of the islet preparation with a subsequent decreased total islet volume infused into the liver may also reduce the likelihood of portal pressure elevation, a risk factor that has been associated with bleeding, in particular, with a second or subsequent islet transplant. The routine use of anticoagulation has likely limited the incidence of portal vein thrombosis, however has been shown to be a factor in the rate of procedural bleeds. Following transplantation, liver transaminases typically are elevated; however routinely return to the normal range within 4 wk[50]. The long-term consequences of intra-hepatic islet infusion are not yet known. The Edmonton group has reported changes consistent with fatty liver in 8 of their first 36 patients on magnetic resonance imaging following transplant.

ImmunosuppressionSince 1999, in the US the majority of islet transplant programs (> 90%) use Sirolimus and Tacrolimus in combination as maintenance immunosuppression (Edmonton Protocol). All programs used one or more induction immunosuppression agents at the time of the first islet infusion, the most common being a non-depleting monoclonal anti-CD25 antibody. Complications related to immunosuppression therapy were the second most common severe event reported by CITR (29.6%). Whereas some studies have reported that islet transplant recipients may demonstrate evidence of deterioration of renal function (immunosuppression related) especially where creatinine clearance is already significantly impaired, others have reported no deleterious renal effect following islet transplant[14,47,51].

OUTCOMES OF PANCREATIC ISLETTRANSPLANTATIONCollaborative islet transplant registryIn a survey of all North American transplant programs by CITR, 31 act ive prog rams repor ted 593 i s le t infusion procedures in 319 recipients during the period 1999-2005[47]. CITR has information on 225 of the 319 allograft recipients (71%) and 425 of the 593 infusion

procedures (72%) from 23 participating centers. Sixty-four of the recipients (28.4%) received one islet infusion, 122 (54%) received two, 38 (17%) received three, and one received a total of four islet infusions. Of the 225 recipients, 203 (89%) received an islet transplant alone, while 22 recipients (10%) had previously received a kidney transplant prior to the islet transplant. Insulin independence (off insulin for 14 days or more) was achieved by 69.7% of islet recipients at some point following their last transplant (within two years). Considering all participants, 46.6% remained insulin independent for at least one year following their last transplant and 33.3% were insulin independent at 2 years. Patients who had achieved insulin independence at any point, at one year had a basal C-peptide level (1.1 ng/mL, SD 0.65), and hemoglobin A1c (6%, SD 0.8) in the normal (or near normal) range. Severe hypoglycemic events were shown to be dramatically deceased following the first islet infusion. Greater than 85% of recipients had one or more severe hypoglycemic event prior to transplant, compared to less than 5% in the first year following the transplant.

International trial of the edmonton protocol for islet transplantationIn 2006, a study organized by the Immune Tolerance Network (initiated by the National Institutes of Health) among six North American and three European transplant centers to assess the feasibility and reproducibility of the Edmonton Protocol reported their results[14]. Each center was confined to use the islet isolation technique and immunosuppression protocol as described by the Edmonton group. In the period 2001-2003, 36 patients underwent 77 islet infusion procedures. Eleven of the recipients (31%) received one islet infusion, 9 (25%) received two and 16 (44%) received three infusions. Insulin independence was achieved by 21 patients (58%) at some point, 16 patients (44%) remained insulin independent for one year and 5 (14%) remained insulin-independent at 2 years following their last transplant (35 patients evaluated at 2 years). Although most patients had returned to requiring insulin by two years, C-peptide remained detectable in 70%, demonstrating persistence of islet function. Average hemoglobin A1c levels were in shown to be in the normal range for patients who remained insulin-independent at 2 years, and only slightly elevated above normal when partial graft function was demonstrated. All patients with residual islet function were completely protected from severe hypoglycemic episodes.

Secondary complications of IDDMSuccessful islet transplantation, with correction of glycemic control has only been a clinical reality for a short period of time and there has been little published to-date on the effect of islet transplantation on the secondary complications (microvascular, neurologic and macrovascular diseases) associated with IDDM. Assessment of the long-term safety and effectiveness of islet transplantation for the treatment of IDDM will require the future report of more centers with longer periods of follow-up.

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CONCLUSIONBoth whole pancreas transplantation and pancreatic islet transplantation have been shown to be successful at creating a euglycemic state in a patient with IDDM with preprandial and postprandial blood glucose concentrations and hemoglobin A1c levels comparable to those in the non-diabetic population. Both forms of transplants are also effective at eliminating the occurrence of significant hypoglycemic events (even with only partial islet function evident). Whole pancreas transplantation has also been shown to be effective at maintaining a euglycemic state over a sustained period of time, and thus provides an opportunity for a recipient to benefit from improvement of their blood glucose control (halt or reverse secondary complications of IDDM). Islet transplantation is attractive as a less invasive alternative to whole pancreas transplantat ion and offers the future promise of immunosuppression-free transplantation through pre-transplant culture. Although recent advances have led to an increased rate of obtaining insulin-independence following islet transplantation, further developments are needed to improve the long-term viability and function of the graft to maintain improved glucose control over time.

REFERENCES1 The effect of intensive treatment of diabetes on the

development and progression of long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N Engl J Med 1993; 329: 977-986

2 Kelly WD, Lillehei RC, Merkel FK, Idezuki Y, Goetz FC. Allotransplantation of the pancreas and duodenum along with the kidney in diabetic nephropathy. Surgery 1967; 61: 827-837

3 Sutherland DE, Gruessner RW, Dunn DL, Matas AJ, Humar A, Kandaswamy R, Mauer SM, Kennedy WR, Goetz FC, Robertson RP, Gruessner AC, Najarian JS. Lessons learned from more than 1,000 pancreas transplants at a single institution. Ann Surg 2001; 233: 463-501

4 Robertson RP, Davis C, Larsen J, Stratta R, Sutherland DE. Pancreas and islet transplantation in type 1 diabetes. Diabetes Care 2006; 29: 935

5 Mai ML, Ahsan N, Gonwa T. The long-term management of pancreas transplantation. Transplantation 2006; 82: 991-1003

6 OPTN/SRTR, HHS/HRSA/HSB/DOT. 2005 OPTN/SRTR Annual Report 1995-2004. Avaliable from: URL: http://www.ustransplant.org/annual_reports, 2005

7 Cohen DJ, St Martin L, Christensen LL, Bloom RD, Sung RS. Kidney and pancreas transplantation in the United States, 1995-2004. Am J Transplant 2006; 6: 1153-1169

8 Gruessner AC, Sutherland DE. Pancreas transplant outcomes for United States (US) and non-US cases as reported to the United Network for Organ Sharing (UNOS) and the International Pancreas Transplant Registry (IPTR) as of June 2004. Clin Transplant 2005; 19: 433-455

9 Larsen JL . Pancreas transplantation: indications and consequences. Endocr Rev 2004; 25: 919-946

10 Demartines N, Schiesser M, Clavien PA. An evidence-based analysis of simultaneous pancreas-kidney and pancreas transplantation alone. Am J Transplant 2005; 5: 2688-2697

11 Humar A , Kandaswamy R, Granger D, Gruessner RW, Gruessner AC, Sutherland DE. Decreased surgical risks of pancreas transplantation in the modern era. Ann Surg 2000; 231: 269-275

12 Malaise J, Steurer W, Koenigsrainer A, Mark W, Margreiter R, Van Ophem D, Mourad M, Squifflet JP. Simultaneous pancreas-kidney transplantation in a large multicenter study:

surgical complications. Transplant Proc 2005; 37: 2859-286013 Shapiro AM , Lakey JR, Ryan EA, Korbutt GS, Toth E,

Warnock GL, Kneteman NM, Rajotte RV. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343: 230-238

14 Shapiro AM, Ricordi C, Hering BJ, Auchincloss H, Lindblad R, Robertson RP, Secchi A, Brendel MD, Berney T, Brennan DC, Cagliero E, Alejandro R, Ryan EA, DiMercurio B, Morel P, Polonsky KS, Reems JA, Bretzel RG, Bertuzzi F, Froud T, Kandaswamy R, Sutherland DE, Eisenbarth G, Segal M, Preiksaitis J, Korbutt GS, Barton FB, Viviano L, Seyfert-Margolis V, Bluestone J, Lakey JR. International trial of the Edmonton protocol for islet transplantation. N Engl J Med 2006; 355: 1318-1330

15 Mendez R , Gonwa T, Yang HC, Weinstein S, Jensik S, Steinberg S. A prospective, randomized trial of tacrolimus in combination with sirolimus or mycophenolate mofetil in kidney transplantation: results at 1 year. Transplantation 2005; 80: 303-309

16 Meier-Kriesche HU, Schold JD, Srinivas TR, Howard RJ, Fujita S, Kaplan B. Sirolimus in combination with tacrolimus is associated with worse renal allograft survival compared to mycophenolate mofetil combined with tacrolimus. Am J Transplant 2005; 5: 2273-2280

17 Kobashigawa JA, Miller LW, Russell SD, Ewald GA, Zucker MJ, Goldberg LR, Eisen HJ, Salm K, Tolzman D, Gao J, Fitzsimmons W, First R. Tacrolimus with mycophenolate mofetil (MMF) or sirolimus vs. cyclosporine with MMF in cardiac transplant patients: 1-year report. Am J Transplant 2006; 6: 1377-1386

18 Yang H . Maintenance immunosuppression regimens: conversion, minimization, withdrawal, and avoidance. Am J Kidney Dis 2006; 47: S37-S51

19 Fishman JA , Rubin RH. Infection in organ-transplant recipients. N Engl J Med 1998; 338: 1741-1751

20 Dreno B. Skin cancers after transplantation. Nephrol Dial Transplant 2003; 18: 1052-1058

21 Lutz J, Heemann U. Tumours after kidney transplantation. Curr Opin Urol 2003; 13: 105-109

22 Tydén G, Bolinder J, Solders G, Brattström C, Tibell A, Groth CG. Improved survival in patients with insulin-dependent diabetes mellitus and end-stage diabetic nephropathy 10 years after combined pancreas and kidney transplantation. Transplantation 1999; 67: 645-648

23 Reddy KS , Stablein D, Taranto S, Stratta RJ, Johnston TD, Waid TH, McKeown JW, Lucas BA, Ranjan D. Long-term survival following simultaneous kidney-pancreas transplantation versus kidney transplantation alone in patients with type 1 diabetes mellitus and renal failure. Am J Kidney Dis 2003; 41: 464-470

24 Robertson RP. Consequences on beta-cell function and reserve after long-term pancreas transplantation. Diabetes 2004; 53: 633-644

25 Fiorina P, Venturini M, Folli F, Losio C, Maffi P, Placidi C, La Rosa S, Orsenigo E, Socci C, Capella C, Del Maschio A, Secchi A. Natural history of kidney graft survival, hypertrophy, and vascular function in end-stage renal disease type 1 diabetic kidney-transplanted patients: beneficial impact of pancreas and successful islet cotransplantation. Diabetes Care 2005; 28: 1303-1310

26 Fioretto P, Steffes MW, Sutherland DE, Goetz FC, Mauer M. Reversal of lesions of diabetic nephropathy after pancreas transplantation. N Engl J Med 1998; 339: 69-75

27 Gruessner AC, Sutherland DE. Analysis of United States (US) and non-US pancreas transplants reported to the United network for organ sharing (UNOS) and the international pancreas transplant registry (IPTR) as of October 2001. Clin Transpl 2001: 41-72

28 Genzini T, Marchini GS, Chang AJ, Antunes I, Hayashi A, Abensur H, Kataoka L, Crescentini F, Romão JE, Rangel EB, Perosa M. Influence of pancreas transplantation alone on native renal function. Transplant Proc 2006; 38: 1939-1940

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www.wjgnet.com

29 Coppelli A, Giannarelli R, Vistoli F, Del Prato S, Rizzo G, Mosca F, Boggi U, Marchetti P. The beneficial effects of pancreas transplant alone on diabetic nephropathy. Diabetes Care 2005; 28: 1366-1370

30 Scheider A, Meyer-Schwickerath E, Nusser J, Land W, Landgraf R. Diabetic retinopathy and pancreas transplantation: a 3-year follow-up. Diabetologia 1991; 34 Suppl 1: S95-S99

31 Pearce IA, Ilango B, Sells RA, Wong D. Stabilisation of diabetic retinopathy following simultaneous pancreas and kidney transplant. Br J Ophthalmol 2000; 84: 736-740

32 Kennedy WR, Navarro X, Sutherland DE. Neuropathy profile of diabetic patients in a pancreas transplantation program. Neurology 1995; 45: 773-780

33 Müller-Felber W, Landgraf R, Scheuer R, Wagner S, Reimers CD, Nusser J, Abendroth D, Illner WD, Land W. Diabetic neuropathy 3 years after successful pancreas and kidney transplantation. Diabetes 1993; 42: 1482-1486

34 Navarro X, Sutherland DE, Kennedy WR. Long-term effects of pancreatic transplantation on diabetic neuropathy. Ann Neurol 1997; 42: 727-736

35 Recasens M, Ricart MJ, Valls-Solé J, Caballero A, Fernández-Cruz L, Esmatjes E. Long-term follow-up of diabetic polyneuropathy after simultaneous pancreas and kidney transplantation in type 1 diabetic patients. Transplant Proc 2002; 34: 200-203

36 Tooke JE. Microvascular function in human diabetes. A physiological perspective. Diabetes 1995; 44: 721-726

37 Eberl N, Piehlmeier W, Dachauer S, König A, Land W, Landgraf R. Blood flow in the skin of type 1 diabetic patients before and after combined pancreas/kidney transplantation. Diabetes Metab Res Rev 2005; 21: 525-532

38 Burke GW , Ciancio G, Cirocco R, Markou M, Olson L, Contreras N, Roth D, Esquenazi V, Tzakis A, Miller J. Microangiopathy in kidney and simultaneous pancreas/kidney recipients treated with tacrolimus: evidence of endothelin and cytokine involvement. Transplantation 1999; 68: 1336-1342

39 Zarifian A, Meleg-Smith S, O'donovan R, Tesi RJ, Batuman V. Cyclosporine-associated thrombotic microangiopathy in renal allografts. Kidney Int 1999; 55: 2457-2466

40 Larsen JL, Ratanasuwan T, Burkman T, Lynch T, Erickson J, Colling C, Lane J, Mack-Shipman L, Lyden E, Loseke M, Miller S, Leone J. Carotid intima media thickness decreases after pancreas transplantation. Transplantation 2002; 73: 936-940

41 Morrissey PE, Shaffer D, Monaco AP, Conway P, Madras PN. Peripheral vascular disease after kidney-pancreas transplantation in diabetic patients with end-stage renal disease. Arch Surg 1997; 132: 358-361; discussion 361-362

42 Gross CR, Limwattananon C, Matthees B, Zehrer JL, Savik K. Impact of transplantation on quality of life in patients with diabetes and renal dysfunction. Transplantation 2000; 70: 1736-1746

43 Kiebert GM, van Oosterhout EC, van Bronswijk H, Lemkes HH, Gooszen HG. Quality of life after combined kidney-pancreas or kidney transplantation in diabetic patients with end-stage renal disease. Clin Transplant 1994; 8: 239-245

44 Sureshkumar KK , Mubin T, Mikhael N, Kashif MA, Nghiem DD, Marcus RJ. Assessment of quality of life after simultaneous pancreas-kidney transplantation. Am J Kidney Dis 2002; 39: 1300-1306

45 Najarian JS, Sutherland DE, Matas AJ, Steffes MW, Simmons RL, Goetz FC. Human islet transplantation: a preliminary report. Transplant Proc 1977; 9: 233-236

46 Warnock GL, Kneteman NM, Ryan EA, Rabinovitch A, Rajotte RV. Long-term follow-up after transplantation of insulin-

producing pancreatic islets into patients with type 1 (insulin-dependent) diabetes mellitus. Diabetologia 1992; 35: 89-95

47 CITR, Collaborative Islet Transplant Registry 2006 Scientific Summary. Available from URL: http://www.web.emmes.com/study/isl/reports/reports.htm, 2006

48 Stock PG, Bluestone JA. Beta-cell replacement for type I diabetes. Annu Rev Med 2004; 55: 133-156

49 Paty BW , Senior PA, Lakey JR, Shapiro AM, Ryan EA. Assessment of glycemic control after islet transplantation using the continuous glucose monitor in insulin-independent versus insulin-requiring type 1 diabetes subjects. Diabetes Technol Ther 2006; 8: 165-173

50 Ryan EA, Paty BW, Senior PA, Bigam D, Alfadhli E, Kneteman NM, Lakey JR, Shapiro AM. Five-year follow-up after clinical islet transplantation. Diabetes 2005; 54: 2060-2069

51 Ryan EA, Bigam D, Shapiro AM. Current indications for pancreas or islet transplant. Diabetes Obes Metab 2006; 8: 1-7

52 Ricordi C , Lacy PE, Finke EH, Olack BJ, Scharp DW. Automated method for isolation of human pancreatic islets. Diabetes 1988; 37: 413-420

53 Robertson GS, Chadwick DR, Contractor H, James RF, London NJ. The optimization of large-scale density gradient isolation of human islets. Acta Diabetol 1993; 30: 93-98

54 Merani S, Shapiro AM. Current status of pancreatic islet transplantation. Clin Sci (Lond) 2006; 110: 611-625

55 Hering BJ. Achieving and maintaining insulin independence in human islet transplant recipients. Transplantation 2005; 79: 1296-1297

56 Hering BJ, Kandaswamy R, Ansite JD, Eckman PM, Nakano M, Sawada T, Matsumoto I, Ihm SH, Zhang HJ, Parkey J, Hunter DW, Sutherland DE. Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes. JAMA 2005; 293: 830-835

57 Sutherland DE, Gruessner R, Kandswamy R, Humar A, Hering B, Gruessner A. Beta-cell replacement therapy (pancreas and islet transplantation) for treatment of diabetes mellitus: an integrated approach. Transplant Proc 2004; 36: 1697-1699

58 Johansson H, Lukinius A, Moberg L, Lundgren T, Berne C, Foss A, Felldin M, Källen R, Salmela K, Tibell A, Tufveson G, Ekdahl KN, Elgue G, Korsgren O, Nilsson B. Tissue factor produced by the endocrine cells of the islets of Langerhans is associated with a negative outcome of clinical islet transplantation. Diabetes 2005; 54: 1755-1762

59 Moberg L, Johansson H, Lukinius A, Berne C, Foss A, Källen R, Østraat Ø, Salmela K, Tibell A, Tufveson G, Elgue G, Nilsson Ekdahl K, Korsgren O, Nilsson B. Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation. Lancet 2002; 360: 2039-2045

60 Bennet W, Groth CG, Larsson R, Nilsson B, Korsgren O. Isolated human islets trigger an instant blood mediated inflammatory reaction: implications for intraportal islet transplantation as a treatment for patients with type 1 diabetes. Ups J Med Sci 2000; 105: 125-133

61 Matsumoto S, Noguchi H, Yonekawa Y, Okitsu T, Iwanaga Y, Liu X, Nagata H, Kobayashi N, Ricordi C. Pancreatic islet transplantation for treating diabetes. Expert Opin Biol Ther 2006; 6: 23-37

62 Villiger P, Ryan EA, Owen R, O'Kelly K, Oberholzer J, Al Saif F, Kin T, Wang H, Larsen I, Blitz SL, Menon V, Senior P, Bigam DL, Paty B, Kneteman NM, Lakey JR, Shapiro AM. Prevention of bleeding after islet transplantation: lessons learned from a multivariate analysis of 132 cases at a single institution. Am J Transplant 2005; 5: 2992-2998

S- Editor Liu Y L- Editor Alpini GD E- Editor Liu Y

Meloche RM. Transplantation for the treatment of type 1 diabetes 6355

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Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6356-6364www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

Evaluation of quantitative contrast harmonic imaging to assess malignancy of liver tumors: A prospective controlled two-center study

EM Jung, DA Clevert, AG Schreyer, S Schmitt, J Rennert, R Kubale, S Feuerbach, F Jung

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LIVER CANCER

EM Jung, AG Schreyer, J Rennert, Feuerbach S, Department of Diagnostic Radiology, University Hospital Regensburg, Franz-Josef Strauss-Allee 11, Regensburg 93043, GermanyDA Clevert, Department of Radiology, Ultrasound Section, Großhadern Hospital, Ludwig Maximilian University of Munich, Marchioninistr, 15, München 81377, GermanyS Schmitt, Department of Trauma Surgery, Passau Hospital, Innstraße 76, Passau 94034, GermanyR Kubale, Institutes of Radiology, Nuclear Medicine and Ultrasound, Pirmasens Hospital, GermanyF Jung, Center for Biomaterial Development, Institute of Polymer Research, GKSS, Kantstr, 55, Teltow 14513, Germany Correspondence to: Ernst Michael Jung, MD, Department of Radiology, University of Regensburg, Franz-Josef Strauss-Allee 11, Regensburg 93053, Germany. emjung@gmx.deTelephone: +49-941-9447401 Fax: +49-941-9447402Received: June 29, 2007 Revised: September 10, 2007

AbstractAIM: To establ ish the extent to which contrast enhancement with SonoVue in combination with quantitative evaluation of contrast-medium dynamics facilitates the detection of hepatic tumors.

METHODS: One hundred patients with histologically confirmed malignant or benign hepatic tumor (maximum size 5 cm) were analyzed. Contrast-enhanced ultrasound (bolus injection 2.5 mL SonoVue) was carried out with intermittent breath-holding technique using a multifrequency transducer (2.5-4 MHz). Native vascularization was analyzed with power Doppler. The contrast-enhanced dynamic ultrasound investigation was carried out with contrast harmonic imaging in true detection mode during the arterial, portal venous and late phases. Mechanical index was set at 0.15. Perfusion analysis was performed by post-processing of the raw data [time intensity curve (TIC) analysis]. The cut-off of the gray value differences between tumor and normal liver tissue was established using Receiver Operating Characteristic (ROC) analysis 64-line multi-slice computed tomography served as reference method in all cases. Magnetic resonance tomography was used additionally in 19 cases.

RESULTS: One hundred patients with 59 malignant (43 colon, 5 breast, 2 endocrine metastases, 7 hepatocellular carcinomas and 2 kidney cancers) and 41 benign (15 hemangiomas, 7 focal nodular hyperplasias, 5

complicated cysts, 2 abscesses and 12 circumscribed fatty changes) tumors were included. The late venous phase proved to be the most sensitive for classification of the tumor type. Fifty-eight of the 59 malignant tumors were classified as true positive, and one as false negative. This resulted in a sensitivity of 98.3%. Of the 41 benign tumors, 37 were classified as true negative and 4 as false negative, which corresponds to a specificity of 90.2%. Altogether, 95.0% of the diagnoses were classified as correct on the basis of the histological classification. No investigator-dependency (P = 0.23) was noted.

CONCLUSION: The results show the possibility of accurate prediction of malignancy of hepatic tumors with a positive prognostic value of 93.5% using advanced contrast-enhanced ultrasound. Contrast enhancement with SonoVue in combination with quantitative evaluation of contrast-medium dynamics is a valuable tool to discriminate hepatic tumors.

© 2007 WJG. All rights reserved.

Key words: Liver tumors; Malignant tumor; Contrast harmonic imaging; Quantitative contrast enhancement

Jung EM, Clevert DA, Schreyer AG, Schmitt S, Rennert J, Kubale R, Feuerbach S, Jung F. Evaluation of quantitative contrast harmonic imaging to assess malignancy of liver tumors: A prospective controlled two-center study. World J Gastroenterol 2007; 13(47): 6356-6364

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INTRODUCTIONBenign liver lesions have a prevalence of approximately 20% in the whole population, whereas tumor patients show hepatic metastases in 25%-50% at the time of diagnosis. The liver is the main region for the manifestation of distant metastases in oncological diseases. Therefore, reliable detection of hepatic metastases is a prerequisite for evaluating the prognosis and establishing a therapeutic procedure. To evaluate the prospects of resection or to plan interventional therapy, it is essential to establish the number and location of the lesions. Prior to this, the focal hepatic lesion must be accurately characterized. Since no

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single diagnostic tool has been able to clarify this question with sufficient reliability up to now, histological analysis remains the gold standard. Characterization of focal hepatic lesions with imaging techniques is an important part of radiological diagnosis and is still the focus of scientific research because of technical innovations of existing methods[1-15]. Special attention has been paid to the validation of new methods, especially the further development of contrast-enhanced ultrasound techniques because ultrasound is the most common and best available imaging technique[1-3,5-14].

In a patient with a known primary malignancy, any focal liver lesion seen on non-enhanced ultrasound must be regarded as suspicious of metastasis. However many lesions (25%-50% of lesions ≤ 2 cm) will eventually prove to be benign; next to contrast-enhanced ultrasound, other imaging modalities or biopsies are used to further characterize the lesion. For lesions < 1 cm, the false-negative rate of non-enhanced ultrasound is as high as 80%. Therefore contrast-enhanced ultrasound should be carried out in addition to conventional ultrasound in most cancer patients for definitive liver staging[6-9].

This prospective two-center study investigated the possibility of evaluating the malignancy of liver lesions on the basis of contrast-enhanced harmonic imaging (CHI) and SonoVue, by comparing the dynamics of the contrast agent in tumor lesions with those in the surrounding tissue. The objective of this study was to examine to what extent the detection of liver tumors will be simplified after contrast application with SonoVue, in combination with quantitative measurement of contrast agent dynamics. Special attention was paid to the feasibility of assessing the malignancy of the tumor by quantitative measurement of the tumor tissue contrast compared to the surrounding healthy tissue.

MATERIALS AND METHODSStudy designThis prospective study was performed at two centers. The equipment (Logic 9; GE Healthcare, Milwaukee, WI, USA), transducers (4C probe), data acquisition (PACS connection) and display, as well as the procedure of ultrasound examination were set identically at both centers.

Only patients with histologically confirmed malignant or benign hepatic lesions were included in the study. Tumor sizes were differentiated as follows: < 3 cm, and 3-5 cm. The maximum number of lesions considered was five.

Exclusion criteria were: tumor lesion > 5 cm, and more than five lesions, strong allergic reactions, diseases of liver and kidney with confirmed elevation of laboratory parameters, acute heart failure, acute myocardial infarction, subcutaneous emphysema, meteorism, tachypnea, and aerobilia.

Operational sequence and follow-upA biopsy was taken from the malignant liver tumors to establish the histological result (if necessary, surgical resection or radiofrequency ablation was performed). In case of hemangioma, constant results of ultrasound

examinations over 2 years and addit ional MRI or multiphase CT with constant results over 2 years was required. All contrast-enhanced ultrasound investigations were conducted with a multi-frequency linear transducer (2.5-4 MHz, Logic 9; GE Healthcare). Transmitted energy was reduced to a magnitude of < 30% with a mechanical index (MI) of 0.15. In fundamental B-scan, an optimal depth adjustment with three focus zones was made. After B-scan and analysis of vascularization with power Doppler ultrasound, an intravenous bolus injection of 2.5 mL SonoVue (Altana, Bracco, Konstanz, Germany) was administered through a 20-18 Ga peripheral cubital cannula, followed by a bolus injection of 10 mL NaCl.

Using CHI, the microbubbles of the contrast medium were stimulated to vibrate and their energetic harmonic Doppler frequencies were employed for imaging. In the technique of pulse inversion harmonic imaging (PIHI) of CHI, emission frequencies are digitally encoded. Several pulses, each after a defined period of time, were produced. Then the received echos were subtracted from each other. The harmonic frequencies of contrast-enhanced echos in the flowing blood remained as image information. In the subtraction mode of the background information, the influx of contrast agent and its distribution were imaged, even with very low acoustic energy.

A low MI allowed the real-time evaluation of the contrast-agent enhancement. CHI suppressed the fundamental linear echoes from the liver tissue, whereas the non-linear echoes reflected from the microbubbles remained, which provided the ultrasound signal. The true agent detection mode was also used to process the fundamenta l non- l inear s igna l g enera ted by ultrasound contrast agents that were stronger than the harmonic signal, thereby increasing the specificity of the microbubble-to-tissue ratio. The combination of SonoVue with true agent detection mode using a low MI allowed dynamic enhancement of the blood supply of a liver nodule to be evaluated during the various phases of contrast-agent circulation. Perfusion curves described the ultrasound signal intensity over time after contrast bolus injection in a region of interest (ROI).

Modified ultrasound slices close to the tumor enabled a minimum depth of penetration to be maintained. One turn of the scanner head examined the whole liver during intermittent breath holding. Digital data from cine sequences allowed post-processing of the dynamic tumor blood flow and calculation of 3D data block images. Scanning was carried out during the arterial (< 30 s), portal venous (40-120 s) and late venous (> 120 s) phases in true agent detection mode. Parallel dynamic imaging in fundamental B-scan and recording of the dynamics of contrast agent in the subtraction mode of CHI were performed in the meantime, which greatly facilitated the localization of tumor lesions.

Color-coded Doppler sonography and power Doppler ultrasound were used to evaluate native vascularization. Color enhancement was adjusted to the lowest possible pulse repetition frequency (PRF, < 1000 Hz) and to the best possible, artifact-free color enhancement, in order to avoid artifacts. The complete data of the contrast-agent examination were recorded in up to 5 min. The length of

the specific cine loops in the three stages was a minimum of 20 s each.

Measurements were done in the arterial phase (to quantify arterial vascularization), in the portal venous phase (to asses contrast-agent accumulation, pooling), and up to 5 min in the late phase (to assess the wash-out effect).

Altogether, five measurements were performed in each patient in the ROIs: one measurement within the lesion (T1) and four in the surrounding liver parenchyma at 12, 3, 6 and 9 o'clock positions (G1, G2, G3 and G4), with ROIs not larger than 5 mm. A careful adjustment of the position of the ROIs was effected manually depending on inspiration or expiration. Time intensity curve (TIC) analyses were made off-line and then transferred into an spreadsheet table (Excel 2003; Microsoft, Redmond, WA, USA) for off-line analysis. In addition, digital raw data were recorded and saved.

The different dynamics of the depletion of contrast agent - in comparison to the depletion within normal liver tissue - was used to assess the malignancy of the tumors. For this purpose, the recorded gray scale parameters of the ROIs were averaged over the time of recording, and then the following gray scale differences were calculated:arterial phase: Diffart = T1art - (G1art + G2art + G3art + G4art)/4portal venous phase: Diffpv = T1pv - (G1pv + G2pv + G3pv + G4pv)/4late venous phase: Difflv = T1sv - (G1sv + G2sv + G3sv + G4sv)/4

The malignancy of the tumor was then inferred from the absolute value of the differences. If the absolute value of Diffsv was smaller than -0.4 in the case of a malignant tumor, the assessment was rated as true positive, if the value was greater than -0.4, the assessment was rated as false negative. If the value was greater than -0.4 in the case of a benign tumor, the assessment was rated as true negative, otherwise as false positive. First, the critical value had been ascertained in a small preliminary study, which had shown characteristic contrast-agent dynamics in malignant liver tumors. The determination of the optimal scale value is shown in the section ROC analysis. Figure 1A-C presents an example of contrast-agent dynamics.

An increased wash-out of contrast agent molecules appeared to occur in the malignant tumor vessels rather than in normal hepatic vessels. This was probably caused by arteriovenous shunts within the lesion and additional tumor necrosis, which prevented accumulation of microbubbles in the malignant tumor. Therefore, the tumor was more hypoechoic, especially in the late phase, i.e., the gray value imaging was less distinct. This characteristic behavior could then be used for quantitative evaluation of gray value differences in the diagnosis of liver tumor malignancy.

Ethical concerns The study data were collected within the framework of an external quality control as a registry complying with the principles of the Helsinki/Edinburgh Declaration of 2002. Before each contrast agent application, patients were informed in detail about possible risks such as allergic

reactions. The consent of all patients was obtained prior to the study.

Statistical analysisAll results were presented as the mean ± SD. Specificity, sensitivity, positive and negative prognostic value, as well as diagnostic accuracy were calculated to evaluate their diagnostic significance. Since there were, as a rule, no clinical findings or the history of the patient was not available, the examiner could not establish a pretest probability. In this situation, the resulting sensitivity and specificity of the three diagnostic tests were calculated on

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Figure 1 Gray value progression of the five ROIs over 10 s. A: For a patient with liver metastasis in the arterial phase; B: For the same patient in the portal-venous phase; C: For the same patient in the late venous phase. (red, gray-value progression in the tumor; green, gray-value progression in the surrounding healthy tissue).

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Analysis of contrast-agent dynamics Arterial phase: Of the 59 patients with histologically confirmed malignant tumors, 40 were classified as true positive and 19 as false negative in the arterial phase. Of the 41 patients with histologically classified benign tumor, 22 were classified as true negative and 19 as false positive (Table 1, Figure 1A). In the arterial phase, a sensitivity of 67.8% with a specificity of 53.7 % (pV 67.8%, nV 53.7%) was achieved. Therefore, 62% of the test results were correct.

Portal venous phase: Of the 59 patients with histologically confirmed malignant tumor, 54 were classified as true positive and 5 as false negative in the portal venous phase. Of the 41 patients with histologically classified benign tumor, 31 were classified as true negative and 10 as false positive (Table 2, Figure 1B). This resulted in a sensitivity of 91.5% with a specificity of 75.6%, which gave a pV of 84.4% and nV of 86.1%. The correctly interpreted test evidence therefore amounted to 85.0%.

Late venous phase: Of 59 patients with histologically confirmed malignant tumor, 58 were classified as true positive and 1 as false negative in the late phase. Of 41 patients with histologically confirmed benign tumor, 37 were classified as true negative and 4 as false positive (Table 3, Figure 1C). This resulted in a sensitivity of 98.3% with a specificity of 90.2%, which gave a pV of 93.5% and nV of 97.4%. In the late venous phase, the correctly interpreted test evidence amounted to 95.0%.

A comparison of the diagnostic values of the three phases shows that the best accuracy was achieved in the late venous phase (Table 4).

Stability of diagnostic criteriaThe sensitivity over the course of the study provided a sensitive indicator of the diagnostic value of this method in identifying malignant liver tumors in 100 patients (Figure 2). Only the late venous phase was analyzed since

Table 1 Results based on contrast-enhanced CHI during the arterial phase

Tumor present Tumor not present

Test positive 40 19 59Test negative 19 22 41

59 41 100

Table 2 Results based on contrast-enhanced CHI during the portal-venous phase

Tumor present Tumor not presentTest positive 54 10 64Test negative 5 31 36

59 41 100

Table 3 Results based on contrast-enhanced CHI during the late phase

Tumor present Tumor not present

Test positive 58 4 62Test negative 1 37 38

59 41 100

Table 4 Diagnostic value of quantitative CHI for detection of malignant hepatic tumors in different phases after intravenous application of ultrasound contrast agent

Arterial phase

Portal-venous phase

Late venous phase

Specificity (%) 53.7 75.6 90.2Sensitivity (%) 67.8 91.5 98.3Positive prognostic value 67.8 84.4 93.5Negative prognostic value 53.7 86.1 97.4

the basis of the following table[17]:Disease existent Disease non-existent

Test positive a b a + bTest negative c d c + d

a + c b + dWhere a is the number of patients with existing disease and a positive test result (true positive); b is the number of patients with no disease, but a positive test result (false positive); c is the number of patients with existing disease and a negative test result (false negative); d is the number of patients with no disease and a negative test result (true negative); a/(a + c): sensitivity: true positive results/number of affected patients; d/(b + d): specificity: true negative results / number of healthy patients.

Using the Bayes formula it was possible to calculate the probability[17] that disease was present, on the basis of a positive result of the diagnostic test (positive prognostic value: pV).

pV = true positive results(true positive results + false positive results)

It was also possible to calculate the probability of no disease on the basis of a negative test result (negative prognostic value: nV).

pV = true negative results(true negative results + false negative results)

RESULTSPatient characteristicsAltogether, 100 consecutive patients (43 women, 57 men, aged 25-83 years; mean 57 years) with suspicion of hepatic tumor were included in the study. Fifty-nine of these suffered from a malignant tumor (43 metastases of colon carcinoma, 5 metastases of breast cancer, 2 endocrine metastases, 7 HCC metastases, and 2 renal cell carcinoma), 41 patients had a benign tumor (9 hemangioma, 6 high-flow hemangioma, 7 nodular hyperplasia, 5 complicated cysts, 2 abscess, and 12 circumscribed fatty degeneration of the liver (focal hyposteatosis) with constant results over a minimum of 2 years. Maximum tumor size was 5 cm.

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it showed the most sensitive results. Sensitivity over the course of the trial was cumulatively calculated after every 10 patients. This showed that sensitivity very clearly approached a stable final value. This meant that sensitivity would not have changed significantly if more patients were included. Therefore, a valid evaluation could be made. The other diagnostic parameters showed similar results.

ROC analysisThe test results depended on the cut-off value of the gray value differences that were chosen. By modifying the cut-off value by means of an ROC analysis, it was possible to attain the optimum selectivity by improving sensitivity and specificity[18]. Figure 5 shows the respective specificity and sensitivity for the late venous phase for different limiting values (varying the cut-off values from +5 to -5). Usually, the greater the distance from the points to the diagonal, the better the diagnostic test. It is evident that the point marked in red in the ROC diagram was the one with

the most sensitivity and specificity. This optimum cut-off value was easy to calculate [cut-off valueopt = 0.5 × (specificity + sensitivity)/2] (Figure 3). The optimal cut-off value between the two groups of patients was readily established. The best selectivity for the two groups was at a cut-off value of -0.4 (Figure 4).

Center dependenceThe diagnostic parameters were calculated and compared for the two centers (Table 5). Variations in the parameters were based on one single case in the whole study with a false-positive value in only one center, and a single case was still too small for a separate description. The quality of testing did not differ between the two centers: 47 out of 50 patients were classified correctly in center 1, and 48 out of 50 in center 2. There was therefore no center-dependence (P = 0.229) (Figure 5A-F).

DISCUSSIONAll patients with suspected hepatic lesions, who were sent for further diagnostic workup to the two different centers, were consecutively included in the study. First, an ultrasound examination of the liver was undertaken using B-scan with a high-resolution multifrequency probe, resulting in a complete digital data set of the whole liver. Then, vascular ultrasound with power Doppler was performed to assess the vascularization of tumor lesions detected by the B-scan. After a bolus injection of 2.5 mL SonoVue, quantitative dynamic contrast-enhanced ultrasound was performed in low MI Technique for the arterial, portal-venous and late venous phase. As for

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Figure 3 ROC diagram showing the specificity and sensitivity in the late venous phase for different limiting values (varying the cut-off values from +5 to -5). A greater distance from the diagonal line indicates that a diagnostic test had a higher reliability. The black point in the ROC diagram corresponds to the best sensitivity and specificity.

Figure 4 Distribution of the characteristic gray-value differences in the late venous phase for patients with benign (black) or malignant (red) hepatic tumor.

Table 5 Specificity and sensitivity for both testing centers

Center 1 n = 50

Center 2 n = 50

Total n = 100

Specificity (%) 85.7 95.0 90.2Sensitivity (%) 100 96.7 98.3Negative prognostic value 100 95.0 97.4Positive prognostic value 90.6 96.7 93.5

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Figure 5 True agent detection mode of CHI with TIC analysis. A: Malignant lesion in the arterial phase. Arterial enhancement of the tumor margin - metastasis of beast cancer; B: Malignant lesion in the portal-venous phase. Lower enhancement of the tumor - metastasis of beast cancer; C: Malignant lesion in the late venous phase (> 100 s). Lower enhancement of the tumor - metastasis of beast cancer; D: Benign lesion in the arterial phase. Lower enhancement of the tumor-adenoma histological confirmed in the early arterial phase; E: Benign lesion in the portal-venous phase. Enhancement of the tumor - adenoma histological confirmed-similar to normal liver tissue; F: Benign lesion in the late venous phase. Enhancement of the tumor-adenoma histological confirmed - similar to normal liver tissue.

the Contrast Harmonic Imaging (CHI) technique, Pulse Inversion Harmonic Imaging (PIHI) with simultaneous data acquisition of the B-image and the contrast-enhanced perfusion image in True Agent Detection mode was used. Data were stored with dynamic and cine sequences up to a maximum of 60 s. Contrast -agent dynamics were represented in terms of the digital raw data of the gray values, exported into a spreadsheet table, and evaluated by an external institution without any knowledge of imaging and the clinical data. The histological result was only communicated after the end of the study, so that diagnostic criteria were analyzed without any previous knowledge.

An information bias was therefore precluded (which is especially important for radiological imaging), which enabled the prognostic values to be calculated according to Bayes[18]. Diagnostic tests were therefore evaluated in the same way as in the specific clinical situation.

Conventional ultrasound methods are limited for depicting and characterizing focal liver lesions by a low contrast between the lesion and normal parenchyma. The use of specific contrast agents for ultrasound improves the diagnostic value of conventional ultrasound and enables a complete diagnosis of liver lesions. Solutions for a complete ultrasound diagnosis have been proposed

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with the introduction of second-generation contrast agents and the use of low MI techniques, such as pulse inversion, which allows a continuous sweep with the same bubbles[1-3,5-10]. The support of gray-scale images is essential to delineate the lesions better and to check the perfect matching of contrast-enhanced and B-mode images. The availability of the true agent detection mode is able to overcome these limitations by higher signal sensitivity and simultaneous acquisition of gray scale and contrast-enhanced images.

SonoVue provides strong and persistent harmonic resonance at low MI (≤ 0.2), with which minimal or no bubble destruction occurs. This allows for continuous real-time imaging of a lesion during its vascular phase. With real-time low-MI imaging, the dynamic enhancement pattern and the vascular morphology of a lesion is assessed during the arterial phase (10-20 s until 25-50 s after bolus injection) and portal-venous phase (30-45 s until 120 s after bolus injection). The delayed phase (> 120 s after bolus injection) is particularly useful for the detection of, as they show as non-enhancing defects. Characterization is also improved by the late phase, as the vast majority of benign lesions show contrast uptake in this phase[5,7,8]. Our results show that by means of CHI and subsequent quantitative gray-value analysis of contrast-agent dynamics in the late venous phase, it is possible to assess malignant liver tumors with a sensitivity of 98.3% with an nV of 97.4%.

High-resolution digital contrast-enhanced ultrasound techniques have considerably improved the assessment of malignant and benign tumor lesions in the liver[1-12]. Under optimal examination conditions that enable imaging of the entire liver, it is possible to attain a diagnostic reliability comparable to that of contrast-enhanced MRI with a liver-specific contrast agent, and an even higher diagnostic certainty of > 90%, compared with contrast-enhanced multi-detector-spiral CT[1,4,15]. Using ultrasound contrast agents, a signal amplification of up to 10 dB is feasible. This allows a better differentiation between regular liver tissue and malignant tissue within the liver, which has an increased contrast wash-out in the portal-venous and, especially, in the late phase. The perfusion curves in healthy liver tissue show a slow increase, which reaches a plateau at the portal-venous phase, followed by a very slow decrease. In most benign tumors, enhancement can also be detected in the late phase after contrast agent injection. In cases of malignancy, the decrease begins in the portal-venous phase in most cases after slow marginal arterial enhancement. Tumor lesions of HCC, l iver adenoma, high-f low hemangioma and focal nodular hyperplasia can show an early arterial enhancement in the first 30 s in perfusion curves[4,6,8,12,13]. These lesions may be masked in the portal-venous phase. Therefore, the most important phase for tissue differentiation is the late phase, because malignant tissue shows a contrast-agent wash-out in the late phase, whereas regular tissue still has a slowly descending plateau. This is the explanation for the significant differences of the gray-value analysis of the contrast-enhanced TIC analysis in the late phase.

Using a continuously acquired contrast-enhanced dynamic CHI, the diagnostic accuracy in characterization

of benign lesions, such as partially thrombosed or high flow hemangioma, focal nodular hyperplasia or local fatty and regenerative changes, is superior to that of B-scan or contrast-enhanced spiral CT[1,2,5,8,10].

The general problem for liver ultrasound still remains unchanged. The subdiaphragmatic liver segments Ⅳa and Ⅷ are difficult to visualize, which makes it very difficult to assess a lesion within these segments. Investigation of only part of the liver was possible owing to the high attenuation of ultrasound especially in the cranial and dorsal parts of the liver where the position of the liver was very high under the diaphragm or when patients had difficulty holding their breath. Secondly, fatty areas or cirrhosis of the liver, as well as lesions location deeper than 10 cm, diminish the penetration of contrast-specific imaging modes, which results in a decreased signal noise[1,5,8-10]. Another major problem for all imaging modalities such as contrast-enhanced CT or MRI remains the detection and characterization of lesions smaller than 5 mm[7].

SonoVue is a pure intravascular blood-pool contrast agent without a specific liver phase. The microbubbles help to visualize small parenchymal vessels. Malignant liver lesions such as metastases or HCC lesions are characterized by demarcation during the late phase. SonoVue late phase is based on visualization of parenchymal vessels. It is possible for very small HCC lesions, hemangiona or adenoma with vascular architecture comparable to that of the liver parenchyma to be missed in the detection study. Hypervascularization is a feature of HCC with a diameter > 2 cm. This may explain why the early phase of enhancement might not be effective for some very small HCC lesions. Early arterial enhancement can be found in 76%-96% of HCC lesions, and homogeneous enhancement in the late phase in 3%-30% of the patients[4,6,8,12,13].

The detection rate still remains examiner-dependent, even with low-MI contrast-agent techniques. In our study, we were able to demonstrate the feasibility of objective digital raw data analysis, which was not influenced by different examiners in our two-center approach. Therefore, dedicated software for computer-aided diagnosis can be developed, which allows the user an objective assessment of benign or malignant liver lesions based on digital raw data.

Contrast-enhanced ultrasound (CEUS) reveals typical patterns of contrast enhancement in the different lesion histotypes, and provides equivalent accuracy to CT and MRI in focal liver lesion characterization. CEUS should be considered in every patient with a known malignancy with proven or suspected liver metastases at baseline ultrasound during preoperative staging or postoperative follow-up.

Without dynamic evaluation of contrast enhancement, in approximately 10% of cases focal liver lesions remain indeterminate even after microbubble injection, and therefore Gadobenate-Dimeglumine (Gd-BOPTA) enhanced MRI should be employed, followed when necessary by ultrasound-guided biopsy. Standard cross-sectional imaging procedures including multislice CT or Gd-BOPTA-enhanced MRI should be referenced in every case not explorable by ultrasound, or in patients with no evidence of liver metastases at CEUS and with clinically

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suspected liver metastases, such as in cases with increased serum levels of tumor markers.

Using dynamic CHI, the malignancy of hepatic tumors can be predicted with a positive prognostic value of 93.5%. CHI with SonoVue in combination with dynamic quantitative evaluation of contrast-agent dynamics is a valuable tool for discrimination.

COMMENTSBackgroundLiver lesions are a common diagnostic problem in medical imaging. Contrast-enhanced ultrasound is a recently introduced new modality in lesion assessment. In our study the quantitative evaluation of raw ultrasound data was assessed compared with histology as a gold standard.

Innovations and breakthroughsBased on this raw data evaluation, new computer-assisted algorithms can be created, which allow automated liver lesion diagnosis.

Applications For contrast-enhanced ultrasound-based lesion evaluation, three phases (arterial, portal-venous, and late phase) are mandatory. Computer-based dynamic analysis of raw digital ultrasound data facilitates lesion characterization.

Peer reviewIn this study, the authors review their experience of CHI and its diagnostic value in patients with space-occupying liver lesions. They conclude that using dynamic CHI, the malignancy of hepatic tumors can be predicted with a pV of 93.5%. CHI with SonoVue in combination with dynamic quantitative evaluation of contrast-agent dynamics is a valuable tool for discrimination.

REFERENCES1 Hohmann J, Albrecht T, Oldenburg A, Skrok J, Wolf KJ.

Liver metastases in cancer: detection with contrast-enhanced ultrasonography. Abdom Imaging 2004; 29: 669-681

2 Albrecht T, Blomley MJ, Burns PN, Wilson S, Harvey CJ, Leen E, Claudon M, Calliada F, Correas JM, LaFortune M, Campani R, Hoffmann CW, Cosgrove DO, LeFevre F. Improved detection of hepatic metastases with pulse-inversion US during the liver-specific phase of SHU 508A: multicenter study. Radiology 2003; 227: 361-370

3 Bartolozzi C, Donati F, Cioni D, Procacci C, Morana G, Chiesa A, Grazioli L, Cittadini G, Cittadini G, Giovagnoni A, Gandini G, Maass J, Lencioni R. Detection of colorectal liver metastases: a prospective multicenter trial comparing unenhanced MRI, MnDPDP-enhanced MRI, and spiral CT. Eur Radiol 2004; 14: 14-20

4 Boozari B, Lotz J, Galanski M, Gebel M. Diagnostic imaging of liver tumours. Current status. Internist (Berl) 2007; 48: 8, 10-2, 14-6, 18-20

5 Dietrich CF, Kratzer W, Strobe D, Danse E, Fessl R, Bunk A, Vossas U, Hauenstein K, Koch W, Blank W, Oudkerk M, Hahn D, Greis C. Assessment of metastatic liver disease in patients with primary extrahepatic tumors by contrast-enhanced sonography versus CT and MRI. World J Gastroenterol 2006; 12: 1699-1705

6 Bruix J, Sherman M, Llovet JM, Beaugrand M, Lencioni R, Burroughs AK, Christensen E, Pagliaro L, Colombo M, Rodés J. Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona-2000 EASL conference. European Association for the Study of the Liver. J Hepatol 2001; 35: 421-430

7 Bleuzen A, Huang C, Olar M, Tchuenbou J, Tranquart F. Diagnostic accuracy of contrast-enhanced ultrasound in focal lesions of the liver using cadence contrast pulse sequencing. Ultraschall Med 2006; 27: 40-48

8 von Herbay A , Haeuss inger D , Gregor M, Vogt C .

Characterization and detection of hepatocellular carcinoma (HCC): comparison of the ultrasound contrast agents SonoVue (BR 1) and Levovist (SH U 508A)--comparison of SonoVue and Levovist in HCC. Ultraschall Med 2007; 28: 168-175

9 Konopke R, Bunk A, Kersting S. The role of contrast-enhanced ultrasound for focal liver lesion detection: an overview. Ultrasound Med Biol 2007; 33: 1515-1526

10 Oldenburg A, Hohmann J, Foert E, Skrok J, Hoffmann CW, Frericks B, Wolf KJ, Albrecht T. Detection of hepatic metastases with low MI real time contrast enhanced sonography and SonoVue. Ultraschall Med 2005; 26: 277-284

11 D'Onofrio M, Rozzanigo U, Masinielli BM, Caffarri S, Zogno A, Malagò R, Procacci C. Hypoechoic focal liver lesions: characterization with contrast enhanced ultrasonography. J Clin Ultrasound 2005; 33: 164-172

12 Ogawa S, Kumada T, Toyoda H, Ichikawa H, Kawachi T, Otobe K, Hibi T, Takeshima K, Kiriyama S, Sone Y, Tanikawa M, Hisanaga Y, Yamaguchi A, Isogai M, Kaneoka Y, Washizu J. Evaluation of pathological features of hepatocellular carcinoma by contrast-enhanced ultrasonography: comparison with pathology on resected specimen. Eur J Radiol 2006; 59: 74-81

13 Wu W, Chen MH, Yin SS, Yan K, Fan ZH, Yang W, Dai Y, Huo L, Li JY. The role of contrast-enhanced sonography of focal liver lesions before percutaneous biopsy. AJR Am J Roentgenol 2006; 187: 752-761

14 Krix M, Plathow C, Essig M, Herfarth K, Debus J, Kauczor HU, Delorme S. Monitoring of l iver metastases after stereotactic radiotherapy using low-MI contrast-enhanced ultrasound--initial results. Eur Radiol 2005; 15: 677-684

15 Regge D, Campanella D, Anselmetti GC, Cirillo S, Gallo TM, Muratore A, Capussotti L, Galatola G, Floriani I, Aglietta M. Diagnostic accuracy of portal-phase CT and MRI with mangafodipir trisodium in detecting liver metastases from colorectal carcinoma. Clin Radiol 2006; 61: 338-347

16 Mamdani B. The Helsinki Declaration, 2000, and ethics of human research in developing countries. Indian J Med Ethics 2004; 1: 94-95

17 Touitou Y, Smolensky MH, Portaluppi F. Ethics, standards, and procedures of animal and human chronobiology research. Chronobiol Int 2006; 23: 1083-1096

18 Linnert K. A Review on the methodology for assessing diagnostic tests. Clin Chem 1988; 34: 1379-1386

19 Delorme S, Krix M, Albrecht T. Ultrasound contrast media--principles and clinical applications. Rofo 2006; 178: 155-164

20 Krix M, Plathow C, Kiessling F, Herth F, Karcher A, Essig M, Schmitteckert H, Kauczor HU, Delorme S. Quantification of perfusion of liver tissue and metastases using a multivessel model for replenishment kinetics of ultrasound contrast agents. Ultrasound Med Biol 2004; 30: 1355-1363

21 Quaia E. Microbubble ultrasound contrast agents: an update. Eur Radiol 2007; 17: 1995-2008

22 Quaia E, Bartolotta TV, Midiri M, Cernic S, Belgrano M, Cova M. Analysis of different contrast enhancement patterns after microbubble-based contrast agent injection in liver hemangiomas with atypical appearance on baseline scan. Abdom Imaging 2006; 31: 59-64

23 Kim SH, Lee JM, Lee JY, Han JK, An SK, Han CJ, Lee KH, Hwang SS, Choi BI. Value of contrast-enhanced sonography for the characterization of focal hepatic lesions in patients with diffuse liver disease: receiver operating characteristic analysis. AJR Am J Roentgenol 2005; 184: 1077-1084

24 Quaia E, Palumbo A, Rossi S, Degobbis F, Cernic S, Tona G, Cova M. Comparison of visual and quantitative analysis for characterization of insonated liver tumors after microbubble contrast injection. AJR Am J Roentgenol 2006; 186: 1560-1570

25 Leen E, Ceccotti P, Kalogeropoulou C, Angerson WJ, Moug SJ, Horgan PG. Prospective multicenter trial evaluating a novel method of characterizing focal liver lesions using contrast-enhanced sonography. AJR Am J Roentgenol 2006; 186: 1551-1559

26 Vilana R, Bianchi L, Varela M, Nicolau C, Sánchez M, Ayuso

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COMMENTS

C, García M, Sala M, Llovet JM, Bruix J, Bru C. Is microbubble-enhanced ultrasonography sufficient for assessment of response to percutaneous treatment in patients with early hepatocellular carcinoma? Eur Radiol 2006; 16: 2454-2462

27 Quaia E, D'Onofrio M, Palumbo A, Rossi S, Bruni S, Cova M. Comparison of contrast-enhanced ultrasonography versus baseline ultrasound and contrast-enhanced computed tomography in metastatic disease of the liver: diagnostic performance and confidence. Eur Radiol 2006; 16: 1599-1609

28 Bryant TH, Blomley MJ, Albrecht T, Sidhu PS, Leen EL, Basilico R, Pilcher JM, Bushby LH, Hoffmann CW, Harvey CJ, Lynch M, MacQuarrie J, Paul D, Cosgrove DO. Improved characterization of liver lesions with liver-phase uptake of

liver-specific microbubbles: prospective multicenter study. Radiology 2004; 232: 799-809

29 Delorme S, Krix M, Albrecht T. Ultrasound contrast media--principles and clinical applications. Rofo 2006; 178: 155-164

30 Jung EM, Kubale R, Jungius KP, Jung W, Lenhart M, Clevert DA. Vascularization of liver tumors - preliminary results with Coded Harmonic Angio (CHA), phase inversion imaging, 3D power Doppler and contrast medium-enhanced B-flow with second generation contrast agent (Optison). Clin Hemorheol Microcirc 2006; 34: 483-497

31 Jung EM , Kubale R, Jungius KP. Vascularization and perfusion of hepatocellular carcinoma: assessment with contrast-enhanced ultrasound using perflutren protein-type A microspheres. Clin Hemorheol Microcirc 2005; 33: 63-73

S- Editor Zhu LH L- Editor Kerr C E- Editor Lu W

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Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6365-6369www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

Analysis of the expression of coxsackievirus and adenovirus receptor in five colon cancer cell lines

Yassan Abdolazimi, Majid Mojarrad, Mehrdad Pedram, Mohammad Hossein Modarressi

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COLORECTAL CANCER

Yassan Abdolazimi, Majid Mojarrad, Mehrdad Pedram, Mohammad Hossein Modarressi, Department of Medical Genetics, Medical Sciences/University of Tehran, Tehran, IranMohammad Hossein Modarressi, Reproductive Biotechnology Research Center, Avesina Research Institute, Tehran, IranSupported by the Funds from Medical Sciences/University of Tehran and Iranian Ministry of Health and Medical EducationCorrespondence to: Dr. Mohammad Hossein Modarressi, Department of Medical Genetics, Medical Sciences/University of Tehran, PO Box 14155-6447, Tehran, Iran. modaresi@tums.ac.irTelephone: +98-21-88953005 Fax: +98-21-66404577Received: July 17, 2007 Revised: September 11, 2007

AbstractAIM: To investigate the expression of coxsackievirus and adenovirus receptor (CAR) and adenovirus-mediated reporter gene transfer in five human colon cancer cell lines.

METHODS: Expression of CAR-specific mRNA and protein was analyzed by reverse transcriptase polymerase chain reaction and Western blotting, respectively. Adenovirus-based gene delivery was evaluated by infection of cells with adenoviral vector carrying the green fluorescent protein (GFP) gene.

RESULTS: All the colon cancer cell lines examined (HT29, LS180, SW480, SW948 and SW1116) expressed CAR full-length mRNA and an alternatively-spliced variant that lacks the transmembrane coding exon. All cell lines were detected as CAR-positive by Western blot analysis. Further, all cells we examined were efficiently infected with adenoviral vector-GFP.

CONCLUSION: The data indicated that the five colon cancer cell lines tested expressed adenovirus primary receptor and could be efficiently infected by adenoviral vectors. Therefore, these cell lines will be useful for adenovirus-based gene transfer and research.

© 2007 WJG. All rights reserved.

Key words: Coxsackievirus and adenovirus receptor; Adenoviral infection; Gene therapy

Abdolazimi Y, Mojarrad M, Pedram M, Modarressi MH. Analysis of the expression of coxsackievirus and adenovirus receptor in five colon cancer cell lines. World J Gastroenterol

2007; 13(47): 6365-6369

http://www.wjgnet.com/1007-9327/13/6365.asp

INTRODUCTIONColorectal cancer is the third most common cancer and among the three top fatal malignancies. In 2007, there were an estimated 153 760 new cases and 52 180 deaths from colorectal cancer, which corresponds to approximately 10% of all newly diagnosed cancers in the United States[1,2]. Gene therapy has been considered a potential innovative approach for the treatment of colorectal cancer[3,4]. To date, over 600 gene therapy clinical trials have been initiated in the US, 60% of which are cancer-related[5]. Currently, adenoviral vectors are the most frequently administered vectors[6]. This is because of their advantageous features such as a broad range of cell targets, the ability to be produced in high titers, and their accommodation of relatively large segments of DNA[7,8].

Adenovir us ini t iates infect ion as wel l as does adenovector-mediated gene transfer by attachment of the fiber knob to a cell surface receptor, the coxsackievirus and adenovirus receptor (CAR)[9]. CAR, the primary high-affinity receptor for adenovirus, is a 46-ku transmembrane glycoprotein and belongs to the immunoglobulin superfamily[10,11]. The human CAR gene, CXADR, encodes a 365-amino acid protein, and four pseudogenes have been reported on chromosomes 15, 18 and 21 (two copies)[12]. Alternatively spliced variants of CAR have also been identified[13]. Thoelen et al has described three splice variants (β-, δ- and δ-transcripts) of the CAR gene in addition to the normal α-transcript.

Expression of CAR has been studied in numerous cell lines including those of head and neck carcinoma[14], renal cell carcinoma[15], bladder cancer[16], ovarian cancer[17,18], cervical cancer[19], lung and pancreatic cancer[20], prostate cancer[21] and glioma[22]. In all these studies, high expression of CAR correlated with increased adenoviral infection efficiency; cells lacking or expressing low levels of CAR were resistant to adenovirus infection.

Since CAR expression has not been studied in colon cancer cell lines, we examined the expression of CAR at the mRNA and protein levels, along with its splice variants in five human colon cancer cell lines. Adenovirus-mediated reporter gene transfer was also evaluated.

MATERIALS AND METHODSCell linesFive human colon cancer cell lines HT29, SW480, SW948, SW1116 and LS180, as well as the human embryonic kidney (HEK) cell line (CAR-positive) and Chinese hamster ovary (CHO) cells (CAR-negative) (ATCC numbers: HTB-38, CCL-228, CCL-237, CCL-233, CL-187, CRL-1573, CCL-61) were obtained from the National Cell Bank of Iran (NCBI). Cells were cultured in RPMI 1640, Dulbecco’s Modified Eagle’s Medium, Minimum Essential Medium, or L-15 medium. All cells were maintained with 100 U/mL penicillin, 100 µg/mL streptomycin and 100 mL/L fetal bovine serum (FBS). Cell lines were cultured either in 10-cm culture plates or in flasks at 37℃ and in 50 mL/L CO2 in a humidified incubator.

RT-PCRTotal cellular RNA was extracted from each cell line by Biozol reagent (Bioflux, Japan). Two micrograms of total RNA were converted to cDNA by reverse transcription, using Moloney murine leukemia virus reverse transcriptase (Fermentas, Lithuania) and random primers (Roche, Ger many) as descr ibed previously [23], with minor modifications. Since pseudogenes have been identified for the CAR gene, the CAR mRNA sequence was aligned with the pseudogenes (multiple alignment at http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.htm). CAR primers (CARf, 5'-CGTGCTCCTGTGCGGAGTAGT-3'; CARr, 5'-GACCCATCCTTGCTCTGTGCT-3') were designed in exons 1 and 7 with intervening introns of approximately 51 Kb, in a way that would align only with CAR mRNA and mismatched the pseudogenes in at least one nucleotide at the 3'-ends. Phosphoglucomutase-1 (PGM-1), human house keeping gene primers (PGMf, 5'-TCCGACTGAGCGGCACTGGGAGTGC-3'; PGMr, 5'-GCCCGCAGGTCCTCTTTCCCTCACA-3') or murine β-actin house keeping gene primers (actinf, 5'-GAACCCTAAGGCCAACCGTGA-3'; actinr, 5'-AGGAAGAGGATGCGGCAGTGG-3') were used as internal controls.

The PCR protocol consisted of an initial denaturation for 5 min at 94℃; followed by 32 cycles of denaturation for 30 s at 94℃, annealing for 30 s at 62℃, and extension for 60 s at 72℃. The final cycle had a prolonged extension time of 7 min at 72℃. PCR products were analyzed by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide and visualized under UV light. The length of the expected product was 1068 (α-transcript) or 806 (β-transcript) bp for the CAR gene and 382 and 374 bp for the PGM1 and β-actin genes, respectively.

Cloning Amplification products of the CAR β-transcript were cloned using InsTAclone PCR Cloning Kit (Fermentas) following the manufacturer’s instructions. In brief, PCR products were ligated into a pTZ57R/T vector and transformed by a heat shock method into competent Escherichia coli. Transformants were selected in LB medium containing ampicillin, X-gal and IPTG. The presence of the expected insert was confirmed by PCR and sequencing.

ImmunoblottingFor immunoblotting analysis, cells were lysed for 1 h at 4℃ in lysis buffer containing 50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 10 mL/L Nonidet-P40 (NP40), 0.1 mL/L SDS and one tablet of complete, mini, EDTA-free protease inhibitor cocktai l (Roche, Germany) per 10 mL lysis buffer. After the removal of cell nuclei by centrifugation, protein concentrations were measured by the Bradford protein assay. Equal amounts of protein (15 µg) from each cell lysate were diluted with SDS loading buffer, heated for 5 min at 95℃ and electrophoresed on a 10% SDS-polyacrylamide gel. The separated proteins were then electrotransferred to a nitrocellulose membrane, which was blocked with 50 g/L non-fat dried milk in Tris buffered saline-Tween (TBST) buffer to block non-specific binding sites. The blot was then incubated with polyclonal rabbit anti-CAR primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1/1000 dilution in TBST and 0.01 g/mL BSA) with gentle shaking for 1 h at room temperature. After extensive washing, polyclonal anti-rabbit horseradish peroxidase-conjugated secondary antibody (1/1000 dilution in TBST and 0.01 g/mL BSA) was applied for 1 h at room temperature with gentle shaking. Bands were visualized using an ECL kit (Amersham) according to the manufacturer’s instructions.

Adenoviral vectorAdenoviral vector expressing the green fluorescent protein (GFP) gene driven by the cytomegalovirus promoter[24] was used to assess the colon cancer cell lines infected with adenoviral vector. Virus titer was determined by optical absorbance at A260 (1 A260 unit = 1012 particles/mL).

Adenovirus-mediated GFP gene transfer To evaluate reporter gene transfer by the adenovirus vector, five colon cancer cell lines were plated in 24-well culture plates at a density of 5 × 104 cells/well and grown at 37℃ in a humidified incubator. After overnight incubation, cells were infected with Ad5-GFP at a multiplicity of infection (MOI) of 50 virus particles/cell. Two hours later, cells were washed twice with PBS and 600 µL fresh complete medium was added to every well. After 36 h, cells were evaluated for GFP expression by fluorescence microscopy.

RESULTSCAR mRNA expression in colon cancer cell linesWe first examined the expression of CAR by RT-PCR. Integrity was evaluated by running RNA on 1% agarose gel and the quantity of RNA was measured spectrophotometrically. cDNA was then synthesized and CAR specific sequences amplified using CAR-specific primers (CARf, CARr). As shown in Figures 1A and 2, CARf and CARr primers generated a seven-exon-encompassing fragment with the expected length of 1068 bp in all the colon cancer cell lines. These primers could not amplify the pseudogenes (Figure 2, lane 1), and therefore the bands observed in lanes 2-6 were generated by just the amplification of cDNA. Any probable

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contamination of cDNA with genomic DNA could be disregarded.

CAR protein expression in colon cancer cell linesWe examined the expression of CAR by Western blotting. For immunoblotting, cell lines were cultured, harvested, lysed in appropriate lysis buffer and finally analyzed with anti-CAR antibodies. As shown in Figure 3, a band of about 46 ku was detected in HEK, HT-29, Ls-180, SW480, SW948 and SW1116 cell lines but was not observed in the negative control CHO cells.

Alternatively spliced variant of CARAfter electrophoresis and staining of RT-PCR products, an additional fragment of 806 bp was also observed (Figure 2). This shorter band produced by CAR-specific primers was cloned and sequenced, which revealed that this fragment was the result of an alternative splicing event between exons 4 and 7, lacking exons 5 and 6, of the CAR normal transcript, and was therefore the previously determined β-splice variant. All the colon cell lines studied expressed the β-splice variant although the band intensity of the splice variant was low when compared with the intensity of the normal transcript.

Detection of GFP gene expression in colon cancer cell linesAs shown in Figure 4, Ad5-GFP vector entered the cells

and expression of the GFP gene indicated the efficient infection of these cells by the adenoviral vector.

DISCUSSIONGene therapy is a therapeutic modality for malignant cancers. For this, the recombinant adenovectors Ad2 and Ad5 derived from the human adenovirus subgroup C are attractive gene transfer vehicles for cancer gene therapy[25,26]. As therapeutic efficacy has been demonstrated to correlate with the ability of adenovirus to enter target cells, expression of CAR has been studied extensively.

Several tumor cell lines including those for ovary cancer [17], head and neck carc inoma[14], renal ce l l carcinoma[15], prostate[21] and bladder[16] cancer have shown relatively low CAR expression; although high expression of CAR mRNA has been reported in osteosarcoma cell lines[27]. In the present study, CAR expression was investigated in five colon cancer cell lines, and it was shown that CAR was expressed in all of them, although some variations in band intensity of RT-PCR products were observed among the different cell lines (Figure 1B). The mismatch between specific primers and CAR pseudogenes made us certain that only cDNA could act as the source of amplification in RT-PCR. The existence of CAR pseudogenes has apparently not been taken into account in previous studies that have investigated CAR expression[18,19,29].

Alternatively spliced products of the CAR gene have been identified; β-variant mRNA has been detected in multiple human tissues such as heart, brain, lung, liver, kidney and pancreas[10], and has also shown to be expressed in osteosarcoma cell lines[27], HeLa cells[28] and musculoskeletal tumors[29]. The β-transcript is predicted to encode a 252-amino-acid protein that can be released

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Figure 2 Expression of CAR in colon cancer cell lines. Two micrograms of total RNA prepared from several different human colon cancer cell lines were subjected to RT-PCR analysis. RT-PCR revealed β-transcript expression in addition to the normal CAR gene transcripts.

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Figure 3 Western blot analysis of CAR protein in colon cancer cell lines. Total protein from five human colon cancer cell lines was separated by SDS-PAGE, transferred to nitrocellulose membranes, and reacted with the correct anti-CAR antibodies.

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Figure 1 A: Multiplex RT-PCR of expression of coxsackievirus and adenovirus receptor, PGM and β-actin (internal controls); B: CAR mRNA level in different cell lines. 1-6 refer to HEK (positive control), HT-29 (colon), LS180 (colon), SW480 (colon), SW948 (colon) and SW1116 (colon) cells, respectively.

A

from cells to the culture medium because of the absence of a transmembrane domain[28]. CAR cytoplasmic and transmembrane domains are not necessary for virus attachment[30], so the anticipated β-variant protein might be as effective as the full-length variant in binding to adenovirus. In the present study, the β-transcript was demonstrated to be present in colon cell lines, in addition to the normal α-transcript.

To eva lua te whether CAR mRNA express ion correlated with CAR protein expression, Western blotting was carried out. CAR protein was shown to be present in the colon cell lines investigated; therefore, they could be considered as CAR-positive on the basis of CAR protein expression. In addition, no alternative protein band that could be attributed to the β-transcript-encoded protein was observed. Western blotting was carried out using a commercial polyclonal antibody against amino acids 1 to 300 of the original protein and cell lysates. Thus, the absence of any alternative signal (β-variant protein) could be explained as very low or undetectable intracellular level of this shorter variant and its secretion from the cell surface. However, β-variant protein is not detected in extracts of HeLa cells that express both β- and normal variants[28]. Therefore, the potential translation of CAR alternative transcript and its proposed regulatory role in antivirus defense mechanisms[28] remains to be confirmed.

Experiments with the Ad5 vector carrying the GFP reporter gene showed that the colon cancer cell lines studied were sensitive to adenovirus infection, and after 36 h GFP gene expression was easily detected by fluorescence microscopy.

In conclusion, the results of this study suggested that adenovirus-based gene delivery was efficiently applied to the five colon cancer cell lines. However, to clarify the efficiency of adenoviral-mediated gene transfer in colon

tumors, further studies will be required to explore the expression of CAR.

COMMENTSBackgroundVectors based on human adenovirus serotypes 2 and 5 show increasing promise as gene delivery vehicles, and currently adenoviral vectors are the most commonly administered vectors for cancer gene therapy. Adenovirus initiates gene transfer by attachment to cell surface receptor, the coxsackievirus and adenovirus receptor (CAR).

Research frontiersReports show that the efficiency of adenoviral infection is dependent on the expression of CAR on target cells. However, adenovirus-mediated gene transfer is in practice hindered by the relatively low expression of CAR on tumor cells.

Innovations and breakthroughsThis is believed to be the first report to examine the expression of CAR in human colon cancer cell lines at the mRNA and protein level.

Applications These results might be of potential value in adenovirus-based gene transfer and research in colon cancer cell lines.

TerminologyGene therapy: to replace a malfunctioning mutated gene with a normal wild-type gene or to express a therapeutic gene in target cells. A prerequisite for efficient gene therapy is to determine a system for delivering the therapeutic gene to target cells. Adenovirus: non-enveloped, icosahedral, particles with double stranded DNA genome that in humans, is divided into six species (A-F) and 51 serotypes. CAR: primary cellular receptor for attachment of both coxsackie B virus and adenovirus subgroup C to target cells.

Peer reviewThis manuscript by Yassan Abdolazimi and co-authors is presented in a clear style.

Figure 4 Sensitivity of colon cancer cells to adenovirus infection. Cells were plated in 24-well culture plates and infected with Ad5-GFP at an MOI of 50. After 36 h, cells were evaluated for GFP expression by fluorescence microscopy. GFP was expressed when recombinant adenoviruses infected the cells.

(Negative control)CHO

SW1116

SW948

SW480

LS180HT-29

Positive controlHEK293

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COMMENTS

Experimental details are provided accordingly. Relevant controls were included in the study. The authors’ findings are discussed with the relevant literature.

REFERENCES1 Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer

statistics, 2007. CA Cancer J Clin 2007; 57: 43-662 Gong Z, Xie D, Deng Z, Bostick RM, Muga SJ, Hurley TG,

Hebert JR. The PPAR{gamma} Pro12Ala polymorphism and risk for incident sporadic colorectal adenomas. Carcinogenesis 2005; 26: 579-585

3 Palmer DH, Chen MJ, Kerr DJ. Gene therapy for colorectal cancer. Br Med Bull 2002; 64: 201-225

4 Chen MJ, Chung-Faye GA, Searle PF, Young LS, Kerr DJ. Gene therapy for colorectal cancer: therapeutic potential. BioDrugs 2001; 15: 357-367

5 Palmer DH, Chen MJ, Kerr DJ. Taking Gene Therapy into the Clinic. J Biomed Biotechnol 2003; 2003: 71-77

6 Wilson DR. Viral-mediated gene transfer for cancer treatment. Curr Pharm Biotechnol 2002; 3: 151-164

7 Vorburger SA, Hunt KK. Adenoviral gene therapy. Oncologist 2002; 7: 46-59

8 Mountain A. Gene therapy: the first decade. Trends Biotechnol 2000; 18: 119-128

9 Qin M, Chen S, Yu T, Escuadro B, Sharma S, Batra RK. Coxsackievirus adenovirus receptor expression predicts the efficiency of adenoviral gene transfer into non-small cell lung cancer xenografts. Clin Cancer Res 2003; 9: 4992-4999

10 Tomko RP, Xu R, Philipson L. HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc Natl Acad Sci USA 1997; 94: 3352-3356

11 Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS, Horwitz MS, Crowell RL, Finberg RW. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 1997; 275: 1320-1323

12 Carson SD. Receptor for the group B coxsackieviruses and adenoviruses: CAR. Rev Med Virol 2001; 11: 219-226

13 Thoelen I, Magnusson C, Tågerud S, Polacek C, Lindberg M, Van Ranst M. Identification of alternative splice products encoded by the human coxsackie-adenovirus receptor gene. Biochem Biophys Res Commun 2001; 287: 216-222

14 Li D, Duan L, Freimuth P, O'Malley BW. Variability of adenovirus receptor density influences gene transfer efficiency and therapeutic response in head and neck cancer. Clin Cancer Res 1999; 5: 4175-4181

15 Haviv YS, Blackwell JL, Kanerva A, Nagi P, Krasnykh V, Dmitriev I, Wang M, Naito S, Lei X, Hemminki A, Carey D, Curiel DT. Adenoviral gene therapy for renal cancer requires retargeting to alternative cellular receptors. Cancer Res 2002; 62: 4273-4281

16 Li Y, Pong RC, Bergelson JM, Hall MC, Sagalowsky AI, Tseng CP, Wang Z, Hsieh JT. Loss of adenoviral receptor expression in human bladder cancer cells: a potential impact on the efficacy of gene therapy. Cancer Res 1999; 59: 325-330

17 Kim M, Zinn KR, Barnett BG, Sumerel LA, Krasnykh V, Curiel

DT, Douglas JT. The therapeutic efficacy of adenoviral vectors for cancer gene therapy is limited by a low level of primary adenovirus receptors on tumour cells. Eur J Cancer 2002; 38: 1917-1926

18 Kim JS , Lee SH, Cho YS , Choi J J , Kim YH, Lee JH. Enhancement of the adenoviral sensitivity of human ovarian cancer cells by transient expression of coxsackievirus and adenovirus receptor (CAR). Gynecol Oncol 2002; 85: 260-265

19 Kim JS, Lee SH, Cho YS, Kim YH, Lee JH. Ectopic expression of the coxsackievirus and adenovirus receptor increases susceptibility to adenoviral infection in the human cervical cancer cell line, SiHa. Biochem Biophys Res Commun 2001; 288: 240-244

20 Pearson AS, Koch PE, Atkinson N, Xiong M, Finberg RW, Roth JA, Fang B. Factors limiting adenovirus-mediated gene transfer into human lung and pancreatic cancer cell lines. Clin Cancer Res 1999; 5: 4208-4213

21 Okegawa T, Li Y, Pong RC, Bergelson JM, Zhou J, Hsieh JT. The dual impact of coxsackie and adenovirus receptor expression on human prostate cancer gene therapy. Cancer Res 2000; 60: 5031-5036

22 Fuxe J, Liu L, Malin S, Philipson L, Collins VP, Pettersson RF. Expression of the coxsackie and adenovirus receptor in human astrocytic tumors and xenografts. Int J Cancer 2003; 103: 723-729

23 Mobasheri MB , Modarressi MH, Shabani M, Asgarian H, Sharifian RA, Vossough P, Shokri F. Expression of the testis-specific gene, TSGA10, in Iranian patients with acute lymphoblastic leukemia (ALL). Leuk Res 2006; 30: 883-889

24 Modarressi MH, Cheng M, Tarnasky HA, Lamarche-Vane N, de Rooij DG, Ruan Y, van der Hoorn FA. A novel testicular RhoGAP-domain protein induces apoptosis. Biol Reprod 2004; 71: 1980-1990

25 Young LS, Searle PF, Onion D, Mautner V. Viral gene therapy strategies: from basic science to clinical application. J Pathol 2006; 208: 299-318

26 Glasgow JN, Everts M, Curiel DT. Transductional targeting of adenovirus vectors for gene therapy. Cancer Gene Ther 2006; 13: 830-844

27 Kawashima H, Ogose A, Yoshizawa T, Kuwano R, Hotta Y, Hotta T, Hatano H, Kawashima H, Endo N. Expression of the coxsackievirus and adenovirus receptor in musculoskeletal tumors and mesenchymal tissues: efficacy of adenoviral gene therapy for osteosarcoma. Cancer Sci 2003; 94: 70-75

28 Dörner A, Xiong D, Couch K, Yajima T, Knowlton KU. Alternatively spliced soluble coxsackie-adenovirus receptors inhibit coxsackievirus infection. J Biol Chem 2004; 279 : 18497-18503

29 Gu W, Ogose A, Kawashima H, Ito M, Ito T, Matsuba A, Kitahara H, Hotta T, Tokunaga K, Hatano H, Morita T, Urakawa S, Yoshizawa T, Kawashima H, Kuwano R, Endo N. High-level expression of the coxsackievirus and adenovirus receptor messenger RNA in osteosarcoma, Ewing's sarcoma, and benign neurogenic tumors among musculoskeletal tumors. Clin Cancer Res 2004; 10: 3831-3838

30 Wang X, Bergelson JM. Coxsackievirus and adenovirus receptor cytoplasmic and transmembrane domains are not essential for coxsackievirus and adenovirus infection. J Virol 1999; 73: 2559-2562

S- Editor Zhu LH L- Editor Kerr C E- Editor Liu Y

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Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6370-6378www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

Probiotic bacteria change Escherichia coli -induced gene expression in cultured colonocytes: Implications in intestinal pathophysiology

Pinaki Panigrahi, Gheorghe T Braileanu, Hegang Chen, O Colin Stine

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BASIC RESEARCH

Pinaki Panigrahi, Gheorghe T Braileanu, Departments of Pediatrics, University of Maryland School of Medicine, 22 South Greene Street, Baltimore, MD 21201, United StatesHegang Chen, Department of Epidemiology & Preventive Medicine, University of Maryland School of Medicine, 22 South Greene Street, Baltimore, MD 21201, United StatesO Colin Stine, Genomics Core, University of Maryland School of Medicine, 22 South Greene Street, Baltimore, MD 21201,United States Supported by the Department of Pediatrics and GCRC (M01-RR-16500), University of Maryland Baltimore, with partial funding from NIH grants UO1 HD 40574 and RO1 HD 053719 Correspondence to: Dr. Pinaki Panigrahi, University of Maryland School of Medicine, Department of Pediatrics, 22 South Greene Street, N5W68, Baltimore, MD 21201,United States. ppanigrahi@pol.net Telephone: +1-410-7061803 Fax: +1-410-7060404Received: July 25, 2007 Revised: August 26, 2007

AbstractAIM: To investigate the change in eukaryotic gene expression profile in Caco-2 cells after infection with strains of Escherichia coli and commensal probiotic bacteria.

METHODS: A 19 200 gene/expressed sequence tag gene chip was used to examine expression of genes after infection of Caco-2 cells with strains of normal flora E. coli , Lactobacillus plantarum , and a combination of the two.

RESULTS: The cDNA microarray revealed up-regulation of 155 and down-regulation of 177 genes by E. coli . L. plantarum up-regulated 45 and down-regulated 36 genes. During mixed infection, 27 genes were up-regulated and 59 were down-regulated, with nullification of stimulatory/inhibitory effects on most of the genes. Expression of several new genes was noted in this group.

CONCLUSION: The commensal bacterial strains used in this study induced the expression of a large number of genes in colonocyte-like cultured cells and changed the expression of several genes involved in important cellular processes such as regulation of transcription, protein biosynthesis, metabolism, cell adhesion, ubiquitination, and apoptosis. Such changes induced by the presence of probiotic bacteria may shape the physiologic and pathologic responses they trigger in the host.

© 2007 WJG. All rights reserved.

Key words: Lactobaci l lus ; Escherichia col i ; Gene expression; Probiotic; cDNA microarray

Panigrahi P, Braileanu GT, Chen H, Stine OC. Probiotic bacteria change Escherichia coli -induced gene expression in cultured colonocytes: Implications in intestinal pathophysiology. World J Gastroenterol 2007; 13(47): 6370-6378

http://www.wjgnet.com/1007-9327/13/6370.asp

INTRODUCTIONThere has been an upsurge in clinical trials involving probiotics in gastrointestinal diseases. Although promising, these trials with specific probiotic bacteria have shown variable results, with limited elucidation of the underlying pathophysiology. In real life, these strains never act on the host cells in isolation and over 800 bacterial strains in the adult human colon are engaged in constant cross talk with intestinal epithelial cells. No detailed study so far has attempted to examine the effect of individual probiotic bacteria on host gastrointestinal cells, and the changes during co-infection with other enteric bacteria.

However, a lot of emphasis has recently been given to the normal bacterial flora in the intestine, including many Lactobacillus strains that are considered as probiotics with health-promoting effects on the host. A myriad of effects have been shown by these bacteria, spanning from bacterial killing via secretion of bacteriocins[1], to inhibition of attachment and invasion by pathogenic bacteria[2], and modulation of host inflammatory responses[3]. These commensal strains have been shown to modulate the expression of genes involved in important physiologic functions such as postnatal intestinal maturation, cell growth, proliferation, nutrient absorption, mucosal barrier function, and angiogenesis[4-6]. Multiple laboratory studies have shown beneficial effects of Lactobacillus strains against single pathogenic bacterial strains in in vitro and in vivo systems[7,8]. During the last few years, there has been an exponential increase of clinical trial reports and reviews in the literature pertaining to the utility of probiotics in gastrointestinal and allergic diseases[9-21]. Many small studies utilizing Lactobacillus and Bifidobacteria have shown beneficial effects such as better weight gain and improved

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feeding tolerance[22] in neonates, and efficacy against neonatal necrotizing enterocolitis (NEC)[23-25] and sepsis[24]. Other reports have demonstrated no effect in NEC[26], and in some cases, deterioration of specific conditions with probiotic therapy[25]. Results of clinical trials done by our group have shown a wide range (0%-60%) of colonization rates in newborn infants when three different probiotic strains were used[27]. These mixed and non-reproducible results have raised more questions than providing answers, and have strongly suggested complex interactions among bacterial strains and epithelial cells in the human intestine[7,28-30].

At this time, our understanding of the response of eukaryotic cells (e.g., intestinal cells) is limited to nutrients and local factors[31], and virulence mechanisms involving individual microorganisms. Although contrasting signal transduction mechanisms in bacterial and eukaryotic gene transcription have been described[32], reports on cross talk between bacteria and epithelial cells have focused on single bacterial strains[33]. As a result, the physiologic and pathologic changes in the host cells as a response to multiple bacteria have not been addressed. Since the mammalian gut is colonized with multiple bacterial strains very quickly after birth, it is conceivable that the ultimate effect of probiotic treatment will depend greatly on the presence of other bacteria in the host intestine at that time.

In the current study, we examined the difference between gene expression in intestinal cells in response to infection with a single bacterial strain, compared to that during mixed infection. Caco-2 cells were utilized to discern the effect of Lactobacillus plantarum (the most common Lactobacillus species in humans)[34], Escherichia coli (a common Gram-negative enteric strain) and the combination of the two strains. A high-density cDNA glass microarray and standard techniques were employed to identify bacteria-induced gene expression in this eukaryotic system.

MATERIALS AND METHODSCaco-2 cell culture modelCaco-2 cells, obtained from American Type Culture Collection (ATCC HTB-37), were used at passage 10-12. This human colon-adenocarcinoma-derived cell line has been used extensively for physiologic and enteric bacterial pathogenesis studies[35]. The cells were cultured in a humidified atmosphere containing 5% CO2 at 37℃ in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY, USA with 10% fetal calf serum (Sigma, St. Louis, MO, USA), 2 mmol/L glutamine, 1.0 mmol/L sodium pyruvate, 0.1% non-essential amino acids, 100 U/mL penicillin and 100 μg/mL streptomycin. All experiments were performed without serum or antibiotics in 8-10-d-old cells after they reached confluence.

Bacterial strains E. coli strain 6-1 was isolated from a healthy infant, and has been used previously in in vitro and in vivo studies in our laboratory[36]. This strain does not possess any known virulence genes[37]. We used a human strain of L. plantarum

(ATCC 202195), the species most commonly isolated from humans[34].

Defined bacterial treatment of epithelial cellsCells were washed in PBS and re-fed with experimental DMEM without ser um or ant ib iot ics before the experiments. Following previously described methods in which a maximal effect of Lactobacillus was seen, Caco-2 cells were infected with E. coli and/or L. plantarum at 1:10 multiplicity of infection, and incubated for 2 h[38].

cDNA microarrayFor examination of Caco-2 cell gene expression under our experimental conditions, we used a high-density glass microarray H19K (University Health Network Microarray Centre, Toronto, www.microarrays.ca/home.html) that had 19 200 genes/expressed sequence tags (ESTs). These included fully characterized, partially characterized and some uncharacterized human gene elements. Each gene/EST was printed in duplicate in this array. The genes in the array represented constitutively expressed genes/ESTs and the manufacturer did not include genes that are transiently expressed, such as cytokines and chemokines. In our experiments, we used dye swapping procedures and bioinformatics tools considered as standard techniques that have been reported in similar studies in the past[39].

Sample preparationTotal RNA was extracted from Caco-2 cells grown in 75-cm2 tissue culture flasks using the TRIZOL method (Invitrogen, Carlsbad, CA, USA), following manufacturer’s instructions. RNA samples were treated with RNAse-free DNAse to remove contaminating genomic DNA, examined by 260/280 nm UV absorption ratio (> 1.8) followed by assessment of integrity by running in a 1.2% agarose gel and ethidium bromide staining.

Preparation of fluorescent-labeled cDNA[40], hybridization[41] and signal detection Total mRNA (10 μg) was reversely transcribed using 20 mmol/L dNTP mix including amino-allyl dUTP (AA-dUTP; Sigma) and 400 U SuperScript Ⅱ Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). The resulting aa-cDNA, cleaned with a QIAquick column (Qiagen, Valencia, CA, USA), was coupled with Cy3 or Cy5 dye (Amersham Biosciences, Piscataway, NJ, USA) in the presence of sodium bicarbonate for 1 h in the dark. After adding 10 μL 4 mol/L hydroxylamine and 125 μL buffer PB (Qiagen supplied) to each, the control and treatment samples were combined and cleaned using another QIAquick column. The elute was transferred to a Microcon YM 30 centrifugal filter device (Amicon Millipore, Bedford, MA, USA), and after adding 20 μL cot-1 human DNA (Gibco-BRL), the whole volume was concentrated to 5 μL. Ten microliters of 1 μg/μL poly (A) RNA (Sigma), 1 μL 10 μg/μL tRNA (Gibco-BRL), 4 μL water and 5 μL hybridization buffer (50% formamide, 5 × SSC (3 mol/L sodium chloride, 0.3 mol/L sodium citrate and 0.1% SDS) were added. The array was pretreated at 42℃ for 1 h with hybridization buffer. After overnight

hybridization at 42℃, the slides were washed in 50 mL 2 × SSC and 0.1% SDS at 55℃ for 5 min, once in 0.1 × SSC and 0.1% SDS for 5 min at room temperature (RT), and for 5 min with 0.1 × SSC at RT, air-dried and scanned with 555 nm and 647 nm lasers in a Scan Array 5000 (GSI Lumonics, Novi, MI, USA). Images of the fluorescence intensity for each dye were analyzed using Imagene 4.2 software (Biodiscovery, CA, USA).

RNA from each experimental condition and control Caco-2 cells were hybridized on the same microarray. To eliminate the color bias, duplicate reactions were carried out in which the dyes (Cy3, Cy5) for the control and experimental samples were swapped.

Data interpretationIndividual gene intensity data files for each experimental condition were compared with the control values using the GeneSight 2.1 program (Biodiscovery). After correction for the local background, normalization using all the spots, removal of the outliers, averaging of the replicates and transforming to base 2, each gene was assigned a relative expression value when compared with the control. A twofold or larger difference in the relative gene expression was considered significant.

The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE5874.

Real-time quantitative PCRWe randomly selected eight genes (BMF, CD248, PPM1E, FXYD3, OAS2, FY, CERK and HPSE) from our pool of expressed genes/ESTs that are well characterized in the literature and appear to have some biologic significance. ESTs were not included. Real-time quantitative PCR (Bio-Rad iQ SYBR Green Supermix and iCicler) was done using GAPDH for normalization. The levels of expression detected by microarray were compared with PCR results. The primers used to amplify specific gene segments are presented in Table 1. The relative gene expression was calculated using the comparative ΔΔCT method. Each sample was tested twice in triplicate.

RESULTSGene expression after bacterial infectionAfter 2 h treatment, E. coli , L. plantarum and their combination changed the expression (by twofold) of 332,

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Table 1 Common genes induced by bacterial treatment (Seventeen genes were influenced by both E. coli and L. plantarum and four genes by both E. coli and combination treatment)

Nr. Gene symbol Gene ID NCBI Gene name Location Function Relative fold modification L.p. E.c. Mix

1 GPR34 2857 G protein-coupled receptor 34 Integral to plasmamembrane

G-protein coupled receptor activity 2.43 3.03 -0.53

2 GTPBP4 23560 GTP binding protein 4 Nucleus Ribosome biogenesis - small GTPase mediated signal transduction

2.00 2.91 -0.30

3 TFPI2 7980 Tissue factor pathway inhibitor 2 Extracellularmatrix

Serine-type endopeptidase inhibitor activity

2.10 2.93 -0.27

4 CYP26A1 1592 Cytochrome P450, family 26,subfamily A, polypeptide 1

Membrane Metal ion binding 2.31 2.39 -0.88

5 ZNF35 7584 Zinc finger protein 35 (clone HF.10) Nucleus Transcription factor activity 2.18 2.19 -0.66 6 RTTN 25914 Rotatin required for left-right specification in

mouse embryos 2.21 2.13 -0.58

7 FXYD3 5349 FXYD domain containing ion transport regulator 3

Membrane Chloride channel activity 2.44 2.03 -0.15

8 CYYR1 116159 Cysteine/tyrosine-rich 1 Integral tomembrane

Molecular function unknown -2.30 -2.20 1.05

9 BFAR 512836 Bifunctional apoptosis regulator Apoptosis regulator -2.40 -2.17 1.0610 C19orf4 25789 Chromosome 19 open reading

frame 4Integral tomembrane

Molecular function unknown -2.51 -2.28 0.19

11 KIAA1305 57523 KIAA1305 protein Hypothetical protein -2.19 -2.29 0.5512 PCDH9 5101 Protocadherin 9 Integral to

membraneCell adhesion -2.18 -2.30 0.50

13 IKIP 121457 IKK interacting protein -2.23 -2.33 0.8314 FLJ21963 79611 FLJ21963 protein Hypothetical protein -2.12 -2.37 1.8815 SCRG1 11341 Scrapie responsive protein 1 Extracellular space Nervous system development -2.14 -2.54 0.7416 ULK2 9706 Unc-51-like kinase 2 (C. elegans) Similar to a serine/threonine kinase in

C. elegans-2.46 -2.72 0.26

17 LIFR 3977 Leukemia inhibitory factor receptor Integral to plasmamembrane

Receptor activity -2.26 -2.96 0.29

18 BMF 90427 Bcl-2 modifying factor Sequestrated bymyosin

Apoptotic activator - protein binding 1.00 2.95 2.40

19 CD248 57124 CD248 antigen, endosialin Marker of stromal fibroblasts 0.74 2.31 2.1020 PPM1E 22843 Protein phosphatase 1E (PP2C

domain containing)Phosphatase 0.76 2.33 2.06

21 CARD8 22900 Caspase recruitment domain family,member 8

Involved in NFkB pathway -0.59 -3.26 4.07

E.c., E. coli; L.p., L. plantarum.

81 and 86 genes, respectively, compared to uninfected control Caco-2 cells (Figure 1). After infection with E. coli, 155 genes were up-regulated and 177 were down-regulated (Table 1 and Supplementary Table 1). L. plantarum induced up-regulation of 45 genes and 36 genes were down-regulated (Table 1 and Supplementary Table 2). The combination treatment up-regulated 27 genes and down-regulated 59 (Table 1 and Supplementary Table 3) [Note: The supplementary tables above can be accessed at: http://panigrahipeds.googlepages.com/suppl-tables.pdf; Raw data of all 19 200 genes during each treatment can be accessed from the NCBI/GEO data base (GSE5874) at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=nzyxdkkuwukuytk&acc=GSE5874]. Mixed infection nullified the previously demonstrated stimulatory and inhibitory effects of E. coli on 152 and 177 genes and of L. plantarum on 38 and 26 genes, respectively. Stimulation of 23 and inhibition of 59 genes were noted after mixed infection that was not influenced by either bacterium alone.

There were 21 genes influenced by two different treatment conditions (Table 1). Seventeen genes were affected by E. coli and L. plantarum, and four by E. coli and the combination of bacteria. Genes nos. 1-7 were up-regulated by both E. coli and L. plantarum; and genes nos. 8-17 were down-regulated by both bacteria. For each of the 17 genes in this group, the effects of the individual bacteria were brought to baseline by the combination treatment. In contrast, for three genes BMF, CD248 and PPM1E (nos. 18, 19 and 20 in Table 1), the stimulatory effect of E. coli was maintained after mixed infection with L. plantarum. For one gene (no. 21, CARD8), the 3.26-fold down-regulation by E. coli was reversed in the mixed infection, with demonstration of a four fold increase.

Apart from the specific up- and down-regulation of genes by either E. coli or L. plantarum, and reversal of E. coli-induced effects when L. plantarum was used as a co-infectant, several genes of physiologic importance were noted in our system. Table 2 describes 58 genes under 10 specific categories that were expressed during mixed infection. While the function of a small number of genes was not very well defined, most of the genes could be grouped into important cellular functions. These include genes involved in transcription regulation, RNA processing, protein biosynthesis, and other important processes such as ubiquitination, cell adhesion, proliferation and apoptosis.

Confirmation of selected gene expression by real-time quantitative PCREight genes were randomly tested by quantitative real-

time PCR to verify the expression detected by microarray (Table 3). For each of these genes, RT-PCR confirmed their expression after the three bacterial treatments in the same direction (stimulation or inhibition) as in the microarray experiments (Figure 2).

DISCUSSIONThe infant gut is essentially sterile at birth and is first colonized with Enterobacteriaceae, which change the redox potential in the intestine and allow more microaerophilic and anaerobic species to colonize[42,43]. The latter group, which is comprised primarily of Bifidobacteria and Lactobacillus organisms[44], are considered as normal flora that coexist in the human colon, as new species are introduced to ultimately provide a stable flora in the human gut[45], in which over 800 bacterial species coexist in harmony[46]. In such a healthy state, the intestinal mucosa serves as the first line of defense against infections by providing an important mechanical and immunologic barrier between the host’s internal milieu and the gut environment. These intestinal epithelial cells generate and transmit signals between bacteria and deeper layers in the intestine[47]. In the event of specific infections, epithelial cells express and secrete proinflammatory and chemoattractant cytokines[48] that further transmit signals to the underlying cells in the reticuloendothelial system[47]. The virulence factors and the host responses to these factors in various diseases have been studied in a fair amount of detail (E. coli, Vibrio cholerae, Salmonella and Pseudomonas) using tissue culture and in vivo models, and specific genes and gene functions have been described[49-52]. These experiments have utilized single bacterial strains.

In an attempt to mimic the natural gut environment, communication systems among bacteria have also been studied relatively well. Chemical signals produced and detected by bacteria can be directed at other bacteria and self. This phenomenon, called as quorum sensing, is important for the microorganism’s adaptation to the local environment[53]. This fundamental prokaryotic behavior (among bacteria) is known to affect the symbiotic or antagonistic environment created within the gut milieu. However, the effect of single versus multiple bacterial species on eukaryotic cells has not been addressed in the literature.

The stimulus for us to conduct the current study came from our observation that a large number of probiotic trials have been conducted and reported in the recent past, with almost no basis for selection of the strain, and more importantly, with no data on changes in physiologic or pathologic parameters in the host, other than analysis of the primary and secondary clinical endpoints. Although a live bacterial supplement was used in all of these reported studies, there was also a serious lack of data on the colonizing ability of the probiotic strain and changes in the colonization by other bacteria in the host gut. Since the newborn gut is colonized with a paucity of bacteria (an average 2.5 species in preterm infants)[37,54] that expands to a limited but heterogenous flora by 10 d of age[55], we designed the current simple system to examine the effects of L. plantarum, a common human probiotic strain, and E. coli, the most common colonizing strain in the neonatal

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L.p. = 81

E. coli = 332Mix = 86

17

4

Figure 1 Schematic representation of the genes influenced by each treatment, and the overlapping (common) genes among treatments. The relative gene expression after treatment was > 2-fold compared with the control.

period, on gut cells. We took advantage of a microarray chip that allowed us to examine 19 200 human genes in this simulated microbial gut environment. In this in vitro model, single and combined bacteria were allowed to interact with cultured cells, and our results were analyzed under high-

stringency conditions to identify specific genes expressed during defined bacteria-gut cell interactions.

In our system, we observed a change (up- or down-regulation) in the expression of 333, 81 and 86 genes upon infection with E. coli, L. plantarum and the combined

Table 2 Modulation of gene expression during mixed (E. coli and L. plantarum) infection

Biological process Gene symbol Gene ID NCBI Gene name Fold change

Category 1:Regulation of transcription

HOXD10 3236 Homeobox D10 2.50PHF7 51533 PHD finger protein 7 (Zinc ion binding) 2.44EGR1 1958 Early growth response 1 -2.08TRIM24 8805 Tripartite motif-containing 24 (Zinc ion and DNA binding) -2.15ENO1 2023 Enolase 1, (alpha) (DNA binding) -2.34

Category 2: RNA processing SSB 6741 Sjogren syndrome antigen B (autoantigen La) -2.08FUSIP1 10772 FUS interacting protein (serine/arginine-rich) 1 -2.21NOLA5 10528 Nucleolar protein 5A (56 kDa with KKE/D repeat) -2.27DDX5 1655 DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 -2.71

Category 3:Protein biosynthesis, folding, binding and transport

NEURL 9148 Neuralized homolog (Intracellular protein transport) 2.91WDR36 134430 WD repeat domain 36 2.37MTFMT 123263 Mitochondrial methionyl-tRNA formyltransferase 2.04ARF4 378 ADP-ribosylation factor 4 -2.03CEP57 9702 Centrosomal protein 57 kDa -2.11ETF1 2107 Eukaryotic translation termination factor 1 -2.27HSPA1A 3303 Heat shock 70 kDa protein 1A -2.28LGALS3 3958 Lectin, galactoside-binding, soluble, 3 (galectin 3) -2.75HSPH1 10808 Heat shock 105 kDa/110 kDa protein 1 -2.93HSPA8 3312 Heat shock 70 kDa protein 8 -2.97

Category 4: Structural protein AMPH 273 Amphiphysin (Actin cytoskeleton) 3.04MAP1B 4131 Microtubule-associated protein 1B 2.13ABCC10 89845 ATP-binding cassette, sub-family C (CFTR/MRP), member 10 -2.05SLC26A2 1836 Solute carrier family 26 (sulfate transporter), member 2 -2.06TUBB2A 7280 Tubulin, beta 2A -2.15

Category 5: Metabolism C5orf14 79770 Chromosome 5 open reading frame 14 2.91NAV2 89797 Neuron navigator 2 2.83SLC24A4 123041 Solute carrier family 24 (sodium/potassium/calcium), member 4 2.35PLEKHM2 23207 Pleckstrin homology domain containing, family M, member 2 2.10TWF1 5756 Twinfilin, actin-binding protein, homolog 1 (Tyrosin kinase) -2.01AKR1C1 1645 Aldo-keto reductase 1, member C1 (Bile acid binding) -2.09

HMGCR 3156 3-hydroxy-3-methylglutaryl-Coenzyme A reductase -2.10DC2 58505 DC2 protein (Glycotransferase activity) -2.12GSTA1 2938 Glutathione S-transferase A1 -2.15GAPD 2597 Glyceraldehyde-3-phosphate dehydrogenase -2.16GCLC 2729 Glutamate-cysteine ligase, catalytic subunit -2.21SRM 6723 Spermidine synthase -2.39HSP90AA1 3320 Heat shock protein 90 kDa alpha (cytosolic), class A member 1 -2.46AHCY 191 S-adenosylhomocysteine hydrolase -2.48MAT2A 4144 Methionine adenosyltransferase Ⅱ, alpha -2.74

Category 6: Cell physiology NCF4 4689 Neutrophil cytosolic factor 4, 40 kDa 2.39CYCS 54205 Cytochrome c, somatic -2.02DBI 1622 GABA receptor modulator, acyl-Coenzyme A binding protein -2.25ATP5G3 518 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C3 -2.26HSPA1L 3305 Heat shock 70 kDa protein 1-like -2.26

Category 7: Cell proliferation FOSL1 8061 FOS-like antigen 1 (transcription factor activity) -2.09FGG 2266 Fibrinogen gamma chain -2.36FGG 2244 Fibrinogen beta chain -2.45

Category 8: Cell adhesion NELL2 4753 NEL-like 2 (Calcium ion binding) 2.25ITGB3 3690 Integrin, beta 3 (platelet glycoprotein Ⅲa, antigen CD61) 2.11RHOB 388 Ras homolog gene family, member B -2.19ADRM1 11047 Adhesion regulating molecule 1 -2.25

Category 9: Ubiquitination UBE2N 7334 Ubiquitin-conjugating enzyme E2N (UBC13 homolog, yeast) -2.02UBE2S 27338 Ubiquitin-conjugating enzyme E2S -2.05ANAPC7 51434 Anaphase promoting complex subunit 7 -2.15CACYBP 27101 Calcyclin binding protein -2.18UBA52 7311 Ubiquitin A-52 residue ribosomal protein fusion product 1 -2.29COL6A3 1293 Collagen, type Ⅵ, alpha 3 2.33RPS3A 6189 Ribosomal protein S3A -2.08TWF1 5756 Twinfilin, actin-binding protein, homolog 1 (Tyrosin kinase) -2.01AKR1C1 1645 Aldo-keto reductase 1, member C1 (Bile acid binding) -2.09

Negative value indicates reduction in gene expression.

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treatment, respectively. Our real-time PCR experiments confirmed the modifications demonstrated in the microarray experiments, albeit at a lower level, a phenomenon also reported in other studies[50]. The numbers of unique genes presented in this study are in the range reported in previous studies in which Gram-negative enteric pathogens modified the expression of 0.5%-13% of the genes in epithelial cells[50,56-58], and commensal bacteria induced differential expression of 0.35%-6.2% of examined genes in mouse colonocytes[59]. Our strain of E. coli modified 1.73%, and L. plantarum modified 0.43% of genes. The slightly lower number of genes identified in our 19 200 array may have been due to the use of a non-pathogenic strain of E. coli, a commensal Lactobacillus, and an array that included only constitutively expressed genes. Genes expected to be expressed after a bacterial insult such as pro-inflammatory cytokines were not spotted on this array. Additionally, a slightly low number might have resulted from our conservative choice of a twofold increase in expression as being significant in our analysis.

There are several comparisons that can be made between our results and those of others using a similar approach but with single bacterial infection. For example, from the six genes up-regulated by enteropathogenic E. coli in HeLa cells[49], we found only one (zyxin, a cytoskeletal

protein) to be in common with our microarray results. There was a similar increase (1.72-fold) in expression of this gene when our E. coli strain 6-1 was used to infect Caco-2 cells. Two previous studies with commensal f lora have reported that bacterial reconstitution of germ-free mice increased the expression of the colon-specific serum amyloid A1 gene[60,61]. In our model, serum amyloid A2 gene expression was increased by 2.22-fold. From the 12 genes down-regulated by non-pathogenic bacterial reconstitution of germ-free mice, reported by Fukushima et al[59] in colonic epithelial cells, three were in common with our microarray; selenoprotein P, 3-hydroxy-3 methylglutaryl-coenzyme A synthase and metallothionein. All three were also down-regulated in our combination treatment model. The authors also showed a down-regulation of solute carrier family 20 - member 1. Our results were very similar to this observation in that we also noted a decrease in the expression of other members of the solute carrier families, i.e., family 2, 9, 12, 20, 24, 25 and 35. Fukushima et al[59] have shown overexpression of heat shock protein (60 kDa) in germ-free mice compared to specific pathogen-free rodents that had received treatment with normal mouse flora. We observed a similar phenomenon in our system in which down-regulation of heat shock proteins 75, 105 and

Table 3 Primers used for RT-PCR

Gene Gene IDGene name Gene role Primer Primer sequence 5’-3’

symbol (NCBI)BMF 90427 Bcl2 modifying factor, transcript

variant 1Has a single Bcl2 homology domain 3 (BH3), binds Bclk2 proteins and functions as an apoptotic activator

F GCTTCAGTTGCATTGCAGACCAGTTR AGAGCCCTTGGGAATTCTCACCAT

CD248 57124 CD248 antigen = endosialin A gene regulated by the cell density in vitro. Has a calcium binding domain

F TCAACTACGTTGGTGGCTTCGAGTR AGTTGGGATAATGGGAAGCTGGGT

PPM1E 22843 Protein phosphatase 1E Member of the PP2C family of Ser/Thr phosphatases known to be negative regulators of stress response pathways

F ATGCCTCCATTCACCTCCACGTTAR TGTCATAGAAGCCATCACAGGCCA

FXYD3 5349 FXY domain containing ion transport reg. 3

The protein encoded by this gene may function as a chloride channel or as a chloride channel regulator

FR

AATGCAAGTTTGGCCAGAAGTCCGTTGCATATGAGGTCCCATGGCTGA

OAS2 4939 2’-5’-oligoadenylate synthetase 2 This enzyme family plays a significant role in theinhibition of cellular protein synthesis

F AGAAGCCAACGTGACATCCTCGATR TGCTGGAGTTCAGTGAAGCAGACT

FY 2532 Duffy blood group antigen Helps in leukocyte recruitment to sites ofinflammation by facilitating movement of chemokines across the endothelium

F TGACTCTGCACTGCCCTTCTTCATR TTGACAACAGCAACAGCTTGGACC

CERK 64781 Ceramide kinase Integral to membranes, has roles in arachidonic acid release and production of eicosanoids

F TGAGAAGAAACGGTGGTTGGGTCTR AGCATTTCCGGATGAGGATGAGGT

HPSE 10855 Heparanase Cell surface expression and secretion markedly promote tumor angiogenesis and metastasis

F ACCTTTGCAGCTGGCTTTATGTGGR CTTGCACGCTTGCCATTAACACCT

GAPDH 2597 Glyceraldehyde-3-phosphate dehydrogenase

Used as reference F GACCACAGTCCATGCCATCACR GAGCTTCAGAAAGTGGTCGTTGA

Figure 2 Effect of different bacterial treatments on ex-pression of eight genes assessed by microarray (M) and RT-PCR (R).

BMF CD248 PPMIE FXYD3 OAS2 FY CERK HPSERMRMRMRMRMRMRMRM

5

4

3

2

1

0

-1

-2

-3

-4Fold

incr

ease

com

pare

d w

ith t

he c

ontr

ol L.p.

E.c.

Mix

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an ortholog of mouse heat shock protein 70 kDa were noted after combined bacterial treatment. We observed cytochrome c oxidase subunits Ⅳ isoform 1, Ⅴa, Ⅵb, Ⅵc, Ⅶa, Ⅶb, Ⅶc and Ⅷ to be up-regulated after L. plantarum treatment, similar to that described by Hooper et al, who demonstrated up-regulation of cytochrome c oxidase subunit 1 by Bacteroides, another species also considered as commensal flora. Hooper and colleagues have also shown up-regulation of calmodulin after treatment with Bacteroides[61]. Similar increases in expression were noted for calmodulin 1, 2 and 3, calmodulin-dependent protein kinase and phosphodiesterase in our system.

We observed modulation of multiple genes known to have an impact on cellular and physiologic processes in the eukaryotic system (Table 2). These genes ranged from basic transcriptional regulators to those involved in protein synthesis, cellular metabolism, cell proliferation and apoptosis. During mixed infection, we observed down-regulation of three genes involved in ubiquitination. Ubiquitin-conjugating enzyme E2N, ubiquitin-carrier protein E2-EPF and ubiquitin A-52 residue ribosomal protein fusion product 1 were reduced 2.02, 2.05 and 2.29-fold, respectively. In a recent study that investigated anti-inf lammatory properties of Lactobaci l lus casei , expression of several genes involved in ubiquitination was reduced, including E2N, a gene (common to our system) that was reported to be decreased 2.88-fold[62]. The authors concluded I-kB stabilization via reduced ubiquitination and downstream modulation of inflammatory response driven by NF-kB in Shigella-infected Caco-2 cells. We used a non-pathogenic commensal strain of E. coli in our experiments, and while the aim of the current study was not to assess or examine the effects of L. plantarum during bacterial infection or inflammation, our results strongly suggest that Lactobacillus strains do indeed affect common physiologic pathways in gut cells, which may ultimately shape the host response in health and disease.

In our study, it was important and intriguing to note that the three experimental infections induced quite unique gene-expression profiles. Even the mixed infection with E. coli and L. plantarum had a very small overlap with the expression profiles of the strains when they were used alone. This illustrates how colonization can change the gene expression of host cells as they are exposed to more than one species of bacteria. In real life, the gut cells are exposed to a multitude of bacterial strains, and hence, it may be of limited value to study the effect of infection or colonization by single bacterial species in a clean tissue culture environment, and use the results as the basis for designing treatment or preventive strategies. Using neonatal models of gut colonization, we have previously shown that bacterial ecology (combination of Gram-negative and Gram-positive organisms), rather than individual virulent bacterial strains, plays a more important role in diseases such as NEC[36]. The results of our current study are in line with previous observations, and now provide an additional line of support and offer a possible explanation for the varied results of recent probiotic trials. On a broader scale, this report provides an insight into the complex host response that can be expected at mucosal sites such as the gastrointestinal tract. Based on the results

obtained from tissue culture with only two bacteria in the system, it can be speculated that our findings are only the tip of the iceberg, and the real in vivo picture in mammals will be even more complex. While it is becoming increasing clear that specific Lactobacillus species posses unique health-promoting characteristics[29], knowledge gained from the current study further indicates that a "one strain fits all" approach may not always succeed in the treatment or prevention of specific diseases. A more global approach needs to be taken with proper emphasis on the microbial ecology, while addressing the pathogenesis of unique bacterial diseases in the mammalian intestine at different ages and stages of development.

In the context of in vivo or clinical trial environment, it should be noted that our current model and results do not represent a universal phenomenon, nor provide a comprehensive picture of the human intestine. For example, genes expressed will probably be different if other probiotic strains such as Bifidobacteria and L. casei were used in our system. Similarly, combinations of other aerobic and anaerobic Gram-negative and Gram-positive strains may induce different sets of genes. We can utilize other microarray systems with cytokine and signaling-molecule genes (not spotted in the current 19 200 gene array), when our aim would be to identify modifications in inflammatory mediators. The relative concentrations of each bacterium in the system may also change the gene-expression profile. In the current study, we selected a 1:10 ratio of E. coli to Lactobacillus infecting dose to simulate the human intestinal microflora, in which anaerobic and microaerophilic organisms form the dominant flora[63]. Since enteric bacteria such as E. coli are sometimes present at < 0.1% of the total bacterial population, with a predominance by obligate anaerobes[64], it is not unexpected to observe a different gene-expression profile when a 10-100-fold higher proportion of Lactobacilli are used in the system. Nevertheless, such manipulations and experiments can be done, and despite some limitations, assessment of mRNA-expression profiles by cDNA array analysis can be utilized as a useful technique for expanding our understanding of the colonocyte-bacteria interaction[50].

While it may appear difficult to analyze complex microflora (400-800 species) and their interactions with gut cells in the mature intestine, this is now made feasible with the availability of new techniques. Fluorescent in situ hybridization utilizing bacterial rRNA can identify and quantify major genera of bacteria, even if they are non-culturable in stools[65,66]. Bacterial microarray chips developed during the last year can identify thousands of bacterial species in stools in one experiment[67,68]. Denaturing gradient gel electrophoresis can be utilized to monitor changes in microflora pattern[69,70] over time and after administration of probiotic supplements. Live colonocytes can be isolated from stool samples and used to examine the expression of genes and proteins during different experimental and/or disease states[71,72]. At this juncture, there is a need for the scientific community to engage in careful evaluation of probiotic strains in in vitro and in vivo systems prior to initiation of clinical trials. With the new non-invasive tools at hand, such preclinical

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endeavors, coupled with concurrent examination of changes in the gut flora and host responses during clinical trials, hold great promise in discerning the difference between "snake oil" and "magic bullets" when it comes to the role of probiotic therapy in human medicine.

REFERENCES1 Atrih A, Rekhif N, Michel M, Lefebvre G. Detection of

bacteriocins produced by Lactobacillus plantarum strains isolated from different foods. Microbios 1993; 75: 117-123

2 Bernet MF, Brassart D, Neeser JR, Servin AL. Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria. Gut 1994; 35: 483-489

3 Perdigón G, Maldonado Galdeano C, Valdez JC, Medici M. Interaction of lactic acid bacteria with the gut immune system. Eur J Clin Nutr 2002; 56 Suppl 4: S21-S26

4 Gordon JI, Hooper LV, McNevin MS, Wong M, Bry L. Epithelial cell growth and differentiation. III. Promoting diversity in the intestine: conversations between the microflora, epithelium, and diffuse GALT. Am J Physiol 1997; 273: G565-G570

5 H o o p e r L V , G o r d o n J I . C o m m e n s a l h o s t - b a c t e r i a l relationships in the gut. Science 2001; 292: 1115-1118

6 Hooper LV, Midtvedt T, Gordon JI. How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annu Rev Nutr 2002; 22: 283-307

7 Reid G, Bruce AW, McGroarty JA, Cheng KJ, Costerton JW. Is there a role for lactobacilli in prevention of urogenital and intestinal infections? Clin Microbiol Rev 1990; 3: 335-344

8 de Waard R, Garssen J, Bokken GC, Vos JG. Antagonistic activity of Lactobacillus casei strain shirota against gastrointestinal Listeria monocytogenes infection in rats. Int J Food Microbiol 2002; 73: 93-100

9 Taylor CJ , Mahenthiralingam E. Functional foods and paediatric gastro-intestinal health and disease. Ann Trop Paediatr 2006; 26: 79-86

10 Szajewska H, Setty M, Mrukowicz J, Guandalini S. Probiotics in gastrointestinal diseases in children: hard and not-so-hard evidence of efficacy. J Pediatr Gastroenterol Nutr 2006; 42: 454-475

11 Sazawal S, Hiremath G, Dhingra U, Malik P, Deb S, Black RE. Efficacy of probiotics in prevention of acute diarrhoea: a meta-analysis of masked, randomised, placebo-controlled trials. Lancet Infect Dis 2006; 6: 374-382

12 Saiman L. Strategies for prevention of nosocomial sepsis in the neonatal intensive care unit. Curr Opin Pediatr 2006; 18: 101-106

13 Novak J, Katz JA. Probiotics and prebiotics for gastrointestinal infections. Curr Infect Dis Rep 2006; 8: 103-109

14 Johnston BC, Supina AL, Vohra S. Probiotics for pediatric antibiotic-associated diarrhea: a meta-analysis of randomized placebo-controlled trials. CMAJ 2006; 175: 377-383

15 Duchmann R. The role of probiotics and antibiotics in regulating mucosal inflammation. Adv Exp Med Biol 2006; 579: 219-226

16 Doron S, Gorbach SL. Probiotics: their role in the treatment and prevention of disease. Expert Rev Anti Infect Ther 2006; 4: 261-275

17 Daniel C, Repa A, Wild C, Pollak A, Pot B, Breiteneder H, Wiedermann U, Mercenier A. Modulation of allergic immune responses by mucosal application of recombinant lactic acid bacteria producing the major birch pollen allergen Bet v 1. Allergy 2006; 61: 812-819

18 Chan-Yeung M, Becker A. Primary prevention of childhood asthma and allergic disorders. Curr Opin Allergy Clin Immunol 2006; 6: 146-151

19 Boyle RJ, Tang ML. The role of probiotics in the management of allergic disease. Clin Exp Allergy 2006; 36: 568-576

20 Kalliomäki MA, Isolauri E. Probiotics and down-regulation of the allergic response. Immunol Allergy Clin North Am 2004; 24: 739-752, viii

21 Miraglia del Giudice M, De Luca MG. The role of probiotics in the clinical management of food allergy and atopic dermatitis. J

Clin Gastroenterol 2004; 38: S84-S8522 Kitajima H, Sumida Y, Tanaka R, Yuki N, Takayama H,

Fujimura M. Early administration of Bifidobacterium breve to preterm infants: randomised controlled trial. Arch Dis Child Fetal Neonatal Ed 1997; 76: F101-F107

23 Hoyos AB. Reduced incidence of necrotizing enterocolitis associated with enteral administration of Lactobacillus acidophilus and Bifidobacterium infantis to neonates in an intensive care unit. Int J Infect Dis 1999; 3: 197-202

24 Lin HC, Su BH, Chen AC, Lin TW, Tsai CH, Yeh TF, Oh W. Oral probiotics reduce the incidence and severity of necrotizing enterocolitis in very low birth weight infants. Pediatrics 2005; 115: 1-4

25 Bin-Nun A, Bromiker R, Wilschanski M, Kaplan M, Rudensky B, Caplan M, Hammerman C. Oral probiotics prevent necrotizing enterocolitis in very low birth weight neonates. J Pediatr 2005; 147: 192-196

26 Dani C, Biadaioli R, Bertini G, Martelli E, Rubaltelli FF. Probiotics feeding in prevention of urinary tract infection, bacterial sepsis and necrotizing enterocolitis in preterm infants. A prospective double-blind study. Biol Neonate 2002; 82: 103-108

27 Agarwal R, Sharma N, Chaudhry R, Deorari A, Paul VK, Gewolb IH, Panigrahi P. Effects of oral Lactobacillus GG on enteric microflora in low-birth-weight neonates. J Pediatr Gastroenterol Nutr 2003; 36: 397-402

28 Madsen KL. The use of probiotics in gastrointestinal disease. Can J Gastroenterol 2001; 15: 817-822

29 Isolauri E. Probiotics in human disease. Am J Clin Nutr 2001; 73: 1142S-1146S

30 Vanderhoof JA, Young RJ. Probiotics in pediatrics. Pediatrics 2002; 109: 956-968

31 Reece RJ, Beynon L, Holden S, Hughes AD, Rébora K, Sellick CA. Nutrient-regulated gene expression in eukaryotes. Biochem Soc Symp 2006: 85-96

32 Cashin P, Goldsack L, Hall D, O'Toole R. Contrasting signal transduction mechanisms in bacterial and eukaryotic gene transcription. FEMS Microbiol Lett 2006; 261: 155-164

33 Raupach B, Mecsas J, Heczko U, Falkow S, Finlay BB. Bacterial epithelial cell cross talk. Curr Top Microbiol Immunol 1999; 236: 137-161

34 Ahrné S, Nobaek S, Jeppsson B, Adlerberth I, Wold AE, Molin G. The normal Lactobacillus flora of healthy human rectal and oral mucosa. J Appl Microbiol 1998; 85: 88-94

35 Engle MJ, Goetz GS, Alpers DH. Caco-2 cells express a combination of colonocyte and enterocyte phenotypes. J Cell Physiol 1998; 174: 362-369

36 Panigrahi P, Gupta S, Gewolb IH, Morris JG. Occurrence of necrotizing enterocolitis may be dependent on patterns of bacterial adherence and intestinal colonization: studies in Caco-2 tissue culture and weanling rabbit models. Pediatr Res 1994; 36: 115-121

37 Gupta S, Morris JG, Panigrahi P, Nataro JP, Glass RI, Gewolb IH. Endemic necrotizing enterocolitis: lack of association with a specific infectious agent. Pediatr Infect Dis J 1994; 13: 728-734

38 Panigrahi P, Bamford P, Horvath K, Morris JG, Gewolb IH. Escherichia coli transcytosis in a Caco-2 cell model: implications in neonatal necrotizing enterocolitis. Pediatr Res 1996; 40: 415-421

39 Ichikawa JK, Norris A, Bangera MG, Geiss GK, van 't Wout AB, Bumgarner RE, Lory S. Interaction of pseudomonas aeruginosa with epithelial cells: identification of differentially regulated genes by expression microarray analysis of human cDNAs. Proc Natl Acad Sci USA 2000; 97: 9659-9664

40 DeRisi. Amino-alyl Dye Coupling Protocol. Available from: URL: http://derisilab.ucsf.edu/pdfs/amino-allyl-protocol.pdf. 2001

41 Hasseman J . Microarray labeled probe hybridization. Available from: URL: http://www.tigr.org/tdb/microarray/protocolsTIGR.shtml. 2002

42 Mai V , Morris JG. Colonic bacterial f lora: changing understandings in the molecular age. J Nutr 2004; 134: 459-464

43 Freter R. Control mechanisms of the large-intestinal microflora

Panigrahi P et al . Modulation of eukaryotic gene expression by probiotics 6377

www.wjgnet.com

and its influence on the host. Acta Gastroenterol Latinoam 1989; 19: 197-217

44 Orrhage K, Nord CE. Bifidobacteria and lactobacilli in human health. Drugs Exp Clin Res 2000; 26: 95-111

45 Savage DC. Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol 1977; 31: 107-133

46 Bäckhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science 2005; 307: 1915-1920

47 Kagnoff MF, Eckmann L. Epithelial cells as sensors for microbial infection. J Clin Invest 1997; 100: 6-10

48 J u n g H C , E c k m a n n L , Y a n g S K , P a n j a A , F i e r e r J , Morzycka-Wroblewska E, Kagnoff MF. A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J Clin Invest 1995; 95: 55-65

49 de Grado M, Rosenberger CM, Gauthier A, Vallance BA, Finlay BB. Enteropathogenic Escherichia coli infection induces expression of the early growth response factor by activating mitogen-activated protein kinase cascades in epithelial cells. Infect Immun 2001; 69: 6217-6224

50 Eckmann L, Smith JR, Housley MP, Dwinell MB, Kagnoff MF. Analysis by high density cDNA arrays of altered gene expression in human intestinal epithelial cells in response to infection with the invasive enteric bacteria Salmonella. J Biol Chem 2000; 275: 14084-14094

51 Firoved AM, Deretic V. Microarray analysis of global gene expression in mucoid Pseudomonas aeruginosa. J Bacteriol 2003; 185: 1071-1081

52 Stokes NR, Zhou X, Meltzer SJ, Kaper JB. Transcriptional responses of intestinal epithelial cells to infection with Vibrio cholerae. Infect Immun 2004; 72: 4240-4248

53 Konaklieva MI, Plotkin BJ. Chemical communication--do we have a quorum? Mini Rev Med Chem 2006; 6: 817-825

54 Gewolb IH, Schwalbe RS, Taciak VL, Harrison TS, Panigrahi P. Stool microflora in extremely low birthweight infants. Arch Dis Child Fetal Neonatal Ed 1999; 80: F167-F173

55 Yoshioka H, Iseki K, Fujita K. Development and differences of intestinal flora in the neonatal period in breast-fed and bottle-fed infants. Pediatrics 1983; 72: 317-321

56 Belcher CE, Drenkow J, Kehoe B, Gingeras TR, McNamara N, Lemjabbar H, Basbaum C, Relman DA. The transcriptional responses of respiratory epithelial cells to Bordetella pertussis reveal host defensive and pathogen counter-defensive strategies. Proc Natl Acad Sci USA 2000; 97: 13847-13852

57 Rosenberger CM, Scott MG, Gold MR, Hancock RE, Finlay BB. Salmonella typhimurium infection and lipopolysaccharide stimulation induce similar changes in macrophage gene expression. J Immunol 2000; 164: 5894-5904

58 Pédron T, Thibault C, Sansonetti PJ. The invasive phenotype of Shigella flexneri directs a distinct gene expression pattern in the human intestinal epithelial cell line Caco-2. J Biol Chem 2003; 278: 33878-33886

59 Fukushima K, Ogawa H, Takahashi K, Naito H, Funayama Y,

Kitayama T, Yonezawa H, Sasaki I. Non-pathogenic bacteria modulate colonic epithelial gene expression in germ-free mice. Scand J Gastroenterol 2003; 38: 626-634

60 Ogawa H, Fukushima K, Sasaki I, Matsuno S. Identification of genes involved in mucosal defense and inflammation associated with normal enteric bacteria. Am J Physiol Gastrointest Liver Physiol 2000; 279: G492-G499

61 Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science 2001; 291: 881-884

62 Tien MT, Girardin SE, Regnault B, Le Bourhis L, Dillies MA, Coppée JY, Bourdet-Sicard R, Sansonetti PJ, Pédron T. Anti-inflammatory effect of Lactobacillus casei on Shigella-infected human intestinal epithelial cells. J Immunol 2006; 176: 1228-1237

63 Onderdonk AB. The intestinal microflora and intra-abdominal sepsis. In: Tannock GW. Medical importance of the normal microflora. Dordrecht, Boston, London: Kluwer Academic Publishers, 1999: 164-176

64 Evaldson G, Heimdahl A, Kager L, Nord CE. The normal human anaerobic microflora. Scand J Infect Dis Suppl 1982; 35: 9-15

65 Harmsen HJ, Raangs GC, He T, Degener JE, Welling GW. Extensive set of 16S rRNA-based probes for detection of bacteria in human feces. Appl Environ Microbiol 2002; 68: 2982-2990

66 Mai V, Katki HA, Harmsen H, Gallaher D, Schatzkin A, Baer DJ, Clevidence B. Effects of a controlled diet and black tea drinking on the fecal microflora composition and the fecal bile acid profile of human volunteers in a double-blinded randomized feeding study. J Nutr 2004; 134: 473-478

67 Kostić T, Weilharter A, Rubino S, Delogu G, Uzzau S, Rudi K, Sessitsch A, Bodrossy L. A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens. Anal Biochem 2007; 360: 244-254

68 Palmer C, Bik EM, Eisen MB, Eckburg PB, Sana TR, Wolber PK, Relman DA, Brown PO. Rapid quantitative profiling of complex microbial populations. Nucleic Acids Res 2006; 34: e5

69 Scanlan PD, Shanahan F, O'Mahony C, Marchesi JR. Culture-independent analyses of temporal variation of the dominant fecal microbiota and targeted bacterial subgroups in Crohn's disease. J Clin Microbiol 2006; 44: 3980-3988

70 Suau A. Molecular tools to investigate intestinal bacterial communities. J Pediatr Gastroenterol Nutr 2003; 37: 222-224

71 Nair P, Lagerholm S, Dutta S, Shami S, Davis K, Ma S, Malayeri M. Coprocytobiology: on the nature of cellular elements from stools in the pathophysiology of colonic disease. J Clin Gastroenterol 2003; 36: S84-S93; discussion S94-S96

72 Matsushita H, Matsumura Y, Moriya Y, Akasu T, Fujita S, Yamamoto S, Onouchi S, Saito N, Sugito M, Ito M, Kozu T, Minowa T, Nomura S, Tsunoda H, Kakizoe T. A new method for isolating colonocytes from naturally evacuated feces and its clinical application to colorectal cancer diagnosis. Gastroenterology 2005; 129: 1918-1927

S- Editor Liu Y L- Editor Kerr C E- Editor Liu Y

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Inflammatory cytokines promote inducible nitric oxide synthase-mediated DNA damage in hamster gallbladder epithelial cells

Amane Kitasato, Yoshitsugu Tajima, Tamotsu Kuroki, Ryuji Tsutsumi, Tomohiko Adachi, Takehiro Mishima, Takashi Kanematsu

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BASIC RESEARCH

Amane Kitasato, Yoshitsugu Tajima, Tamotsu Kuroki, Ryuji Tsutsumi, Tomohiko Adachi, Takehiro Mishima, Takashi Kanematsu, Department of Surgery, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8501, JapanCorrespondence to: Amane Kitasato, MD, Department of Surgery, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan. a-kit@bg8.so-net.ne.jpTelephone: + 81-958-497316 Fax: + 81-958-497319Received: January 4, 2007 Revised: September 8, 2007

AbstractAIM: To investigate the link between chronic biliary inf lammation and carcinogenesis using hamster gallbladder epithelial cells.

METHODS: Gallbladder epithelial cells were isolated from hamsters and cultured with a mixture of inflammatory cytokines including interleukin-1β, interferon-γ, and tumor necrosis factor-α. Inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) generation, and DNA damage were evaluated.

RESULTS: NO generation was increased significantly following cytokine stimulation, and suppressed by an iNOS inhibitor. iNOS mRNA expression was demonstrated in the gallbladder epithelial cells during exposure to inflammatory cytokines. Furthermore, NO-dependent DNA damage, estimated by the comet assay, was significantly increased by cytokines, and decreased to control levels by an iNOS inhibitor.

CONCLUSION: Cytokine stimulation induced iNOS expression and NO generation in normal hamster gallbladder epithelial cells, which was sufficient to cause DNA damage. These results indicate that NO-mediated genotoxicity induced by inflammatory cytokines through activation of iNOS may be involved in the process of biliary carcinogenesis in response to chronic inflammation of the biliary tree.

© 2007 WJG. All rights reserved.

Key words: Biliary carcinoma; Inflammation; Inflammatory cytokine; Nitric oxide; Inducible nitric oxide synthase; DNA damage; Gallbladder epithelial cell; Hamster

Kitasato A, Tajima Y, Kuroki T, Tsutsumi R, Adachi T, Mishima T, Kanematsu T. Inflammatory cytokines promote inducible nitric oxide synthase-mediated DNA damage in hamster gallbladder epithelial cells. World J Gastroenterol 2007; 13(47): 6379-6384

http://www.wjgnet.com/1007-9327/13/6379.asp

INTRODUCTIONIt is well known that chronic local inflammation increases the risk for cancer development in several organs, such as colon, lung, pancreas, and esophagus[1,2]. Similarly, biliary carcinoma develops under chronic inflammatory conditions involving the biliary epithelium in the setting of gallstone disease, congenital choledochal cyst, pancreaticobiliary maljunction, or primary sclerosing cholangitis [3,4]. Moreover, recent reports have described the occurrence of secondary biliary carcinomas in patients with persistent reflux cholangitis after bilioenterostomy, transduodenal sphincteroplasty, or endoscopic sphincterotomy for both benign and malignant diseases of the liver, bile duct, and pancreas[5-7]. However, the molecular mechanisms of biliary carcinogenesis as a consequence of chronic biliary inflammation remain unclear. We have previously demonstrated that persistent reflux cholangitis after bilioenterostomy accelerates biliary carcinogenesis through activation of biliary epithelial cell kinetics in hamsters[8-10]. Furthermore, more severe cholangitis is associated with a high occurrence of biliary carcinoma. However, the molecular mechanisms by which chronic biliary inflammation increases the risk of biliary carcinogenesis are obscure, similar to the clinical occurrence in humans. Meanwhile, we have established a method for culturing biliary epithelial cells from the hamster using a collagen gel technique[11].

Chemically reactive oxidants, radicals, and electrophilic mediators, such as hydrogen peroxide and oxyradicals, nitric oxide, malondialdehyde, 4-hydroxynonenal, or eicosanoids, are produced during inflammation, and these chemical mediators are known to induce a variety of biological reactions[2]. Recently, attention has been focused on nitric oxide (NO) as an endogenous mutagen, an angiogenesis factor, and an inhibitor of apoptosis[12]. NO is a free radical that is synthesized from L-arginine by the family of nitric oxide synthases (NOSs). Three

isoforms of the NOSs have been isolated: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS)[13,14]. Although nNOS and eNOS are present constitutively, iNOS is induced in inflamed tissues and generates relatively large amounts of NO, compared with nNOS and eNOS[13-15]. Cytokine and/or lipopolysaccharide stimulation is known to induce iNOS expression in macrophages, hepatocytes, and many other cell types including certain epithelial cells[16-19]. Moreover, iNOS expression and generation of NO in inflamed tissues have been postulated to potentiate epithelial cells to malignant transformation through the ability of NO to promote mutagenic changes in DNA through DNA oxidization and protein nitrosylation[20,21].

In this study, we investigated the role of iNOS activation, NO generation, and DNA damage as the link between chronic inflammation and biliary carcinogenesis, by utilizing normal hamster gallbladder epithelial cells cultured with inflammatory cytokines.

MATERIALS AND METHODSAnimalsFive-week-old female Syrian golden hamsters (Shizuoka Laboratory Animal Center, Shizuoka, Japan) were used. The animals were kept under standard laboratory conditions in the Laboratory Animal Center for Biochemical Research at Nagasaki University Graduate School of Biochemical Sciences. All experiments were performed following the Guidelines for Animal Experimentation of Nagasaki University.

Isolation and culture of biliary epithelial cells from hamstersBiliary epithelial cells were isolated from the biliary tree of hamsters as previously described[11]. After laparotomy, inferior vena cava was paracentesed with a 22G needle, and the liver was perfused in situ with 100 mL Ca2+

- and Mg2+ - free phosphate-buffered saline (CMF-PBS) containing 10 mmol/L 2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) at pH7.4, and 1 mmol/L ethylene glucol-bis (E-aminoethylether) N, N, N', N'-tetraacetic acid (EGTA) for 10 min at 37℃. The vena cava was clamped above the diaphragm, and the perfusing solution was drained via the incised portal vein, followed by perfusion with 100 mL Hanks' balanced salt solution (GIBCO, Grand Island, NY) containing 50 mmol/L HEPES and 0.04% collagenase (Nittazeratin, Osaka, Japan) for 10 min at 37℃. After perfusion with the collagenase solution, the liver, gall bladder, and extrahepatic bile duct were removed en bloc. The biliary tree was isolated and separated into the intrahepatic and extrahepatic bile ducts and the gall bladder in CMF-PBS containing 0.1 mmol/L EGTA. The biliary fragments were minced.

The biliary fragments were embedded on collagen gel plated (Collagen Gel Culture kit; Nittazeratin, Osaka, Japan) 60-mm petri dishes with 2 mL of an ice-cold mixture of collagen solution composed of 0.3% acid solution collagen (Cellmatrix TypeⅠ-A), 10 × HamF12, and 0.8 N NaOH at 8:1:1 dilution. After incubation at 37℃ with 5% CO2 and 95% humidity for 20-30 min,

collagen gels were overlaid with 5 mL of culture medium composed of Dulbecco's modified Eagle medium/HamF12 medium (DMEM/HamF12, GIBCO) and 10% fetal bovine serum (GIBCO). After incubation of biliary fragments for 7-10 d, the epithelial cells extended widely on the surface of the gel, while the mesenchymal cells progressed toward the inside of the gel. The biliary epithelial cells were isolated from the peripheral region of cellular sheets (Figure 1).

Addition of inflammatory cytokinesGallbladder epithelial cells isolated from hamsters were used in this study because of its higher cellular activity compared to other biliary epithelial cells. Resuspended gallbladder epithelial cells (1 × 105 cells/mL) were plated on collagen-coated plates. After incubation for 24 h, the epithelial cells were prepared for three different

Figure 1 Isolation of hamster biliary epithelial cells. A: Phase contrast microscopy revealing a small amount of hamster gallbladder epithelial cells growing on the collagen gels 24 h after culture (× 100); B: High magnification of hamster gallbladder epithelial cells demonstrating cuboidal cells around the biliary fragments (× 400); C: Phase contrast microscopy showing widely extended gallbladder epithelial cells on the surface of the gel 7 d after culture (× 40).

C

B

A

Fragments

Fragments

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10

0

experimental protocols: incubation with culture medium alone (control group), incubation with a cytokine mixture known to increase iNOS expression in other cell types[18,22] consisting of human recombinant interleukin (IL) 1-β (0.5 ng/mL), interferon (IFN)-γ (5 ng/mL), and tumor necrosis factor (TNF)-α (250 ng/mL) (CM group), or incubation with the same cytokine mixture and an iNOS inhibitor L-N (G)-monomethyl arginine (L-NMMA, 0.03 mmol/L) (CM + L-NMMA group). These human recombinant cytokines and L-NMMA were obtained from the Sigma Chemical Co. (St. Louis, MO). Gallbladder epithelial cells in each group were incubated at 37℃ for 24 h, and then processed for the following analyses.

Measurement of NO2- and NO3

- in the mediumTo determine the amount of NO produced by gallbladder epithelial cells, nitrite (NO2

-) and nitrate (NO3-) levels were

measured in the culture media by high performance liquid chromatography with a NOx analyzer (ENO-10; Eicom, Kyoto)[23].

RT-PCRiNOS mRNA was amplified using a nested RT-PCR method[24]. The sequences of primers (Invitrogen Life Technologies, Carlsbad, CA) used in this study are shown in Table 1[24-26].

Total RNA was extracted from gallbladder epithelial cells using a RNA extraction kit (ISOGEN; Nippon Gene, Tokyo). Reverse transcription and PCR amplification were performed with a RNA PCR kit Ver.3.0 (Takara Shuzo, Tokyo, Japan). After reverse transcription, the first amplification was performed using 1 µmol/L of the cDNA, together with PCR primers NOS-590F and NOS-893R. The PCR conditions were one cycle of denaturing at 95℃ for 2 min, followed by a touch-down protocol consisting of 18 cycles of 15 s at 95℃, 30 s at 60℃, and 1 min at 72℃, then 25 cycles of 15 s at 95℃, 30 s at 42℃ minus 1℃ per cycle, 1 min at 72℃, and a final extension at 72℃ for 10 min[24]. Next, 1 µL of the cDNA from this amplification was reamplified using the PCR primers specific for iNOS (NOS40F and NOS40R)[24]. The PCR conditions were an initial denaturation at 95℃ for 2 min, followed by a touch-down protocol consisting of 13 cycles of 15 s at 95℃, 30 s at 70℃ minus 1℃ per cycle, 1 min at 72℃, then 30 cycles of 15 s at 95℃, 30 s at 57℃, 1 min at 72℃, and a final extension at 72℃ for 10 min[24]. As a control, amplification of mRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed[24,26],

and the PCR conditions were the same as those described for the first nested PCR amplification. The amplification products were resolved by electrophoresis on 3% (w/v) agarose gels containing 0.1 µg/mL ethidium bromide and photographed under UV trans-illumination.

Comet assay (single cell gel electrophoresis assay)Comet assay was performed as described previously[27,28]. The gallbladder epithelial cells in each group were resuspended at 1 × 105 cells/mL in ice cold CMF-PBS and combined with molten LMAgarose (Trevigen, Gaitherburg, MD) at a ratio of 1:10 (v/v). The sample was immediately pipetted onto a frosted microscope slide (CometSlide; Trevigen, Gaitherburg, MD). The slides were placed flat at 4℃ in the dark for 10 min, immersed in prechilled lysis solution (Trevigen, Gaitherburg, MD), and left at 4℃ for 30 min to remove cellular proteins, leaving DNA as nucleoids. The slides were then immersed in an alkaline solution (pH > 13, 0.3 mol/L NaOH and 0.001 mol/L EDTA) for 30 min to denature the DNA and hydrolyze sites of damage. The samples were electrophoresed for 10 min and stained with SYBR green I (Trevigen, Gaitherburg, MD) according to the manufacturers instructions. At least 75 cells on each slide, randomly selected by fluorescence microscopy, were analyzed using National Institutes of Health image (Netscape Navigator) with the comet analysis macro (comet 1.4 macro)[29].

Statistical analysisValues for NO2

- + NO3- levels and proportion of DNA

damage were expressed as mean ± SE. For statistical analysis, ANOVA was used to compare non-repeated measurements between groups, followed by Bonferroni correction for paired comparison. P < 0.05 was considered statistically significant.

RESULTSGeneration of NO2

- and NO3-

The amount of NO measured by high performance liquid chromatography in each group is shown in Figure 2. The concentration of NO2

- + NO3- in the media was

Table 1 Oligonucleotide primers used for RT-PCR in this study

Primers Sequences ReferencesNOS-590F GGYTGGTACATGRGCACYGAGATYGG Cox et al[25], 2001NOS-893R AAGGCRCARAASTGDGGRTA Cox et al[25], 2001NOS40F GCAGGATGGGAAACTGAGGCCCAG Ramirez-Emiliano

et al[24], 2005NOS40R TGAACAAGGCAGCCAGGTCCCGG Ramirez-Emiliano

et al[24], 2005GAPDHF TCCCTCAAGATTGTCAGCAA Liu et al[26], 1993GAPDHR AGATCCACAACGGATACATT Liu et al[26], 1993

NO

2- /NO

3- (µm

ol/L

)

Control CM L-NMMA

P < 0.001 P < 0.001

Figure 2 High performance liquid chromatography showing significantly elevated NO2

- + NO3- levels in the CM group compared with the control group, similar NO

generation in the CM + L-NMMA group and control group, being significantly lower than that in the CM group.

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10.35 ± 0.47 µmol/L in the control group (n = 26), 11.06 ± 0.18 µmol/L in the CM group (n = 26), and 10.46 ± 0.18 µmol/L in the CM + L-NMMA group (n = 27). NO generation was significantly higher in the CM group than in the control group (P < 0.001). Meanwhile, NO generation in the CM + L-NMMA group and control group was similar, and significantly lower than that in the CM group (P < 0.001).

RT-PCR RT-PCR/touch down amplification of iNOS mRNA in gallbladder epithelial cells is shown in Figure 3. iNOS mRNA (183 bp) expression was demonstrated in the CM, while none of the control cells expressed iNOS mRNA.

Comet assayRepresentative fluorescent micrograph images evaluated by single cell gel electrophoresis using comet assay are shown in Figure 4A. In contrast to the intact spherical nuclei observed in the control group, the cells treated with cytokine mixture demonstrated a comet tail indicative of DNA damage. In the CM + L-NMMA group, the cells also exhibited intact spherical nuclei. The ratio of DNA exhibiting comet tails evaluated by using NIH image was 2.14% ± 0.59% in the control group, 5.14% ± 0.69% in the CM group, and 1.70% ± 0.62% in the CM + L-NMMA group (Figure 4B). The level of DNA damage in the CM group was significantly higher than that in the control group (P < 0.001), and decreased to the control level after the addition of L-NMMA.

DISCUSSIONThe induction of iNOS expression and NO production has previously been described in a variety of precancerous or cancerous lesions, in which the NO level of production is in proportion to the degree of malignancy[30,31]. Recently, Jaiswal et al[27,32] reported that human cholangiocarcinomas exhibit intensive immunohistochemical staining for iNOS and that cholangiocarcinoma cell lines stimulated by inflammatory cytokines and cholangiocyte cell lines transfected with iNOS produce large amounts of NO due to iNOS expression, which result in both oxidative DNA damage and inhibition of the excision DNA repair process. In the present study, we used primary epithelial cells isolated from the gallbladder because these normal epithelial cells should be suitable for estimating the involvement of iNOS and NO in biliary carcinogenesis, especially in the initiation of biliary carcinoma in response to chronic inflammation.

Following stimulation with a mixture of inflammatory

cytokines of IL-1β, IFN-γ, and TNF-α, NO generation was significantly increased in the gallbladder epithelial cells. Furthermore, the production of NO in the presence of inflammatory cytokines was completely suppressed by the addition of an iNOS inhibitor, L-NMMA. Moreover, the expression of iNOS mRNA in the gallbladder epithelial cells was clearly demonstrated in the presence of cytokine-stimulation using nested RT-PCR, but not in the absence of stimulatory cytokines. These findings indicate that inflammatory cytokines promote iNOS expression and NO generation in normal epithelial cells of hamster gallbladder.

NO has the ability to directly oxidize DNA, causing mutagenic changes[20,21]. Although NO also contributes to intracellular communication, inhibition of apoptosis, and enhancement of vascular dilatation, permeability, and neovascularization[33,34], DNA damage may be an essential and initial process involved in the malignant transformation of a wide variety of epithelial cells, and DNA damage in the individual cell can be detected using the highly sensitive comet assay[27,28,32]. In this assay, damaged single- and double-stranded DNA within the nucleus are allowed to migrate toward the anode, by an alkaline hydrolysis process during electrophoresis, resulting in the appearance of a “comet tail”. In our study, the comet assay clearly demonstrated that the proportion of DNA moving to the comet tail, i.e., indicating DNA damage, was significantly higher in the cytokine mixture group than in the control group, and that the increased DNA damage was completely inhibited by L-NMMA. These data indicate that inflammatory cytokines stimulate iNOS-mediated DNA damage in normal gallbladder

GAPDH (309 bp)

iNOS (183 bp)

GAPDH iNOS GAPDH iNOS

Control CM

Figure 3 iNOS mRNA expression in gallbladder epithelial cells in the CM group and no iNOS mRNA expression in the control group.

A

7.5

5.0

2.5

0.0D

NA

dam

age

(% D

NA

in t

ail)

P < 0.001 P < 0.001

Control CM L-NMMA

B

Figure 4 NO-dependent DNA damage evaluated by comet assay. A: Fluorescence microscopy showing the apparent comet tail in gallbladder cells stimulated by the cytokine mixture and intact spherical nuclei of the cells in the control group and CM + L-NMMA group; B: Analysis of the proportion of DNA migrated to comet tails using NIH image showing a significantly higher level of DNA damage in the CM group than in the control group, which decreased to the control level after the addition of L-NMMA.

a b cControl CM CM + L-NMMA

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epithelial cells. Although almost all DNA oxidative breaks can be excised by multiple excision DNA repair processes before mutations occur[35], DNA damage can lead to p53-mediated cell growth arrest and apoptosis, and the accumulation of p53 protein can repress the transcription of iNOS[36]. Furthermore, a recent report has shown that the tumor suppressant organization of normal p53 protein is inhibited in the presence of large amounts of NO in inflamed tissues[37]. In consideration of these facts, the results of our study suggest that NO-mediated oxidative DNA damage produced by inflammatory cytokines through iNOS expression is involved in an initiation process linking chronic biliary inflammation to malignant transformation.

Inflammatory cytokines released in the inflamed tissues initiate the induction of iNOS, resulting in increased NO production. As demonstrated in our study, iNOS-mediated NO production is sufficient to induce DNA damage in normal biliary epithelial cells. In association with persistency of biliary inflammation, the accumulation of NO-mediated genotoxicity may initiate the malignant transformation of epithelial cells lining the biliary tree. Our hamster models of both in vivo and in vitro tumorigenesis will enhance the understanding of the mechanisms of inflammation-related biliary carcinogenesis.

ACKNOWLEDGMENTSThe authors would like to thank Dr. S Maeda, Department of Pharmacology 1, Nagasaki University Graduate School of Biomedical Sciences, for technical assistance and advice in measuring NO.

COMMENTSBackgroundIt is well known that biliary carcinoma develops under chronic inflammatory conditions involving the biliary epithelium. We have previously demonstrated that persistent reflux cholangitis after bilioenterostomy accelerates biliary carcinogenesis through activation of biliary epithelial cell kinetics in hamsters. However, the cellular mechanisms of biliary carcinogenesis in response to inflammation remain unclear.

Research frontiersIn the present study, we used primary epithelial cells isolated from the gallbladder of hamsters. These normal epithelial cells should be suitable for estimating the involvement of iNOS and NO in biliary carcinogenesis, especially in initiation of biliary carcinoma in response to chronic inflammation.

Innovations and breakthroughsIn normal hamster gallbladder epithelial cells, cytokine stimulation induced iNOS expression and NO generation which was sufficient to cause DNA damage. The results suggest that NO-mediated oxidative DNA damage produced by inflammatory cytokines through iNOS expression is involved in an initiation process linking chronic biliary inflammation to malignant transformation.

ApplicationsOur hamster models of both in vivo and in vitro tumorigenesis will enhance the understanding of the mechanisms of inflammation-related biliary carcinogenesis.

Peer reviewThe authors investigated the role of inducible nitric oxide (iNOS) in DNA damage using cultured gallbladder epithelial cells. They speculated that the DNA damage would be related to biliary carcinogenesis. The data were interesting.

REFERENCES1 Ohshima H, Bartsch H. Chronic infections and inflammatory

processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutat Res 1994; 305: 253-264

2 Fitzpatrick FA. Inflammation, carcinogenesis and cancer. Int Immunopharmacol 2001; 1: 1651-1667

3 Baumann R, Uettwiller H, Duclos B, Jouin H, Kerschen A, Adloff M, Weill JP. Congenital cystic dilatation of the common bile duct, anomaly of the biliopancreatic junction and cancer of the bile ducts. Gastroenterol Clin Biol 1987; 11: 849-855

4 Fujii H, Yang Y, Tang R, Kunitomo K, Itakura J, Mogaki M, Matsuda M, Suda K, Nobukawa B, Matsumoto Y. Epithelial cell proliferation activity of the biliary ductal system with congenital biliary malformations. J Hepatobiliary Pancreat Surg 1999; 6: 294-302

5 Tocchi A, Mazzoni G, Liotta G, Lepre L, Cassini D, Miccini M. Late development of bile duct cancer in patients who had biliary-enteric drainage for benign disease: a follow-up study of more than 1,000 patients. Ann Surg 2001; 234: 210-214

6 Hakamada K, Sasaki M, Endoh M, Itoh T, Morita T, Konn M. Late development of bile duct cancer after sphincteroplasty: a ten- to twenty-two-year follow-up study. Surgery 1997; 121: 488-492

7 Tanaka M, Takahata S, Konomi H, Matsunaga H, Yokohata K, Takeda T, Utsunomiya N, Ikeda S. Long-term consequence of endoscopic sphincterotomy for bile duct stones. Gastrointest Endosc 1998; 48: 465-469

8 Kitajima T, Tajima Y, Onizuka S, Matsuzaki S, Matsuo K, Kanematsu T. Linkage of persistent cholangitis after bilioenterostomy with biliary carcinogenesis in hamsters. J Exp Clin Cancer Res 2000; 19: 453-458

9 Kitajima T , Tajima Y, Matsuzaki S, Kuroki T, Fukuda K, Kanematsu T. Acceleration of spontaneous biliary carcinogenesis in hamsters by bilioenterostomy. Carcinogenesis 2003; 24: 133-137

10 Tajima Y, Eto T, Tsunoda T, Tomioka T, Inoue K, Fukahori T, Kanematsu T. Induction of extrahepatic biliary carcinoma by N-nitrosobis(2-oxopropyl)amine in hamsters given cholecystoduodenostomy with dissection of the common duct. Jpn J Cancer Res 1994; 85: 780-788

11 Asakawa T, Tomioka T, Kanematsu T. A method for culturing and transplanting biliary epithelial cell from syrian golden hamster. Virchows Arch 2000; 436: 140-146

12 Facchetti F, Vermi W, Fiorentini S, Chilosi M, Caruso A, Duse M, Notarangelo LD, Badolato R. Expression of inducible nitric oxide synthase in human granulomas and histiocytic reactions. Am J Pathol 1999; 154: 145-152

13 Geller DA, Billiar TR. Molecular biology of nitric oxide synthases. Cancer Metastasis Rev 1998; 17: 7-23

14 Marletta MA. Nitric oxide synthase: aspects concerning structure and catalysis. Cell 1994; 78: 927-930

15 Michel T, Feron O. Nitric oxide synthases: which, where, how, and why? J Clin Invest 1997; 100: 2146-2152

16 Mannick JB, Asano K, Izumi K, Kieff E, Stamler JS. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell 1994; 79: 1137-1146

17 Hoffman RA, Zhang G, Nüssler NC, Gleixner SL, Ford HR, Simmons RL, Watkins SC. Constitutive expression of inducible nitric oxide synthase in the mouse ileal mucosa. Am J Physiol 1997; 272: G383-G392

18 Geller DA, Nussler AK, Di Silvio M, Lowenstein CJ, Shapiro RA, Wang SC, Simmons RL, Billiar TR. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci USA 1993; 90: 522-526

19 Geller DA, Di Silvio M, Nussler AK, Wang SC, Shapiro RA, Simmons RL, Billiar TR. Nitric oxide synthase expression is induced in hepatocytes in vivo during hepatic inflammation. J Surg Res 1993; 55: 427-432

20 Tamir S, Burney S, Tannenbaum SR. DNA damage by nitric oxide. Chem Res Toxicol 1996; 9: 821-827

21 Filep JG, Lapierre C, Lachance S, Chan JS. Nitric oxide co-operates with hydrogen peroxide in inducing DNA

Kitasato A et al . iNOS-mediated DNA damage in hamster gallbladder epithelial cells 6383

www.wjgnet.com

COMMENTS

fragmentation and cell lysis in murine lymphoma cells. Biochem J 1997; 321 (Pt 3): 897-901

22 Nüssler AK, Geller DA, Sweetland MA, Di Silvio M, Billiar TR, Madariaga JB, Simmons RL, Lancaster JR. Induction of nitric oxide synthesis and its reactions in cultured human and rat hepatocytes stimulated with cytokines plus LPS. Biochem Biophys Res Commun 1993; 194: 826-835

23 Archer S. Measurement of nitric oxide in biological models. FASEB J 1993; 7: 349-360

24 Ramírez-Emiliano J, González-Hernández A, Arias-Negrete S. Expression of inducible nitric oxide synthase mRNA and nitric oxide production during the development of liver abscess in hamster inoculated with Entamoeba histolytica. Curr Microbiol 2005; 50: 299-308

25 Cox RL, Mariano T, Heck DE, Laskin JD, Stegeman JJ. Nitric oxide synthase sequences in the marine fish Stenotomus chrysops and the sea urchin Arbacia punctulata, and phylogenetic analysis of nitric oxide synthase calmodulin-binding domains. Comp Biochem Physiol B Biochem Mol Biol 2001; 130: 479-491

26 L i u S , A d c o c k I M , O l d R W , B a r n e s P J , E v a n s T W . Lipopolysaccharide treatment in vivo induces widespread tissue expression of inducible nitric oxide synthase mRNA. Biochem Biophys Res Commun 1993; 196: 1208-1213

27 Jaiswal M, LaRusso NF, Burgart LJ, Gores GJ. Inflammatory cytokines induce DNA damage and inhibit DNA repair in cholangiocarcinoma cells by a nitric oxide-dependent mechanism. Cancer Res 2000; 60: 184-190

28 Duthie SJ, McMillan P. Uracil misincorporation in human

DNA detected using single cel l gel e lectrophoresis . Carcinogenesis 1997; 18: 1709-1714

29 Helma C, Uhl M. A public domain image-analysis program for the single-cell gel-electrophoresis (comet) assay. Mutat Res 2000; 466: 9-15

30 Szabó C, Ohshima H. DNA damage induced by peroxynitrite: subsequent biological effects. Nitric Oxide 1997; 1: 373-385

31 Ambs S, Bennett WP, Merriam WG, Ogunfusika MO, Oser SM, Harrington AM, Shields PG, Felley-Bosco E, Hussain SP, Harris CC. Relationship between p53 mutations and inducible nitric oxide synthase expression in human colorectal cancer. J Natl Cancer Inst 1999; 91: 86-88

32 Jaiswal M, LaRusso NF, Shapiro RA, Billiar TR, Gores GJ. Nitric oxide-mediated inhibition of DNA repair potentiates oxidative DNA damage in cholangiocytes. Gastroenterology 2001; 120: 190-199

33 Pérez-Sala D, Rebollo A. Novel aspects of Ras proteins biology: regulation and implications. Cell Death Differ 1999; 6: 722-728

34 García-Cardeña G, Folkman J. Is there a role for nitric oxide in tumor angiogenesis? J Natl Cancer Inst 1998; 90: 560-561

35 Wood RD, Mitchell M, Sgouros J, Lindahl T. Human DNA repair genes. Science 2001; 291: 1284-1289

36 Ambs S, Hussain SP, Harris CC. Interactive effects of nitric oxide and the p53 tumor suppressor gene in carcinogenesis and tumor progression. FASEB J 1997; 11: 443-448

37 Calmels S, Hainaut P, Ohshima H. Nitric oxide induces conformational and functional modifications of wild-type p53 tumor suppressor protein. Cancer Res 1997; 57: 3365-3369

S- Editor Liu Y L- Editor Zhu LH E- Editor Lu W

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(0.31 ± 0.08 EU/mL vs control 0.09 ± 0.03 EU/mL), alanine transferase (ALT, 219.1 ± 17.4 U/L vs control 5.9 ± 2.2 U/L) and portal vein pressure. Compared with normal control animals, the number of total cells in bronchoalveolar lavage fluid (BALF) of the cirrhotic rats at the 8th wk was not changed, but the number of macrophages and the ratio of macrophages to total cells were increased by nearly 2-fold, protein expression of inducible nitric oxide synthase (iNOS) and endothelial nitr ic oxide synthase (eNOS) started to increase significantly at the 4th wk, and reached its peak at the 8th wk in the lung of cirrhotic rats. The increase of iNOS expression appeared to be quicker than that of eNOS. NO2

-/NO3- was also increased, which was correlated to

the increase of iNOS (r = 0.7699, P < 0.0001) and eNOS (r = 0.5829, P < 0.002). mRNA expression of eNOS and iNOS was highly consistent with their protein expression.

CONCLUSION: Progression and severity of HPS as indicated by both increased pulmonary capillaries and histological changes are closely associated with LPS levels and progression of hepatic dysfunction as indicated by increased levels of ALT and portal vein pressure. Intestinal endotoxemia plays a central role in the development of HPS in the cirrhotic rat model by inducing NO and/or CO.

© 2007 WJG. All rights reserved.

Key words: Endotoxin; Alcohol; Nitric oxide synthase; Hemeoxygenase-1; Capillary; Macrophage; Cirrhosis; Hepatopulmonary syndrome

Zhang HY, Han DW, Su AR, Zhang LT, Zhao ZF, Ji JQ, Li BH, Ji C. Intestinal endotoxemia plays a central role in development of hepatopulmonary syndrome in a cirrhotic rat model induced by multiple pathogenic factors. World J Gastroenterol 2007; 13(47): 6385-6395

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INTRODUCTIONHepatopulmonary syndrome (HPS) develops when arterial oxygenation deficiency occurs due to intrapulmonary vascular dilatations that are often associated with severe hepatic disease[1]. Recent studies support that the presence

BASIC RESEARCH

Intestinal endotoxemia plays a central role in development of hepatopulmonary syndrome in a cirrhotic rat model induced by multiple pathogenic factors

Hui-Ying Zhang, De-Wu Han, Ai-Rong Su, Li-Tong Zhang, Zhong-Fu Zhao, Jing-Quan Ji, Bao-Hong Li, Cheng Ji

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Hui-Ying Zhang, Zhong-Fu Zhao, Jing-Quan Ji, Bao-Hong Li, Department of Pathophysiology, Changzhi Medical College, Changzhi 046000, Shanxi Province, ChinaDe-Wu Han, Institute of Hepatology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, ChinaAi-Rong Su, Department of Physiology, Bengbu Medical College, Bengbu 233030, Anhui Province, ChinaLi-Tong Zhang, Department of Gynaecology, Shanxi Modern Women Hospital, Taiyuan 030012, Shanxi Province, ChinaCheng Ji, Department of Medicine, University of Southern California, Los Angeles, CA 90033, United StatesSupported by the Awards to University Academic Leaders Granted by the Government of Shanxi Province of China to Hui-Ying Zhang and partially by the US National Institute of Health, NIAAA, Grant R01 AA014428 to Cheng Ji Correspondence to: Hui-Ying Zhang, MD, Professor, Depa-rtment of Pathophysiology, Changzhi Medical College, 161 Jie Fang Dong Jie, Changzhi 046000, Shanxi Province, China. zhhy2001@hotmail.comTelephone: +86-355-3031235 Fax: +86-355-3034065Received: July 4, 2007 Revised: September 27, 2007

AbstractAIM: To characterize the correlation between severity of hepatopulmonary syndrome (HPS) and degree of hepatic dysfunction, and to explore how intestinal endotoxemia (IETM) affects the development of HPS in cirrhotic rats.

METHODS: Male Wister rats were fed with a diet containing maize flour, lard, cholesterol, and alcohol and injected subcutaneously with CCl4 oil solution every two days for 8 wk to induce typical cirrhosis and development of HPS. The animals were also given a nitric oxide (NO) production inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME) intraperitoneally, and an iNOS inhibitor, aminoguanidine hydrochloride (AG) via gavage daily from the end of the 4th wk to the end of the 6th or 8th wk, or a HO-1 inhibitor, zinc protoporphyrin (ZnPP) intraperitoneally 12 h prior to killing. Blood, liver and lung tissues were sampled.

RESULTS: Histological deterioration of the lung paralleled to that of the liver in the cirrhotic rats. The number of pulmonary capillaries was progressively increased from 6.1 ± 1.1 (count/filed) at the 4th wk to 14.5 ± 2.4 (count/filed) at the 8th wk in the cirrhotic rats. Increased pulmonary capillaries were associated with increased blood levels of lipopolysaccharide (LPS)

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of HPS significantly increases the mortality of cirrhotic patients, particularly those with decompensated liver disease[2,3].

The pathogenes i s and prec i se mechan ism of pulmonary vascular abnormalities in HPS are the fields of active investigation. So far, studies have been focused on characterizing the increased pulmonary production of vasodilator substances, most notably nitric oxide (NO). Pulmonary NO overproduction in human HPS, as assessed by exhaled NO levels, has also been reported[4,5]. It was reported that HPS patients showed short term improvements in oxygenation after acute administration of L-NAME (a specific nitric oxide synthase inhibitor) or methylene blue (an inhibitor of cyclic guanosine monophosphate generation)[6-8], indicating that pulmonary NO and cyclic guanosine monophosphate production can lead to changes in pulmonary vascular tone, thus affecting oxygenation. However, the role of pulmonary NO in lung oxygenation is still controversial. Inhalation of L-NAME could not acutely improve intrapulmonary vasodilatation[9], and sequential inhibition of the nitric oxide-cyclic guanosine monophosphate pathway by curcumin (diferuloylmethane), terlipressin and methylene blue could not improve the intrapulmonary shunt, and both hypoxaemia and orthodeoxia were substantially worsened after treatment with the above three durgs[10], strongly suggesting that factors other than nitric oxide synthase (NOS)-derived NO effects on vascular tone contribute to HPS. It has also been shown that the levels of COHb indicative of CO production are increased in cirrhotic patients who develop HPS compared to the cirrhotic patients who do not develop HPS and correlate with gas exchange abnormalities, suggesting that CO may also contribute to human HPS[11].

The sequelae of endotoxemia are often fatal. The lung appears to be an organ sensitive to endotoxemia and many distant-focus infections can be detected as a result of pulmonary dysfunction[12,13]. Patients with hepatic cirrhosis have an elevated plasma level of lipopolysacchride (LPS)[14], which is a leading causative factor for cirrhotic complications, such as hepatorenal syndrome[15-18] and hepatic encephalopathy[19].

We have previously developed a rat model characteristic of cirrhosis and HPS that can be non-invasively induced by multiple pathogenic factors including high fat diet, alcohol, cholesterol, corn flour and CCl4[20]. A significantly increased level of LPS in plasma is closely related to the deceased blood oxygen content and intrapulmonary vascular dilatation in the rat model, suggesting that intestinal endotoxemia (IETM) and its accompanying cytokines, such as TNF-α, play a role in the pathogenesis of HPS.

This study was to further characterize the relation between sever i ty of HPS and deg ree of hepat ic dysfunction, and to explore how IETM affects the development of HPS in cirrhotic rats. The results show that the severity of HPS parallels to the levels of blood LPS and progression of hepatic dysfunction and IETM plays a central role in the development of HPS in cirrhotic rats.

MATERIALS AND METHODSAnimals and reagents Male Wister rats, weighing 220-250 g, were obtained from the Animal Center of Shanxi Medical University. All animals received human care during the study. The study was approved by the Ethical and Research Committee of the Shanxi Medical University. Experiments were performed following the institutional guideline for animal research. Cirrhosis and HPS were induced in the rats as previously described[20]. In brief, male Wister rats were fed with a complex diet containing maize flour, lard, cholesterol, and alcohol for 8 wk, and injected subcutaneously with CCl4 oil solution every two days over the experimental period to induce typical cirrhosis and development of HPS. The animals were also given a NO production inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME) intraperitoneally, 5 mg/kg per day, or an inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine hydrochloride (AG) via gavage, 100 mg/kg per day from the end of the 4th wk to the end of the 6th or 8th wk, or 50 mmol/kg hemeoxygenase-1 (HO-1) inhibitor, zinc protoporphyrin (ZnPP) intraperitoneally 12 h prior to killing. The animals were randomized into four groups: (1) normal control group (fed with standard diet), (2) 4-wk group (fed with the complex diet for 4 wk), (3) 6-wk group (fed with the complex diet for 6 wk), and (4) 8-wk group (fed with the complex diet for 8 wk). Blood, liver and lung tissues were sampled from the rats.

L imulus amebocyte lysate (LAL) reagent , for determination of LPS in plasma, was provided by Shanghai Yi Hua Scientific, Inc (Shanghai, China). Tumor necrosis factor α (TNF-α) radioimmunoassay kit was provided by Radioimmunity Institute of PLA General Hospital (Beijing, China). Detecting kit for NO2

-/NO3- was provided by

Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Polyclone anti- endothelial nitric oxide synthase (eNOS) and anti- inducible nitric oxide synthase (iNOS) were purchased from Beijing Zhongshan Biotic Reagent Company (Beijing, China). Immunohistochemistry kit for hemeoxygenase-1 (HO-1) was obtained from Wuhan Boshide Bioengeering Institue (Wuhan, China). TRIzol reagent was bought from the Life Technologies Company (California, USA). Reverse transcriptase polymerase chain reaction (RT-PCR) primers for β-actin, eNOS, and iNOS were synthesized by Shanghai Jiebeist Gene Company, LTD (Shanghai, China). NO inhibitor L-NAME, specific iNOS inhibitor AG, and HO-1 inhibitor ZnPP were obtained from Sigma Chemical Co (St. Louis, MO).

Measurement of LPS, TNF-α, alanine transferase , malondialdehyde in plasma, NO2

-/NO3- and COHb in

pulmonary homogenate and portal vein pressure The contents of LPS, TNF-α, alanine transferase (ALT), malondialdehyde (MDA) in plasma, NO2

-/NO3- (indirectly

reflecting NO) and COHb (indirectly reflecting CO) in pulmonary homogenate were measured with the different kits following their manufacturers'instructions. The method described by MacPhail et al[21] was modified for

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measurement of portal vein pressure (PVP) in which 18-gauge over-the-needle catheter was inserted into portal vein and connected to a water manometer.

Histology and calculation of capillaries Samples from the liver and lung were collected and fixed immediately in 100 g/L phosphate-buffered formaldehyde overnight. After paraffin embedding, serial 4-mm thick sections were prepared and stained with hematoxylin and eosin (HE). Images (× 100) of liver and lung sections were captured with Olympus microscope equipped with a digital camera. The number of capillaries was counted in 6 fields per slide per group (n = 6) with the Image-Pro plus full automatism analysis system.

Number of cells in bronchoalveolar lavage fluidBronchoalveolar lavage was performed with 0.9% sterile NaCl. The number of total cells and macrophages in bronchoalveolar lavage fluid (BALF) was counted respectively with a hemocytometer and the ratio of macrophages to total cells was calculated.

Dynamic study of eNOS, iNOS and HO-1 protein expression in lung architectureSamples from the right lobe of lung were collected into 100 g/L phosphate-buffered formaldehyde and fixed overnight. Serial 4-mm thick sections were prepared after the samples were dehydrated in graded ethanol solutions, cleared in chloroform, and embedded in paraplast. Expression of eNOS, iNOS and HO-1 was analyzed with immunohistochemical staining and quantified with the Image-Pro plus full automatism analysis system in a blind fashion.

Dynamic study of eNOS and iNOS mRNA expression in lungRT-PCR was employed to detect the mRNA expression of eNOS and iNOS genes in lung. The primer sequences used are as follows: eNOS (435 bp)[22]: 5'-CTGCTGCCCGAGATATCTTC-3', 5'-AAGTAAGTGAGAGCCTGGCGCA-3'; iNOS (388 bp)[23]: 5'-AGCATCACCCCTGTGTTC CACCC-3', 5'-TGGGGCAGTCTCCATTGCCA-3'; β-actin (630 bp, as control)[24]: 5'-GATGGTGGGTAT GGGTCAGAAGGAC-3', 5'-GCTCATTGCCGATAG TGA TGA CT-3'.

Total RNA was extracted from 50-100 mg snap-frozen left lung tissue following TRIzol protocol. Following precipitation, the RNA was resuspended in RNase-free buffer, the concentration was determined by measuring the ultra-violet light absorbance at 260 nm and 280 nm and the purity of RNA was estimated from the ratio of A260/A280.

Single-stranded complementary DNA (cDNA) was synthesized from the total RNA. Two mg RNA was mixed with 0.5 mg oligo (dT), pre-incubated in 15 mL of diethylpyrocarbonate (DEPC) at 70℃ for 5 min, and rapidly chilled on ice. Five mL of M-MLV 5 × reaction buffer, 1.25 mL of dNTP (10 mmol/L, each), 25 units of rRNasin ribonuclease inhibitor, 200 units of M-MLV RT and DEPC-treated water were added into the annealed

primer/template to a final volume of 25 mL. The reaction solution was incubated at 42℃ for 60 min and terminated by placing it on ice after denaturation at 85℃ for 5 min. The resulting cDNA was used as a template for subsequent PCR.

The PCR mixture contained 5 mL of 10 × Taq buffer, 1 mL of dNTP (10 mmol/L, each), 1 mL of gene specific primers, 2.0 units of Taq DNA polymerase and 1 mL of cDNA in a total volume of 50 mL. Thirty-five cycles of amplification were performed with an initial incubation at 94℃ for 3 min and a final extension at 72℃ for 7 min, each cycle consisted of denaturation at 94℃ for 1 min, annealing at 64℃ for 1 min and extension at 72℃ for 1 min. The quantities of cDNA producing equal amounts of β-actin-PCR-product were used in PCR with the primers for iNOS and eNOS. Following RT-PCR, 10 mL samples of amplified products was resolved by electrophoresis on 1.2% agarose gel and stained with ethidium bromide. The level of each PCR product was semi-quantitatively evaluated using a digital camera and an image analysis system (Alpha Innotech Co., USA), and normalized to β-actin.

Statistical analysisData were evaluated by analysis of variance, multiple comparisons between groups were performed with SPSS software. All values are reported as mean ± SD. P < 0.05 was considered statistically significant.

RESULTSComparison of histological features of both liver and lung Pulmonary pathological changes were consistent with hepatic pathological changes towards the same direction, namely, pulmonary pathological changes became progressively exacerbated as liver injury progressed. In normal liver architecture, sinusoids and cord located in order around central veins (Figure 1A). Derangement of hepatic cord and central lobular necrosis with infiltration of inflammatory cells, cytoplasm rarefaction in liver cells with balloon-like alteration and steatosis, and increased fiber amount in interlobular area were found in the 4th wk group (Figure 1B). Furthermore, more typical balloon-like alteration and steatosis, fiber network formation, further aggravated infiltration of inflammatory cells were observed in the 6th wk group (Figure 1C). False lobule formation, secondary degeneration and necrosis in liver cells with infiltration of inflammatory cells were demonstrated in the 8th wk group (Figure 1D). Thin alveoli wall with disseminated pulmonary phagocytes was observed in the normal control group (Figure 2A). Thick alveoli wall with infiltration of phagocytes and neutrophils was observed in the 4th wk group (Figure 2B). Dilated capillaries and increased capillary density in further wider septum, and partially decreased air space in their size with infiltration of phagocytes and neutrophils were found in the 6th wk group (Figure 2C). Obviously dilated capillaries and increased number of capillaries, massive enlarged phagocytes within wider septa and air spaces with focal narrower air spaces were found in the 8th wk group (Figure 2D).

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Changes in pathological parameters The levels of LPS, TNF-α, ALT and MDA in plasma were progressively increased over the period of 8 wk. PVP was significantly elevated in the 4th wk and persisted up to the 8th wk (Table 1).

We have previously reported that the pulmonary capillaries are dilated in the lung of cirrhotic rats based on Tc99MAA labeling and scanning[20]. To know if proliferation of capillaries also occurs during the development of HPS, we counted the number of capillaries. The number of pulmonary capillaries was progressively increased in cirrhotic

rats (Figure 3), which was positively related to the increased levels of LPS, TNF-α, ALT and MDA in plasma as well as to the NO and CO in pulmonary homogenate (Table 2).

The number of cells in bronchoalveolar lavage fluids could reflect the function of lung. Compared with normal control animals, the total number of cells was not changed. However, the number of macrophages and the ratio of macrophages to total number of cells were significantly increased in BALF of cirrhotic rats at the 8th wk, strongly suggesting that macrophages could play a role in the pathogenesis of HPS (Table 3).

Figure 1 Liver histology of HPS (HE, × 100) in normal control group (A), 4th wk group (B), 6th wk group (C) and 8th wk group (D).

A B

C D

A B

C D

Figure 2 Lung histology of HPS (HE, × 200) in normal control group (A), 4th wk group (B), 6th wk group (C) and 8th wk group (D).

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Levels of NO2-/NO3

- , eNOS and iNOS in lung Immunohistochemistry displayed that eNOS protein expression in pulmonary architecture started to increase significantly at the 4th wk and reached its peak at the 8th wk (Table 4). The change in iNOS protein expression was similar to that of eNOS, but significant differences were detected between the 4th and 6th wk as well as between the 6th and 8th wk, showing a progressive ascending. NO2

-/NO3

- was nearly increased by 3.5-fold at the 4th wk, by 5-fold at the 6th wk, and by more than 7-fold at the 8th wk.

The increased NO2-/NO3

- was more relevant to the expression of iNOS (r = 0.7699; P < 0.01) than to that of eNOS (r = 0.5829, P < 0.002).

The expression of eNOS and iNOS was distributed differently in the lung tissue stained with immunohisto-chemistry. In normal lung, eNOS was expressed in pulmonary capillary endothelium. In contrast, eNOS was expressed not only in the endothelium but also in typeⅠand Ⅱ epithelial cells and macrophages, and the expression was progressively increased in the lung of cirrhotic rats over the experimental period (Figure 4). iNOS was located mainly in macrophages and type Ⅱ epithelial cells during the development of HPS.

At mRNA levels, the expression of eNOS and iNOS in the lung was highly consistent with their protein expression (Table 5, Figure 5). Plasma endotoxin was significantly correlated with the expression of eNOS and iNOS over the 8 wk period (Table 6), suggesting that IETM might induce the expression of eNOS and/or iNOS in the development of HPS.

Effects of NOS or HO-1 inhibitors on expression of HO-1 or NOS in lung The level of COHb and expression of HO-1 in the lung of cirrhotic rats began to increase at the 4th wk and reached their peak at the 8th wk (Table 7). The location of

Table 1 Levels of endotoxin, TNF-α, ALT and MDA in plasma and PVP (mean ± SD, n = 6)

Groups Endotoxin (Eu/mL) TNF-α (ng/mL) ALT (u/L) MDA (nmol/mL) PVP (H2O cm)

Normal 0.09 ± 0.03 0.53 ± 0.16 5.85 ± 2.24 0.06 ± 0.03 10.31 ± 1.164th wk 0.19 ± 0.03a 1.37 ± 0.35a 56.24 ± 14.73a 0.10 ± 0.05 17.54 ± 1.84a

6th wk 0.24 ± 0.04a 1.68 ± 0.23a 53.50 ± 22.10a 0.18 ± 0.03a,c 15.44 ± 3.11a,c

8th wk 0.31 ± 0.08a,e 2.42 ± 0.38a,e 219.10 ± 17.37a,e 0.26 ± 0.06a,e 18.38 ± 2.53a,e

aP < 0.05 vs normal group; cP < 0.05 vs 4th wk group; eP < 0.05 vs 6th wk group.

Table 2 Correlation analysis (n = 6)

Groups r P

LPS vs quantities of pulmonary capillaries 0.626 < 0.001TNF-α vs quantities of pulmonary capillaries 0.644 < 0.001ALT vs quantities of pulmonary capillaries 0.556 < 0.01MDA vs quantities of pulmonary capillaries 0.691 < 0.0002CO vs quantities of pulmonary capillaries 0.432 < 0.01NO vs quantities of pulmonary capillaries 0.725 < 0.0001

Table 3 Changes of cell numbers in bronchoalveolar lavage fluid (mean ± SD, n = 6)

Groups Total cell numbers (/mL)

Macrophages (/mL)

Ratio of macrophage/total cell numbers

Normal control 582.7 ± 74.5 184.1 ± 5.4 0.32 ± 0.058th wk 619.2 ± 116.5 310.8 ± 27.5a 0.51 ± 0.06a

aP < 0.05 vs normal control group.

Table 4 Levels of NO2-/NO3-, expression of eNOS and iNOS in lung (mean ± SD, n = 6)

Groups NO2-/NO3

-

(mmol/g protein)eNOS (IOD) iNOS (IOD)

Normal control 3.6 ± 1.7 2385.6 ± 752.4 4214.8 ± 1783.74th wk 12.6 ± 3.7a 6448.9 ± 922.7a 8498.7 ± 2703.7a

6th wk 18.9 ± 5.5a,c 8008.9 ± 2812.7a 12831.6 ± 4901.2a,c

8th wk 26.9 ± 6.4a,e 14704.7 ± 5779.9a,e 19173.4 ± 2401.2a,e

aP < 0.05 vs normal group; cP < 0.05 vs 4th wk group; eP < 0.05 vs 6th wk group.

Table 5 Levels of eNOS and iNOS mRNA expression in lung (mean ± SD, n = 6)

Groups eNOS iNOS

Normal control 0.62 ± 0.07 0.22 ± 0.014th wk 0.65 ± 0.08a 0.98 ± 0.03a

6th wk 0.68 ± 0.07a 1.23 ± 0.03a,c

8th wk 0.75 ± 0.08a,e 1.46 ± 0.05a,e

aP < 0.05 vs normal group; cP < 0.05 vs 4th wk group; eP < 0.05 vs 6th wk group.20

15

10

5

0 A B C D

Num

ber

of p

ulm

onar

y ca

pilla

ries

(c

ount

/fiel

d)

Figure 3 Change in the number of pulmonary capillaries in normal control group (A), 4th wk group (B), 6th wk group (C) and 8th wk group (D). aP < 0.05 B, C, D vs A; aP < 0.05 D vs B.

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HO-1 was mainly found in pulmonary type Ⅱ epithelial cells, macrophages, and capillary endothelium (Figure 6). The levels of plasma LPS and TNF-α were closely correlated with COHb and expression of HO-1 (Table 8).

The expression of HO-1 in cirrhotic rats was decreased by 3-fold at the 6th wk when the non-specific inhibitor (L-NAME) of NOS was administrated and by 5-fold when the specific inhibitor (AG) of iNOS was administrated (Table 9). Similar effects were also detected at the 8th wk.

Inhibition of HO-1 expression specifically by ZnPP in cirrhotic rats decreased significantly the expression of eNOS and iNOS (Table 10), indicating that both CO and NO were involved in the development of HPS.

DISCUSSIONIn this study, dynamic alterations in plasma endotoxin were found to be closely associated with the expression

Figure 4 Immunohistochemistry of NOS expressions in the lung of rats with their eNOS and iNOS stained brown (× 200).

Control Control

4th wk 4th wk

6th wk 6th wk

8th wk 8th wk

eNOS iNOS

Table 6 Analysis of correlation (n = 6)

Pairs r P value

Plasma endotoxin vs eNOS protein 0.6348 P < 0.01Plasma endotoxin vs eNOS mRNA 0.7490 P < 0.01Plasma endotoxin vs iNOS protein 0.5710 P < 0.01Plasma endotoxin vs iNOS mRNA 0.8980 P < 0.01Plasma TNF-α vs eNOS protein 0.8354 P < 0.01Plasma TNF-α vs iNOS protein 0.8538 P < 0.01

Table 7 Changes of COHb and HO-1 protein in lung (mean ± SD, n = 6)

Groups COHb (mg/g protein) HO-1 protein (IOD)

Normal Control 0.13 ± 0.06 1515.18 ± 981.504th wk 0.27 ± 0.06a 7391.47 ± 674.97a

6th wk 0.29 ± 0.06a 11 157.03 ± 6093.39a

8th wk 0.43 ± 0.98a,e 24 867.59 ± 11 054.95a,e

aP < 0.05 vs normal group; eP < 0.05 vs 6th wk group.

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of eNOS, iNOS, HO-1, the number of capillaries, the histological changes characteristic of HPS in cirrhotic

rats, demonstrating that the function of lung and liver is simultaneously deteriorated, both CO and NO are involved in pulmonary vascular abnormalities, pulmonary macrophages play a role in HPS of hepatic cirrhosis rats.

Whether the presence and severity of HPS are associated with the severity of underlying liver disease remains unclear. It was reported that HPS occurs more common in advanced cirrhosis[25-29]. HPS can also occur in acute[30] and chronic noncirrhotic hepatitis[31,32]. In patients with hypoxic hepatitis, intrapulmonary vasodilatation indicative of HPS frequently occurs and can be reversed

2000

1000

750

500

250

A M 1 2 3 4

630 bp (β-actin)435 bp (eNOS)

bp2000

1000

750

500

250

B M 1 2 3 4

630 bp (β-actin)

388 bp (iNOS)

bp

Figure 5 Expre-s s i o n o f e N O S mRNA (A) and iNOS mRNA (B ) in ra t lung. M: DNA size marker; 1: control; 2: 4th wk group; 3: 6th wk group; 4: 8th wk group.

Figure 6 Immunohistochemistry of HO-1 expressions in lung of rats with HO-1 stained brown (× 200).

C 4th wk

6th wk 8th wk

Table 8 Correlation analysis (n = 6)

Groups r P

LPS vs COHb 0.876 < 0.01LPS vs HO-1 0.802 < 0.01TNF-α vs COHb 0.755 < 0.01TNF-α vs HO-1 0.796 < 0.05

Table 9 Effect of L-NAME and AG on HO-1 expression (mean ± SD, n = 6; IOD)

Groups 6th wk 8th wk

Cirrhotic control 11 157.03 ± 6093.39 24 867.59 ± 11054.95L-NAME + cirrhosis 3455.13 ± 1370.03a 11 063.48 ± 426.48a

AG + cirrhosis 2286.59 ± 6181.20a 9828.32 ± 799.51a

aP < 0.05 vs cirrhotic control group.

Table 10 Specific effect of ZnPP on expression of eNOS and iNOS (mean ± SD, n = 6)

aP < 0.05 vs cirrhotic control group.

Groups eNOS (IOD) iNOS (IOD)

6th wk cirrhotic control 13 206.7 ± 8490.9 9401.1 ± 691.96th wk ZnPP 2025.7 ± 6097.1a 3500.3 ± 237.4a

8th wk cirrhotic control 21 198.1 ± 11 865.3 26 419.8 ± 3560.58th wk ZnPP 2406.3 ± 9117.9a 3522.8 ± 356.5a

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after normalization of hepatic dysfunction[33], indicating that HPS is interrelated with underlying liver disease. In contrast, other studies reported that HPS occurs both in extrahepatic portal venous obstruction[34] and in hepatic venous outflow obstruction without hepatic cirrhosis[35], suggesting that severe liver dysfunction and cirrhosis are not absolutely associated with the development of HPS. In the present study, the characteristic histological changes in both liver and lung during the development of HPS were observed in cirrhotic rats, indicating that there is a paralleling deterioration of lung and liver function, which supports our hypothesis that hepatic pathological alteration is a substantial basis for the development of HPS in cirrhotic rats.

Oxygenation of red blood cells occurs in the alveolar-capillary network where the pulmonary capillary bed and alveoli come together in the alveolar wall achieving optimal gas exchange. Because the diameter of pulmonary capillaries (about 6 mm) is slightly smaller than that of red blood cells (8 mm), red blood cells must flow through in a single file and change their shape as they pass through the pulmonary capillaries. This shape change ensures a minimal distance between oxygen in alveoli and hemoglobin in red cells. Passive changes in the diameters of pulmonary capillaries are caused by alterations in pre- and post-capillary resistance. Injection of micro-opaque gelatin into pulmonary arteries of autopsy specimens from patients with cirrhosis has documented the presence of pre-capillary pulmonary vascular dilatations and direct arteriovenous communications[36]. The resulting consequence is the distending of pulmonary capillaries with increased blood leading to incomplete penetration of oxygen through dilated vessels that abut upon alveoli and true anatomic shunts in which deoxygenated blood is directly shunted into arterial blood bypassing gas exchange units. Therefore, it is believed that hypoxemia occurs as a result of one (or the combination of several) of the following mechanisms: (1) ventilation-perfusion mismatching (reflecting excess perfusion for a given ventilation), (2) diffusion-perfusion impairment (due to an increased oxygen diffusion distance from alveoli to hemoglobin across the dilated vessels), and (3) true intrapulmonary anatomical shunts[37-42]. It was reported that vascular abnormalities in HPS are associated with the increased number of dilated pre-capillaries, capillaries and pre-capillary arteriovenous communications[43]. In experimental HPS, the increased number of pulmonary vascular capillaries can be observed under electron microscope[44], but no detailed analysis is available. Lung perfusion imaging with Tc99MAA can show broadened pulmonary capillaries in rats with HPS[20]. Furthermore, in the present study, a number of progressively increased pulmonary capillaries were found as cirrhosis progresses in rats, confirming the probability of remodeling, angiogenesis or vasculogenesis proposed by Gomez et al[9]. The increased number of pulmonary capillaries is positively correlated with both dynamically increased levels of LPS, TNF-α, and MDA in plasma, further demonstrating that LPS plays a central role in the development of HPS in cirrhotic rats[20]. The increased number of pulmonary

capillaries is also positively correlated with dynamically increased ALT. Moreover, a clinical syndrome similar to HPS has been observed in congenital disorders without liver injury in which either hepatic venous blood flow does not reach the lung [45] or portal venous blood reaches the inferior vena cava without passing through the liver[46], indicating that factors either produced or metabolized in the liver can modulate the pulmonary vasculature and play a key role in maintaining the normal pulmonary vascular integrity. In addition, liver transplantation can reverse the ventilation-perfusion mismatch seen in HPS and restore normoxaemia[47]. These findings further support our notion that hepatic pathological alteration is a substantial basis for the development of HPS.

The role of NO in cirrhotic hyperdynamic circulation and induction of iNOS in endothelial cells by cytokines and endotoxin has been suggested as the mechanism[48], which soon appears as a very likely candidate for HPS[49]. However, the involvement of NOS isoform in increased NO production in the lung is controversial[50-53]. In the present study, the expression of eNOS and iNOS started to increase at the 4th wk and was closely related to the increased plasma LPS. Interestingly, the expression of iNOS not eNOS was significantly increased at the 6th wk compared to that at the 4th wk and the increased levels of nitric and nitrate in pulmonary homogenate exhibited a more significant linear correlation with iNOS than with eNOS, suggesting that iNOS may be more important in the development of HPS. In addition, a significantly increased quantity of macrophages in bronchoalveolar lavage fluid was observed during HPS in cirrhotic rats. These results demonstrate that the elevated NO results preferably from iNOS of pulmonary macrophages and, to a lesser extent, from eNOS in the lung of cirrhotic rats. Pulmonary macrophages may be a leading contributor to the development of HPS. Meanwhile, COHb in homogenate and HO-1 expression in the lung were increased over the 8 wk period of experiment. The increased COHb and HO-1 were positively correlated with the increased plasma LPS and TNF-α level. Since increased HO-1 expression in intravascular macrophages and carbon monoxide overproduction are also reported in a model of common bile duct ligation in rats[52], it would be interesting to investigate the interplay of NOS/NO pathway and HO-1/CO pathway in pulmonary vascular abnormalities in cirrhotic rats. For this purpose, L-NAME (a non-selective inhibitor of NOS), AG (a selective inhibitor of iNOS), or ZnPP (a selective inhibitor of HO-1) were administrated in cirrhotic rats. Interestingly, we observed a mutual inhibition, i.e., L-NAME or AG inhibited HO-1 expression, whereas ZnPP inhibited eNOS and iNOS expression. Histologically, AG inhibitor likely inhibited the enlargement of diameters of the dilated pulmonary capillaries at the 6th wk but there was no obvious change in the number of capillaries (data not show). Therefore, CO may be another important mediator in the development of HPS, HO-1/CO and NOS/NO pathways may be interrelated in pathogenesis of intrapulmonary vascular abnormalities in cirrhotic rats.

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The underlying mechanisms are required to be further investigated.

Hypoxia is very important in inducing expression of the vascular endothelial growth factor (VEGF)[54], which specifically regulates endothelial cell growth and differentiation and also acts as a survival factor for endothelial cells[55]. It is generally believed that hypoxia is a consequence of intrapulmonary vascular dilatation in the presence of IETM. Since hypoxia induces NO generation[56-58] and NO induces expression of VEGF[59-62], it is conceivable that blood vessel remodeling and angiogenesis or vasculogenesis occurs during the development of HPS in cirrhotic rats. However, it is worth mentioning that CO may also be a contributing factor in regulating the expression of VEGF since CO levels are correlated with the number of capillaries.

In summary, IETM plays a central role in the develo-pment of HPS under conditions of hepatic cirrhosis[63,64]. Therefore, strategies against LPS should be made for preventing the development of HPS.

ACKNOWLEDGMENTSThe authors thank Mr. Yuan-Chang Zhao and Professor Rui-Ling Xu for their technical assistance. Dr. Hui-Ying Zhang is currently a visiting scholar at the GI/Liver Division of USC Keck School of Medicine, Los Angeles, USA.

COMMENTSBackgroundHepatopulmonary syndrome (HPS) develops when arterial oxygenation deficiency occurs due to intrapulmonary vascular dilatation often associated with severe hepatic disease. Recent studies support that the presence of HPS significantly increases mortality in cirrhosis patients, particularly in those with decompensated liver disease. The pathogenesis of HPS remains to be elucidated. Some complications of cirrhosis, including hepatorenal syndrome, hepatic encephalopathy and HPS are closely associated with intestinal endotoxemia (IETM) in both experimental and clinical investigations. We further explored the pathogenesis of HPS on the basis of the preceding research in a cirrhotic rat model induced by multiple pathogenic factors.

Research frontiersOne of the characteristics of hepatopulmonary syndrome is intrapulmonary vascular dilation which severely affects pulmonary gas exchange leading to hypoxia and increased mortality of cirrhotic patients. The present study focused on clarifying the pathogenesis of HPS, particularly the mechanism of intrapulmonary vasodilation. Clinically, many studies have been performed on evaluating the prevalence, etiology, clinical features, early diagnosis, treatment and prognosis of this syndrome worldwide.

Innovations and breakthroughsIn this study, we further characterized the relation between severity of HPS and degree of hepatic dysfunction, and explored how IETM affects the development of HPS in cirrhotic rats. Our results indicate that the severity of HPS is directly associated with the level of blood LPS and progression of hepatic dysfunction, and IETM plays a central role in the development of HPS.

ApplicationsAll of our findings in the present study further confirm that IETM plays a central role in the development of HPS under conditions of hepatic cirrhosis. Therefore, the strategy against LPS is certainly effective both in preventing the development of HPS and in its treatment.

Terminology Angiogenesis: a natural process in the body that involves the growth of new blood vessels. It can occur in many diseases, such as coronary artery disease, peripheral artery disease and stroke when blood supply and oxygen are insufficient in tissues. It denotes the formation of new blood vessels from pre-existing ones. Vasculogenesis: the process of blood vessel formation occurring by a de novo production of endothelial cells. Microvasculature remodeling: alterations in a blood vessel network resulting from arteriogenesis and angiogenesis. Briefly, arteriogenesis is an increased arterial diameter while angiogenesis is an increased number of capillaries either by sprouting from or splitting existing capillaries. External events stimulate these two types of vessel growth through a combination of mechanical and chemical pathways.

Peer reviewThis manuscript is very interesting. The topic is original for a basic science study and the structure of material and methods is outstanding. Results are clearly explored and discussion widely covers the topic even for humans.

REFERENCES1 Rodríguez-Roisin R , Krowka MJ, Hervé P, Fallon MB.

Pulmonary-Hepatic vascular Disorders (PHD). Eur Respir J 2004; 24: 861-880

2 Schenk P , Schöniger-Hekele M, Fuhrmann V, Madl C, Silberhumer G, Müller C. Prognostic significance of the hepatopulmonary syndrome in patients with cirrhosis. Gastroenterology 2003; 125: 1042-1052

3 Swanson KL, Wiesner RH, Krowka MJ. Natural history of hepatopulmonary syndrome: Impact of liver transplantation. Hepatology 2005; 41: 1122-1129

4 Cremona G, Higenbottam TW, Mayoral V, Alexander G, Demoncheaux E, Borland C, Roe P, Jones GJ. Elevated exhaled nitric oxide in patients with hepatopulmonary syndrome. Eur Respir J 1995; 8: 1883-1885

5 Rolla G, Brussino L, Colagrande P, Scappaticci E, Morello M, Bergerone S, Ottobrelli A, Cerutti E, Polizzi S, Bucca C. Exhaled nitric oxide and impaired oxygenation in cirrhotic patients before and after liver transplantation. Ann Intern Med 1998; 129: 375-378

6 Rolla G , Bucca C, Brussino L. Methylene blue in the hepatopulmonary syndrome. N Engl J Med 1994; 331: 1098

7 Brussino L, Bucca C, Morello M, Scappaticci E, Mauro M, Rolla G. Effect on dyspnoea and hypoxaemia of inhaled N(G)-nitro-L-arginine methyl ester in hepatopulmonary syndrome. Lancet 2003; 362: 43-44

8 Schenk P, Madl C, Rezaie-Majd S, Lehr S, Müller C. Methylene blue improves the hepatopulmonary syndrome. Ann Intern Med 2000; 133: 701-706

9 Gómez FP, Barberà JA, Roca J, Burgos F, Gistau C, Rodríguez-Roisin R. Effects of nebulized N(G)-nitro-L-arginine methyl ester in patients with hepatopulmonary syndrome. Hepatology 2006; 43: 1084-1091

10 Almeida JA, Riordan SM, Liu J, Galhenage S, Kim R, Bihari D, Wegner EA, Cranney GB, Thomas PS. Deleterious effect of nitric oxide inhibition in chronic hepatopulmonary syndrome. Eur J Gastroenterol Hepatol 2007; 19: 341-346

11 A r g u e d a s M R , D r a k e B B , K a p o o r A , F a l l o n M B . Carboxyhemoglobin levels in cirrhotic patients with and without hepatopulmonary syndrome. Gastroenterology 2005; 128: 328-333

12 Cody CS , DL Dunn . Endotoxins and septic shock. In: Handbook of Mediators in Septic Shock, edited by Neugebauer EA, and Holady JW. Boca Raton, FL: CRC Press, 1993: 1-38

13 Vito L, Dennis RC, Weisel RD, Hechtman HB. Sepsis presenting as acute respiratory insufficiency. Surg Gynecol Obstet 1974; 138: 896-900

14 Zhao LF, Han DW. Clinical significance of endotoxemia in patients with liver disease. Shijie Huaren Xiaohua Zazhi 1999; 7: 391-393

15 Liu JC, Han DW and Wang R. Influence of endothelin on renal

Zhang HY et al. Role of IETM in HPS of cirrhotic rats 6393

www.wjgnet.com

COMMENTS

function in rats with cirrhosis. Zhongguo Binglishengli Zazhi 1998; 14: 717-719

16 Wang R, Han DW, Liu JC and Zhao YC. Studies on the pathogenesis of renal insufficiency in acute hepatic failure. Zhongguo Binglishengli Zazhi 1999; 7: 482-486

17 Wang R, Liu JC, Han DW and Zhao YC. The dynamic changes of renal LTC4 in acute hepatic failure. Zhongguo Binglishengli Zazhi 1998; 14: 720-722

18 Wang R, Liu JC and Han DW. Eddects of endothelin on renal insufficiency in acute hepatic failure. Zhongguo Binglishengli Zazhi 1999; 15: 154-156

19 Li XQ , Han DW, Xu RL, Zhao YC, MA XH.. Hepatic encephalopathy in cirrhotic rats induced by endotoxin injection. Zhongguo Binglishengli Zazhi 1999; 15: 151-153

20 Zhang HY, Han DW, Zhao ZF, Liu MS, Wu YJ, Chen XM, Ji C. Multiple pathogenic factor-induced complications of cirrhosis in rats: a new model of hepatopulmonary syndrome with intestinal endotoxemia. World J Gastroenterol 2007; 13: 3500-3507

21 MacPhail CM, Monnet E, Gaynor JS. Evaluation of splanchnic oxygen extraction ratio as an index of portal vein pressure in dogs. Am J Vet Res 2002; 63: 15-18

22 Murphy PR, Limoges M, Dodd F, Boudreau RT, Too CK. Fibroblast growth factor-2 stimulates endothelial nitric oxide synthase expression and inhibits apoptosis by a nitric oxide-dependent pathway in Nb2 lymphoma cells. Endocrinology 2001; 142: 81-88

23 Wu F, Park F, Cowley AW, Mattson DL. Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney. Am J Physiol 1999; 276: F874-F881

24 Nanji AA, Miao L, Thomas P, Rahemtulla A, Khwaja S, Zhao S, Peters D, Tahan SR, Dannenberg AJ. Enhanced cyclooxygenase-2 gene expression in alcoholic liver disease in the rat. Gastroenterology 1997; 112: 943-951

25 Schenk P, Fuhrmann V, Madl C, Funk G, Lehr S, Kandel O, Müller C. Hepatopulmonary syndrome: prevalence and predictive value of various cut offs for arterial oxygenation and their clinical consequences. Gut 2002; 51: 853-859

26 Martínez GP, Barberà JA, Visa J, Rimola A, Paré JC, Roca J, Navasa M, Rodés J, Rodriguez-Roisin R. Hepatopulmonary syndrome in candidates for liver transplantation. J Hepatol 2001; 34: 651-657

27 Abrams GA, Jaffe CC, Hoffer PB, Binder HJ, Fallon MB. Diagnostic utility of contrast echocardiography and lung perfusion scan in patients with hepatopulmonary syndrome. Gastroenterology 1995; 109: 1283-1288

28 Krowka MJ, Wiseman GA, Burnett OL, Spivey JR, Therneau T, Porayko MK, Wiesner RH. Hepatopulmonary syndrome: a prospective study of relationships between severity of liver disease, PaO(2) response to 100% oxygen, and brain uptake after (99m)Tc MAA lung scanning. Chest 2000; 118: 615-624

29 Whyte MK , Hughes JM, Peters AM, Ussov W, Patel S, Burroughs AK. Analysis of intrapulmonary right to left shunt in the hepatopulmonary syndrome. J Hepatol 1998; 29: 85-93

30 Regev A, Yeshurun M, Rodriguez M, Sagie A, Neff GW, Molina EG, Schiff ER. Transient hepatopulmonary syndrome in a patient with acute hepatitis A. J Viral Hepat 2001; 8: 83-86

31 Silverman A, Cooper MD, Moller JH, Good RA. Syndrome of cyanosis, digital clubbing, and hepatic disease in siblings. J Pediatr 1968; 72: 70-80

32 Teuber G, Teupe C, Dietrich CF, Caspary WF, Buhl R, Zeuzem S. Pulmonary dysfunction in non-cirrhotic patients with chronic viral hepatitis. Eur J Intern Med 2002; 13: 311-318

33 Fuhrmann V, Madl C, Mueller C, Holzinger U, Kitzberger R, Funk GC, Schenk P. Hepatopulmonary syndrome in patients with hypoxic hepatitis. Gastroenterology 2006; 131: 69-75

34 Gupta D, Vijaya DR, Gupta R, Dhiman RK, Bhargava M, Verma J, Chawla YK. Prevalence of hepatopulmonary syndrome in cirrhosis and extrahepatic portal venous obstruction. Am J Gastroenterol 2001; 96: 3395-3399

35 De BK, Sen S, Biswas PK, Sanyal R, Majumdar D, Biswas J. Hepatopulmonary syndrome in inferior vena cava obstruction responding to cavoplasty. Gastroenterology 2000; 118: 192-196

36 Berthelot P, Walker JG, Sherlock S, Reid L. Arterial changes in the lungs in cirrhosis of the liver--lung spider nevi. N Engl J Med 1966; 274: 291-298

37 Rodriguez-Roisin R, Roca J, Agusti AG, Mastai R, Wagner PD, Bosch J. Gas exchange and pulmonary vascular reactivity in patients with liver cirrhosis. Am Rev Respir Dis 1987; 135: 1085-1092

38 Mélot C, Naeije R, Dechamps P, Hallemans R, Lejeune P. Pulmonary and extrapulmonary contributors to hypoxemia in liver cirrhosis. Am Rev Respir Dis 1989; 139: 632-640

39 Hedenstierna G , Söderman C, Eriksson LS, Wahren J. Ventilation-perfusion inequality in patients with non-alcoholic liver cirrhosis. Eur Respir J 1991; 4: 711-717

40 Thorens JB, Junod AF. Hypoxaemia and liver cirrhosis: a new argument in favour of a “diffusion-perfusion defect”. Eur Respir J 1992; 5: 754-756

41 Crawford AB, Regnis J, Laks L, Donnelly P, Engel LA, Young IH. Pulmonary vascular dilatation and diffusion-dependent impairment of gas exchange in liver cirrhosis. Eur Respir J 1995; 8: 2015-2021

42 Krowka MJ, Porayko MK, Plevak DJ, Pappas SC, Steers JL, Krom RA, Wiesner RH. Hepatopulmonary syndrome with progressive hypoxemia as an indication for liver transplantation: case reports and literature review. Mayo Clin Proc 1997; 72: 44-53

43 Rodríguez-Roisin R, Agustí AG, Roca J. The hepatopulmonary syndrome: new name, old complexities. Thorax 1992; 47: 897-902

44 Schraufnagel DE, Malik R, Goel V, Ohara N, Chang SW. Lung capillary changes in hepatic cirrhosis in rats. Am J Physiol 1997; 272: L139-L147

45 Lee J, Menkis AH, Rosenberg HC. Reversal of pulmonary arteriovenous malformation after diversion of anomalous hepatic drainage. Ann Thorac Surg 1998; 65: 848-849

46 Alvarez AE , Ribeiro AF, Hessel G, Baracat J , Ribeiro JD. Abernethy malformation: one of the etiologies of hepatopulmonary syndrome. Pediatr Pulmonol 2002; 34: 391-394

47 Eriksson LS, Söderman C, Ericzon BG, Eleborg L, Wahren J, Hedenstierna G. Normalization of ventilation/perfusion relationships after liver transplantation in patients with decompensated cirrhosis: evidence for a hepatopulmonary syndrome. Hepatology 1990; 12: 1350-1357

48 Vallance P, Moncada S. Hyperdynamic circulation in cirrhosis: a role for nitric oxide? Lancet 1991; 337: 776-778

49 R o l l a G . I s n i t r i c o x i d e t h e u l t i m a t e m e d i a t o r i n hepatopulmonary syndrome? J Hepatol 2003; 38: 668-670

50 Fallon MB, Abrams GA, Luo B, Hou Z, Dai J, Ku DD. The role of endothelial nitric oxide synthase in the pathogenesis of a rat model of hepatopulmonary syndrome. Gastroenterology 1997; 113: 606-614

51 Luo B, Abrams GA, Fallon MB. Endothelin-1 in the rat bile duct ligation model of hepatopulmonary syndrome: correlation with pulmonary dysfunction. J Hepatol 1998; 29: 571-578

52 Zhang J, Ling Y, Luo B, Tang L, Ryter SW, Stockard CR, Grizzle WE, Fallon MB. Analysis of pulmonary heme oxygenase-1 and nitric oxide synthase alterations in expe-rimental hepatopulmonary syndrome. Gastroenterology 2003; 125: 1441-1451

53 Nunes H, Lebrec D, Mazmanian M, Capron F, Heller J, Tazi KA, Zerbib E, Dulmet E, Moreau R, Dinh-Xuan AT, Simonneau G, Hervé P. Role of nitric oxide in hepatopulmonary syndrome in cirrhotic rats. Am J Respir Crit Care Med 2001; 164: 879-885

54 Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992; 359: 843-845

55 Risau W. Mechanisms of angiogenesis. Nature 1997; 386: 671-674

56 Shaul PW, North AJ, Brannon TS, Ujiie K, Wells LB, Nisen PA, Lowenstein CJ, Snyder SH, Star RA. Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung. Am J Respir Cell Mol Biol

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www.wjgnet.com

1995; 13: 167-17457 Melillo G, Musso T, Sica A, Taylor LS, Cox GW, Varesio L.

A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. J Exp Med 1995; 182: 1683-1693

58 Coulet F, Nadaud S, Agrapart M, Soubrier F. Identification of hypoxia-response element in the human endothelial nitric-oxide synthase gene promoter. J Biol Chem 2003; 278: 46230-46240

59 Hood JD , Meininger CJ, Ziche M, Granger HJ. VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells. Am J Physiol 1998; 274: H1054-H1058

60 Papapetropoulos A, García-Cardeña G, Madri JA, Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest 1997; 100: 3131-3139

61 Uhlmann S, Friedrichs U, Eichler W, Hoffmann S, Wiedemann

P. Direct measurement of VEGF-induced nitric oxide production by choroidal endothelial cells. Microvasc Res 2001; 62: 179-189

62 Ghiso N, Rohan RM, Amano S, Garland R, Adamis AP. Suppression of hypoxia-associated vascular endothelial growth factor gene expression by nitric oxide via cGMP. Invest Ophthalmol Vis Sci 1999; 40: 1033-1039

63 Sztrymf B, Rabiller A, Nunes H, Savale L, Lebrec D, Le Pape A, de Montpreville V, Mazmanian M, Humbert M, Hervé P. Prevention of hepatopulmonary syndrome and hyperdynamic state by pentoxifylline in cirrhotic rats. Eur Respir J 2004; 23: 752-758

64 Rabiller A, Nunes H, Lebrec D, Tazi KA, Wartski M, Dulmet E, Libert JM, Mougeot C, Moreau R, Mazmanian M, Humbert M, Hervé P. Prevention of gram-negative translocation reduces the severity of hepatopulmonary syndrome. Am J Respir Crit Care Med 2002; 166: 514-517

S- Editor Liu Y L- Editor Wang XL E- Editor Ma WH

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Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6396-6403www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

Two distinct pathways of p16 gene inactivation in gallbladder cancer

Hiroyuki Tadokoro, Takako Shigihara, Tomomi Ikeda, Masaru Takase, Masafumi Suyama

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CLINICAL RESEARCH

Hiroyuki Tadokoro, Masafumi Suyama, Department of Gastroenterology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 136-0075, JapanTakako Shigihara, Tomomi Ikeda, Biochemical Research Center, Division of Molecular and Biochemical Research, Juntendo University School of Medicine, Tokyo 136-0075, JapanMasaru Takase, Department of Human Pathology, Juntendo University School of Medicine, Tokyo 136-0075, JapanCorrespondence to: Hiroyuki Tadokoro, MD, Department of Gastroenterology, Juntendo Tokyo Koto Geriatric Medical Center, 3-3-20 Shinsuna, Koto-ku, Tokyo 136-0075,Japan. heroyou27@yahoo.co.jpTelephone: +81-356-323111 Fax: +81-356-323728Received: July 19, 2007 Revised: September 12, 2007

AbstractAIM: To examine the mechanism of inactivation of the p16 gene in gallbladder cancer, and to investigate p16 alterations and their correlation with clinicopathological features.

METHODS: Specimens were collected surgically from 51 patients with gallbladder cancer. We evaluated the status of protein expression, loss of heterozygosity (LOH), homozygous deletion and promoter hypermethylation using immunohistochemistry, microsatellite analysis, quantitative real-time polymerase chain reaction (PCR) and methylation-specific PCR, respectively. In addition, mutations were examined by direct DNA sequencing.

RESULTS: Homozygous deletions of the p16 gene exon2, LOH at 9p21-22, p16 promoter hypermethylation, and loss of p16 protein expression were detected in 26.0% (13/50), 56.9% (29/51), 72.5% (37/51) and 62.7% (32/51), respectively. No mutations were found. LOH at 9p21 correlated with the loss of p16 protein expression (P < 0.05). Homozygous deletion of the p16 gene, a combination LOH and promoter hypermethylation, and multiple LOH at 9p21 were significantly correlated with the loss of p16 protein expression (P < 0.05). LOH at 9p21 and promoter hypermethylation of the p16 gene were detected in 15.4% (2/13) and 92.3% (12/13) of the tumors with homozygous deletion of the p16 gene, respectively. P16 alterations were not associated with clinicopathological features.

CONCLUSION: Our results suggest that LOH and homozygous deletion may be two distinct pathways in the inactivation of the p16 gene. Homozygous deletion, a

combination of LOH and promoter hypermethylation, and multiple LOH are major mechanisms of p16 inactivation in gallbladder cancer.

© 2007 WJG. All rights reserved.

Key words: Gallbladder cancer; Homozygous deletion; Loss of heterozygosity; p16; Quantitative real time PCR

Tadokoro H, Shigihara T, Ikeda T, Takase M, Suyama M. Two distinct pathways of p16 gene inactivation in gallbladder cancer. World J Gastroenterol 2007; 13(47): 6396-6403

http://www.wjgnet.com/1007-9327/13/6396.asp

INTRODUCTIONGallbladder carcinoma is a highly malignant neoplasm with a poor prognosis, and most patients are diagnosed at an already advanced stage[1-3]. Gallbladder carcinoma is a relatively common cancer and is the sixth highest cause of cancer death in Japanese women.

Several genes have been implicated in the tumorigenesis of gallbladder cancers, including K-ras, cerbβ2, p53[4,5], p16INK4a/CDKN2, and the fragile histidine triad (FHIT)[6]. Genetic alterations in the 9p21 chromosomal region have been linked to malignant progression.

The p16 gene, located on chromosome 9p21, encodes a critical negative regulator of cell cycle progression and is inactivated in various cancers. The p16 gene is an important tumor suppressor gene, which interacts strongly with cyclin-dependent kinases 4 and 6, and inhibits their ability to interact with cyclin D[7]. p16 induces cell cycle arrest at G1 and G2/M checkpoints, which blocks cells from phosphorylating retinoblastoma protein 1, and prevents cells from exiting the G1 phase of the cell cycle[8]. p16 can act as a negative regulator of normal cell proliferation. Inactivation of the p16 gene plays an important role in tumorigenesis. p16 inactivation by loss of heterozygosity (LOH) and point mutations has been reported in biliary tract cancers[9] and intrahepatic cholangiocarcinoma[10].

Aberrant promoter methylation is an important mechanism in silencing cancer-related genes during the process of carcinogenesis. Epigenetic inactivation of tumor suppressor genes has been commonly reported in various tumors[11]. Promoter hypermethylation, as

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well as gene deletions and point mutations, has been shown to be a major mechanism of p16 inactivation[12,13]. Hypermethylation of the CpG islands of the p16 gene promoter region has been reported in various types of tumor.

The main modes of p16 gene inactivation in gallbladder carcinoma are known to include LOH, mutation and hypermethylation[12,14]. Homozygous deletion of the p16 gene has not previously been investigated in gallbladder cancer. Therefore, we sought to comprehensively study genetic and epigenetic alterations of p16, including homozygous deletion of the p16 gene, and the relationship between these abnormalities and clinicopathological features.

MATERIALS AND METHODSTissue specimensParaffin-embedded tissue samples were obtained from 51 patients who underwent surgical resection at Juntendo University School of Medicine, Japan, between April 1996 and April 2005. Gallbladder carcinoma patients consisted of 25 women and 26 men, ranging in age from 36 to 94 (mean, 65.1) years. Their tumors consisted of 46 adenocarcinomas and five adenosquamous carcinomas. The adenocarcinomas included 39 well-to-moderately differentiated and seven poorly differentiated tumors. Most of the patients had advanced gallbladder carcinoma, with invasion of the subserosa (n = 22, 43.1%) and serosa (n = 18, 35.3%), while the other 11 patients (19.6%) had early gallbladder carcinoma (mucosa or muscularis propria invasion). All histological slides were reviewed by M.T. and H.T. and were classified based on the WHO classification of gallbladder carcinoma. Medical records were available for all patients.

Immunohistochemical analysisImmunohistochemistry was performed using anti-p16 (F-12; 1:500 dilution; Santa Cruz Biochemistry, Santa Cruz, CA, USA) and an automated slide staining system (NexES IHC; Ventana, AZ, USA), according to the manufacturer’s instructions. P16 immunostaining was performed within 2 weeks of sectioning, because reactivity decreased over time after preparation. Normal lymphocytes and intrahepatic bile ducts were positive controls for p16. The percentage of positive nuclei was scored as follows: -, 0%-10%, + 1%-25%, ++, 25%-50%, and +++, > 50% positive cells. Scores of +, ++ and +++ were considered to represent positive immunostaining, while - was deemed to be negative.

DNA extractionFormalin-fixed, paraffin-embedded tissue blocks were used. Serial 10-μm sections were cut from each block and stained with hematoxylin and eosin to locate the tumor and non-neoplastic tissue before DNA extraction. Sections were cut, deparaffinized, and microdissected with an 18-gauge needle. The microdissected tissues were digested overnight at 55℃ in buffer (1% Tween 20, 10 mmol/L Tris-HCl, pH 8, 1 mmol/L EDTA, and 100 μg/mL proteinase K). The lysates were heated at 95℃ for 10

min and stored at 4℃ until analyzed by polymerase chain reaction (PCR).

Methylation assayDNA methylation was investigated using an EZ DNA methylation kit (Zymo Research, CA, USA), according to the manufacturer’s protocol. Microdissected genomic DNA (1 ng) was denatured with M-dilution buffer at 37℃ for 15 min, followed by incubation with CT conversion reagent at 50℃ for 16 h in the dark. After treatment, the DNA was purified using M-binding buffer, incubated with M-desulphonation buffer at room temperature for 15 min, washed with wash buffer, and finally resuspended in M-elution buffer.

Primers for the p16 gene were 5'-TTATTAGAGGGTGGGGTGGATTGT-3' (sense) and 5'-CCACCTAAATCAACCTCCAACCA-3' (antisense) for the unmethylated reactions, and 5'-TTATTAGAGGGTGGGGCGGATCGC-3' (sense) and 5'-GACCCCGAACCGCGACCGTAA-3' (antisense) for the methylated reactions, as described previously[15]. PCR reactions were started by denaturation at 95℃ for 5 min, followed by 40-45 cycles of 94℃ for 30 s, 65℃ (for methylated p16) or 60℃ (for unmethylated p16) for 45 s, and 72℃ for 60 s, with a final extension at 72℃ for 10 min. PCR products were analyzed by electrophoresis on a 3% agarose gel, and visualized by ethidium bromide staining. DNA from the Raji cell line was used as a positive control. Distilled water was used as a negative control.

LOH analysisPaired normal and tumor DNA samples were amplified by hot-start PCR, using locus-specific flanking primer pairs for five fluorescently labeled microsatellite markers, D9S171-FAM, D9S1748-FAM, D9S942-NEX, D9S974-NED, and D9S1749-NED (Figure 1). Primer sequences were obta ined f rom the NCBI UniSTS database (http://www.ncbi.nlm.nih.gov/). Markers mapping to the chromosome 9p21-22 region were used. D9S1748, D9S942 and D9S974 are within a coding sequence of the p16 gene. D9S1749 is telomeric to p16 and D9S171 is centromeric to p16. PCR was performed with an initial denaturation at 95℃ for 15 min, followed by 36 cycles of denaturation at 95℃ for 30 s, annealing at 58℃ (D9S171, D9S1748, D9S942, D9S974) or 55℃ (D9S1749) for 45 s, and extension at 72℃ for 60 s, and final extension at 72℃ for 10 min. After PCR, samples were diluted at 1:7 in formamide, heated to 95℃ for 2 min, chilled on ice, and analyzed with Genescan software on an ABI PRISM 310 genetic analyzer (PE-Applied Biosystems, Foster City, CA, USA). Allelic ratios in both normal and tumor samples were calculated and compared. The area under each peak, representing each allele in the microsatellite pair, was obtained. LOH was defined as a > 50% reduction in the tumor peak compared to that of the corresponding normal tissue. Additional bands that were not seen on normal DNA, but were observed in tumor samples, were considered evidence of microsatellite instability (MSI).

Detection of homozygous deletion of p16 exon2 Homozygous gene deletion and gene dosage of p16 exon2

in gallbladder cancer were determined using a TaqMan-based real-time PCR method. Briefly, p16 gene exon2 and the GAPDH gene were amplified in a monoplex assay. The primer sequences for detecting p16 exon2 were 5'-AGCTTCCTTTCCGTCATGC-3' (sensze) and 5'-TCATGACCTGCCAGAGAGAA-3' (antisense). The primer sequences for detecting the GAPDH gene were 5'-GCATCCTGGGCTACACTGAG-3' (sense) and 5'-AGGTGGAGGAGTGGGTGTC-3' (antisense). The probe sequence for p16 gene exon2 was FAM-TGGCTCTG, and the probe sequence for the GAPDH gene was FAM-CTCCTCTG (the three FAM-labeled probes were manufactured by Roche Applied Science, Mannheim, Germany). The real-time PCR was performed in a 25 μL final volume containing 12.5 μL of Premix Ex Taq (Perfect Real Time; Takara, Kyoto, Japan), 50 ng DNA template, 10 μmol/L of each primer, 10 μmol/L Universal Library Probe, 0.5 μL ROX reference dye (50 ×), and 5.75 μL distilled water. The thermal cycling conditions on the ABI PRISM 7500 instrument were set to 10 s at 95℃, followed by 40 cycles of 5 s at 95℃, alternating with 34 s at 60℃. DNA from lymphocytes isolated from a paraffin block was used as a positive control. All reactions were done in triplicate. Normalized gene dosage ratios were interpreted as follows: 0-0.3, homozygous deletion; 0.31-0.69, under-representation (of the test gene, relative to the reference

gene); 0.7-1.49, retention of copy number; and > 1.5 over-representation.

Mutation analysisMutation analysis was performed for the p16 gene (exons 1α, 2 and 3). Primer sequences and PCR conditions were as described previously[16,17] (Table 1). Amplification was performed using a Perkin Elmer GeneAmp 9600 Thermal cycler. After visualizing the PCR products in a 3% agarose gel, an aliquot (5 μL) of the PCR product was treated at 37℃ for 15 min with 1 μL ExoSAP-IT (GE Healthcare Biosciences, Piscataway, NJ, USA), followed by inactivation at 80℃ for 15 min. Part of this mix (6 μL) was directly sequenced using a BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems Japan, Chiba) on an automated sequencer (ABI PRISM 3100 Genetic analyzer; Perkin-Elmer, Japan).

Statistical analysisFrequency distributions were analyzed by the χ2 test. Correlations were examined between alterations of p16 and p16 expression, or between alterations of p16 and clinicopathological parameters. P < 0.05 was deemed statistically significant.

RESULTSImmunohistochemical analysisLoss of p16 protein expression occurred in 62.7% (32/51) of gallbladder cancer patients. Staining for p16 was weak in 13.7% (7/51) of patients, moderate in 17.6% (9/51), and strong in only 9.8% (5/51) of patients (Figure 1). There was no significant difference in p16 protein expression according to patient age, gender, tumor stage, T factor, N factor or histology (Table 2).

p16 statusLOH at 9p21-22 was detected in at least one marker in 56.9% (29/51) of patients. LOH occurred in 11.8%, 27.5%, 11.8%, 13.7% and 39.2% of patients who were positive for the markers D9S171, D9S1748, D9S942, D9S974 and D9S1749, respectively. Approximately 29.4% of the cases presented with LOH in a single marker, 11.8% with LOH in two markers, and 15.7% with LOH in three or more markers. LOH at the three markers D9S1748,

200 μm

200 μm

200 μm

C

B

A

Figure 1 Immunohistochemical staining of p16. A: Tumor cells showing diffuse strong positive staining of p16; B: Tumor cells showing diffuse moderate staining; C: Tumor cells showing partly weak staining.

Table 1 The primer sequence of the p16 genes

Exon Primer sequence Size(bp)

Annelingtemperature (℃)

Exon1α F: 5'-GAGAGGGGGAGAGCAGGCAG-3' 250 58R: 5'-GCAAACTTCGTCCTCCAGAGT-3'

Exon2-1 F: 5'-AGCTTCCTTTCCGTCATGC-3' 203 56R: 5'-GCAGCACCACCAGCGTG-3'

Exon2-2 F: 5'-AGCCCAACTGCGCCGAC-3' 147 58R: 5'-CCAGGTCCACGGGCAGA-3'

Exon2-3 F: 5'-TGGACGTGCGCGATGC-3' 189 56R: 5'-GGAAGCTCTCAGGGTACAAATT C-3'

Exon3 F: 5'-CCGGTAGGGACGGCAAGAGA-3' 169 58R: 5'-CTGTAGGACCCTCGGTGACTGAT GA-3'

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D9S942 and D9S974 within a coding sequence of the p16 gene was 37.3% in total. Representative examples of LOH at D9S942 and D91748 are shown in Figure 2.

Gene dosage of p16 exon 2 was successfully measured in 50 of 51 cases. Homozygous deletion of p16 exon2 was detected in 13 of 50 (26%) tumors. Overall the p16 gene was altered by homozygous deletion and LOH in 56.9% (29/51) of the tumors, indicating that alterations at this locus are involved in the vast majority of the tumors. In our analysis, LOH at 9p21-22 and homozygous deletion of p16 exon 2 were not associated with demographic variables such as age, gender, tumor histology and stage, T factor or N factor (Table 2).

p16 methylation statusHypermethylation of the p16 gene was observed in 72.5% (37/51) of the patients (Figure 3). The relationship between

p16 hypermethylation and various clinicopathological features was analyzed statistically. There was no significant correlation with the clinicopathological parameters assessed, including age, gender, tumor stage, T factor or N factor (Table 2). P16 methylation was found in 37 of 51 gallbladder cancer patients (72.5%), and loss of p16 protein expression was detected in 20 of the 37 tumors (54.1%) showing p16 hypermethylation. Our data showed that P16 protein expression was not significantly correlated with p16 hypermethylation.

p16 mutationsIn tumors without homozygous deletions, exons 1α, 2 and 3 were amplified. In five cases, which were non-informative for the markers investigated, constitutive DNA was not available. No mutations were detected in exons 1α, 2 or 3.

Table 2 Association between p16 alterations and clinicopathological variables in gallbladder cancers

p16 protein expression Methylation of p16INK4a LOH at 9p21-22 Homozygous deletion of p16 gene

Clinicopathological variables Absent Present P Absent Present P Retension LOH P Absent Present Pn n n n n n n n

Age < 65 13 7 0.789 5 15 0.753 10 10 0.427 13 6 0.481 > 65 19 12 9 22 12 19 24 7Gender Female 14 11 0.329 6 19 0.588 10 15 0.657 20 4 0.148 Male 18 8 8 18 12 14 17 9Tumor type Adenocarcinoma 28 18 0.401 14 32 0.148 19 27 0.423 35 10 0.068 Adenosquamous 4 1 0 5 3 2 2 3Differentiation grade Well-Moderate 22 17 0.144 12 27 0.907 18 21 0.115 29 10 0.16 Poor 6 1 2 5 1 6 6 0Stage 0 and ⅠA, ⅠB 15 13 0.135 9 19 0.407 15 13 0.097 20 7 0.99 ⅡA and ⅡB 17 6 5 18 7 16 17 6T factor Tis and T1 6 6 0.296 3 9 0.828 8 4 0.060 11 1 0.11 T2 and T3 26 13 11 28 14 25 26 12N factor N0 21 16 0.150 9 28 0.416 19 18 0.054 28 8 0.329 N1 11 3 5 9 3 11 9 5

All P values were revealed by χ2-test.

Figure 2 Representative example of the results of microsatellite analysis showing LOH at D9S1748 and LOH at D9S942 (right, case 25; left, case 45). The scales on the top and left side of each figure represent the size (bp) and the intensity, respectively. N: Normal; T: Tumor.

100 bp 120 bp 100 bp 120 bp

D9S1748 D9S942

800

400

0

1200

800

400

0

1200

800

400

0

800

400

0

T

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Association between p16 protein expression andalterations of p16We investigated the association between p16 protein expression and P16 alterations. LOH at 9p21-22 was not associated with the loss of p16 expression. However, LOH at three genes (D9S1748, D9S942 and D9S974) within a coding sequence of p16 was correlated with the loss of p16 expression (P < 0.05) (Tables 3 and 4). Furthermore, homozygous deletion of the p16 gene, a combination of LOH at 9p21 and promoter hypermethylation of the p16 gene, multiple LOH at 9p21 correlated with the loss of p16 protein expression (P < 0.05) (Table 5). Loss of p16 protein expression was detected in nine of 13 tumors with homozygous deletion. LOH at 9p21 was detected in only two of 13 cases with homozygous deletion, while promoter hypermethylation of the p16 gene was detected in 12 of 13 cases with homozygous deletion.

DISCUSSIONWe examined the mechanisms of p16 inactivation and the relationship between p16 alterations and clinicopathological features in gallbladder cancer. p16 alterations have been evaluated individually in previous studies, but our study is believed to be the first attempt to evaluate homozygous deletion of the p16 gene. We sought to carry out a comprehensive study of p16 gene status, investigating gene dosage, allelic status, hypermethylation, mutation, protein expression, and clinicopathological features in gallbladder cancer.

Loss of p16 protein expression has been examined in various types of cancer, including gallbladder cancer. We examined p16 protein expression by immunohistochemistry in 51 cases of gallbladder cancer. Loss of p16 protein expression has been reported to range between 24 and 76% in gallbladder cancer[14,18-20,23]. In our study, the loss of p16 expression was detected in 32 of 51 (62.7%) patients with gallbladder cancer. In addition, the loss of p16 expression has been correlated with tumor progression or

with decreased survival among patients with carcinoma of the lung, pancreas and esophagus, and malignant melanoma[21,22,24]. The correlation between p16 expression and clinicopathological factors is controversial. Ma et al[15] have reported that decreased expression of p16 is correlated with pathological grade and tumor progression in gallbladder carcinoma. However, Shi et al[16] have reported that loss of p16 protein expression is not significantly associated with any clinicopathological factors or survival. Quan et al[20] have reported that the loss of p16 expression is not associated with pathological grade. We also failed to find any association between the loss of p16 expression and clinicopathological parameters.

The mechanisms of inactivation of the p16 gene are homozygous deletion, LOH, promoter hypermethylation, rearrangement, and intragenic mutation.

Homozygous deletions are important for complete inactivation of tumor suppressor genes[13]. Previous investigators have evaluated homozygous deletion of the p16 gene in a small series of biliary tract and gallbladder cancer cell lines. Ku et al[25] have reported that homozygous deletion of the p16 gene was detected in three of six (50%) gallbladder cell lines. Yoshida et al[9] have reported that homozygous deletion of the p16 gene was detected in one of two gallbladder cell lines and in two biliary tract cell lines. Caca et al[26] have reported that homozygous deletion of the p16 gene was detected in eight of nine (88.8%) biliary tract cell lines, but homozygous deletion of the p16 gene was not detected in 21 biliary tract cancers. Homozygous deletion of the p16 gene has not previously been examined in gallbladder cancer. Our study is believed to be the first report to evaluate homozygous deletion of the p16 gene. We employed quantitative real-time PCR to evaluate homozygous deletion. In our series, homozygous deletion of the p16 gene was detected in 13 of 50

Table 5 Association between p16 immunohistochemistry and p16 alterations in gallbladder cancers

p16 immunohistochemistryPositive Negative P

Retension 3 3Hypermethyaltion 9 6LOH 1 3LOH (multiple) + Hypermethylation 2 8Multiple LOH 0 3Homozygous deletion 4 9 P < 0.05

LOH: Loss of heterozygosity; Multiple LOH: LOH in 9p21 at more than two loci.

Table 3 Association between p16 protein expression and 9p21-22

p16 protein expressionNegative Positive P

LOH (-) 12 10LOH (+) 20 9 0.292

OH was estiomated by allelic status at D9S171, D9S1748, D9S942, D9S974, D9S1749. LOH: Loss of heterozygosity.

Table 4 Association between p16 protein expression and LOH at 9p21

p16 protein expressionNegative Positive P

LOH (-) 16 16LOH (+) 16 3 0.0146

LOH was estiomated by allelic status at D9S1748, D9S942, D9S974.

200 bp

M M M M M M MU MU U U U U U UT11 T12 T13 T14 T15 T16 P N

Figure 3 Methylation status of the p16INK4a gene by methylation-specific PCR. PCR products amplified by unmethylated (U) and methylated (M) specific primers. P: Positive control; N: Negative control. Methyaltion band is at 150 bp and unmethylated band is at 234 bp.

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cases (26%). Previous studies have demonstrated that homozygous deletion of tumor suppressor genes plays an important role in the development and progression of some malignancies. However, in our series, homozygous deletion of the p16 gene was not associated with clinicopathological features.

Loss of p16 expression is correlated with homozygous deletion of the p16 gene in gallbladder cancer and other malignancies. Eight of nine biliary tract cell lines with homozygous deletion of the p16 gene showed loss of p16 expression, as reported by Caca et al[26]. In our series, loss of p16 expression correlated with homozygous deletion of the p16 gene in nine of 13 tumors. In four cases, homozygous deletion of the p16 gene did not correlate with p16 protein expression. Four tumors with homozygous deletion of the p16 gene displayed moderate to strong positive staining in immunohistochemistry. These tumors showed diffuse positive staining in some areas and partial or complete loss of p16 staining in other areas. The areas which showed loss of p16 expression were not captured during microdissection, and consequently the tumors were scored as having homozygous deletion. Previous reports have shown that the loss of p16 protein expression does not always correlate with homozygous deletion of the p16 gene[27,28].

P romote r hyper methy l a t ion o f p16 l e ads to inactivation of the gene in various cancers. In gallbladder cancer, the frequency of p16 promoter hypermethylation ranges from 24% to 56%[12,29-31]. In our study, p16 hypermethylation was found in 72.5% (37/51) of the tumors. The frequency in our study was comparatively higher than that in previous studies. Previous studies have revealed that the frequency of p16 promoter hypermethylation is not associated with tumor progression and clinicopathological characteristics[30,31]. Similarly, we found that p16 hypermethylation was not associated with any clinicopathological features. Some investigators have demonstrated that p16 hypermethylation is correlated with the loss of p16 expression in intraductal carcinoma of the liver, lung cancer, hepatocellular carcinoma and esophageal cancer[32-35]. In the present study, 20 of 30 (66.6%) cases with p16 promoter hypermethylation showed a loss of p16 expression. However, there was no correlation between promoter hypermethylation and the loss of p16 expression.

We failed to detect any p16 mutations in the present study. Previous studies have shown a frequency of p16 mutation of 0%-80% in gallbladder and biliary tract cancer and cell lines[9,12,14,25]. Ueki et al have reported that 13 of 53 (24.5%) cases of gallbladder cancer showed non-silent p16 gene mutations. Kim et al have reported that p16 mutations were detected in four of 13 (30.7%) patients with gallbladder cancer. Yoshida et al have reported that eight of 10 cases of gallbladder cancer showed p16 point mutations. These studies did not examine homozygous deletion of the p16 gene. Ku et al have reported that homozygous deletion of the p16 gene was found in three of six biliary tract cell lines, but no p16 mutation was found in the remaining three biliary tract cell lines, which did not show homozygous deletion. Caca et al[26] have reported that p16 mutations were not found in three

biliary tract cell lines and 21 biliary tract cancers, which did not show homozygous deletion of the p16 gene. In the present study, a p16 mutation was not found in any of the cases analyzed. These results suggest that the p16 mutation is associated with homozygous deletion of the p16 gene.

LOH at 9p21 has been detected in different types of tumors and cell lines[36,37]. The frequency of LOH at 9p21 in gallbladder carcinoma ranges from 38 to 60%[5,38,39]. Previous studies have demonstrated that LOH at 9p21 correlates with the loss of p16 expression in various types of cancer[22,40]. We also investigated the association between p16 protein expression and LOH at 9p21-22 in gallbladder cancer. Although an association between LOH at 9p21-22 and p16 protein expression was not found in our study, LOH at three genes (D9S1748, D9S942 and D9S974) which are located within a coding sequence of p16, correlated with loss of p16 protein expression. The mode of p16 silencing may be explained by a modification of Knudson’s two-hit model[41]. In cases which show the loss of p16 protein expression, LOH or promoter hypermethylation may have occurred in only one allele, and other mechanisms may also have been involved in other alleles.

In conclusion, we investigated comprehensively the mechanisms of inactivation of the p16 gene in gallbladder cancer, and the association between p16 alterations and clinicopathologic features. Although the mutation of p16 is a rare event in gallbladder cancer, homozygous deletion, LOH and promoter hypermethylation were frequent events. LOH at 9p21 correlated with loss of p16 protein expression. In addition, homozygous deletion of the p16 gene, combination of LOH and promoter hypermethylation, and multiple LOH at 9p21 significantly correlated with loss of p16 protein expression (P < 0.05). LOH at 9p21 was detected in only two of 13 cases with homozygous deletion, while promoter hypermethylation of the p16 gene was detected in 12 of 13 cases with homozygous deletion. Promoter hypermethylation of the p16 gene may have occurred as an earlier event, followed by homozygous deletion as a later event in cases of homozygous deletion. LOH and homozygous deletion may be two distinct pathways for inactivating the p16 gene in gallbladder cancer.

Our results suggest that multiple alterations of the p16 gene imply multiple mechanisms for the inactivation of the p16 gene in gallbladder cancer. The mechanisms may be important for the diagnosis and treatment of this disease.

ACKNOWLEDGMENTSWe thank Katsumi Miyahara, Yasuko Toi and Akemi Koyanagi for their technical assistance.

REFERENCES1 Offerhaus GJA. Tumors of the gallbladder, extrahepatic bile

ducts and ampulla of vater. atlas of tumor pathology. J Clin Pathol 2001; 54: 816

2 Lazcano-Ponce EC, Miquel JF, Muñoz N, Herrero R, Ferrecio C, Wistuba II, Alonso de Ruiz P, Aristi Urista G, Nervi F. Epidemiology and molecular pathology of gallbladder cancer. CA Cancer J Clin 2001; 51: 349-364

Tadokoro H et al . Two distinct pathways of p16 gene inactivation 6401

www.wjgnet.com

3 Misra S, Chaturvedi A, Misra NC, Sharma ID. Carcinoma of the gallbladder. Lancet Oncol 2003; 4: 167-176

4 Wistuba II, Gazdar AF, Roa I, Albores-Saavedra J. p53 protein overexpression in gallbladder carcinoma and its precursor lesions: an immunohistochemical study. Hum Pathol 1996; 27: 360-365

5 Wistuba II, Sugio K, Hung J, Kishimoto Y, Virmani AK, Roa I, Albores-Saavedra J, Gazdar AF. Allele-specific mutations involved in the pathogenesis of endemic gallbladder carcinoma in Chile. Cancer Res 1995; 55: 2511-2515

6 Wistuba II, Ashfaq R, Maitra A, Alvarez H, Riquelme E, Gazdar AF. Fragile histidine triad gene abnormalities in the pathogenesis of gallbladder carcinoma. Am J Pathol 2002; 160: 2073-2079

7 Sherr CJ. Cancer cell cycles. Science 1996; 274: 1672-16778 Weinberg RA. The retinoblastoma protein and cell cycle

control. Cell 1995; 81: 323-3309 Yoshida S, Todoroki T, Ichikawa Y, Hanai S, Suzuki H, Hori M,

Fukao K, Miwa M, Uchida K. Mutations of p16Ink4/CDKN2 and p15Ink4B/MTS2 genes in biliary tract cancers. Cancer Res 1995; 55: 2756-2760

10 Tannapfel A, Benicke M, Katalinic A, Uhlmann D, Köckerling F, Hauss J, Wittekind C. Frequency of p16(INK4A) alterations and K-ras mutations in intrahepatic cholangiocarcinoma of the liver. Gut 2000; 47: 721-727

11 Esteller M , Corn PG, Baylin SB, Herman JG. A gene hypermethylation profile of human cancer. Cancer Res 2001; 61: 3225-3229

12 Ueki T, Hsing AW, Gao YT, Wang BS, Shen MC, Cheng J, Deng J, Fraumeni JF, Rashid A. Alterations of p16 and prognosis in biliary tract cancers from a population-based study in China. Clin Cancer Res 2004; 10: 1717-1725

13 Cairns P, Polascik TJ, Eby Y, Tokino K, Califano J, Merlo A, Mao L, Herath J, Jenkins R, Westra W. Frequency of homozygous deletion at p16/CDKN2 in primary human tumours. Nat Genet 1995; 11: 210-212

14 Kim YT, Kim J, Jang YH, Lee WJ, Ryu JK, Park YK, Kim SW, Kim WH, Yoon YB, Kim CY. Genetic alterations in gallbladder adenoma, dysplasia and carcinoma. Cancer Lett 2001; 169: 59-68

15 Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996; 93: 9821-9826

16 Gazzeri S, Gouyer V, Vour'ch C, Brambilla C, Brambilla E. Mechanisms of p16INK4A inactivation in non small-cell lung cancers. Oncogene 1998; 16: 497-504

17 Iwato M, Tachibana O, Tohma Y, Arakawa Y, Nitta H, Hasegawa M, Yamashita J, Hayashi Y. Alterations of the INK4a/ARF locus in human intracranial germ cell tumors. Cancer Res 2000; 60: 2113-2115

18 Ma HB, Hu HT, Di ZL, Wang ZR, Shi JS, Wang XJ, Li Y. Association of cyclin D1, p16 and retinoblastoma protein expressions with prognosis and metastasis of gallbladder carcinoma. World J Gastroenterol 2005; 11: 744-747

19 S h i Y Z , H u i A M , L i X , T a k a y a m a T , M a k u u c h i M . Overexpression of retinoblastoma protein predicts decreased survival and correlates with loss of p16INK4 protein in gallbladder carcinomas. Clin Cancer Res 2000; 6: 4096-4100

20 Quan ZW, Wu K, Wang J, Shi W, Zhang Z, Merrell RC. Association of p53, p16, and vascular endothelial growth factor protein expressions with the prognosis and metastasis of gallbladder cancer. J Am Coll Surg 2001; 193: 380-383

21 Gerdes B, Ramaswamy A, Ziegler A, Lang SA, Kersting M, Baumann R, Wild A, Moll R, Rothmund M, Bartsch DK. p16INK4a is a prognostic marker in resected ductal pancreatic cancer: an analysis of p16INK4a, p53, MDM2, an Rb. Ann Surg 2002; 235: 51-59

22 Sabah M, Cummins R, Leader M, Kay E. Loss of heterozygosity of chromosome 9p and loss of p16INK4A expression are associated with malignant gastrointestinal stromal tumors. Mod Pathol 2004; 17: 1364-1371

23 Parwani AV, Geradts J, Caspers E, Offerhaus GJ, Yeo CJ,

Cameron JL, Klimstra DS, Maitra A, Hruban RH, Argani P. Immunohistochemical and genetic analysis of non-small cell and small cell gallbladder carcinoma and their precursor lesions. Mod Pathol 2003; 16: 299-308

24 Tanaka R, Wang D, Morishita Y, Inadome Y, Minami Y, Iijima T, Fukai S, Goya T, Noguchi M. Loss of function of p16 gene and prognosis of pulmonary adenocarcinoma. Cancer 2005; 103: 608-615

25 Ku JL, Yoon KA, Kim IJ, Kim WH, Jang JY, Suh KS, Kim SW, Park YH, Hwang JH, Yoon YB, Park JG. Establishment and characterisation of six human biliary tract cancer cell lines. Br J Cancer 2002; 87: 187-193

26 Caca K, Feisthammel J, Klee K, Tannapfel A, Witzigmann H, Wittekind C, Mössner J, Berr F. Inactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct cancers and bile tract cancer cell lines. Int J Cancer 2002; 97: 481-488

27 DeHaan RD, Kipp BR, Smyrk TC, Abraham SC, Roberts LR, Halling KC. An assessment of chromosomal alterations detected by fluorescence in situ hybridization and p16 expression in sporadic and primary sclerosing cholangitis-associated cholangiocarcinomas. Hum Pathol 2007; 38: 491-499

28 Oda Y, Yamamoto H, Takahira T, Kobayashi C, Kawaguchi K, Tateishi N, Nozuka Y, Tamiya S, Tanaka K, Matsuda S, Yokoyama R, Iwamoto Y, Tsuneyoshi M. Frequent alteration of p16(INK4a)/p14(ARF) and p53 pathways in the round cell component of myxoid/round cell liposarcoma: p53 gene alterations and reduced p14(ARF) expression both correlate with poor prognosis. J Pathol 2005; 207: 410-421

29 House MG, Wistuba II, Argani P, Guo M, Schulick RD, Hruban RH, Herman JG, Maitra A. Progression of gene hypermethylation in gallstone disease leading to gallbladder cancer. Ann Surg Oncol 2003; 10: 882-889

30 Tozawa T , Tamura G, Honda T, Nawata S, Kimura W, Makino N, Kawata S, Sugai T, Suto T, Motoyama T. Promoter hypermethylation of DAP-kinase is associated with poor survival in primary biliary tract carcinoma patients. Cancer Sci 2004; 95: 736-740

31 Takahashi T, Shivapurkar N, Riquelme E, Shigematsu H, Reddy J, Suzuki M, Miyajima K, Zhou X, Bekele BN, Gazdar AF, Wistuba II. Aberrant promoter hypermethylation of multiple genes in gallbladder carcinoma and chronic cholecystitis. Clin Cancer Res 2004; 10: 6126-6133

32 Ishikawa A, Sasaki M, Sato Y, Ohira S, Chen MF, Huang SF, Oda K, Nimura Y, Nakanuma Y. Frequent p16ink4a inactivation is an early and frequent event of intraductal papillary neoplasm of the liver arising in hepatolithiasis. Hum Pathol 2004; 35: 1505-1514

33 Wu MF, Cheng YW, Lai JC, Hsu MC, Chen JT, Liu WS, Chiou MC, Chen CY, Lee H. Frequent p16INK4a promoter hypermethylation in human papillomavirus-infected female lung cancer in Taiwan. Int J Cancer 2005; 113: 440-445

34 Matsuda Y, Ichida T, Matsuzawa J, Sugimura K, Asakura H. p16(INK4) is inactivated by extensive CpG methylation in human hepatocellular carcinoma. Gastroenterology 1999; 116: 394-400

35 Bian YS, Osterheld MC, Fontolliet C, Bosman FT, Benhattar J. p16 inactivation by methylation of the CDKN2A promoter occurs early during neoplastic progression in Barrett's esophagus. Gastroenterology 2002; 122: 1113-1121

36 Fountain JW, Karayiorgou M, Ernstoff MS, Kirkwood JM, Vlock DR, Titus-Ernstoff L, Bouchard B, Vijayasaradhi S, Houghton AN, Lahti J. Homozygous deletions within human chromosome band 9p21 in melanoma. Proc Natl Acad Sci USA 1992; 89: 10557-10561

37 Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA. Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 1994; 368: 753-756

38 Matsuo K, Kuroki T, Kitaoka F, Tajima Y, Kanematsu T. Loss of heterozygosity of chromosome 16q in gallbladder carcinoma. J Surg Res 2002; 102: 133-136

39 Hidaka E, Yanagisawa A, Sakai Y, Seki M, Kitagawa T,

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Setoguchi T, Kato Y. Losses of heterozygosity on chromosomes 17p and 9p/18q may play important roles in early and advanced phases of gallbladder carcinogenesis. J Cancer Res Clin Oncol 1999; 125: 439-443

40 Mariatos G, Gorgoulis VG, Zacharatos P, Kotsinas A, Vogiatzi T, Rassidakis G, Foukas P, Liloglou T, Tiniakos D, Angelou N, Manolis EN, Veslemes M, Field JK, Kittas C. Expression of p16(INK4A) and alterations of the 9p21-23 chromosome region

in non-small-cell lung carcinomas: relationship with tumor growth parameters and ploidy status. Int J Cancer 2000; 89: 133-141

41 Elenitoba-Johnson KS, Gascoyne RD, Lim MS, Chhanabai M, Jaffe ES, Raffeld M. Homozygous deletions at chromosome 9p21 involving p16 and p15 are associated with histologic progression in follicle center lymphoma. Blood 1998; 91: 4677-4685

S- Editor Zhu LH L- Editor Kremer M E- Editor Liu Y

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Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6404-6409www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

Left-sided gallbladder: Its clinical significance and imaging presentations

Sheng-Lung Hsu, Tai-Yi Chen, Tung-Liang Huang, Cheuk-Kwan Sun, Allan M Concejero, Leo Leung-Chit Tsang, Yu-Fan Cheng

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CLINICAL RESEARCH

Sheng-Lung Hsu, Tai-Yi Chen, Tung-Liang Huang, Leo Leung-Chit Tsang, Yu-Fan Cheng, Department of Diagnostic Radiology, Chang Gung Memorial Hospital - Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan, ChinaCheuk-Kwan Sun, Allan M Concejero, Department of Surgery, Chang Gung Memorial Hospital - Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan, ChinaSupported by Project Grant NHRI-GT-EX92-9228SP from the National Health Research Institutes and NSC 93-2314-B-182A-084 from the National Science Council, Taiwan, ChinaCorrespondence to: Yu-Fan Cheng, MD, Department of Diagnostic Radiology, Chang Gung Memorial Hospital - Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan, 123 Ta-Pei Road, Niao-Sung, Kaohsiung, 833, Taiwan, China. cheng.yufan@msa.hinet.netTelephone: +886-7-7317123-3027 Fax: +886-7-7331415Received: September 24, 2006 Revised: September 16, 2007

AbstractAIM: To assess the importance of preoperat ive diagnosis and presentation of left-sided gallbladder using ultrasound (US), CT and angiography.

METHODS: Retrospective review of 1482 patients who underwent enhanced CT scanning was performed. Left-sided gallbladder was diagnosed if a right-sided ligamentum teres was present. The image presentations on US, CT and angiography were also reviewed.

RESULTS: Left-sided gallbladder was diagnosed in nine patients. The associated abnormalities on CT imaging included portal vein anomalies, absence of umbilical portion of the portal vein in the left lobe of the liver, club-shaped portal vein in the right lobe of the liver, and difficulty in identifying segment Ⅳ. Angiography in six of nine patients demonstrated abnormal portal venous system (trifurcation type in four of six patients). The main hepatic arteries followed the portal veins in all six patients. The segment Ⅳ artery was identified in four of six patients using angiography, although segment Ⅳ was difficult to define on CT imaging. Hepatectomy was performed in three patients with concomitant liver tumor and the diagnosis of left-sided gallbladder was confirmed intraoperatively.

CONCLUSION: Left-sided gallbladder is an important clinical entity in hepatectomy due to its associated portal

venous and biliary anomalies. It should be considered in US, CT and angiography images that demonstrate no definite segment Ⅳ, absence of umbilical portion of the portal vein in the left lobe, and club-shaped right anterior portal vein.

© 2007 WJG. All rights reserved.

Key words: Left-sided gal lbladder; Angiography; Computed tomography; Ultrasound

Hsu SL, Chen TY, Huang TL, Sun CK, Concejero AM, Tsang LLC, Cheng YF. Left-s ided gal l b ladder: I ts clinical significance and imaging presentations. World J Gastroenterol 2007; 13(47): 6404-6409

http://www.wjgnet.com/1007-9327/13/6404.asp

INTRODUCTIONLeft-sided gallbladder is defined as a gallbladder located to the left side of the ligamentum teres. This anomaly can be divided into three anatomic abnormalities: a situs inversus, an ectopic left-sided gallbladder, and a right-sided ligamentum teres[1-4]. Left-sided gallbladder with right-sided ligamentum teres has been a rare anomaly since Hochstetter’s first description in 1886. Its characteristic description is a “gallbladder lying over the left side of the ligamentum teres”[1,4,5]. The associated anomalies with left-sided gallbladder include portal vein anomalies, biliary system anomalies, and segment Ⅳ atrophy[5-9]. The complex anomalies associated with left-sided gallbladder are important during hepatectomy, particularly in living donor liver transplantation. Preoperative survey of the anomalies of the portal triad is important in minimizing the incidence of postoperative biliary and vascular complications. Unknowingly, ligation of the left branch of the portal vein and bile duct that contribute to three-quarters of the liver hemodynamics will cause hepatic failure, biliary congestion and leakage[7,8,10-12]. This series reports nine cases of left-sided gallbladder with right-sided ligamentum teres. Its associated anomalies and significance during hepatectomy are identified and discussed.

MATERIALS AND METHODSWe reviewed 1528 enhanced CT scans performed

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for survey of liver tumors, other abdominal tumors, retroperitoneal tumors and abdominal pain at Chang Gung Memorial Hospital, Kaohsiung, Taiwan from 1999 to 2004. Forty-six patients were excluded because the gallbladder position or fossa could not be determined from previous hepatectomy or cholecystectomy. Of the remaining 1482 patients, nine were diagnosed to have left-sided gallbladder due to the presence of a right-sided ligament teres on enhanced CT. Among these patients, one underwent right hepatectomy and cholecystectomy for segment Ⅳ hepatocellular carcinoma (HCC). The other two patients underwent left lateral segmentectomy and partial hepatectomy for left lateral segment HCC, respectively.

RESULTSNine patients from a screening population of 1482 patients (0.6%) were diagnosed with left-sided gallbladder with enhanced CT. Ultrasound (US) was performed in eight of the nine patients, and angiography was done because of liver tumors in six patients. The clinical status and imaging findings are listed in Table 1. US demonstrated no umbilical portion of the portal vein over the left lobe of the liver in eight patients, of whom, four had an abnormal club-shaped portal vein over the right side of the liver (Figure 1A), whereas the other four patients did not clearly show a dilated portion of the portal vein on the right side of the liver. Enhanced CT demonstrated absence of the umbilical portion of the portal vein over the left lobe of the liver, and an abnormal umbilical portion of the portal vein over the right lobe of the liver, as in the US findings. Segment Ⅳ of the liver was difficult to define on CT imaging in all nine patients (Figure 1B). Angiography was performed in six of the nine patients. In four patients, the portal veins ramified to the right and left portal tributaries, and the left tributary of the portal vein diverged into lateral and medial portions. The medial portion veered to the ventral side and formed the umbilical portion, which finally joined the ligamentum teres (trifurcation type) (Figure 1C). The other two patients had the portal veins ramified to the

right, and small left portal tributaries. In these patients, the right portal vein formed the umbilical portion with some tributaries, and finally joined the ligamentum teres. The type of portal vein ramification could not be defined from conventional descriptions in these two patients (Figure 2). The main hepatic arteries ran alongside with the main portal venous tributaries in all six patients, of whom four had well-defined segment Ⅳ artery (Figure 3). One patient underwent right hepatectomy and cholecystectomy due to HCC over the right lobe of the liver. Another two patients receive left lateral segmentectomy. In these cases, the left-sided gallbladder was well-demonstrated intraoperatively (Figure 1D).

DISCUSSIONLeft-sided gallbladder with right-sided ligamentum teres is rarely reported. However, with advances in diagnostic imaging modalities, such as US, CT, MRI and angiography, left-sided gallbladder is increasingly being reported to be associated with right-sided ligamentum teres, accompanied by abnormal intrahepatic portal venous branching. An initial impression of left-sided gallbladder is made when the ligamentum teres is deviated to the right side, and a right-sided umbilical portion of the portal vein is present with difficulty in defining segment Ⅳ anatomy[5,6,8]. Our survey of nine cases is the largest report to date, and revealed an incidence of 0.6% of this anomaly, which is consistent with the previously reported incidence of 0.1%-1.2%[5,7,8,13,14].

There are several explanations for the development of left-sided gallbladder without situs inversus. First, the normal gallbladder bud may migrate to the left lobe instead of the right, and lies on the left side of the ligamentum teres[15]. In this situation, the portal vein, biliary tree and hepatic artery should be in the normal location and classified into ectopic gallbladder. Second, the gallbladder may develop directly from the left hepatic duct, and is accompanied by failure in development of the normal structure on the right side[7,15]. Our literature review

Table 1 Patient profiles and imaging results

Patientnumber

Clinical status and history

Club-shaped form portalvein (ultrasound finding1)

Left lateral segment of liver (computed tomography finding2)

Segment Ⅳ artery (angiogram finding)

Portal vein configuration(angio-portogram finding)

1 HCV, HCC, right hepatectomy

Over right lobe liver Hypertrophic From RHA Trifurcation type

2 HCC, HBV and HCV Not clear Normal size Not obvious Other type 3 Right lobe HCC Over right lobe liver Mild hypertrophic From RHA Trifurcation type 4 Right lobe HCC, HBV Over right lobe liver Normal size > From RHA Trifurcation type 5 Hysterectomy,

abdominal painNot clear Normal size NA Not available

6 Liver tumor, HBV Not clear Mild hypertrophic NA Not available 7 Multiple HCC Over right lobe liver Normal size NA Not available 8 HCC, left lateral

partial segmentectomyNot clear Normal size From LHA Trifurcation type

9 HCC, left lateralsegmentectomy

NA Normal size Not obvious Other type

1No umbilical portion over left lobe liver in all patients whose US results were available; 2Club-shaped form portal vein over right side of the liver, no umbilical portion over left lobe of the liver. No defined segment Ⅳ in any patients. HBV: Hepatitis B carrier; HCV: Hepatitis C carrier; NA: Not available; RHA: Right hepatic artery.

showed[5-7,11,15,16] only one case of left-sided gallbladder in which the cystic duct originated from the left hepatic duct[16]. The proposal of left-sided gallbladder development due to a second gallbladder originating directly from the left hepatic duct was not favored in those cases. Third, the left umbilical vein disappears and right umbilical vein partly remains. The peripheral (or placental) and central portions of the latter develop into the ligamentum teres and ligamentum venosum, respectively (Matsumoto's hypothesis)[13,17]. According to this hypothesis, the right umbilical portion should lie to the right of the gallbladder bed, but the gallbladder bed lay astride the ligamentum teres, as reported in four previous cases[7,18]. Nevertheless, this explanation also does not fit with our cases. Fourth, the gallbladder is on the left side of the ligamentum teres simply because the latter deviated to the right[5,7,16]. Maetini et al[7] have stated that there are three types of portal

vein anomalies with the umbilical portion supplying the right anterior portion. In type A, the umbilical portion is to the right of the Cantlie line, whereas the umbilical portion lies on and to the left of the Cantlie line in types B and type C, respectively (Figure 4). They have also found that in the cases of left-sided gallbladder, the right posterior segmental duct joins the common duct distal to the bifurcation, and the aberrant right anterior segmental duct lies in the groove for the ligamentum teres, at which it forms a reverse U-shaped segment similar to the portal vein (Figure 5). They believe that the gallbladder on the left side of the ligamentum teres is there simply because the latter deviated to the right. As the right lobe of the liver becomes atrophied, we propose that the ligamentum teres moves from the left (type C) to the right (type A).

Figure 1 A 50-year-old woman with chronic hepatitis C. Right trise-gmentectomy and cholecy-stectomy were performed due to the right lobe liver tumor. A: US demonstrated a club-shaped portal vein (arrow) over the right lobe of the liver and no umbilical portion over the left lobe of the liver; B: Enhanced CT demonstrated enlarged left lateral segment and a club-shaped portal vein (arrow) over the right side of the liver; C: Angiography demonstrated trifurcation type of the portal vein; D: Intraoperative photography demo-nstrated gallbladder over left side of ligamentum teres and adhesions on the left lateral segment (arrow).

DC

BA

Figure 2 A 55-year-old man with HCC, chronic hepatitis B and C. Angio-portography demonstrated bifurcation with a small branch of the left portal vein (black arrow), segmental Ⅳ branch of the portal vein (white arrow), and a club-shaped form of the right portal vein with many branches.

Figure 3 A 59-year-old woman with HCC in the segment VI of the liver and chronic hepatitis C. Angio-portography with substrate on arterial phase. The hepatic arteries (white vessels) followed the portal veins (black vessels). The segmental Ⅳ artery (arrow) was demonstrated. Right anterior side liver parenchyma was supplied from the segmental Ⅳ artery. The left hepatic artery (double arrows) originated from the common hepatic artery near the orifice of the gastroduodenal artery.

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The gallbladder moves to the left of the ligamentum teres, and the umbilical portion occupies the right side of liver.

Left-sided gallbladders have previously been reported to be associated with portal vein anomalies, biliary system anomalies, and segment Ⅳ atrophy. The associated portal vein anomalies are significant in hepatectomy, split liver transplantation, or living donor liver hepatectomy in transplantation, and can be classified into three types: the trifurcation type, bifurcation type and the other type, in which portal vein ramification cannot be defined from conventional descriptions (Figure 6)[5,19]. Intrahepatic arteries in left-sided gallbladders have not previously been described in detail[2,5]. In principle, the hepatic arteries follow the portal venous tributaries but the hepatic arterial variations and dissociation between the intrahepatic bile duct and portal vein have been reported[5,11,20,21]. In our six patients, in general, the hepatic arteries followed the portal venous tributaries (Figure 3). Angiography demonstrated the segment Ⅳ hepatic artery (Figures 3 and 7) in four of six patients, although segment Ⅳ was difficult to define on CT imaging. Segment Ⅳ atrophy is unlikely and we believe that the segment is embedded in the right lobe of the liver, and the true right lobe of the liver is relatively hypoplastic. Hypoplasia of the right lobe of the liver contributes to the ligamentum teres and umbilical portion being deviated to the right and therefore, causing a left-sided gallbladder. The dividing line between segment Ⅳ and the right lobe of the liver was not visualized, while segment Ⅳ was embedded in the right lobe of the liver. Although biliary tree anatomy was not evaluated in our patients, the bile duct and arterial branches appear to follow the portal venous tributaries of left-sided gallbladders[5,7,8,11]. If segment Ⅳ is embedded in the right lobe of the liver, the segment Ⅳ bile duct

originated from left main intrahepatic duct ran downward and ventrally formed a reverse U-shaped right anterior segmental bile duct because of the intrahepatic duct following the portal vein. The segment Ⅳ bile duct can reasonably explain the formation of a reverse U-shaped right anterior segmental duct that joins the left hepatic duct and lies in the groove for the ligamentum teres, as reported by Maetani et al and Newcombe et al[7,22,23].

A left-sided gal lbladder is an important entity during liver resection, particularly in left hepatectomy for tumors, complicated hepatolithiasis, and in liver transplantation[7,8,24]. Our experience of one patient who underwent hepatectomy for liver tumor in the segment Ⅳ with diagnosis of left-sided gallbladder and trifurcation type of portal vein anomaly aided in planning for resection (Figure 1A-D). As a result, an extended right instead of a left hepatectomy was performed. If this anomaly had not been recognized preoperatively, left hepatectomy could have been performed, and the portal vein supplying three-quarters of the liver may have been ligated, leading to

Figure 4 Maetini et al have described three types of portal vein anomalies with the umbilical portion supplying the right anterior portion. In type A, the umbilical portion is over the right side of the Cantlie line. In type B, the umbilical portion lies on the Cantlie line. In type C, the umbilical portion arises from the left portal vein with anterior segmental branches. As the ligamentum teres moves from the type C to the type A, the gallbladder moves to the left of the ligamentum teres.

CBA

Figure 5 The right anterior segmental bile duct ran downward and ventrally formed a reverse U-shape (arrow), which followed the right anterior portal vein and lay in the groove for the ligamentum teres in left-sided gallbladder. Maetani et al believe that the gallbladder on the left side of the ligamentum teres is simply because the latter deviated to the right.

A B

Figure 6 A: The trifurcation type of portal vein anomaly in a left-sided gallbladder; B: Bifurcation type of portal vein anomaly in a left-sided gallbladder. The abnormal intrahepatic portal venous branches could not be determined into these two types was divided into the other type.

Figure 7 A 64-year-old man with HCC. The segmental Ⅳ artery (arrow) from the left hepatic artery is demonstrated.

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hepatic failure, biliary congestion or bile leakage[7,8,11,12]. In two other patients, a tumor was located over the lateral segment; left hepatectomy was considered risky and only a left lateral segmental or partial hepatectomy was acceptable in both cases (Figure 8). All patients were discharged uneventfully post-hepatectomy.

Prior knowledge of a left-sided gallbladder is also important in living donor liver transplantation. In right lobe living donor liver transplantation, right lobe graft harvesting encounters double right portal veins in a patient with a left-sided gallbladder. Hepatic venoplasty or vein graft interposition for the reconstruction of the portal veins must be performed, thereby contributing to the complexity of surgery. In left lobe living donor liver transplantation, left-sided gallbladders with trifurcation-type portal vein anomaly are more technically demanding because only the left lateral segment can be used, and separation of the right anterior portal and left portal vein is mandatory[12,25-27]. The volume of the left lateral segment instead of the whole lateral lobe is calculated to determine left liver volume suitable for donation. In some cases, there is no common trunk of the left portal veins, which leads to difficulty in portal vein reconstruction and the possibility of portal vein thrombosis[8].

Anatomical variations associated with left-sided gallbladders are also important in split liver trans-plantation[12,25-27]. There are two ways of splitting the liver parenchyma suitable for transplantation: (1) dividing the right and left lobes of the liver into anatomical right or left lobes; and (2) separating the liver between the left medial segment and left lateral segment. During division of the right and left lobes of the liver, the portal venous pedicle is separated at the hepatic hilar region, with the left portal vein free and the right portal vein continuous with the main portal vein, or the double right portal veins free and the left portal vein continuous with the main portal vein. Hepatic venoplasty or vein graft interpositions are necessary to reconstruct these anomalous vessels. When separating the liver between the left medial and left lateral segments, the portal venous pedicle is usually divided at the hepatic hilar region, with the left portal vein free and the right portal vein continuous with the main portal vein. This conventional approach can preserve the right lobe, but the volume of left lateral segment may not be sufficient for donation from donors with a left-sided gallbladder. Preoperative evaluation of the left lateral

segment volume is, therefore, critical in donors with a left-sided gallbladder.

Right anterior hepatic duct joining the left hepatic duct is a common variation in left-sided gallbladders. Its angle is too acute for possible dilatation in the treatment of the postoperative residual hepatolithiasis, with eventual stenting through the T-tube tract. Percutaneous transhepatic cholangioscopic lithotomy via the left intrahepatic duct should be considered and can provide a more favorable anatomy[24,28,29]. Left-sided gallbladders are overlapped by both the ligamentum teres and falciform ligaments, as well as by the left lobe of the liver. Although laparoscopic cholecystectomy can be performed with the standard port sites with a falciform lift, more medial positioning of the gallbladder-retracting port, and placement of the right-hand operating port to the left of the midline is suggested for laparoscopic removal of left-sided gallbladders[6,30,31].

In conclusion, left-sided gallbladders are a rare anomaly. Judicious preoperative planning is important to identify associated anomalies necessary for the safe conduct of laparoscopic cholecystectomy, hepatectomy, split liver transplantation, or living donor liver hepatectomy in transplantation. If a left-sided gallbladder is found on enhanced CT imaging, further study such as angiography, CT angiography or MRI studies can provide more detailed vascular and even biliary anomalies for preoperative planning. Although left-sided gallbladders with segment Ⅳ atrophy have been reported[5,6,8], we propose that segment Ⅳ is not atrophic but embedded in the hypoplastic right lobe. However, not all cases in our study were defined by CT and angiography.

COMMENTSBackgroundLeft-sided gallbladders with right-sided ligamentum teres are a rare anomaly associated portal vein anomalies, biliary system anomalies, and the absence of segment Ⅳ. The associated anomalies are important entities during liver resection, complicated hepatolithiasis, and in liver transplantation. Preoperative diagnosis of left-sided gallbladders with associated anomalies is indispensable to minimize the incidence of operative complications.

Research frontiersAlthough preoperative diagnosis of left-sided gallbladders is important, there are only a few reported case series of left-sided gallbladders. We assessed the imaging presentation on US, CT and angiography of left-sided gallbladders, which was diagnosed if a right-sided ligamentum teres was present on CT.

Innovations and breakthroughsThis study of nine cases is the largest reported to date with detailed imaging presentation by US, CT and angiography. The etiology of left-sided gallbladders was investigated with the imaging presentations of hepatic vein anomalies, hepatic artery anomalies, biliary anomalies and the absence of segment Ⅳ of the liver.

Applications Awareness of the special imaging presentations of left-sided gallbladders on US, CT and angiography is important to make an accurate diagnosis. Identification of the associated anomalies will prevent dangerous maneuvers in hepatectomy, split liver transplantation, or living donor liver hepatectomy in transplantation.

TerminologyA left-sided gallbladder is defined as a gallbladder located to the left side of the ligamentum teres. The anomalies associated with left-sided gallbladders, including

BA

Figure 8 Trifurcation type of portal vein anomaly. A: If left hepatectomy is performed, the portal veins draining three-quarters of the liver parenchyma will be ligated. Hepatic necrosis may occur over right anterior portion of the liver parenchyma and contribute to hepatic failure; B: Left lateral segmentectomy or right hepatectomy is safe in this type portal vein anomaly.

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COMMENTS

portal vein anomalies, biliary system anomalies, and segment Ⅳ atrophy, were reported. Segment Ⅳ is one of three hepatic segments that constitute the left lobe of the liver. It lies between the falciform ligament and the vertical plane of the right hepatic vein, and is demarcated on the diaphragmatic surface of the liver by a line extrapolated from the fossa for the gallbladder to the inferior vena cava; the medial segment is supplied by medial branches of the umbilical part of the left branch of the portal vein.

Peer reviewThe authors assessed the importance of preoperative diagnosis and presentation of left-sided gallbladders using US, CT and angiography. They concluded that left-sided gallbladders are an important clinical entity in hepatectomy, because of their associated portal venous and biliary anomalies. It should be considered in US, CT and angiography images that demonstrate no definite segment Ⅳ, absence of umbilical portion of the portal vein in the left lobe, and club-shaped right anterior portal vein.

REFFERENCES 1 Hochstetter F. Anomalien der Pfortader und der Nabelvene in

Verbindung mit Defect oder Linkslage der Gallenblase. Arch Anat Entwick 1886: 369-384

2 Chuang VP. The aberrant gallbladder: angiographic and radioisotopic considerations. AJR Am J Roentgenol 1976; 127: 417-421

3 Meilstrup JW , Hopper KD, Thieme GA. Imaging of gallbladder variants. AJR Am J Roentgenol 1991; 157: 1205-1208

4 Fujita N, Shirai Y, Kawaguchi H, Tsukada K, Hatakeyama H. Left-sided gallbladder on the basis of a right-sided round ligament. Hepatogastroenterology 1998; 45: 1482-1484

5 Nagai M, Kubota K, Kawasaki S, Takayama T, BandaiY. Are left-sided gallbladders really located on the left side? Ann Surg 1997; 225: 274-280

6 Idu M, Jakimowicz J, Iuppa A, Cuschieri A. Hepatobiliary anatomy in patients with transposition of the gallbladder: implications for safe laparoscopic cholecystectomy. Br J Surg 1996; 83: 1442-1443

7 Maetani Y, Itoh K, Kojima N, Tabuchi T, Shibata T, Asonuma K, Tanaka K, Konishi J. Portal vein anomaly associated with deviation of the ligamentum teres to the right and malposition of the gallbladder. Radiology 1998; 207: 723-728

8 Asonuma K, Shapiro AM, Inomata Y, Uryuhara K, Uemoto S, Tanaka K. Living related liver transplantation from donors with the left-sided gallbladder/portal vein anomaly. Transplantation 1999; 68: 1610-1612

9 Baba Y, Hokotate H, Nishi H, Inoue H, Nakajo M. Intrahepatic portal venous variations: demonstration by helical CT during arterial portography. J Comput Assist Tomogr 2000; 24: 802-808

10 Ikoma A , Tanaka K, Hamada N, Honbo K, Yamauchi T, Ishizaki N, Taira A, Mukai M. Left-sided gallbladder wi th accessory l iver accompanied by in t rahepat i c cholangiocarcinoma. Nihon Geka Gakkai Zasshi 1992; 93: 434-436

11 Kaneoka Y, Yamaguchi A, Isogai M, Harada T. Hepatectomy for cholangiocarcinoma complicated with right umbilical portion: anomalous configuration of the intrahepatic biliary tree. J Hepatobiliary Pancreat Surg 2000; 7: 321-326

12 Hwang S , Lee SG, Park KM, Lee YJ, Ahn CS, Kim KH, Moon DB, Ha TY, Cho SH, Oh KB. Hepatectomy of living donors with a left-sided gallbladder and multiple combined anomalies for adult-to-adult living donor liver transplantation. Liver Transpl 2004; 10: 141-146

13 Matsumoto H. A newer concept of segmentation of the liver. Jpn J Med Ultrasonics 1986; 48: 551-552

14 Kuwayama M, Takeuuchi K, Tsuruoka N, Inoue Y, Shiroyama

M, Tsuru C, Nakanishi S. Ultrasonic evaluation of the different branching patterns of the portal vein in the hepatic hilum with special reference to anomalous branching patterns. Jpn J Med Ultrasonics 1989; 16: 346-353

15 Gross R. Congenital anomalies of the gallbladder. A review of 148 cases, with report of a double gallbladder. Arch Surg 1936; 32: 131-162

16 Ozeki Y , Onitsuka A, Hayashi M, Sasaki E. Left-sided gallbladder: report of a case and study of 26 cases in Japan. Nihon Geka Gakkai Zasshi 1987; 88: 1644-1650

17 Kubo S, Lee S, Yamamoto T, Edagawa A, Kinoshita H. Left-sided gallbladder associated with anomalous branching of the portal vein detected by sonography. Osaka City Med J 2000; 46: 95-98

18 Ozeki Y, Onituka A, Hino A. Anomalous branching of intrahepatic portal vein associated with anomalous position of round ligament. Kanzou 1989; 30: 372-378

19 Yang DM, Kim H, Kang JH, Park CH, Chang SK, Jin W, Kim HS. Anomaly of the portal vein with total ramification of the intrahepatic portal branches from the right umbilical portion: CT features. J Comput Assist Tomogr 2005; 29: 461-463

20 Gruttadauria S, Foglieni CS, Doria C, Luca A, Lauro A, Marino IR. The hepatic artery in liver transplantation and surgery: vascular anomalies in 701 cases. Clin Transplant 2001; 15: 359-363

21 Cheng YF , Huang TL, Chen CL, Sheen-Chen SM, Lui CC, Chen TY, Lee TY. Anatomic dissociation between the intrahepatic bile duct and portal vein: risk factors for left hepatectomy. World J Surg 1997; 21: 297-300

22 Newcombe JF, Henley FA. Left-Sided Gallbladder. A Review of the Literature and a Report of a Case Associated with Hepatic Duct Carcinoma. Arch Surg 1964; 88: 494-497

23 Nakanishi S, Shiraki K, Yamamoto K, Koyama M, Nakano T. An anomaly in persistent right umbilical vein of portal vein diagnosed by ultrasonography. World J Gastroenterol 2005; 11: 1179-1181

24 Cheng YF, Chen TY, Ko SF, Huang CC, Huang TL, Weng HH, Lee TY, Sheen-Chen SM. Treatment of postoperative residual hepatolithiasis after progressive stenting of associated bile duct strictures through the T-tube tract. Cardiovasc Intervent Radiol 1995; 18: 77-81

25 Deshpande RR, Heaton ND, Rela M. Surgical anatomy of segmental liver transplantation. Br J Surg 2002; 89: 1078-1088

26 Cheng YF, Huang TL, Lee TY, Chen TY, Chen CL. Variation of the intrahepatic portal vein; angiographic demonstration and application in living-related hepatic transplantation. Transplant Proc 1996; 28: 1667-1668

27 Rocca JP, Rodriguez-Davalos MI, Burke-Davis M, Marvin MR, Sheiner PA, Facciuto ME. Living-donor hepatectomy in right-sided round-ligament liver: importance of mapping the anatomy to the left medial segment. J Hepatobiliary Pancreat Surg 2006; 13: 454-457

28 Cheng YF, Lee TY, Sheen-Chen SM, Huang TL, Chen TY. Treatment of complicated hepatolithiasis with intrahepatic biliary stricture by ductal dilatation and stenting: long-term results. World J Surg 2000; 24: 712-716

29 Puente SG, Bannura GC. Radiological anatomy of the biliary tract: variations and congenital abnormalities. World J Surg 1983; 7: 271-276

30 Donthi R, Thomas DJ, Sanders D, Schmidt SP. Report of laparoscopic cholecystectomy in two patients with left-sided gallbladders. JSLS 2001; 5: 53-56

31 Reddy PK, Subramanian RV, Yuvaraja S. Laparoscopic cholecystectomy for left-sided gallbladder (sinistroposition). JSLS 2005; 9: 356-357

S- Editor Zhu LH L- Editor Kerr C E- Editor Lu W

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Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6410-6413www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

New method for long-term monitoring of intragastric pH

Shouko Ono, Mototsugu Kato, Yuji Ono, Masahiro Asaka

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RAPID COMMUNICATION

Shouko Ono, Mototsugu Kato, Division of Endoscopy, Hokkaido University Hospital, Sapporo 060-8648, JapanYuji Ono, Masahiro Asaka, Department of Gastroenterology, Hokkaido University Graduate School of Medicine, Sapporo 060-8648, JapanCorrespondence to: Mototsugu Kato, Division of Endoscopy, Hokkaido University Hospital Nishi-5, Kita-14, Kita-ku, Sapporo 060-8648, Japan. m-kato@med.hokudai.ac.jpTelephone: +81-11-7161161 Fax: +81-11-7067867 Received: June 20, 2007 Revised: September 13, 2007

AbstractAIM: Consecutive monitoring of intragastric pH using the Bravo® capsule.

METHODS: We put threads through a Bravo® capsule and then affixed it to the gastric wall by endoscopic hemoclipping in seven subjects. Study data were uploaded to a computer via Datalink every 48 h. In this way, repeated monitoring of intragastric pH was undertaken.

RESULTS: All subjects were able to monitor gastric pH over a 1-wk period, and f ive for > 2 wk. No complications were encountered during the monitoring. After pH monitoring, we safely retrieved the capsule endoscopically.

CONCLUSION: Clipping a Bravo® capsule onto the gastric wall enabled long-term intragastric pH monitoring. This is a methodological report of pH monitoring over a period of > 2 wk.

© 2007 WJG. All rights reserved.

Key words: Gastric pH; Ambulatory monitoring; Bravo system

Ono S, Kato M, Ono Y, Asaka M. New method for long-term monitoring of intragastric pH. World J Gastroenterol 2007; 13(47): 6410-6413

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INTRODUCTIONThe Bravo® pH monitoring system is a catheter-less system. It has been reported to be useful clinically because it enables monitoring without pain while the

subject undertakes daily activities[1-5]. The Bravo® capsule is attached to the esophageal mucosa by suction via the delivery system, and it is usually eliminated spontaneously within 1 wk[2]. According to manufacturer Medtronic, the average life of a capsule’s battery is 14 d, and consecutive pH monitoring is theoretically possible until the capsule battery goes flat. We used these characteristics of the Bravo® capsule to develop a new method for long-term monitoring of intragastric pH[6].

MATERIALS AND METHODSSubjects and materialsThe subjects of this study were seven Helicobacter pylori-negative volunteers. The Bravo® pH monitoring system (Medtronic, Shoreview, MI, USA), nylon threads, flexible overtube® (Sumitomo Bakelite, Tokyo, Japan), and stainless steel hemoclips (HX-200L-135; Olympus Optical, Tokyo, Japan) were prepared.

Preparation for Bravo® capsuleFirst, we removed the Bravo® capsule from its delivery system and we tied two nylon threads to the capsule and made each thread into a 2-cm diameter ring. Next, we confirmed a connection between the capsule and the receiver. The capsule was then calibrated in buffer solutions.

Placement of the Bravo® capsuleEndoscopic examinations with sedation were conducted on the subjects and results revealed the absence of any abnormal lesions. The flexible overtube was back-loaded onto the endoscope prior to the procedure. We carefully inserted the flexible overtube® with the tip of the endoscope inside the stomach. The Bravo® capsule was then passed through the tube into the stomach. Using hemoclips, the threads attached to the capsule were fixed on the greater curvature of the upper corpus (Figure 1).

Schedule We administered antacids to determine whether the Bravo® capsule precisely monitored changes in intragastric pH. The subject was administered 150 mg ranitidine hydrochloride orally twice daily at 8:00 and 20:00 h. No limitation of meals and activities was imposed but meal times were set at 7:00, 12:00 and 19:00 h. Alcohol and tobacco were prohibited.

Analysis of pH dataStudy data were uploaded to a computer via Datalink

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7

4

0

(Medtronic) every 48 h and analyzed using basic computer software (EXCEL; Microsoft, Redmond, WA, USA). After monitoring was completed, the fixed Bravo® capsules were removed endoscopically. We cut the threads tied to the capsule using scissors forceps (FS-3L-1; Olympus Optical), and put the capsule in the Roth Retrieval Net TM (United States Endoscopy Group, Mentor, OH, USA). Then, we retrieved the capsule by pulling it into an attachment mounted on the tip of the fiberscope.

EthicsThis study was approved by our hospital ethics committee and informed consent for participation in the study was obtained from all subjects.

RESULTSPlacement of the Bravo® capsules took less than 5 min. No complications or side effects were observed during pH monitoring. All subjects were able to perform normal daily activities. Two of the seven subjects ended gastric pH monitoring because their capsule batteries went flat (at d 7 and 13). The other five succeeded in pH monitoring for 16 consecutive days. During this study, the average rate of captured data per day was 97.7%. A selection of the pH monitoring data is shown in Figure 2.

Gastric acid secretion was suppressed, beginning on the first day of ranitidine intake. However, during the medication period, the percentage time with pH < 4 increased and the median pH decreased. At the time of re-administration, the effect of the drug developed more slowly than at the first time it was given.

Retrieval of the Bravo® capsules took less than 5 min. No capsule suffered any damage.

DISCUSSIONUsing the standard vacuum method, it is more difficult to attach the Bravo® capsule to a large lumen such as the stomach, than to the esophagus. It took us 15 min to place the capsule using the conventional method. The capsule was then eliminated on the fifth day. In previous studies of the Bravo® system, intragastric pH monitoring was carried

out for just 48 h[7-9]. Since an endoscopic procedure and administration of premedication can influence gastric acid secretion[10], a longer period for pH monitoring would be helpful for diagnosis[11-13]. In our study, we were able to monitor gastric pH for at least 1 wk.

We had some difficulty in passing threads through the capsule beforehand, but we needed only about 5 min to affix the capsule under endoscopic guidance[7]. The Bravo® system has been reported to be safe[2,14,15], although there is a report of esophageal perforation during attachment of the capsule[16]. We used a flexible overtube® so that the capsule could pass safely through the cervical esophagus, a physiologic stricture. However, complications with the flexible overtube® have been reported[17], and so care must be taken when inserting it.

In addition, the precision of long-term pH monitoring using an antimony electrode fixed in stomach may create problems. However, in our study, pH rhythm was reflected by the effects of meals and medication, but the precision itself was not influenced. Some studies have suggested that the effects of histamine H2-receptor antagonists are attenuated during continuous treatment; an effect expressed as tolerance or tachyphylaxis[18-21]. Tachyphylaxis of ranitidine was observed in our study. The rates of captured data per day in the esophagus and stomach have been reported as 97.7% to 99.3%[7,14] and 98.3%[7], respectively. In our study, the rate was high and did not decrease over the 16-d period.

After monitoring for 16 d, we endoscopically retrieved the fixed capsules from the stomach. The Bravo® capsule attached to the esophagus by suction was eliminated spontaneously through the gastrointestinal tract. However, we confirmed that a capsule fixed to the stomach for a long period did not suffer any damage.

H2-receptor antagonists and proton-pump inhibitors rapidly and potently suppress gastric acid secretion and are widely used for treatment of acid-related diseases[22-27]. However, they have some clinical weaknesses, tachyphylaxis and nocturnal gastric acid breakthrough[28-30], which have not been resolved. One reason has been the availability of only intermittent pH monitoring, but not long-term continuous monitoring. Our method is useful clinically not only for the diagnosis of acid-related diseases, but also for the elucidation of their problems.

Figure 1 Bravo® capsule in the stomach. The Bravo® capsule was placed onto the gastric wall in the greater curvature of the upper corpus. Threads attached to the capsule were fixed by hemoclips.

pH

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12:00 0:00 12:00 0:00 12:00 0:00 12:00 0:00 12:00

12:00 0:00 12:00 0:00 12:00 0:00 12:00 0:00

d 3 d 4 d 5 d 6 d 7

d 13 d 14 d 15 d 16

Figure 2 Intragastric pH graph monitored by the Bravo® pH capsule. The upper graph shows d 3-7 of pH monitoring, and the lower graph shows d 13-16 after placement of the Bravo® capsule in one subject.

In conclusion, we present a newly developed method for easy and simple long-term monitoring of intragastric pH using the Bravo® pH monitoring system.

ACKNOWLEDGMENTSWe thank Mrs. E Shouji (Department of Gastroenterology and Hematology, Hokkaido University Graduate School of Medicine) and Mr. T Iwasaki (Medical Care Support Division, Hokkaido University Hospital) for endoscopic support.

COMMENTSBackgroundThe Bravo® capsule is usually attached to esophageal mucosa by suction via the delivery system and eliminated spontaneously within 1 wk. However, consecutive pH monitoring is theoretically possible until the capsule battery goes flat.

Research frontiersIt has been reported that an endoscopic procedure and administration of premedication can influence gastric acid secretion when using the Bravo® pH monitoring system. In previous studies of the Bravo® system using its conventional vacuum method of attachment, it was only possible to carry out intragastric pH monitoring for 48 h.

Innovations and breakthroughsClipping of a Bravo® capsule onto the gastric wall enabled intragastric pH monitoring for > 2 wk. The methodology is easy and simple.

Applications Our method is clinically useful, not only for diagnosis of acid-related diseases, but also for the elucidation of tolerance of H2-receptor antagonists and nocturnal gastric acid breakthrough of proton-pump inhibitors.

TerminologyBravo® pH monitoring system is a catheter-less pH monitoring system.

Peer reviewThe authors have reported a modified method for long-term monitoring of intragastric pH using the Bravo capsule fixed on the gastric wall by endoscopic hemoclipping. The contents of the manuscript are reasonable, and this may be a useful method for the elucidation of tolerance to H2-receptor antagonists and nocturnal gastric acid breakthrough of proton-pump inhibitors, as the authors state.

REFERENCES 1 Remes-Troche JM, Ibarra-Palomino J, Carmona-Sánchez RI,

Valdovinos MA. Performance, tolerability, and symptoms related to prolonged pH monitoring using the Bravo system in Mexico. Am J Gastroenterol 2005; 100: 2382-2386

2 Pandolfino JE, Richter JE, Ours T, Guardino JM, Chapman J, Kahrilas PJ. Ambulatory esophageal pH monitoring using a wireless system. Am J Gastroenterol 2003; 98: 740-749

3 Gillies RS, Stratford JM, Booth MI, Dehn TC. Oesophageal pH monitoring using the Bravo catheter-free radio capsule. Eur J Gastroenterol Hepatol 2007; 19: 57-63

4 Pandolfino JE, Schreiner MA, Lee TJ, Zhang Q, Boniquit C, Kahrilas PJ. Comparison of the Bravo wireless and Digitrapper catheter-based pH monitoring systems for measuring esophageal acid exposure. Am J Gastroenterol 2005; 100: 1466-1476

5 Fox M. Bravo wireless versus catheter pH monitoring systems. Gut 2006; 55: 434-435

6 Ono S, Kato M, Ono Y, Nakagawa M, Shimizu Y, Takeda H, Asaka M. A new procedure for 16 consecutive days monitoring of intragastric pH. J Clin Gastroenterol 2008;42:960

7 Yamaguchi T, Seza A, Odaka T, Shishido T, Ai M, Gen S, Kouzu T, Saisho H. Placement of the Bravo wireless pH monitoring capsule onto the gastric wall under endoscopic guidance. Gastrointest Endosc 2006; 63: 1046-1050

8 Pandolfino JE, Schreiner MA, Lee TJ, Zhang Q, Kahrilas PJ. Bravo capsule placement in the gastric cardia: a novel method for analysis of proximal stomach acid environment. Am J Gastroenterol 2005; 100: 1721-1727

9 Mori M, Suzuki H, Masaoka T, Imaeda H, Nomoto Y, Hosoda H, Nishizawa T, Kangawa K, Hibi T. Intravenous ghrelin administration enhances gastric acid secretion - evaluation using wireless pH capsule. Aliment Pharmacol Ther 2006; 24 Suppl 4: 96-103

10 Ward EM, Devault KR, Bouras EP, Stark ME, Wolfsen HC, Davis DM, Nedrow SI, Achem SR. Successful oesophageal pH monitoring with a catheter-free system. Aliment Pharmacol Ther 2004; 19: 449-454

11 Bhat YM, McGrath KM, Bielefeldt K. Wireless esophageal pH monitoring: new technique means new questions. J Clin Gastroenterol 2006; 40: 116-121

12 Ahlawat SK, Novak DJ, Williams DC, Maher KA, Barton F, Benjamin SB. Day-to-day variability in acid reflux patterns using the BRAVO pH monitoring system. J Clin Gastroenterol 2006; 40: 20-24

13 Tseng D, Rizvi AZ, Fennerty MB, Jobe BA, Diggs BS, Sheppard BC, Gross SC, Swanstrom LL, White NB, Aye RW, Hunter JG. Forty-eight-hour pH monitoring increases sensitivity in detecting abnormal esophageal acid exposure. J Gastrointest Surg 2005; 9: 1043-1051; discussion 1051-1052

14 Wenner J , Johnsson F, Johansson J, Oberg S. Wireless oesophageal pH monitoring: feasibility, safety and normal values in healthy subjects. Scand J Gastroenterol 2005; 40: 768-774

15 Marchese M, Spada C, Iacopini F, Familiari P, Shah SG, Tringali A, Costamagna G. Nonendoscopic transnasal placement of a wireless capsule for esophageal pH monitoring: feasibility, safety, and efficacy of a manometry-guided procedure. Endoscopy 2006; 38: 813-818

16 Fajardo NR, Wise JL, Locke GR, Murray JA, Talley NJ. Esophageal perforation after placement of wireless Bravo pH probe. Gastrointest Endosc 2006; 63: 184-185

17 Holderman WH, Etzkorn KP, Patel SA, Harig JM, Watkins JL. Endoscopic findings and overtube-related complications associated with esophageal variceal l igat ion. J Cl in Gastroenterol 1995; 21: 91-94

18 Wilder-Smith C, Halter F, Ernst T, Gennoni M, Zeyen B, Varga L, Roehmel JJ, Merki HS. Loss of acid suppression during dosing with H2-receptor antagonists. Aliment Pharmacol Ther 1990; 4 Suppl 1: 15-27

19 Nwokolo CU, Smith JT, Gavey C, Sawyerr A, Pounder RE. Tolerance during 29 days of conventional dosing with cimetidine, nizatidine, famotidine or ranitidine. Aliment Pharmacol Ther 1990; 4 Suppl 1: 29-45

20 Lachman L, Howden CW. Twenty-four-hour intragastric pH: tolerance within 5 days of continuous ranitidine administration. Am J Gastroenterol 2000; 95: 57-61

21 Komazawa Y, Adachi K, Mihara T, Ono M, Kawamura A, Fujishiro H, Kinoshita Y. Tolerance to famotidine and ranitidine treatment after 14 days of administration in healthy subjects without Helicobacter pylori infection. J Gastroenterol Hepatol 2003; 18: 678-682

22 Welage LS. Overview of pharmacologic agents for acid suppression in critically ill patients. Am J Health Syst Pharm 2005; 62: S4-S10

23 Richardson P, Hawkey CJ, Stack WA. Proton pump inhibitors. Pharmacology and rationale for use in gastrointestinal disorders. Drugs 1998; 56: 307-335

24 Sanders SW. Pathogenesis and treatment of acid peptic disorders: comparison of proton pump inhibitors with other antiulcer agents. Clin Ther 1996; 18: 2-34; discussion 1

25 Langtry HD, Markham A. Rabeprazole: a review of its use in acid-related gastrointestinal disorders. Drugs 1999; 58: 725-742

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COMMENTS

26 Adachi K, Komazawa Y, Mihara T, Azumi T, Fujisawa T, Katsube T, Furuta K, Kinoshita Y. Comparative study of the speed of acid-suppressing effects of oral administration of cimetidine and famotidine. J Gastroenterol Hepatol 2005; 20: 1012-1015

27 Abe Y, Inamori M, Togawa J, Kikuchi T, Muramatsu K, Chiguchi G, Kawamura H, Kobayashi N, Kirikoshi H, Sakaguchi T, Takamura T, Nakajima A, Ueno N, Sekihara H. The comparative effects of single intravenous doses of omeprazole and famotidine on intragastric pH. J Gastroenterol 2004; 39: 21-25

28 Peghini PL, Katz PO, Bracy NA, Castell DO. Nocturnal recovery of gastric acid secretion with twice-daily dosing of proton pump inhibitors. Am J Gastroenterol 1998; 93: 763-767

29 Katsube T, Adachi K, Kawamura A, Amano K, Uchida Y, Watanabe M, Kinoshita Y. Helicobacter pylori infection influences nocturnal gastric acid breakthrough. Aliment Pharmacol Ther 2000; 14: 1049-1056

30 Katz PO , Anderson C, Khoury R, Castell DO. Gastro-oesophageal reflux associated with nocturnal gastric acid breakthrough on proton pump inhibitors. Aliment Pharmacol Ther 1998; 12: 1231-1234

S- Editor Zhu LH L- Editor Kerr C E- Editor Lu W

Ono S et al . Long-term intragasric pH monitoring 6413

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Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6414-6418www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

Personality factors and profiles in variants of irritable bowel syndrome

Alireza Farnam, Mohammad H Somi, Firouz Sarami, Sara Farhang, Sanaz Yasrebinia

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RAPID COMMUNICATION

Alireza Farnam, Mohammad H Somi, Firouz Sarami, Sara Farhang, Sanaz Yasrebinia, Liver and Gastrointestinal Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, IranCorrespondence to: Sara Farhang, Liver and Gastrointestinal Diseases Research Center, Imam Hospital, University Ave, Tabriz, East Azerbaijan, Iran. sfarhang@yahoo.comTelephone: +98-411-3367473 Fax: +98-411-3367499Received: July 23, 2007 Revised: September 12, 2007

AbstractAIM: To study the association between irritable bowel syndrome (IBS) variants (constipation, diarrhea, or both) and personality traits in non-psychiatric patients.

METHODS: IBS was diagnosed using the Rome Ⅱ diagnostic criteria after exclusion of organic bowel pathology. The entry of each patient was confirmed following a psychiatric interview. Personality traits and the score of each factor were evaluated using the NEO Five Factor Inventory.

RESULTS: One hundred and fifty patients were studied. The mean age (± SD) was 33.4 (± 11.0) year (62% female). Subjects scored higher in neuroticism (26.25 ± 7.80 vs 22.92 ± 9.54, P < 0.0005), openness (26.25 ± 5.22 vs 27.94 ± 4.87, P < 0.0005) and conscientiousness (32.90 ± 7.80 vs 31.62 ± 5.64, P < 0.01) compared to our general population derived from universities of Iran. Our studied population consisted of 71 patients with Diarrhea dominant-IBS, 33 with Constipation dominant-IBS and 46 with Altering type-IBS. Scores of conscientiousness and neuroticism were significantly higher in C-IBS compared to D-IBS and A-IBS (35.79 ± 5.65 vs 31.95 ± 6.80, P = 0.035 and 31.97 ± 9.87, P = 0.043, respectively). Conscientiousness was the highest dimension of personality in each of the variants. Patients with C-IBS had almost similar personality profiles, composed of higher scores for neuroticism and conscientiousness, with low levels of agreeableness, openness and extraversion that were close to those of the general population.

CONCLUSION: Differences were observed between IBS patients and the general population, as well as between IBS subtypes, in terms of personality factors. Patients with constipation-predominant IBS showed similar personality profiles. Patients with each subtype of IBS may benefit from psychological interventions, which

can be focused considering the characteristics of each subtype.

© 2007 WJG. All rights reserved.

Key words: Irritable bowel syndrome; Personality; Conscientiousness; Neuroticism; Openness; Constipation-predominant

Farnam A, Somi MH, Sarami F, Farhang S, Yasrebinia S. Personality factors and profile in variants of irritable bowel syndrome. World J Gastroenterol 2007; 13(47): 6414-6418

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INTRODUCTIONIrritable bowel syndrome (IBS) is a common functional gastrointestinal disorder with a wide variety of presentations that include abdominal pain, bloating, disturbed defecation (constipation and/or diarrhea) or alternating bowel habits, and the absence of any detectable organic pathological process[1]. Symptom-based criteria along with limited medical evaluation are used for diagnosis. Treatment is challenging because of the heterogeneity of the presenting symptoms, together with the unclear pathophysiology of the disorder. To date, treatment strategies are focused on specific symptoms, potential underlying disorders in stress responsiveness, and predisposing psychological features.

Regardless of the unclear etiology of the syndrome, it is commonly accepted as being a disorder closely influenced by affective factors. IBS seems to be influenced by psychosocial stressors and psychiatric comorbidity. The incidence of mood and anxiety disorders has been well studied in IBS patients. The high rates of psychiatric co-morbidity in IBS patients indicate that the affective symptoms may be specific and integral to the syndrome, rather than be a specific syndrome related to a chronic intestinal disease[2]. This has resulted in recommendations on how to best detect and integrate treatments to achieve better outcomes for these patients[3], and has led to significant improvements. Patients and physicians might benefit from detailed identification and psychotherapeutic intervention in patients with comorbid psychiatric disorders (as opposed to psychodynamic personality dysfunction).

However, the underlying personality structure might be misinterpreted at the present time because of the

Farnam A et al . Personality in IBS subtypes 6415

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poor quality and dubious results of early research done in the area of personality factors as they relate to IBS[4]. The five-factor model of personality defines personality traits in terms of five basic dimensions: extraversion, which incorporates talkativeness, assertiveness and activity level; agreeableness, which includes kindness, trust and warmth; conscientiousness, which includes organization, thoroughness and reliability; neuroticism versus emotional stability, which includes nervousness, moodiness and temperamentality; and openness to experience, which incorporates imagination, curiosity and creativity. This model has been widely accepted because the structure of traits in it is consistent among highly diverse cultures with various languages, and between men and women, and older and younger adults[5].

Patients are often subclassified by their predominant bowel habits, that is, constipation-predominant, diarrhea-predominant, or alternating diarrhea and constipation. Patients with IBS share basic pathophysiological features, regardless of bowel habits; however, differences in perception, autonomic function, and symptom characteristics between constipation-predominant and diarrhea-predominant patients have been described[6-8]. Psychological treatment has been reported to be more effective for diarrhea- than constipation-predominant patients[9]. The association between psychological features and specific symptoms of IBS has been minimally explored. We hypothesized that personality traits are also associated with the dominant symptom of IBS. We sought to assess the distribution of personality traits in IBS patients in our cohort as a first step, and then define any relationship with dominant symptoms.

MATERIALS AND METHODSContinuous patients attending our university outpatient clinics with a diagnosis of IBS were included. Diagnosis was established after a stool examination, clinical evaluation and endoscopy (in some cases) by a gastroenterologist using the ROME Ⅱ criteria for IBS. All patients were clinically investigated to identify the presence of “alarm factors”. Patients were excluded if an organic cause of the condition were possible, or if there were a history of serious somatic disease. All patients gave informed written consent. Demographic information, severity and course of illness, abdominal pain severity over previous weeks, bowel habits and gastrointestinal symptoms were obtained. Patients described whether their symptoms arose with stress and if/how they disrupted their daily activities. Patients were divided into three groups: constipation-predominant (C-IBS), diarrhea-predominant (D-IBS) and altering diarrhea or constipation (A-IBS), according to their self-explanation of recent symptoms.

Patients were referred to the first author, who was blinded to characteristics of IBS, for psychiatric and psychosomatic assessments. A history or current symptoms of any DSM-Ⅳ psychiatric diagnosis[11] on axis Ⅰ or seizure disorders led to exclusion from the study.

Personality dimensions in non- psychiatric IBS patients were evaluated by the NEO Five-Factor Inventory (NEO-FFI), a 60-item questionnaire which usually requires 10-15

min to complete. This questionnaire is rated on a five-point scale to yield scores in five major domains of personality and requires a sixth-grade reading level. Scores of five personality factors measured by NEO have previously been described in a survey of an Iranian population of all universities[10].

Statistical analysisData were analyzed using the SPSS Statistical package ver.13 (SPSS, Chicago, IL, USA). Means ± SD were used to describe continuous variables and proportions for categorical data. Conditions were met for using two-tailed Student’s t test and Chi-square test, which were applied when appropriate. Within- and between-group comparisons were performed using ANOVA. The Bonferroni inequality test was used to affirm that significance was not reached by chance alone. Overall significance was set at 0.05.

RESULTSOne hundred and fifty patients age, mean ± SD was 33.4 ± 11.0 year (62% female), with IBS by ROME Ⅱ criteria were enrolled in the study. Bowel problems that were provoked by distress in > 80% of patients interrupted daily activities in up to 70%.

NEO-FFI showed a significantly higher level of neuroticism and conscientiousness and a lower level of openness in the non-psychiatric IBS patients. Table 1 shows mean scores for five personality factors in our patients compared to the Iranian general population (by NEO FFI)[12]. Women with IBS had significantly higher levels of neuroticism, conscientiousness and extraversion compared to men (P = 0.032, 0.003 and 0.037 in that order) (Figure 1).

Our study population consisted of 71 patients with D-IBS, 33 with C-IBS and 46 with A-IBS. Patient occupation, educational level and marriage status had similar patterns among the groups. Symptoms reported by patients with C-IBS and A-IBS were more related to stressors (P = 0.004).

The score for conscientiousness was significantly higher in C-IBS (35.79 ± 5.65) than D-IBS (31.95 ± 6.80) and A-IBS (31.97 ± 9.87) (P = 0.035 and 0.043). Neuroticism had a higher score in C-IBS compared to the other groups (P = 0.044). Conscientiousness was the highest dimension of personality in each of the variants: 42% of C-IBS, 55% of D-IBS and 47% of A-IBS patients.

Personality profiles were somewhat capricious in

Table 1 Mean (SD) scores of five personality factors measured by FFI in patients with irritable bowel syndrome compared to the Iranian general population

IBS patients General population POpenness 26.25 (5.22) 27.94 (4.87) < 0.0005Conscientiousness 32.90 (7.80) 31.62 (5.64) < 0.0005Extraversion 27.06 (6.09) 26.89 (6.15) 0.733Agreeableness 28.97 (6.74) 32.90 (7.00) 0.344Neuroticism 26.25 (7.80) 22.92 (9.54) < 0.0005

patients with A-IBS and D-IBS. Whereas, patients with C-IBS had mostly similar personality profiles (Figure 2), which showed higher scores for neurotic ism and conscientiousness, a low level of agreeableness, along with openness and extraversion close to those of the general population.

DISCUSSIONThe present study was designed to investigate personality characteristics of non-psychiatric IBS patients considering their IBS subtype. Emotional states and personality traits may affect the physiology of the gut[13], and play a role in how symptoms are experienced and interpreted, and can thus influence treatment[14,15]. This can be an important issue when considering a management strategy to achieve a better outcome for an IBS patient. The prevalence of IBS has been reported to be 18.4% in the general Iranian population[16].

The five-factor model provides a dimensional account of the structure of normal personality traits, dividing personality into five broad dimensions. There has been little research examining the biological correlates of the dimensions and very little in known about the personality structure in IBS patients. Neuroticism and aggression are reported to be higher in patients with functional gastrointestinal disease without psychiatric comorbidity, and personality traits are believed to influence pain reporting[17]. A low level of neuroticism and little concealed aggressiveness is reported to predict treatment outcome with antidepressants in non-psychiatric patients, which are most prominent in women. These personality dimensions are better predictors of outcome than serotonergic sensitivity[18].

Differences between male and female patients with IBS have been reported; the significant differences found here in the traits of neuroticism, extraversion and conscientiousness were consistent with other studies that suggest women consistently score higher than men on self-reported trait

anxiety[19,20]. The data for non-psychiatric individuals drawn from a pool prepared for standardization of the Iranian version of NEO PI-R are among the limitations of the present study. For a more appropriate comparison, the control individuals in future studies might be beneficially selected from the member of patients' family.

Studies on personality dimensions according to subtypes of IBS are limited. Similar personality dimensions (by the Minnesota Multiphasic Personality Inventory) have been reported in subgroups of IBS patients with predominant constipation and for those with predominant diarrhea[21].

In the current study, C-IBS patients scored higher on neuroticism and conscientiousness. Neuroticism is a personality trait characterized by overstated reactivity to physiological changes. According to Costa and McCrae[5], people with elevated scores on the neuroticism dimension are emotionally unstable with overwhelmingly negative emotions. Neuroticism is related to emotional intelligence, which involves emotional regulation, motivation, and interpersonal skills. Hans Eysneck theorized that neuroticism is a function of activity in the limbic system, and research suggests that people who score high on neuroticism have a more reactive sympathetic nervous system, and are more sensitive to environmental stimulation[22]. Centrally targeted medications, such as anxiolytics and low-dose tricyclic antidepressants, which involve inhibitory effects on the sensitivity of emotional motor system[23] are widely used for C-IBS patients[24] as they can balance the neuroticism dimension of such patients.

Individuals with high self-consciousness are not at ease with others, are sensitive to ridicule, and prone to feelings of inferiority. This is compatible with a lack of success in completing the “anal stage” of Freud’s theory of psychosexual development, which is supposed to result in an obsession with perfection and control. Such patients may benefit from selective serotonin re-uptake inhibitors which increase the extracellular level of the neurotransmitter serotonin.

Personality disorders can be conceptualized as extreme variants of the normal personality dimensions[25]. Excluding such disorders, we sought the personality make up that can introduce vulnerable profiles for IBS in a cross-sectional manner. Research has focused on incorporating various forms of psychotherapy in the hope of alleviating symptoms. Psychological interventions

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Figure 1 Scores of personality factors in irritable bowel syndrome patients, defined by dominant symptom. Box plots show distribution of the scores, defining means, minimum, maximum, range, interquartile range and cases out of 95% confidence intervals( as1). O: Openness to Experience; C: Conscientiousness; E: Extraversion; A: Agreeableness; N: Neuroticism.

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Figure 2 Personality profiles in patients with C-IBS. 1: Openness to Experience; 2: Conscientiousness; 3: Extraversion; 4: Agreeableness; 5: Neuroticism.

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have been aimed at individuals and at groups and include insight-oriented psychotherapy, hypnotherapy, behavior therapy and group psychotherapy. Such interventions try to address various unconscious conflicts in the subject and thereby help them re-establish a sense of emotional stability[26], modify maladaptive behavior and seek new solutions to problems[27], incorporate multi-component cognitive-behavioral therapy treatment programs[28], and have reported significant improvement in symptoms. It is felt that the profession of counseling psychology, which looks to develop wellness, strength and resources within individuals, has the potential to make a unique contribution to the prevention and alleviation of IBS[29]. Presently, the effectiveness of psychological treatments in IBS is being reviewed[30] in the light of conflicting evidence that supports the use of psychological treatment, an inadequate methodology for randomized controlled trials in this area, and the limited evidence of how to improve the global health of IBS patients with drug therapy[31]. The present study presents added evidence of the differences between subgroups of IBS. As such, this may help to focus management plans in each subgroup to obtain better outcomes.

ACKNOWLEDGMENTSWe are grateful to Mrs. Maryam Akbari for her helpful assistance.

COMMENTSBackgroundIrritable bowel syndrome (IBS) is a common functional gastrointestinal disorder that is commonly accepted as a disorder closely related to psychological factors. Patients might benefit from detailed identification and psychotherapeutic interventions.

Research frontiersMood and anxiety disorders are well studied in IBS patients but information about any underlying personality structure is lacking. Additionally, differences in subtypes of IBS (according to dominant symptom) have been described in perception, autonomic function, and symptom characteristics, which are recommended for evaluation in IBS patients according to their presenting symptoms. This report elucidates personality characteristics of IBS patients according to their dominant symptom using a well-designed personality questionnaire.

Innovations and breakthroughsIBS patients scored higher on neuroticism, openness and conscientiousness compared to the general Iranian population. Conscientiousness was the highest dimension of personality in each of the variants. The scores for conscientiousness and neuroticism were significantly higher in C-IBS compared to D-IBS and A-IBS. An analogous personality profile was noted in patients with C-IBS composed of the higher scores of neuroticism and conscientiousness, a low level of agreeableness, and with openness and extraversion similar to those of the general population.

Applications Overstated personality dimensions of IBS patients (especially the constipation-dominant subtype) can be balanced by the use of well-known medications, like anxiolytics, low-dose tricyclic antidepressants and selective serotonin re-uptake inhibitors. Given the added evidence of the differences between subtypes of IBS and vulnerable personality profiles, future studies on drug or psychotherapy may achieve more reliable outcomes.

TerminologyIBS is a gastrointestinal disorder that has a wide variety of presentations that

include abdominal pain, bloating, disturbed defecation (constipation and/or diarrhea or alternating bowel habits) and the absence of any detectable organic pathological process. NEO-FFI is a questionnaire with a five-point scale to yield scores in five major domains of personality: openness to experience, conscientiousness, extraversion, agreeableness and neuroticism

Peer reviewThe results and conclusions of the paper are reliable. The presentation is adequate and easy to read. There are no ethics problems. This is a good manuscript with an interesting approach to the subject.

REFERENCES1 Camilleri M, Choi MG. Review article: irritable bowel

syndrome. Aliment Pharmacol Ther 1997; 11: 3-152 Walker EA, Katon WJ, Jemelka RP, Roy-Bryne PP. Comorbidity

of gastrointestinal complaints, depression, and anxiety in the Epidemiologic Catchment Area (ECA) Study. Am J Med 1992; 92: 26S-30S

3 Drossman DA, Corazziari E, Talley NJ, Thompson WG, Whitehead WE. Rome II: a multinational consensus document on functional gastrointestinal disorders. Gut 1999; 45 Suppl II :II1–II81

4 Olden KW. Irritable bowel syndrome: What is the role of the psyche? Dig Liver Dis 2006; 38: 200-201

5 Costa PT, McCrae RR. Stability and change in personality assessment: the revised NEO Personality Inventory in the year 2000. J Pers Assess 1997; 68: 86-94

6 Elsenbruch S , Orr WC. Diarrhea- and constipation-predominant IBS patients differ in postprandial autonomic and cortisol responses. Am J Gastroenterol 2001; 96: 460-466

7 Aggarwal A, Cutts TF, Abell TL, Cardoso S, Familoni B, Bremer J, Karas J. Predominant symptoms in irritable bowel syndrome correlate with specific autonomic nervous system abnormalities. Gastroenterology 1994; 106: 945-950

8 Heitkemper M, Jarrett M, Cain KC, Burr R, Levy RL, Feld A, Hertig V. Autonomic nervous system function in women with irritable bowel syndrome. Dig Dis Sci 2001; 46: 1276-1284

9 Tillisch K, Labus JS, Naliboff BD, Bolus R, Shetzline M, Mayer EA, Chang L. Characterization of the alternating bowel habit subtype in patients with irritable bowel syndrome. Am J Gastroenterol 2005; 100: 896-904

10 Garousi MT. Application of the NEO PIR test and analytic evaluation of its characteristics and factorial structure among Iranian university students. Human Sci Alzahra Uni 2001; 11: 30-38

11 American Psychiatric Association. American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders. 4th ed. Washington DC: American Psychiatric Association, 1994: 320-327

12 Guthrie E, Creed F, Dawson D, Tomenson B. A controlled trial of psychological treatment for the irritable bowel syndrome. Gastroenterology 1991; 100: 450-457

13 Wood JD , Alpers DH, Andrews PL. Fundamentals of neurogastroenterology. Gut 1999; 45 Suppl 2: II6-II16

14 Bennett EJ, Piesse C, Palmer K, Badcock CA, Tennant CC, Kellow JE. Functional gastrointestinal disorders: psychological, social, and somatic features. Gut 1998; 42: 414-420

15 Drossman DA, Creed FH, Olden KW, Svedlund J, Toner BB, Whitehead WE. Psychosocial aspects of the functional gastrointestinal disorders. Gut 1999; 45 Suppl 2: II25-II30

16 Ghannadi K, Emami R, Bashashati M, Tarrahi MJ, Attarian S. Irritable bowel syndrome: an epidemiological study from the west of Iran. Indian J Gastroenterol 2005; 24: 225-226

17 Tanum L, Malt UF. Personality and physical symptoms in nonpsychiatric patients with functional gastrointestinal disorder. J Psychosom Res 2001; 50: 139-146

18 Tanum L, Malt UF. Personality traits predict treatment outcome with an antidepressant in patients with functional gastrointestinal disorder. Scand J Gastroenterol 2000; 35: 935-941

19 Fock KM, Chew CN, Tay LK, Peh LH, Chan S, Pang EP.

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COMMENTS

Psychiatric illness, personality traits and the irritable bowel syndrome. Ann Acad Med Singapore 2001; 30: 611-614

20 Tran Y, Craig A, Boord P, Connell K, Cooper N, Gordon E. Personality traits and its association with resting regional brain activity. Int J Psychophysiol 2006; 60: 215-224

21 Talley NJ, Phillips SF, Bruce B, Twomey CK, Zinsmeister AR, Melton LJ. Relation among personality and symptoms in nonulcer dyspepsia and the irritable bowel syndrome. Gastroenterology 1990; 99: 327-333

22 Sato T. The Eysenck Personality Questionnaire Brief Version: factor structure and reliability. J Psychol 2005; 139: 545-552

23 Drossman DA, Camilleri M, Mayer EA, Whitehead WE. AGA technical review on irritable bowel syndrome. Gastroenterology 2002; 123: 2108-2131

24 Jailwala J, Imperiale TF, Kroenke K. Pharmacologic treatment of the irritable bowel syndrome: a systematic review of randomized, controlled trials. Ann Intern Med 2000; 133: 136-147

25 Saulsman LM, Page AC. The five-factor model and personality disorder empirical literature: A meta-analytic review. Clin Psychol Rev 2004; 23: 1055-1085

26 Creed F, Guthrie E. Psychological treatments of the irritable bowel syndrome: a review. Gut 1989; 30: 1601-1609

27 Creed F , Guthrie E, Ratcliffe J, Fernandes L, Rigby C, Tomenson B, Read N, Thompson DG. Does psychological treatment help only those patients with severe irritable bowel syndrome who also have a concurrent psychiatric disorder? Aust N Z J Psychiatry 2005; 39: 807-815

28 Svedlund J , S jödin I , Dotevall G, Gil lberg R. Upper gastrointestinal and mental symptoms in the irritable bowel syndrome. Scand J Gastroenterol 1985; 20: 595-601

29 Blanchard EB, Schwarz SP, Radnitz CR. Psychological assessment and treatment of irritable bowel syndrome. Behav Modif 1987; 11: 348-372

30 De Wit N, Zijdenbosch I, van der Heijden G, Quartero O, Rubin G. Psychological treatments for the management of irritable bowel syndrome. Cochrane Database Syst Rev 2007; 2: CD006442

31 Quartero AO, Meineche-Schmidt V, Muris J, Rubin G, de Wit N. Bulking agents, antispasmodic and antidepressant medication for the treatment of irritable bowel syndrome. Cochrane Database Syst Rev 2005: CD003460

S- Editor Zhu LH L- Editor Kerr C E- Editor Liu Y

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Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6419-6424www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

Prevalence of fatty liver disease and its risk factors in thepopulation of South China

Yong-Jian Zhou, Yu-Yuan Li, Yu-Qiang Nie, Jin-Xiang Ma, Lun-Gen Lu, Sheng-Li Shi, Min-Hu Chen, Pin-Jin Hu

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RAPID COMMUNICATION

Yong-Jian Zhou, Yu-Yuan Li, Min-Hu Chen, Pin-Jin Hu, Department of Gastroenterology, The First Affiliated Hospital, Sun Yat-Sen University of Medical Sciences, Guangzhou 510080, Guangdong Province , ChinaYong-Jian Zhou, Yu-Yuan Li, Yu-Qiang Nie, Sheng-Li Shi, Department of Gastroenterology and Hepatology, First Municipal People’s Hospital of Guangzhou, Guangzhou Medical College, Guangzhou 510180, Guangdong Province, ChinaJin-Xiang Ma, Department of Prevention Medicine, Guangzhou Medical College, Guangzhou 510182, Guangdong Province, ChinaLun-Gen Lu, Department of Gastroenterology, Renji Hospital, Shanghai Institute of Digestive Disease, Shanghai Jiaotong University School of Medicine, Shanghai 200001, ChinaSupported by a Grant from Guangzhou Health Bureau Project, No. 2004-Z001Correspondence to: Yu-Yuan Li, Department of Gastro-enterology and Hepatology, First Municipal People’s Hospital of Guangzhou, No. 1 Panfu Road, Guangzhou 510180, Guangdong Province, China. liyyliyy@tom.comTelephone: +86-20-81048720 Fax: +86-20-81045937Received: July 11, 2007 Revised: September 29, 2007

AbstractAIM: To investigate the population-based prevalence of fatty liver disease (FLD) and its risk factors in Guangdong Province, China.

METHODS: A cross-sectional survey with multiple-stage stratified cluster and random sampling of inhabitants over 7-year-old was performed in 6 urban and rural areas of Guangdong Province, China. Questionnaires, designed by co-working of epidemiologists and hepatologists, included demographic characteristics, current medication use, medical history and health-relevant behaviors, i.e., alcohol consumption, smoking habits, dietary habits and physical activities. Anthropometric measurements, biochemical tests and abdominal ultrasonography were carried out.

RESULTS: Among the 3543 subjects, 609 (17.2%) were diagnosed having FLD (18.0% males, 16.7% females, P > 0.05). Among them, the prevalence of confirmed alcoholic liver disease (ALD), suspected ALD and nonalcoholic fatty liver disease (NAFLD) were 0.4%, 1.8%, and 15.0%, respectively. The prevalence rate (23.0%) was significantly higher in urban areas than (12.9%) in rural areas. After adjustment for age, gender and residency, the standardized prevalence of FLD in adults was 14.5%. Among them, confirmed

ALD, suspected ALD and NAFLD were 0.5%, 2.3%, and 11.7%, respectively, in adults and 1.3% (all NAFLD) in children at the age of 7-18 years. The overall prevalence of FLD increased with age in both genders to the peak of 27.4% in the group of subjects at the age of 60-70 years. The prevalence rate was significantly higher in men than in women under the age of 50 years (22.4% vs 7.1%, P < 0.001). However, the opposite phenomenon was found over the age of 50 years (20.6% vs 27.6%, P < 0.05). Multivariate and logistic regression analysis indicated that male gender, urban residency, low education, high blood pressure, body mass index, waist circumference, waist to hip ratio, serum triglyceride and glucose levels were the risk factors for FLD.

CONCLUSION: FLD, especially NAFLD, is prevalent in South China. There are many risk factors for FLD.

© 2007 WJG. All rights reserved.

Key words: Fatty liver disease; Prevalence; Epidemiology; Risk factors

Zhou YJ, Li YY, Nie YQ, Ma JX, Lu LG, Shi SL, Chen MH, Hu PJ. Prevalence of fatty liver disease and its risk factors in the population of South China. World J Gastroenterol 2007; 13(47): 6419-6424

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INTRODUCTIONFatty liver disease (FLD), defined as lipid accumulation exceeding the normal range of 5% of liver wet weight, is a chronic disorder, which includes alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD). FLD encompasses a morphological spectrum consisting of hepatic steatosis and steatohepatitis which can progress to cirrhosis and hepatocellular carcinoma[1]. In the past, excess alcohol consumption accounted for most cases of FLD, but recently nonalcoholic causes of fatty liver have attracted considerable attention. In addition to alcohol consumption, factors such as insulin resistance (IR), oxidative stress, mitochondrial dysfunction, immune deregulation, and adipokines play an important role in the pathogenesis of FLD[2,3].

FLD has become a common problem in both developed and developing countries. According to the

Office for National Statistics in the United Kingdom, liver disease mainly FLD ranks the fifth most common cause of death after heart disease, stroke, pulmonary disease and cancer[4]. In recent years, due to alterations in life style and dietary habits, the incidence of FLD has increased dramatically in China. It is thus of importance to assess the epidemiological features of FLD in this country in order to facilitate its prevention and treatment. Although some epidemiological studies concerning FLD have been reported in China, most of them were based on clinical settings or on health check-up groups, which do not represent population data. So far only one population-based study from Shanghai, an eastern city of China, has been published in English[5]. The data from rural areas and other parts of this country are still lacking.

In this study, we investigated the prevalence of FLD in population of South China and identified its major risk factors. Since the random sampling method and high response rate limited the selection and responder biases, our results reflect the population-based data in both urban and rural areas.

MATERIALS AND METHODSStudy design Guangdong Province with a population of about 85 millions (mainly Han nationality people) is located in an economically advanced region in South China. Its capital city, Guangzhou, has 8-million residents. The heterogeneity of economy, geography, and culture in different areas inside the province influences the life styles of the people.

Between April and November of 2005, a cross-sectional survey with multiple-stage stratified cluster and random sampling was performed on the basis of a previous analysis of the population distribution in this province[6,7]. In order to represent the data in general population, sampling clusters were randomly drawn from urban and rural areas all over the province. Six areas with 4365 residents over 7 years of age were selected, i.e., 2 communities from downtown of Guangzhou city (Yuanzhongyuan community of the Liwan district and the Xinyi community of the Baiyun district, with a total of 1436 urban residents) and four units within the province, i.e., a village in central area of the province (Huadu county, 822 rural residents), a village in southern area (Huizhou county, 1037 rural residents), a village in western area (Zhanjiang county, 351 rural residents) and a community in northern area (Shaoguan city, 719 urban residents).

Of the 4365 potential participants, 3903 agreed to take part in this investigation with a responder rate of 89.6%. Among them, 3543 subjects (90.6%, 1311 males and 2232 females, at the age of 7-100 years) provided their complete information, accounting for approximately 44.3/100 000 of the total population of Guangdong Province[7]. Interview, physical examination, laboratory assessment and ultrasonographic examination were performed for each subject on the same day at a mobile examination center. The study was approved and supported by Guangzhou Health Bureau. Written consent was obtained from each participant.

Interview and physical examinationA face-to-face interview was carried out by specially trained post-graduate students of Guangzhou Medical College and supervised by experienced investigators. Standard questionnaires, designed by co-working of epidemiologists and hepatologists, included the following items: demographic characteristics, current medication use, medical history and health-relevant behaviors, i.e., alcohol consumption, smoking habits, dietary habits and physical activities. Physical examination covered body height, weight, blood pressure, waist circumference (WC), waist-to-hip ratio (WHR) and routine anthropometric parameters in healthy check-up.

Biochemical tests Intravenous blood samples were collected from fasting subjects by routine methods. Fasting serum glucose levels and lipid profiles were measured with an automatic chemistry- immuno-analyzer (Olympus Corporation, Tokyo, Japan). Serum asparatate aminotransferase (AST), and alanine aminotransferase (ALT), serum bilirubin (BIL) and albumin levels were measured by standard laboratory methods. Tests for the serum makers of hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV) were also carried out.

UltrasonographyReal-time ultrasonography (US) of the upper abdominal organs was performed for each subject by 2 experienced physicians using a scanner equipped with a 3.5 mmHz transducer (Siemens Adama, German). The physicians performing the US were unaware of the clinical and laboratory results.

Diagnostic criteriaFLD, NAFLD and ALD were diagnosed according to the guidelines for diagnosis and treatment of nonalcoholoic and alcoholoic fatty liver diseases issued by Fatty Liver and Alcoholic Liver Disease Study Group of the Chinese Liver Disease Association[8,9], which were adapted from the American Gastroenterological Association[10]. Briefly, the diagnosis of FLD, NAFLD and ALD was based on the combination of medical history, clinical symptoms, laboratory and US findings. Viral hepatitis and other chronic liver diseases needed to be ruled out. Liver biopsy was taken when the diagnosis was suspected. In this epidemiological study, only 12 subjects underwent biopsy. In most cases FLD was diagnosed and staged by US findings. ALD was diagnosed when a subject fulfilling the FLD criteria drank more than 40 g (male) or 20 g (female) alcohol per day over 5 years. Suspected ALD was defined when the alcohol consumption was 20-40 g (male) or 10-20 g (female). NAFLD was diagnosed when the alcohol consumption was less than these amounts.

The US diagnostic patterns of FLD were based on the presence of a ‘bright’ l iver (brightness and posterior attenuation) with stronger echoes in the hepatic parenchyma than in the renal parenchyma, vessel blurring and narrowing of the lumen of hepatic veins in the absence of findings suggestive of other chronic liver

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diseases. The degree of FLD evaluated by US reflected the degree of steatosis. A liver displaying increased but homogenous dot reflection, blurred but still discernible vascular profiles and weak portal vein echogenicity was defined as early-stage. A medium-stage FLD was characterized by disappearance of the portal vascular wall and slightly increased liver volume. Patients with nebulous optical appearance in the liver, little portal vein echogenicity in more than 1/3-1/2 of the liver area and increased liver volume were considered to be in the advanced stage[11-13].

Obesity was categorized according to the body mass index (BMI) criteria for Asians issued by the Regional Office for Western Pacific Region of the World Health Organization[14]. Subjects with BMI ≥ 25 were considered as obese, BMI ≥ 23 but < 25 as overweight, BMI ≥ 18.5 but < 23 as normal, BMI < 18.5 as underweight. Central (or abdominal) obesity was estimated by waist circumference (WC) and waist-to-hip ratio (WHR). A WHR value ≥ 0.9 (male) or ≥ 0.8 (female) was considered as central obesity. Dyslipidemia was considered when having one of following serum lipid profiles: total cholesterol (TC) ≥ 5.72 mmol/L, triglycerides (TG) ≥ 1.70 mmol/L, high-density lipoprotein cholesterol (HDL-C) < 0.91 mmol/L, and low-density lipoprotein cholesterol (LDL-C) ≥ 3.64 mmol/L. Subjects with their fasting serum glucose (FSG) value of ≥ 7.0 mmol/L and/or with a history of diabetes were considered to have diabetes mellitus. Hypertension was defined as systolic or diastolic blood pressure above 140 mmHg or 90 mmHg, respectively. Smokers in this study were defined as those smoking more than one cigarette each day for at least one year or more than 20 packages of cigarettes in total. Alcohol drinkers were defined as those drinking more than 2 times each month for more than 5 years. Heavy tastes meant the habits of preferring salted and spiced foods. Extraverted character meant the active personal characters of subjects. The definitions of urban and rural areas were in accordance with the publication of the Fifth Chinese National Population Census in 2000[7].

Statistical analysisData were examined with the SPSS 13.0 for Windows. P < 0.05 was considered statistically significant. Exposure ratio comparison and multivariate regression analyses were performed to evaluate the risk factors.

RESULTSAge and gender-specific prevalence of FLD Data are shown in Tables 1 and 2. Of the 3543 subjects,

609 (17.2%) were diagnosed having FLD, among them 367 (10.4%), 218 (6.1%) and 24 (0.7%) fell into early, medium and advanced degrees by US staging. The overall prevalence of FLD increased to the peak of 27.4% in the group at the age of 60- years and then decreased in both genders (P < 0.01). There was no prevalent difference between males and females (18.0% vs 16.7%, P > 0.05). However, FLD was more common in males than in females under 50 years of age. An opposite trend was noted over 50 years of age (P < 0.05). The prevalence of FLD in children at the age of 7-18 years (5/379, 1.3%) differed significantly from that in adults (604/3164, 19.1%, P < 0.001). The overall prevalence of ALD, suspected ALD and NAFLD was 0.4%, 1.8% and 15%, respectively, accounting for 2.3%, 10.7% and 87.0% of the 609 FLD subjects, respectively. In subgroup comparison, the prevalence of confirmed ALD and suspected ALD was significantly higher in males (0.9% and 4.0%) than in females (0.1% and 0.5%, P < 0.001). Nevertheless, the prevalence of NAFLD was lower in males than in females (13.0% vs 16.1%, P < 0.05).

Prevalence of FLD in urban and rural areasThe prevalence of FLD was significantly lower in rural areas than in urban areas (12.9% vs 23.0%, P < 0.001), mainly due to the difference in NAFLD (11.1% vs 20.3%, P < 0.001). A slight difference was observed in rural areas between ALD and suspected ALD subjects compared with urban areas, but it was not significant (P > 0.05) (Table 3).

Prevalence of FLD and serum lipid profilesHigh serum levels of TG, TC, LDL-C and FSG were all positively associated with FLD (P < 0.01), but HDL-C did not reach significance (P > 0.05) (Table 4). The prevalence of FLD in dyslipidemia subjects was 3.65 (95% CI = 2.87- 4.65) folds higher than that in normal controls. High TG levels were the most prominent factor for dyslipidemia

Table 1 Age and sex-specific prevalence of fatty liver disease

Total Male Female P n n (%) n n (%) n n (%)

7- 379 5 (1.3) 210 4 (1.9) 169 1 (0.6) 0.26518- 349 20 (5.7) 129 14 (10.9) 220 6 (2.7) 0.00230- 477 53 (11.1) 155 31 (20.0) 322 22 (6.8) 0.00040- 667 101 (15.1) 193 46 (23.8) 474 55 (11.6) 0.00050- 809 214 (26.5) 230 53 (23.0) 579 161 (27.8) 0.16660- 551 151 (27.4) 277 65 (23.5) 274 86 (31.4) 0.03770- 311 65 (20.9) 117 23 (19.7) 194 42 (21.6) 0.676Total 3543 609 (17.2) 1311 236 (18.0) 2232 373 (16.7) 0.326

Confirmed ALD Suspected ALD NAFLD Total (FLD) n n (%) n (%) n (%) n (%)

Male 1311 12 (0.9) 53 (4.0) 171 (13.1) 236 (18.0)Female 2232 2 (0.1) 12 (0.5) 359 (16.1) 373 (16.7)Total 3543 14 (0.4) 65 (1.8) 530 (15.0) 609 (17.2)P < 0.001 < 0.001 < 0.05 > 0.05

Table 2 Sex-specific prevalence in fatty liver disease subgroups

FLD: Fatty liver disease; ALD: Alcoholic liver disease; NAFLD: Non-alcoholic fatty liver disease.

Table 3 Residence-specific prevalence in FLD subgroups

FLD: Fatty liver disease; ALD: Alcoholic liver disease; NAFLD: Non-alcoholic fatty liver disease.

Confirmed ALD Suspected ALD NAFLD Total (FLD) n n (%) n (%) n (%) n (%)

Rural 2047 11 (0.5) 27 (1.3) 227 (11.1) 265 (12.9)Urban 1496 3 (0.2) 38 (2.5) 303 (20.3) 344 (23.0)Total 3543 14 (0.4) 65 (1.8) 530 (15.0) 609 (17.2)P < 0.001

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with an odd ratio (OR) of 4.49 (95% CI = 3.56-5.66). The OR of high TC was 1.95 (95% CI = 1.56-2.44). The OR of elevated FSG and combined dyslipidemia was 4.93 (95% CI = 3.55-6.84) and 6.64 (95% CI = 4.83-9.12), respectively.

Adjusted prevalence of FLD After adjustment for gender, age and residence area according to the demographic characteristics of the Fifth Chinese Census[7], the age-, gender- and residency- specific standardized prevalence of FLD was 14.5%. Confirmed ALD, suspected ALD and NAFLD were 0.5%, 2.3% and 11.7% respectively in adults (over 18 years of age). The overall prevalence of FLD was 11.3% and confirmed ALD, suspected ALD and NAFLD were 0.4%, 1.8% and 9.1% respectively, in all people over 7 years of age.

Exposure ratio and logistic regression analysisThe exposure ratio (%) analysis of anthropometric and clinical parameters in adults (above 18 years of age, n = 3164) with and without FLD was carried out to screen for the relevant factors for FLD (Table 5). The ratios of all variables for FLD and controls were significantly different (P < 0.01). In order to identify the risk factors, we further

performed a multivariate regression logistic analysis with probability for entry 0.05 and removal 0.1 (Table 6). Male gender, urban inhabitation, hypertension, high BMI, WC, WHR, serum TG, and FSG were found to be independent risk factors for FLD, and education was a protective factor for FLD. Alcoholic consumption and heavy tastes in food were marginally but not significantly related to FLD. Smoking and extraverted character were excluded from the risk factors for FLD (P > 0.05).

DISCUSSIONFLD is a common disease. NAFLD has been increasingly recognized as the most common liver disease in Western countries. So far no accurate incidence is available. The prevalent data obtained from clinical series and autopsy studies suggest that 20%-30% of individuals in the Western world have FLD. In general population, the prevalence of NAFLD ranges 3%-24% in the world[15-17], 20%-25% in Italy[18], 30% in Israel[19], 16% in Korea[20], 14% in Japan[21] and 15% (18% for FLD) in Shanghai, China[25]. The discrepancy among the studies is probably due to the methods of sample selection, modalities used for diagnosis and diversity of life styles and dietary habits in different areas. Since China is a developing country, it is not surprising that the standardized prevalence of FLD (14.5%) and NAFLD (11.7%) is lower in China than in developed countries. The urban prevalence is lower in Guangdong Province than in Shanghai city[5]. It seems that the rapid modernization of Shanghai has brought about its inhabitants more metabolic problems than that of Guangdong Province. Our results in adults and children are comparatively close to those in Japan, probably because the inhabitants in South China share the similar dietary habits (sea food preference) with Japanese[21]. In this study, only 24 cases (0.7% in general population) had advanced stage of FLD diagnosed by US, accounting for 3.9% of the 609 FLD cases. Although US is reasonably accurate as compared with biopsy, it cannot provide precise information about the histological features associated with disease progression especially inflammation[15]. The results in this study roughly reflect the lower severity of FLD in this area.

Table 4 Serum levels and lipid profiles in FLD patients and controls (mean ± SE)

n TG (mmol/L)

TC (mmol/L)

HDL-C (mmol/L)

LDL-C (mmol/L)

FSG (mmol/L)

FLD 609 2.81 ± 2.55 5.52 ± 1.11 1.72 ± 1.25 3.06 ± 0.91 6.01 ± 2.68Controls 2934 1.63 ± 1.59 5.19 ± 1.07 1.67 ± 0.57 2.90 ± 0.81 4.95 ± 1.56P < 0.001 < 0.01 > 0.05 < 0.01 < 0.01

FLD: Fatty liver disease; TC: Total cholesterol; TG: Triglycerides; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol.

Table 5 Exposure ratios of variables in adult FLD patients compared with controls

n Male Urban Primaryschool andbelow

Extravertedcharacter

Alcoholdrinkers

Smokers

Total3164

n (%) n (%) n (%) n (%) n (%) n (%)

FLD604

232(38.41)

344 (57.0)

245(40.6)

214(35.4a)

141(23.3)

160(26.5a)

Controls2560

869(33.9)

1140 (44.5)

783(30.6)

787 (30.7)

457(17.9)

553 (21.6)

χ2 4.295 30.282 22.112 4.966 9.619 6.665n Heavy

tastesOverweight Obesity Hypertension WHR WC

Total3164

n (%) n (%) n (%) n (%) n (%) n (%)

FLD604

208 (34.4)

478 (79.4)

120(19.9)

287 (47.6)

531 (87.9)

432 (71.5)

Controls2560

689(26.9)

650(25.5)

79(3.1)

658 (25.7)

1469 (57.8)

558(21.8)

χ2 13.616 616.708 235.812 110.141 189.387 560.357

aP < 0.05 vs controls and all the others; P < 0.01 vs controls. FLD: Fatty liver disease; WC: Waist circumference; WHR: Waist-to-hip ratio.

Table 6 Multivariate regression logistic analysis for FLD (n = 3543)

Variables β SE χ2 P OR OR 95%CI

Male 0.628 0.218 5.361 0.002 0.765 0.321-0.921Urban 0.212 0.128 28.219 < 0.001 0.332 0.211-0.291Education level -0.351 0.105 11.124 0.001 0.704 0.573-0.865Hypertension 0.362 0.158 5.277 0.022 1.436 1.055-1.957BMI 1.234 0.130 90.488 < 0.001 3.435 2.664-4.430WHR 0.811 0.228 12.648 < 0.001 2.250 1.439-3.518WC 1.017 0.174 33.987 < 0.001 2.765 1.964-3.892TG 0.623 0.158 15.535 < 0.001 1.864 1.367-2.540FSG 0.788 0.129 37.187 < 0.001 2.200 1.708-2.835Alcohol-drinking 0.373 0.212 3.112 0.078 1.452 0.959-2.198Heavy tastes 0.307 0.169 3.286 0.070 1.360 0.975-1.895

FLD: Fatty liver disease; BMI: Body mass index; WHR: w-to-hip ratio; WC: Waist circumference; TC: Total cholesterol; FSG: Fasting serum glucose.

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There are conflicting results regarding the relation of NAFLD to age and gender[15-18]. Bedogni et al[18] found that the prevalence of NAFLD increases with age in both genders and then significantly decreases over 66 years of age[18]. However, it was reported that hepatic triglyceride is correlated with age only in white women[16]. The variations between studies can be attributed to the differences in social, cultural and environmental backgrounds among the target subjects. In this study, the highest prevalence of FLD (mainly NAFLD) was observed in the sixth decade of life. The prevalence of FLD in males increased stably with age, and steadily from 50-60 years of age in females. The peak prevalence was observed in females (31.4%) at the age of 60 years, which was 20 years later than that in males (23.8%), which might be due to the menopausal status and lack of physical exercise in this period of time.

In developed countries, alcoholism, obesity and diabetes are the most common causes for FLD. Alcohol drinking plays a role in FLD[22,23]. In this study, only 0.5% adults in Guangdong Province suffered from ALD, reflecting the low alcohol consumption in South China. For this reason, multivariate analyses failed to demonstrate the role of alcohol drinking in FLD (P = 0.078). NAFLD is a manifestation of metabolic syndrome in liver, including obesity, type 2 diabetes, hypertension and dyslipidemia[15,24-28]. Metabolic syndrome, a common disorder in Western countries, has become a severe problem in China due to alterations in life styles and dietary habits. In this study, the prevalence of FLD increased with BMI and HWR, an index for central obesity. These findings are consistent with the reported data[5,19,29,30]. Our results indicate that dyslipidemia, especially high serum level of TG is a major risk factor for FLD, which also agrees with the results in most studies that the role of TG is more important than TC in the pathogenesis of metabolic syndrome and NAFLD[18,23]. When the serum level of TC in this area is relatively low, it fails to reach the significant level as a risk factor for FLD. The education background is related to the prevalence of FLD, probably because the people with a higher education level have paid more attention to their health and they are more likely to adjust their diet habits and exercise for the prevention of obesity.

There are several methodological limitations in this study. First, although participants were randomly selected, the enrollment of a greater proportion of females and elderly subjects in the study may bring about bias to the results. Second, the large number of immigrant population in Guangdong Province, especially in cities may have also confounded the study. In order to limit these biases, the prevalence rate was standardized for age, gender and residency areas. Multivariate regression analyses were performed to strengthen the reliability of our findings. Third, although histology remains the gold standard for the pathological diagnosis of FLD[31,32], imaging modalities like ultrasonography with a reasonably high sensitivity can identify FLD[32-35]. In this study, the diagnosis of fatty liver was based on ultrasonography. For the ethical reason, it is impossible to perform biopsy in an epidemiological study. In addition, the influence of exercise was not taken into

account in this study. In summary, FLD, especially NAFLD, is common

in South China and many risk factors are related to the pathogenesis of this metabolic syndrome.

COMMENTSBackgroundFatty liver disease (FLD) has become a common problem in both developed and developing countries. In recent years, due to alterations in life style and dietary habits, the incidence of FLD has increased dramatically in China. It is thus of importance to assess the epidemiological features of FLD in this country in order to facilitate its prevention and treatment. Although some epidemiological studies concerning FLD have been reported in China, most of them were based on clinical settings or on health check-up groups, which do not represent the population data. So far only one population-based study from Shanghai, an eastern city of China, has been published in English. The data from rural areas and other parts of this country are still lacking.

Research frontiersFLD encompasses a morphological spectrum consisting of hepatic steatosis and steatohepatitis which can progress to cirrhosis and hepatocellular carcinoma. In the past, excess alcohol consumption accounted for most cases of FLD, but recently nonalcoholic causes for fatty liver have attracted considerable attention. In addition to alcohol consumption, factors such as insulin resistance (IR), oxidative stress, mitochondrial dysfunction, immune deregulation, and adipokines play an important role in the pathogenesis of FLD. Although FLD has been reported worldwide, it is difficult to determine the true prevalence because of problems in interpreting data from various studies due to referral bias, population heterogeneity, study design, imaging modalities, and liver biopsies. These studies on the FLD epidemiology have improved the detailed knowledge about the prevalence of FLD and the metabolic risk factors associated regional (e.g., urban vs rural) and ethnic differences, as they may provide clues to pathogenesis and individual risk factors for liver disease and metabolic complications. Further studies are also required to confirm the impact of fatty liver on chronic liver diseases.

Innovations and breakthroughsThe incidence of FLD is likely to rise steadily in China in urban areas owing to the westernization of the diet, excessive food intake, increased elderly population, changes in life style and lack of exercise. Because the majority of people live in rural areas of China, it is necessary to investigate the prevalence of FLD in China. In this study, since the random sampling method and high response rate limited the selection and responder biases, our results reflect the population-based data and the major risk factors for FLD in both urban and rural areas. This study assessed the prevalence of and risk factors for NAFLD in China.

Applications With regards to increasing obesity, FLD, especially NAFLD, is believed to be the most common form of chronic liver diseases, which can progress to cirrhosis. Because there is no proven management for NAFLD, identification of important risk factors for this condition will provide valuable information on both risk stratification and development of risk-reduction strategies.

TerminologyFatty liver disease (FLD), defined as lipid accumulation exceeding the normal range of 5% of liver wet weight, is a kind of chronic disorders, including alcoholic liver disease (ALD) and nonalcoholic fatty liver diseases (NAFLD). Briefly, the diagnosis of FLD, NAFLD and ALD is based on the combination of medical history, clinical symptoms, laboratory and imaging modalities (ultrasonography, computed tomography scans, and magnetic resonance imaging). Viral hepatitis and other chronic liver diseases need to be ruled out. ALD is diagnosed when a subject fulfilling the FLD criteria drinks more than 40 g (male) or 20 g (female) alcohol per day over 5 years. NAFLD is diagnosed when alcohol consumption is less than 40 g (male) or 20 g (female).

Peer reviewThis is an interesting, well written and generally well designed study. Although it does not identify fundamentally new or unexpected findings with respect to the incidence of FLD, the data highlighting the nonalcohol - related incidence of FLD

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in developing areas are important.REFERENCES 1 Reddy JK, Rao MS. Lipid metabolism and liver inflammation.

II. Fatty liver disease and fatty acid oxidation. Am J Physiol Gastrointest Liver Physiol 2006; 290: G852-G858

2 Crabb DW, Galli A, Fischer M, You M. Molecular mechanisms of alcoholic fatty liver: role of peroxisome proliferator-activated receptor alpha. Alcohol 2004; 34: 35-38

3 Machado M, Cortez-Pinto H. Non-alcoholic steatohepatitis and metabolic syndrome. Curr Opin Clin Nutr Metab Care 2006; 9: 637-642

4 Williams R. Global challenges in liver disease. Hepatology 2006; 44: 521-526

5 Fan JG, Zhu J, Li XJ, Chen L, Lu YS, Li L, Dai F, Li F, Chen SY. Fatty liver and the metabolic syndrome among Shanghai adults. J Gastroenterol Hepatol 2005; 20: 1825-1832

6 Chen M, Xiong L, Chen H, Xu A, He L, Hu P. Prevalence, risk factors and impact of gastroesophageal reflux disease symptoms: a population-based study in South China. Scand J Gastroenterol 2005; 40: 759-767

7 Bu X. Data of the fifth Chinese census (2000) in Guangdong province. Guangzhou: Press of Chinese Statistics, 2001: 1-45

8 Fatty Liver and Alcoholic Liver Disease Study Group of the Chinese Liver Disease Association. Guideline for diagnosis and treatment of non-alcohol fatty liver disease. Zhonghua Ganzangbing Zazhi 2006; 14: 161-163

9 Fatty Liver and Alcoholic Liver Disease Study Group of the Chinese Liver Disease Association. Guideline for diagnosis and treatment of alcohol liver disease. Zhonghua Ganzangbing Zazhi 2006; 14: 164-166

10 American Gastroenterological Association medical position statement: nonalcoholic fatty liver disease. Gastroenterology 2002; 123: 1702-1704

11 Zelber-Sagi S , Nitzan-Kaluski D, Halpern Z, Oren R. Prevalence of primary non-alcoholic fatty liver disease in a population-based study and its association with biochemical and anthropometric measures. Liver Int 2006; 26: 856-863

12 Lonardo A, Bellini M, Tartoni P, Tondelli E. The bright liver syndrome. Prevalence and determinants of a "bright" liver echopattern. Ital J Gastroenterol Hepatol 1997; 29: 351-356

13 Joseph AE, Saverymuttu SH, al-Sam S, Cook MG, Maxwell JD. Comparison of liver histology with ultrasonography in assessing diffuse parenchymal liver disease. Clin Radiol 1991; 43: 26-31

14 Anuurad E, Shiwaku K, Nogi A, Kitajima K, Enkhmaa B, Shimono K, Yamane Y. The new BMI criteria for asians by the regional office for the western pacific region of WHO are suitable for screening of overweight to prevent metabolic syndrome in elder Japanese workers. J Occup Health 2003; 45: 335-343

15 Neuschwander-Tetri BA , Caldwell SH. Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference. Hepatology 2003; 37: 1202-1219

16 Browning JD, Szczepaniak LS, Dobbins R, Nuremberg P, Horton JD, Cohen JC, Grundy SM, Hobbs HH. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology 2004; 40: 1387-1395

17 Clark JM. The epidemiology of nonalcoholic fatty liver disease in adults. J Clin Gastroenterol 2006; 40 Suppl 1: S5-S10

18 Bedogni G, Miglioli L, Masutti F, Tiribelli C, Marchesini G, Bellentani S. Prevalence of and risk factors for nonalcoholic fatty liver disease: the Dionysos nutrition and liver study. Hepatology 2005; 42: 44-52

19 Amarapurkar DN, Hashimoto E, Lesmana LA, Sollano JD, Chen PJ, Goh KL. How common is non-alcoholic fatty liver disease in the Asia-Pacific region and are there local differences? J Gastroenterol Hepatol 2007; 22: 788-793

20 Park SH, Jeon WK, Kim SH, Kim HJ, Park DI, Cho YK, Sung IK, Sohn CI, Keum DK, Kim BI. Prevalence and risk factors of non-alcoholic fatty liver disease among Korean adults. J Gastroenterol Hepatol 2006; 21: 138-143

21 Nomura H, Kashiwagi S, Hayashi J, Kajiyama W, Tani S, Goto M. Prevalence of fatty liver in a general population of Okinawa, Japan. Jpn J Med 1988; 27: 142-149

22 Porta EA, Hartroft WS, De la Iglesia FA. Hepatic changes associated with chronic alcoholism in rats. Lab Invest 1965; 14: 1437-1455

23 Klatskin G . Alcohol and its relation to liver damage. Gastroenterology 1961; 41: 443-451

24 Marchesini G , Marzocchi R, Agostini F, Bugianesi E. Nonalcoholic fatty liver disease and the metabolic syndrome. Curr Opin Lipidol 2005; 16: 421-427

25 Farrell GC, Larter CZ. Nonalcoholic fatty liver disease: from steatosis to cirrhosis. Hepatology 2006; 43: S99-S112

26 Angulo P. Nonalcoholic fatty liver disease. N Engl J Med 2002; 346: 1221-1231

27 Chitturi S, Abeygunasekera S, Farrell GC, Holmes-Walker J, Hui JM, Fung C, Karim R, Lin R, Samarasinghe D, Liddle C, Weltman M, George J. NASH and insulin resistance: Insulin hypersecretion and specific association with the insulin resistance syndrome. Hepatology 2002; 35: 373-379

28 Yoon KH, Lee JH, Kim JW, Cho JH, Choi YH, Ko SH, Zimmet P, Son HY. Epidemic obesity and type 2 diabetes in Asia. Lancet 2006; 368: 1681-1688

29 Church TS, Kuk JL, Ross R, Priest EL, Biltoft E, Blair SN. Association of cardiorespiratory fitness, body mass index, and waist circumference to nonalcoholic fatty liver disease. Gastroenterology 2006; 130: 2023-2030

30 Ruhl CE, Everhart JE. Epidemiology of nonalcoholic fatty liver. Clin Liver Dis 2004; 8: 501-519, vii

31 Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, Unalp-Arida A, Yeh M, McCullough AJ, Sanyal AJ. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005; 41: 1313-1321

32 McCullough AJ. The epidemiology and risk factors of NASH. In: Farrell GC, George J, de la M Hall P, McCullough AJ, editors. FattyLiver Disease: NASH and Related Disorders. Malden, MA: Blackwell Publishing, 2005; 23-37

33 Gore RM. Diffuse liver disease . In: Gore RM, Levine MS, Laufer I,eds. Textbook of Gastrointestinal Radiology. Philadelphia: Saunders, 1994: 1968-2017

34 Yajima Y, Ohta K, Narui T, Abe R, Suzuki H, Ohtsuki M. Ultrasonographical diagnosis of fatty liver: significance of the liver-kidney contrast. Tohoku J Exp Med 1983; 139: 43-50

35 Fusamoto H , Suzuki K, Hayashi N, Sasaki Y, Kono M, Kasahara A, Kamada T. Obesity and liver disease: evaluation of fatty infiltration of the liver using ultrasonic attenuation. J Nutr Sci Vitaminol (Tokyo) 1991; 37 Suppl: S71-S77

S- Editor Zhu LH L- Editor Wang XL E- Editor Lu W

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by microscopy,reduction of VEGF production induced by ribozyme resulted in a significantly higher cell differentiation and less proliferation vigor in xenografted tumor.

CONCLUSION: Anti-hVEGF hairpin ribozyme can effectively inhibit VEGF expression and growth of hepatocarcinoma in vitro and in vivo . VEGF is functionally related to cell proliferation, differentiation and tumori-genesis in hepatocarcinoma.

© 2007 WJG. All rights reserved.

Key words: Vascular endothelial growth factor; Angi-ogenesis; Hairpin ribozyme; Hepatocarcinoma; Gene therapy

Li LH, Guo ZJ, Yan LL, Yang JC , Xie YF, Sheng WH, Huang ZH, Wang XH. Antitumor and antiangiogenic activities of anti-vascular endothelial growth factor hairpin ribozyme in human hepatocellular carcinoma cell cultures and xenografts. World J Gastroenterol 2007; 13(47): 6425-6432

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INTRODUCTIONRapidly growing tumors routinely outstrip their supply of oxygen and nutrients, and induction of new blood vessels is critical to sustain neoplastic proliferation[1-4]. A number of growth factors have been identified as potential positive regulators of angiogenesis. Among them, vascular endothelial growth factor (VEGF) appears to have a central role in the angiogenic process. VEGF not only is the target of many proangiogenic factors but also regulates molecules that are implicated in endothelial proliferation. It has been suggested that VEGF may be a proximate angiogenic factor through which others act. In fact, over-expression of VEGF is the characteristic of most malignant tumors including hepatocarcinoma[5-9], which is regulated in response to hypoxia[7,10-12] and highly related to microvessel density of cancer, grade of malignance and metastasis[13-15]. It now appears that VEGF also has autocrine functions acting as a survival factor for tumor cells protecting themselves from stresses such as hypoxia, chemotherapy and radiotherapy[16]. Consequently, anti-

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Antitumor and antiangiogenic activities of anti-vascular endothelial growth factor hairpin ribozyme in human hepatocellular carcinoma cell cultures and xenografts

Li-Hua Li, Zi-Jian Guo, Ling-Ling Yan, Ji-Cheng Yang, Yu-Feng Xie, Wei-Hua Sheng, Zhao-Hui Huang, Xue-Hao Wang

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Li-Hua Li, Zi-Jian Guo, Ling-Ling Yan, Zhao-Hui Huang, Oncology Institute of Wuxi, the Fourth Affiliated Hospital of Soochow University, 200 Huihe Road, Wuxi 214062, Jiangsu Province, ChinaJi-Cheng Yang, Yu-Feng Xie, Wei-Hua Sheng, Department of Cellular and Molecular Biology, Medical School, Soochow University, Soochow 215007, Jiangsu Province, China Xue-Hao Wang, Department of Liver and Biliary Duct Surgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, ChinaSupported by the Grant from Calling for Tenders by Key Subject of Jiangsu Province, No. WK200221Correspondence to: Zi-Jian Guo, Oncology Institute of Wuxi, the Fourth Affiliated Hospital of Soochow University, 200 Huihe Road, Wuxi 214062, Jiangsu Province, China. gzjwxsy@sina.comTelephone: +86-510-88682119 Fax: +86-510-88683507Received: August 17, 2007 Revised: September 19, 2007

AbstractAIM: To study the effectiveness and mechanisms of anti- human vascular endothelial growth factor (hVEGF) hairpin ribozyme on angiogenesis, oncogenicity and tumor growth in a hepatocarcinoma cell line and a xenografted model.

METHODS: The artificial anti-hVEGF hairpin ribozyme was transfected into hepatocarc inoma cel l l ine SMMC-7721 and, subsequently, polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were performed to confirm the ribozyme gene integration and transcription. To determine the effects of ribozyme ,VEGF expression was detected by semiquantitative RT-PCR and enzyme liked immunosorbent assay (ELISA). MTT assay was carried out to measure the cell proliferation. Furthermore,the transfected and control cells were inoculated into nude mice respectively, the growth of cells in nude mice and angiogenesis were observed.

RESULTS: VEGF expression was down-regulated sharply by ribozyme in transfected SMMC-7721 cells and xenografted tumor. Compared to the control group, the transfected cel ls grew slower in cel l cultures and xenografts, and the xenograft formation was delayed as well. In addition, the microvessel density of the xenografted tumor was obviously declined in the transfected group. As demonstrated

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VEGF therapies are being actively investigated as potential anti-cancer treatment modalities, either as alternatives or adjuncts to conventional chemo or radiation therapy. Clinical trials using anti-VEGF mAbs such as bevacizumab have validated the efficacy of this therapeutic approach but have also revealed adverse effects.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers worldwide, being the third cause for cancer-related deaths. As a malignant solid tumor, HCC is characterized by fast infiltrating growth, early metastasis, high-grade malignancy, and poor therapeutic efficacy[17]. It is a highly vascular tumor dependenting on neovascularization and a rapidly developing malignancy[18]. The VEGF level in HCC tissues is significantly higher than that in distal cancerous tissues[6]. The abnormal expression levels of VEGF in sera of HCC patients are directly correlated with the metastasis and recurrence of tumors[5,19,20]. Since HCC is insensitive to conventional chemotherapeutics and its prognosis is poor, it is important to explore an antiangiogenesis method for HCC characterized by a high vascularity. Up to now, many strategies for gene therapy have been developed. Ribozymes are a group of catalytically active nucleic acids capable of site-specific cleavage of target mRNAs, thus decreasing mRNA expression and inhibiting the function of target gene[21]. Both hairpin and hammerhead ribozymes are the most commonly used gene therapy models. In contrast to hammerhead ribozyme, the hairpin ribozyme reaction requires an environment closer to human physiology. The hairpin ribozyme uses a catalytic mechanism that does not require metals for cleavage or ligation of substrate RNA. In this regard, it is presently unique among RNA catalysts. The hairpin ribozyme has been approved for its use in gene therapy.

T h e r o l e o f V E G F i n H C C p r o l i f e r a t i o n , differentiation and tumorigenesis is unclear. The present study was to study the effectiveness and mechanisms of hairpin ribozyme targeting human VEGF (hVEGF) mRNA on exon 3 on VEGF expression, angiogenesis, oncogenicity and tumor growth in HCC cell cultures and xenografts.

MATERIALS AND METHODSCell line and cell cultures Human hapatocarcinima cell l ine SMMC-7721 was maintained in our laboratory. Cells were incubated with RPMI 1640 medium (GIBCO BRL, Carlsbad, CA, USA) supplemented with 10% heat-inactivated calf serum (Sijiqing, Hangzhou, China), 100 U/mL penicillin G sodium and 100 μg/mL streptomycin sulfate in a humidified atmosphere containing 50 mL/L CO2 at 37℃.

Synthesis of anti-human VEGF hairpin ribozyme and recombinant plasmidsThe secondary structure of ribozyme was obtained by the ribozyme- aided design software, and the primers of hairpin ribozyme were synthesized (Shengong, Shanghai, China) based on the third exon of hVEGF as previously described[22]. The sequences of specific primers of ribozyme used are as follows: forward: 5'-GATCCCTGAT

AAGAATCCAACCAGAGAAACACACGTTGTGGTATATTACCTGGTAG-3' and reverse: 5'-AATTCTACCAGGTAATATACCACAACGTGTGTTTCTCTGGTTGGATTCTTATCAGG-3'. Ribozyme was synthesized in a 50 μL reaction system, which was mixed with 1 μL (25 μmol/L) primers, 5 μL NaCl buffer and 43 μL ddH2O. Since the gene of anti- hVEGF hairpin ribozyme only has a 50 bp product, after incubation at 94℃ for 2 min followed by an-nealing of the two primers at room temperature, ribozyme was acquired.

After plasmid pcDNA3.1+ (Invitrogen, San Diego, CA) was digested with restriction endonucleases BamHI and EcoRI (TakaRa, Japan), the purified products of anti- hVEGF hairpin ribozyme were subcloned into them, thus, the recombinant eukaryotic expression plasmid pcDNA3.1+/ribozyme (abbreviated as pcDNA3.1+/RZ) was constructed.

Preparation and identification of SMMC-7721/RZ transgenic cellsSMMC-7721 cells were seeded in 6-well plates at 2 × 105 cells per well and cultured as described above. The recombinant eukar yot ic express ion plasmid pcDNA3.1+/RZ was transfected into SMMC-7721 cells by LipofetamineTM2000 (Invitrogen, San Diego, CA) following the manufacturer's instructions, and positive clones were selected with G418, then SMMC-7721/RZ transgenic cells were obtained. At the same time, two control groups were established: the SMMC-7721 cell control (abbreviated as SMMC-7721 cell) and the SMMC-7721 cells transfected with blank vector of pcDNA3.1+ control (abbreviated as SMMC-7721/pcDNA3.1+ cells).

Integration and transcription of the objective gene in SMMC-7721/RZ transgenic cells were identified by polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR). Genomic DNA was extracted from cells in 3 groups using genomic DNA (Shengong, Shanghai, China). PCR was performed to detect the integration of ribozyme gene in SMMC-7721/RZ transgenic cells. The ribozyme primers (forward: 5'-GGACTTTCCTACTTGGCAGTACATC-3', reverse: 5'-CCACAACGTGTGTTTCTCTGGTTGG-3') were used in this study. Genomic DNA template was amplified with an initial denaturation at 94℃ for 2 min, then 30 cycles at 94℃ for 50 s, at 58℃ for 50 s and at 72℃ for 45 s, followed by at 72℃ for 10 min. To evaluate transcription of the ribozyme gene in SMMC-7721/RZ transgenic cells, RT-PCR was performed. Total RNA was extracted from cells using UNIQ column total RNA extraction kits (Shengong, Shanghai, China). First strand cDNA was synthesized from 2 μg of total RNA with an oligo (dT)18-primer in 20 μL of first strand reaction mix at 37℃ for 1 h. Thirty cycles of PCR were done. First strand cDNA was employed as template and the sequences of specific primers of ribozyme are as follows: forward: 5'-TAG AGAACCCACTGCTTACTGGCT-3', and reverse: 5'- CCACAACGTGTGTTTCTCTGGTTGG-3'.

RT-PCR and ELISA for VEGF expression SMMC-7721/RZ cells were seeded in 24-well plates at

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1 × 105 cells per well. The SMMC-7721 cell control and the SMMC-7721/pcDNA3.1+ cell control were established. Cells were cultured for 72 h, and then the supernatant was harvested. Total RNA was isolated from cells, and then RT-PCR was performed to detect the transcription level of VEGF mRNA in cells. Data were presented and normalized to β-actin. The sequences of PCR primers of VEGF and β-actin are as follows: VEGF (forward: 5 ' - T G G TAG AG T T C AT G G AT G T C TAT C A - 3 ' , reverse: 5'-GCATGGTGATGTTGGACTCCTCA-3'), β-actin (forward: 5'-TGCGTGACATTAAGGAGAAG-3', reverse: 5'-CTGCATCCTGTCGGCAATG-3'). The expression of VEGF protein in supernatant was detected by ELISA assay following the manufacturer's instructions (Jingmei, Shanghai, China).

Cell proliferation detection by MTT assaySMMC-7721/RZ cells were seeded in 96-well plates at 1 × 104 cells per well. Three control groups were established as follows: non-cell control group, SMMC-7721 cell control group and SMMC-7721/pcDNA3.1+ cell control group. Cells were cultured for 24, 48, 72, 96 h respectively, and viability was assessed using 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) (SIGMA, St. Louis. USA) at a final concentration of 0.5 mg/mL. Cells were incubated for 2 h, the medium was aspirated, and the cells were dissolved in acidic isopropanol (90% isopropanol, 0.5% sodium dodecyl sulfate, 40 mmol/L HCl). Optical density was read on a microplate reader at 570 nm using the isopropanol as blank. The inhibitory rate (IR) was calculated as follows: IR (%) = (1-absorbance of the treated wells)/(absorbance of the control wells) × 100%.

Human hepatocarcinoma xenograft experimentsFemale athymic BLAB/c nude mice, 4-5 wk of age, were purchased from Shanghai Experimental Animal Company, Chinese Academy of Sciences (Shanghai, China) and housed in a pathogen-free facility. In vivo studies approved by the Fourth Affiliated Hospital of Soochow University Animal Care Committee, were conducted in accordance with the institutional and China guidelines. Subconfluent SMMC-7721/RZ transgenic cells or SMMC-7721/pcDNA3.1+ cells were suspended in PBS and injected subcutaneously into the right flanks (2 × 106/0.1 mL) of 5 nude mice in each treatment group. Tumor size was measured every other day using an external caliper. Tumor volumes (V) were determined following the equation: V = (L × W2) × 0.5, where L is the length and W is the width of tumor. The standard for tumor formation was the diameter of tumors > 0.5 cm. When the experiment was terminated at the 5th wk after tumor formation, the mice were sacrificed. The tumors were isolated, photographed, snap frozen in liquid nitrogen and stored at -80℃, or fixed in formalin for subsequent multiple assays.

Histologic and immunohistochemical assayTumor tissues were formalin-fixed and paraffin-embedded. After deparaffinized and rehydrated, the slides were stained with hematoxylin and eosin (HE) in succession, and finally mounted and visualized under microscope. The number

of karyokinesis and pathologic karyokinesis was calculated in each visual field, and ten visual fields were selected randomly in each section.

Immunostaining was performed on 5-μm thick sections. Serial sections of paraffin-embedded tumor tissue were dewaxed and hydrated. After antigen retrieval, its endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution for 5-10 min. The sections were washed in distilled water, and soaked in pH 7.4 phosphate-buffered saline (PBS) for 5 min. Nonspecific binding was blocked by incubation with 3% normal goat serum for 10 min. The sections were incubated with primary antibody, rabbit anti-human VEGF polyclonal antibody or rat anti-murine cell determinant (CD34) monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz), at a dilution of 1:100 for 1-2 h at 37℃. Anti-CD34 was used as a pan-endothelial marker for microvessel density (MVD) analysis. After washed with PBS, the sections were incubated for 30 min at 37℃ with biotinylated secondary antibody and then with horseradish peroxidase (HRP)-conjugated streptavidin for 30 min at 37℃. The color was developed with a Vector DAB substrate kit for 1 min and 30 s and counterstained with hematoxylin. All sections were analyzed by conventional light microscopy and digital photography.

MVD was evaluated according to the CD34 endothelial cell immunostaining. For the microvessel counting, positivelystained CD34 was counted in ten visual fields of each slide. Any endothelial cluster positive for CD34 (brown yellow staining) was considered a single countable microvessel.

Statistical analysisAll statistical analyses were carried out by the Statistical Package for the Social Sciences (SPSS) software, Version 10.0 for Windows. All values such as tumor volume and weight, karyokinesis and microvessel, were expressed as mean ± SD. Chi-square test was used for rate contrast. P < 0.05 was considered statistically significant.

RESULTSConstruction and identification of recombinant eukaryotic expression plasmid pcDNA3.1+/RZ When products from annealing of the two specific primers of ribozyme were run on agarose gels and visualized with ethidium bromide staining, an amplicon of 50 bp of anti-hVEGF hairpin ribozyme (including cohesive terminal of BamHI and EcoRI) was shown. After cleaved by BamHI and EcoRI, the recombinant eukaryotic expression plasmid pcDNA3.1+/RZ generated an amplicon of 50 bp, revealing that anti-hVEGF hairpin ribozyme was successfully subcloned into pcDNA3.1+. Recombinant plasmids harboring ribozyme sequences in the pcDNA3.1+ backbone were checked by restriction enzyme analysis and sequencing. As shown in Figure 1A, the sequence of the gene subcloned into pcDNA3.1+ was identical to that of the synthetic gene designed according to Genebank, indicating that the recombinant eukaryotic

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expression plasmid pcDNA3.1+/RZ was successfully constructed.

Integration and transcription of anti-hVEGF hairpin ribozyme in SMMC-7721/RZ cellsThe forward and reverse primers were designed based on the sequence of pcDNA3.1+ and anti-hVEGF hairpin ribozyme. PCR amplification of ribozyme in genomic DNA of SMMC-7721/RZ cells generated an amplicon of 400 bp (Figure 1B). Using the primers designed based on the sequence of pcDNA3.1+ transcriptional start site and ribozyme, RT-PCR showed that PCR amplification of ribozyme in cDNA of SMMC-7721/RZ cells generated an amplicon of 140 bp (Figure 1C). In contrast, these amplicons were not found in SMMC-7721 cells and SMMC-7721/pcDNA3.1+ cells, demonstrating that the gene of anti-hVEGF hairpin ribozyme transfected into SMMC-7721 cells could be integrated with SMMC-7721 cell genome and transcripted into corresponding mRNA efficiently.

VEGF transcription and expressionAs the ultimate goal we sought to achieve was the reduction of VEGF secretion into extracellular compartment by SMMC-7721 cells, we investigated if transfection of ribozyme has a direct effect on VEGF secretion by SMMC-7721 cells. As demonstrated in Figure 2A, VEGF protein expression level detected by ELISA in transgenic SMMC-7721/RZ group was 363.64 ± 19.68 ng/L,

which was significantly lower than that in SMMC-7721 cell group (1358.69 ± 49.81 ng/L) and SMMC-7721/pcDNA3.1+ group (1369.57 ± 32.61 ng/L) (P < 0.01). There was no significant difference between SMMC-7721 cell group and SMMC-7721/pcDNA3.1+ group (P > 0.05). These results indicate that ribozyme inhibited VEGF expression (more than 73%) in HCC cells in vitro.

To determine whether the inhibitory effect of ribozyme on VEGF expression is at the transcriptional level, VEGF mRNA expression in tumor cells was detected by RT-PCR. The products were run on 1% agarose gels and visualized with ethidium bromide staining. SMMC-7721/RZ cells and two control group cells generated an amplicon of 195 bp. By normalizing to β-actin, the level of VEGF mRNA expression in SMMC-7721/RZ cells was remarkably lower than that in two control groups (Figure 2B), suggesting that ribozyme could reduce VEGF expression by inhibiting its transcription.

Proliferation of transgenic cellsAs shown in Figure 2C-D, after cells were cultured for 24, 48, 72 and 96 h, the proliferation rate for SMMC-7721/RZ cells was significantly lower than that for SMMC-7721 cells and SMMC-7721/pcDNA3.1+ cells (P < 0.01). On the third day, ribozyme-mediated growth inhibition rate reached 78.6%. There was no significant difference in proliferation of SMMC-7721 cells and SMMC-7721/pcDNA3.1+ cells (P > 0.05). The results reveal that ribozyme inhibited proliferation of HCC cells in vitro.

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Figrue 1 Construction and identification of recombinant eukaryotic expression plasmid pcDNA3.1+/RZ. A: Sequencing result of the anti-hVEGF hairpin ribozyme gene subcloned into pcDNA3.1+; B: Integration of the ribozyme gene detected by PCR (1: SMMC-7721 cells; 2: SMMC-7721/pcDNA3.1+cells; 3: SMMC-7721/RZ cells; M: DL2000 marker); C: Transcription of the ribozyme gene detected by RT-PCR (1: SMMC-7721/RZ cells; 2: SMMC-772 cells; 3: SMMC-7721/ pcDNA3.1+ cells; M: DL2000 marker).

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Formation and growth of xenografted tumor To determine the impact of anti-hVEGF hairpin ribozyme on tumorigenicity in vivo, control and ribozyme-transduced cells were transplanted subcutaneously into BLAB/c nude mice. Histology and immunohistochemistry displayed different levels of VEGF expression and angiogenesis on sections of xenografted and control tumor tissues. As expected, tumors appeared on d 14 (± 6.3) and d 7 (± 2.6) in SMMC-7721/RZ group and SMMC-7721/pcDNA3.1+ group, respectively (P < 0.01). The ribozyme transgenic cells resulted in tumor formation with lower levels of VEGF expression and angiogenesis and delayed tumor formation on 17.2 ± 13.7 d compared with 8.6 ± 3.3 d in control cells (P < 0.01). Formation of large, red (vascular) tumors was observed in SMMC-7721/pcDNA3.1+ cells of all mice. However, formation of small white (avascular) tumors was found after inoculation with SMMC-7721/RZ cells. The tumor volume and weight at the time of sacrifice in SMMC-7721/RZ group (0.19 ± 0.0085 cm3, 0.26 ± 0.076 g) were significantly lower than those in SMMC-7721/pcDNA3.1+ group (0.59 ± 0.019 cm3, 0.74 ± 0.050 g, P < 0.01). The inhibitory effects of anti-hVEGF hairpin ribozyme on HCC xenograft growth in nude mice were comparable and significant (P < 0.01).

Histologic and immunohistochemical findingsAs shown in Figure 3A, different shape and size of tumor cells were found in SMMC-7721/pcDNA3.1+ group. Their nuclei were large with a disproportional ratio of karyoplasm. Nuclear chromatin was condensed. Several basophilia chromatospherites were seen. Normal karyokinesis (8.26 ± 0.55/visual field) and pathologic karyokinesis (8.52 ± 0.17/visual field) were frequently

observed. In contrast, the shape and size of tumor cells in SMMC-7721/RZ group were coincident. Nuclear chromatin was puff. Normal karyokinesis (3.18 ± 0.23/visual field) and pathologic karyokinesis (3.3 ± 0.37/visual field) were significantly less than those in SMMC-7721/pcDNA3.1+ group (P < 0.01).

Immunohistochemical staining showed VEGF as light brown particles. VEGF chromatosis was distinctly less or/and weaker in SMMC-7721/RZ group than in SMMC-7721/pcDNA3.1+ group (Figure 3B).

The control mice displayed well vascularized tumors. Tumor blood vessels and their wall were abundant in the control group. In SMMC-7721/RZ group, however, tumor blood vessels were seldom seen with vessel wall frame collapsed. As shown in Figure 3C, a significant difference in the relative intensity of immunostaining for anti-CD34 antibody (an endothelial marker) was observed between the SMMC-7721/RZ group and the control group, indicating that vascularization was decreased in SMMC-7721/RZ group after transfection of anti-hVEGF hairpin ribozyme. MVD in the sections was significantly lower in SMMC-7721/RZ group (3.82 ± 0.88) than in the control group (7.02 ± 0.14, P < 0.01).

DISCUSSIONIt was reported that ribozyme is used to inhibit cellular targets [23-25]. Among them is the r ibozyme-based therapeutics for cancer which might be devised to inhibit tumor growth or to prevent its metastasis. Compared to other gene technologies, the superiorities of ribozyme are as follows: (1) ribozyme has dual effects of cleavage and blockade, (2) it cannot be easily hydrolyzed by nucleic acid enzymes because it can bind to target RNA to

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Figure 2 Effects of anti-hVEGF hairpin ribozyme on expression of VEGF and proliferation of SMMC-7721 cells in vitro. A: ELISA validation of VEGF expression; B: RT-PCR validation of VEGF mRNA transcription in SMMC-7721 group(1), SMMC-7721/pcDNA3.1+ group(2) and SMMC-7721/RZ group(3), (M: DL2000 marker); C: Influence of r ibozyme on SMMC-7721 cell proliferation; D: Influence of ribozyme on inhibitory rate for proliferation of SMMC-7721 cells.

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form a stable helix structure, (3) ribozyme molecule can destruct multiple target RNAs and be used repeatedly[26]. Biochemical characterization has shown that hairpin ribozyme is one of the most efficient ribozymes.

In order to devise effective anti-hVEGF hairpin ribozymes, we performed a sequence analysis of hVEGF mRNA and studied its secondary structure. We took into account the ideal site of hairpin ribozyme cleavage and its substrates in exon 3 of a GUC since this site is more easy for hairpin ribozyme to approach and less affected by the secondary structure. A fragment of the ribozyme gene and its sequence were verified by digestion of pcDNA3.1+/RZ with BamHI and EcoRI and sequencing. Our results show that the recombinant eukaryotic expression vector pcDNA3.1+/RZ could be successfully constructed. Stable integration and transcription of the ribozyme gene in SMMC-7721 cells were confirmed by PCR and RT-PCR. When introduced into hepatocarcinoma cell line SMMC-7721, anti-hVEGF hairpin ribozyme suppressed not only VEGF mRNA transcription but also VEGF protein expression. VEGF protein expression was inhibited by approximately 73% in SMMC-7721 cells transfected

by ribozyme. These results reveal that the constructed ribozyme can selectively inhibit VEGF gene expression in human hepatocarcinoma cells. Ciafre et al [27] have developed an anti-VEGF hammerhead ribozyme targeting the 5' part of human VEGF mRNA. Transfection of U87 human glioblastoma cells with plasmid vectors encoding for this ribozyme resulted in a strong (-56%) reduction of VEGF secretion in the extracellular medium. We believe that our ribozyme has a better biological activity in selectively inhibiting VEGF expression. The successful use of ribozymes as therapeutic agent depends upon many factors, e.g., category of ribozymes (hairpin and hammerhead ribozymes), relative amount of active ribozymes in cells, their co-localization with target RNAs, structural features of transcripts influencing accessibility to specific target sites, catalytic efficiency, interaction of target RNAs with proteins, and intracellular stability of targeted RNAs and ribozymes.

Little is known about the role of VEGF in HCC cell proliferation, differentiation and tumorigenesis. One of the more intriguing aspects of our results is that after cells were cultured for 24, 48, 72 and 96 h, the proliferation

Figure 3 HE staining of tumor tissue (A), immunohistochemical staining of VEGF antibody (B) and CD34 antibody (C) for assay of their positive particles.

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rate for SMMC-7721/RZ cells was significantly lower than that for SMMC-7721 cells and blank vector cells, and the inhibitory rate of ribozyme for the growth of SMMC-7721 cells was 21.4% on the third day. Apoptosis peak was detected by flow cytometry in SMMC-7721/RZ cells, the rate of apoptosis was 11.0% (data not shown), suggesting that the expression of anti-hVEGF hairpin ribozyme can significantly reduce VEGF secretion by transfected cells and inhibit cell growth and apoptosis in hepatocarcinoma cells. In other words, VEGF signaling can prolong the survival of HCC cells that may be independent of angiogenesis. Since Masood et al [28] reported that the expression of Flt-1 and KDR, the receptors of VEGF, is high in AIDS-Kaposi sarcoma cells and primary tumor tissues. Similar results have been observed in several tumors, by researchers in succession[29-31]. To inhibit VEGF expression or to block VEGF binding to its receptor results in growth inhibition of tumor cell lines, such as melanoma and ovarian carcinoma cell lines expressing VEGF receptors. In sharp contrast, cel l l ines not expressing VEGFRs show no response[28]. VEGF addition causes phosphorylation of mitogen-activated protein kinase as well as VEGF receptor, and induces proliferation and migration of lung cancer cells[32]. Our results are consistent with those previously observed. These findings suggest that tumor cells secrete VEGF which not only induces angiogenesis but also acts as an autocrine growth factor for carcinoma cells defined by the expression of VEGF receptors[33]. Our data suggest that HCC cells express specific VEGF receptors which respond to autocrine VEGF, thus activating signaling pathways that impede apoptosis and promote cell proliferation.

Because results on cell lines often represent a distorted and incomplete picture of the in situ physiopathology of cancer where the tumor microenvironment and neovascularization play a critical role in tumor growth and progression, we expanded our study to matched primary tumors using xenograft models. Oncogenicity is a critical index to judge malignant proliferation. The transfected SMMC-7721 cells were transplanted into nude mice. VEGF expression and microvascularization were distinctly decreased in SMMC-7721/RZ cell group when compared with SMMC-7721/pcDNA3.1+ cell group. Furthermore, tumor formation was delayed and its growth was significantly slowed down by ribozyme. Furthermore, reduced VEGF production induced by ribozyme resulted in higher cell differentiation, less cyto-heteromorphism and proliferation in the ribozyme transfected group, as demonstrated by microscopy, suggesting that VEGF is functionally related to cell differentiation directly or indirectly by regulating expression of certain genes. Our data reveal that VEGF plays a basic role in the induction of tumorigenicity, and underlines the importance of inhibiting its production. Moreover, Xu et al[34] found that down-regulation of VEGF gene by introducing anti-VEGF hairpin ribozyme gene into leukemia cell line K562 can alter the gene expression profiles. Among the 4096 gene clones on the microarray, 191 were detected to have the marked changes with 104 down-regulated and 87 up-regulated, showing that they are functionally related to cell cycle progression, gene replication, metabolism, cell

apoptosis, cell signal transduction, and oncogenes[34]. All these findings indicate that VEGF is a key molecule for tumor progression, although it is unclear how VEGF expression regulates various genes.

In human hepatocarcinoma tumor xenograft expe-riment, although there was a significant difference in P value between the control and ribozyme groups, tumor formation was observed in the ribozyme group. To further decrease tumor formation, in combined with anti-VEGF ribozyme, it is necessary to explore ribozymes targeting other genes, which contribute to hepatocarcinoma tumorigenesis such as N- ras, C-myc and IGF-Ⅱ.

In conclusion, VEGF promotes tumorigenesis and angiogenesis, and anti-hVEGF hairpin ribozyme can efficiently inhibit the VEGF expression and growth of hepatocarcinoma in vitro and in vivo. In VEGFRs-expressing tumors such as HCC, VEGF inhibition may be attributed to inhibition of tumor angiogenesis and direct effects on tumor cell proliferation/survival. Anti-hVEGF hairpin ribozyme may be a candidate for HCC gene therapy. However, its clinical application needs further study.

COMMENTSBackgroundAngiogenesis, known as the development and proliferation of new blood vessels, is vital for the growth of tumors. Vascular endothelial growth factor (VEGF) is an essential cytokine in the regulation of angiogenesis. Little is known about the role of VEGF in hepatocellular carcinoma (HCC) proliferation, differentiation and tumorigenesis. The present study was to study the effectiveness and mechanisms of anti-human VEGF (hVEGF) hairpin ribozyme on VEGF expression, oncogenicity and tumor growth in hepatocarcinoma cell line and xenografted model.

Research frontiersHCC is a highly vascular tumor depending on neovascularization and insensitive conventional chemotherapeutics. It is important to explore antiangiogenesis methods for HCC. Hairpin ribozyme has been used in gene therapy.

Innovations and breakthroughsTo our knowledge, this is the first report that anti-hVEGF hairpin ribozyme can effectively inhibit VEGF expression and growth of hepatocarcinoma in vitro and in vivo. VEGF inhibition can inhibit tumor angiogenesis and tumor growth that may be dependent and independent of angiogenesis in HCC. Our results also suggest that VEGF is functionally related to cell proliferation, differentiation and tumorigenesis.

Applications Anti-hVEGF hairpin ribozyme can be used as a therapeutic agent for hepa-tocarcinoma.

TerminologyRibozymes: a group of catalytically active nucleic acids capable of site-specific cleavage of target mRNAs, thus decreasing mRNA expression and inhibiting the function of target gene.

Peer reviewIn this paper, the role of anti-VEGF hairpin ribozyme in antitumor and antigangiogenic activities is described. The experiments were well designed. The data support that VEGF plays its role through angiogenesis and other pathways directly leading to carcinoma.

REFERENCES1 Carmeliet P, Jain RK. Angiogenesis in cancer and other

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COMMENTS

diseases. Nature 2000; 407: 249-2572 Folkman J. The role of angiogenesis in tumor growth. Semin

Cancer Biol 1992; 3: 65-713 Ribatti D. The crucial role of vascular permeability factor/

vascular endothelial growth factor in angiogenesis: a historical review. Br J Haematol 2005; 128: 303-309

4 Nishida N , Yano H, Nishida T, Kamura T, Kojiro M. Angiogenesis in cancer. Vasc Health Risk Manag 2006; 2: 213-219

5 Yao DF, Wu XH, Zhu Y, Shi GS, Dong ZZ, Yao DB, Wu W, Qiu LW, Meng XY. Quantitative analysis of vascular endothelial growth factor, microvascular density and their clinicopathologic features in human hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int 2005; 4: 220-226

6 Iavarone M, Lampertico P, Iannuzzi F, Manenti E, Donato MF, Arosio E, Bertolini F, Primignani M, Sangiovanni A, Colombo M. Increased expression of vascular endothelial growth factor in small hepatocellular carcinoma. J Viral Hepat 2007; 14: 133-139

7 Horrée N, van Diest PJ, van der Groep P, Sie-Go DM, Heintz AP. Hypoxia and angiogenesis in endometrioid endometrial carcinogenesis. Cell Oncol 2007; 29: 219-227

8 Möbius C, Demuth C, Aigner T, Wiedmann M, Wittekind C, Mössner J, Hauss J, Witzigmann H. Evaluation of VEGF A expression and microvascular density as prognostic factors in extrahepatic cholangiocarcinoma. Eur J Surg Oncol 2007; 33: 1025-1029

9 El-Gohary YM, Silverman JF, Olson PR, Liu YL, Cohen JK, Miller R, Saad RS. Endoglin (CD105) and vascular endothelial growth factor as prognostic markers in prostatic adenocarcinoma. Am J Clin Pathol 2007; 127: 572-579

10 Hlatky L, Tsionou C, Hahnfeldt P, Coleman CN. Mammary fibroblasts may influence breast tumor angiogenesis via hypoxia-induced vascular endothelial growth factor up-regulation and protein expression. Cancer Res 1994; 54: 6083-6086

11 Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992; 359: 843-845

12 Gratzinger D, Zhao S, Marinelli RJ, Kapp AV, Tibshirani RJ, Hammer AS, Hamilton-Dutoit S, Natkunam Y. Microvessel density and expression of vascular endothelial growth factor and its receptors in diffuse large B-cell lymphoma subtypes. Am J Pathol 2007; 170: 1362-1369

13 Kopfstein L, Veikkola T, Djonov VG, Baeriswyl V, Schomber T, Strittmatter K, Stacker SA, Achen MG, Alitalo K, Christofori G. Distinct roles of vascular endothelial growth factor-D in lymphangiogenesis and metastasis. Am J Pathol 2007; 170: 1348-1361

14 Nikiteas NI, Tzanakis N, Theodoropoulos G, Atsaves V, Christoni Z, Karakitsos P, Lazaris AC, Papachristodoulou A, Klonaris C, Gazouli M. Vascular endothelial growth factor and endoglin (CD-105) in gastric cancer. Gastric Cancer 2007; 10: 12-17

15 Djordjevic G, Mozetic V, Mozetic DV, Licul V, Ilijas KM, Mustac E, Oguic R, Fuckar Z, Jonjic N. Prognostic significance of vascular endothelial growth factor expression in clear cell renal cell carcinoma. Pathol Res Pract 2007; 203: 99-106

16 Byrne AM, Bouchier-Hayes DJ, Harmey JH. Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF). J Cell Mol Med 2005; 9: 777-794

17 Orito E, Mizokami M. Hepatitis B virus genotypes and hepatocellular carcinoma in Japan. Intervirology 2003; 46:

408-41218 Tabor E. Tumor suppressor genes, growth factor genes, and

oncogenes in hepatitis B virus-associated hepatocellular carcinoma. J Med Virol 1994; 42: 357-365

19 Jiang YF, Yang ZH, Hu JQ. Recurrence or metastasis of HCC:predictors, early detection and experimental antiangiogenic therapy. World J Gastroenterol 2000; 6: 61-65

20 Toi M, Matsumoto T, Bando H. Vascular endothelial growth factor: its prognostic, predictive, and therapeutic implications. Lancet Oncol 2001; 2: 667-673

21 Puerta-Fernández E, Romero-López C, Barroso-delJesus A, Berzal-Herranz A. Ribozymes: recent advances in the development of RNA tools. FEMS Microbiol Rev 2003; 27: 75-97

22 Yan RL, Qian XH, Xin XY, Jin M, Hui HX, Wang DT, Wang J. Experimental study of anti-VEGF hairpin ribozyme gene inhibiting expression of VEGF and proliferation of ovarian cancer cells. Ai Zheng 2002; 21: 39-44

23 Vashishta A, Ohri SS, Proctor M, Fusek M, Vetvicka V. Ribozyme-targeting procathepsin D and its effect on invasion and growth of breast cancer cells: an implication in breast cancer therapy. Int J Oncol 2007; 30: 1223-1230

24 Li QX, Tan P, Ke N, Wong-Staal F. Ribozyme technology for cancer gene target identification and validation. Adv Cancer Res 2007; 96: 103-143

25 Jung HS, Lee SW. Ribozyme-mediated selective killing of cancer cells expressing carcinoembryonic antigen RNA by targeted trans-splicing. Biochem Biophys Res Commun 2006; 349: 556-563

26 Rossi JJ. Controlled, targeted, intracellular expression of ribozymes: progress and problems. Trends Biotechnol 1995; 13: 301-306

27 Ciafrè SA, Niola F, Wannenes F, Farace MG. An anti-VEGF ribozyme embedded within the adenoviral VAI sequence inhibits glioblastoma cell angiogenic potential in vitro. J Vasc Res 2004; 41: 220-228

28 Masood R, Cai J, Zheng T, Smith DL, Naidu Y, Gill PS. Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma. Proc Natl Acad Sci USA 1997; 94: 979-984

29 Masood R, Cai J, Zheng T, Smith DL, Hinton DR, Gill PS. Vascular endothelial growth factor (VEGF) is an autocrine growth factor for VEGF receptor-positive human tumors. Blood 2001; 98: 1904-1913

30 Ng IO, Poon RT, Lee JM, Fan ST, Ng M, Tso WK. Microvessel density, vascular endothelial growth factor and its receptors Flt-1 and Flk-1/KDR in hepatocellular carcinoma. Am J Clin Pathol 2001; 116: 838-845

31 Yoshiji H, Kuriyama S, Hicklin DJ, Huber J, Yoshii J, Ikenaka Y, Noguchi R, Nakatani T, Tsujinoue H, Fukui H. The vascular endothelial growth factor receptor KDR/Flk-1 is a major regulator of malignant ascites formation in the mouse hepatocellular carcinoma model. Hepatology 2001; 33: 841-847

32 Tanno S, Ohsaki Y, Nakanishi K, Toyoshima E, Kikuchi K. Human small cell lung cancer cells express functional VEGF receptors, VEGFR-2 and VEGFR-3. Lung Cancer 2004; 46: 11-19

33 Mercurio AM, Bachelder RE, Bates RC, Chung J. Autocrine signaling in carcinoma: VEGF and the alpha6beta4 integrin. Semin Cancer Biol 2004; 14: 115-122

34 Xu WL, Shen HL, Wu ZY, Tang HR, Wang FC. The down regulation of VEGF gene expression may link to change the expression profile of genes in leukemia cell line K562. Zhonghua Yixue Yichuanxue Zazhi 2006; 23: 37-42

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Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6433-6435www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

Complete pathological response following down-staging chemoradiation in locally advanced pancreatic cancer: Challenging the boundaries

AZ Khan, V Pitsinis, SS Mudan

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CASE REPORT

AZ Khan, V Pitsinis, SS Mudan, Department of Gastrointestinal Surgery, Royal Marsden Hospital, Fulham Road, London,United KingdomCorrespondence to: Aamir Z Khan, FRCS Eng (Gen), Department of Gastrointestinal Surgery, Royal Marsden Hospital, Chelsea, London SW3 6JJ,United Kingdom. aamirzkhan@yahoo.comTelephone: +44-207-3528171 Fax: +44-207-8082988Received: September 22, 2006 Revised: September 29, 2007

AbstractPancreat ic cancer is an aggress ive mal ignancy, relatively resistant to chemotherapy and radiotherapy, which usually presents late. Disease specific mortality approaches unity despi te advances in adjuvant therapy. We present the first reported case of complete pathological response following neoadjuvant therapy in a locally advanced pancreatic adenocarcinoma.

© 2007 WJG. All rights reserved.

Key words: Chemotherapy; Pancreatic cancer; Pathology

Khan AZ, Pitsinis V, Mudan SS. Complete pathological response following down-staging chemoradiation in locally advanced pancreatic cancer: Challenging the boundaries. World J Gastroenterol 2007; 13(47): 6433-6435

http://www.wjgnet.com/1007-9327/13/6433.asp

INTRODUCTIONPancreatic cancer is an aggressive intra-abdominal malignancy and approximately 3000 new cases are diagnosed each year in the United Kingdom, most of them usually present with advanced pancreatic cancer and only 10%-20% of patients are resectable at presentation. Surgery with complete resection of the primary lesion offers the only chance of long term survivorship and cure in this cohort of patients. For locally advanced pancreatic cancer, several chemotherapeutic and combined modality regimens have been described, generally with conflicting results, poor response rates, high toxicity, not changing the natural history of the disease. Several novel and new regimens are being tried in the neoadjuvant setting in patients with locally

advanced pancreatic cancer at presentation in the hope of down-staging the disease and rendering it resectable. We present the first case of complete pathological response following down-staging chemotherapy.

CASE REPORTA 69-year-old man presented with nausea, anorexia and weight loss. On investigation, he was diagnosed to have locally advanced adenocarcinoma of the pancreas encasing the superior mesenteric artery and vein with enlarged peri-pancreatic and aortocaval nodes (Figure 1). Final staging after endoscopic ultrasound was T4N1M0. The patient had a past medical history of aortic stenosis and atrial fibrillation for which he took diltiazem daily. Shortly after diagnosis he developed obstructive jaundice which was relieved by an endobiliary stent, which failed to achieve adequate drainage, following which he underwent a double bypass in the form of a gastrojejunostomy and hepatojejunostomy.

After a CT guided biopsy which confirmed invasive adenocarcinoma of the pancreas, he commenced chemotherapy wi th gemci tab ine in combinat ion with oxaliplatin. Upon completion of two cycles of chemotherapy, gemcitabine was replaced with capecitabine due to drug interaction causing breathlessness and he underwent 9 cycles of chemotherapy on the new regime. During chemotherapy surveillance cross-sectional imaging suggested stable disease with normal serum CA19-9 levels. After a 4-wk break he further had 6 cycles of the above combination chemotherapy.

Eighteen months from the initial diagnosis surveillance scans demonst ra ted g ood loca l response to the combination chemotherapy without any distant metastatic disease and he was referred for consolidation radiotherapy, of which he had a total of 25 fractions, which he tolerated well and CT scans suggested further reduction in the disease which now looked resectable (Figure 2).

Based on the remarkable response the pat ient underwent surgery with curative intent in the form of pancreatoduodenectomy approximately 2 years after his initial diagnosis from which he made an uneventful recovery. Histopathology review of the resected specimen showed a complete response with no evidence of the original invasive adenocarcinoma. A follow-up CT scan at 4 mo as shown did not demonstrate any evidence of recurrent disease.

DISCUSSIONPancreatic cancer remains a formidable challenge to clinicians and carries a tumour specific mortality of nearly 100%[1]. Complete resection offers the only hope of cure but is applicable to the minority of patients that present with resectable disease. Approximately 10%-20% of patients present with locally advanced pancreatic cancer without evidence of distant metastatic spread at the time of diagnosis. Prior to the use of neoadjuvant therapy these patients were denied surgery with curative intent and had very short survival times after diagnosis.

Several neoadjuvant regimens have been described to try and downstage locally advanced pancreatic cancer and chemoradiation here is considered as an induction therapy to reduce tumour volume, lymph node disease and extent of vascular involvement[2-5]. In locally advanced pancreatic cancer, complete remissions have not yet been described[6]. Neoadjuvant therapy has its own intrinsic advantages in that it theoretically increases vulnerability of cancer cells because of intact vasculature, better tumour cell oxygenation and probability of sterilizing cells at the resection margin. It also affords a test of the biology of the disease in that patients with progressive disease on chemotherapy can be spared an exploratory laparotomy and trial dissection and managed along the palliative pathway. The risk of pancreatic fistula also seems to be decreased in previously irradiated field[7].

Several regimens have been described and utilized in the neoadjuvant setting for locally advanced pancreatic cancer in an attempt to induce regression of the disease and render it resectable with conflicting results[8]. 5-FU based regimens were originally described but had poor response rates and eventually gave way to gemcitabine based regimens[9]. Combined modality treatments using gemcitabine in combination with other agents like capecitabine, oxaliplatin, bevacizumab and irenotecan have shown promise with prolonged progession-free intervals and better response rates[10-12]. In these settings gemcitobine is used as a radiation sensitizer and combination with external beam radiotherapy remains a popular regimen[13].

Complete pathological response remains an elusive goal in this subset of patients who may then be offered a pancreatoduodenectomy with the aim of long term surviviorship or cure, who if untreated have a very short

life expectancy. The future of combined modality therapy for locally advanced pancreatic cancer will focus on the development of newer biological agents that target specific alterations in the cancer cells including vascular endothelial growth factor, epidermal growth factor and matrix metalloprotineases[14,15].

In conclusion, complete pathological response following down-staging chemotherapy is possible in patients with locally advanced pancreatic cancer and may offer the patient a chance at long term survival or cure.

REFERENCES1 Kim HJ , Czischke K, Brennan MF, Conlon KC. Does

neoadjuvant chemoradiation downstage locally advanced pancreatic cancer? J Gastrointest Surg 2002; 6: 763-769

2 Bramhall SR, Allum WH, Jones AG, Allwood A, Cummins C, Neoptolemos JP. Treatment and survival in 13,560 patients with pancreatic cancer, and incidence of the disease, in the West Midlands: an epidemiological study. Br J Surg 1995; 82: 111-115

3 Sohn TA, Yeo CJ, Cameron JL, Koniaris L, Kaushal S, Abrams RA, Sauter PK, Coleman J, Hruban RH, Lillemoe KD. Resected adenocarcinoma of the pancreas-616 patients: results, outcomes, and prognostic indicators. J Gastrointest Surg 2000; 4: 567-579

4 Cleary SP, Gryfe R, Guindi M, Greig P, Smith L, Mackenzie R, Strasberg S, Hanna S, Taylor B, Langer B, Gallinger S. Prognostic factors in resected pancreatic adenocarcinoma: analysis of actual 5-year survivors. J Am Coll Surg 2004; 198: 722-731

5 Lawrence TS, Chang EY, Hahn TM, Hertel LW, Shewach DS. Radiosensitization of pancreatic cancer cells by 2',2'-difluoro-2'-deoxycytidine. Int J Radiat Oncol Biol Phys 1996; 34: 867-872

6 Willett CG, Lewandrowski K, Warshaw AL, Efird J, Compton CC. Resection margins in carcinoma of the head of the pancreas. Implications for radiation therapy. Ann Surg 1993; 217: 144-148

7 Mancuso A, Calabrò F, Sternberg CN. Current therapies and advances in the treatment of pancreatic cancer. Crit Rev Oncol Hematol 2006; 58: 231-241

8 Hoffman JP , Cooper HS, Young NA, Pendurthi TK. Preoperative chemotherapy of chemoradiotherapy for the treatment of adenocarcinoma of the pancreas and ampulla of Vater. J Hepatobiliary Pancreat Surg 1998; 5: 251-254

9 Wanebo HJ, Glicksman AS, Vezeridis MP, Clark J, Tibbetts L, Koness RJ, Levy A. Preoperative chemotherapy, radiotherapy, and surgical resection of locally advanced pancreatic cancer. Arch Surg 2000; 135: 81-87; discussion 88

10 Louvet C, Labianca R, Hammel P, Lledo G, Zampino MG, André T, Zaniboni A, Ducreux M, Aitini E, Taïeb J, Faroux R, Lepere C, de Gramont A. Gemcitabine in combination

Figure 1 Initial CT scan showing the inoperable mass in the pancreatic head. Figure 2 CT scan after completion of chemotherapy showing the operable disease.

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with oxaliplatin compared with gemcitabine alone in locally advanced or metastatic pancreatic cancer: results of a GERCOR and GISCAD phase III trial. J Clin Oncol 2005; 23: 3509-3516

11 Reni M, Cordio S, Milandri C, Passoni P, Bonetto E, Oliani C, Luppi G, Nicoletti R, Galli L, Bordonaro R, Passardi A, Zerbi A, Balzano G, Aldrighetti L, Staudacher C, Villa E, Di Carlo V. Gemcitabine versus cisplatin, epirubicin, fluorouracil, and gemcitabine in advanced pancreatic cancer: a randomised controlled multicentre phase III trial. Lancet Oncol 2005; 6: 369-376

12 Yip D, Goldstein D. Adding irinotecan to first-line gemcitabine

improves tumour response in advanced pancreatic cancer. Cancer Treat Rev 2005; 31: 236-241

13 M o r n e x F , G i r a r d N , D e l p e r o J R , P a r t e n s k y C . Radiochemotherapy in the management of pancreatic cancer--part I: neoadjuvant treatment. Semin Radiat Oncol 2005; 15: 226-234

14 Dickler A, Abrams RA. Radiochemotherapy in the management of pancreatic cancer--part II: use in adjuvant and locally unresectable settings. Semin Radiat Oncol 2005; 15: 235-244

15 Heidelberger C, Griesbach L, Montag BJ, Mooren D, Cruz O, Schnitzer RJ, Grunberg E. Studies on fluorinated pyrimidines. II. Effects on transplanted tumors. Cancer Res 1958; 18: 305-317

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INTRODUCTIONHepatocel lular carcinoma (HCC) is a very highly invasive tumor that metastasizes hematogenously and lymphogenously to distant sites. Most frequently affected sites are the lungs, regional lymph nodes, bones, and adrenal glands[1]. However, external auditory canal (EAC) metastases from HCC are extremely rare. A review of the literature revealed only five other cases of HCC metastasis to the temporal bone, all of which mainly metastasized in the internal acoustic meatus[2-4]. To the best of our knowledge, the present patient is the first case of HCC metastasis to the EAC.

We report here a rare case of bleeding due to EAC metastasis from HCC. The patient was treated with surgical debulking and high dose rate 192Ir remote afterloading system (RALS) for postoperative intracavitary irradiation.

CASE REPORTA 55-year-old man presented to our department with a three-month history of increasing left otalgia, and hearing loss with recent fresh aural bleeding, in April 2006. He denied any vertigo, and had no evidence of facial palsy. His past medical history revealed that hepatitis B virus-related HCC was diagnosed two years ago. Owing to the tumor’s inoperability and the presence of lung metastases, the patient began systemic chemotherapy with 5-fluorouracil and cisplatin, which was maintained for two years.

Physical examination did not find the left tympanic membrane, because the EAC was completely occupied by a tumor. Right-sided otoscopy was normal. Vestibular examination was unremarkable. There was no lymphade-nopathy, and the remainder of the head and neck and neurologic examinations was unremarkable.

Magnetic resonance imaging showed a well-enhanced tumor lesion within the left EAC. T1- and T2- weighted images revealed that the EAC lesion exhibited a mixture of hypointensity and hyperintensity (Figure 1A). CT imaging showed tumor formation on the posterior wall of the EAC, and a shallow bony erosion (Figure 1B).

A biopsy was performed, and pathologic examination

CASE REPORT

Metastatic hepatocellular carcinoma of the external auditory canal

Ryuji Yasumatsu, Kenji Okura, Yumiko Sakiyama, Makoto Nakamuta, Taisei Matsumura, Satoru Uehara, Tomoya Yamamoto, Shizuo Komune

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Ryuji Yasumatsu, Department of Otorhinolaryngology, Graduate School of Medical Sciences, Kyushu University and Department of Otorhinolaryngology and Clinical Research Institute, National Hospital Organization Kyushu Medical Center, Fukuoka 812-8582, JapanKenji Okura, Tomoya Yamamoto, Department of Otorhino-laryngology and Clinical Research Institute, National Hospital Organization Kyushu Medical Center, Fukuoka 812-8582, JapanYumiko Sakiyama, Makoto Nakamuta, Department of Gastro-enterology and Clinical Research Institute, National Hospital Organization Kyushu Medical Center, Fukuoka 812-8582, JapanTaisei Matsumura, Satoru Uehara, Department of Radiology and Clinical Research Institute, National Hospital Organization Kyushu Medical Center, Fukuoka 812-8582, JapanShizuo Komune, Department of Otorhinolaryngology, Graduate School of Medical Sciences, Kyushu University, JapanCorrespondence to: Ryuji Yasumatsu, Department of Otorhi-nolaryngology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. yasuryuj@qent.med.kyushu-u.ac.jp Telephone: +81-92-6425668 Fax: +81-92-6425685Received: August 29, 2007 Revised: September 28, 2007

AbstractThis report descr ibes a rare case of metastat ic hepatocellular carcinoma (HCC) presenting a huge mass in the left external auditory canal (EAC). The patient was a 55-year-old man with hepatitis B virus-related HCC. He presented to our department with a three-month history of increasing left otalgia, and hearing loss with recent fresh aural bleeding. Histopathologic examination indicated that the tumor was secondary to HCC. Although external irradiation was not effective, the tumor was treated with surgical debulking and high dose rate 192 Ir remote afterloading system (RALS) for postoperative intracavitary irradiation. A review of the literature revealed only five other cases of HCC metastasis to the temporal bone, all of which mainly metastasized in the internal acoustic meatus. The present case is the first report of HCC metastasis to the EAC.

© 2007 WJG. All rights reserved.

Key words: Hepatocellular carcinoma; Metastasis; External auditory canal

Yasumatsu R, Okura K, Sakiyama Y, Nakamuta M, Matsumura T, Uehara S, Yamamoto T, Komune S. Metastatic hepatocellular carcinoma of the external auditory canal.

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taken through the EAC suggested metastasis of HCC with histopathologic features similar to the primary lesion (Figure 2). The biopsy of the mass produced much bleeding, which was stopped with local pressure.

First, the patient received external irradiation (total dose, 30 Gy) to treat the EAC lesion. Despite the external irradiation therapy, the tumor increased in size, and was continuously bleeding. Therefore, it was decided to change the treatment from external irradiation to surgical debulking and 192Ir RALS. We planned to proceed with the debulking surgery for the progressive disease followed by transcatheter arterial embolization by means of superselective catherization.

Angiography was performed to delineate blood supply to the mass, and to embolize any feeding vessels. The external carotid artery was cannulated via the femoral artery, using the Seldinger technique. Evidence of vascular blush in the region of the left EAC was observed, and the

Yasumatsu R et al. HCC of the external auditory canal 6437

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posterior auricular artery was identified for embolization (Figure 3).

Following angiography, surgical debulking was performed under general anesthesia and a 6-Fr catheter for the 192Ir remote afterloader system was inserted into the left EAC (Figure 4A and B). A total dose of 15 Gy (10 Gy/Fr/wk) was given over a one-week period. When the irradiation treatment was completed in May 2006, otoscopy revealed tumor remission. Neither radiation-induced dermatitis nor otitis media with effusion was observed. Although the patient remained clinically free of disease in his left EAC (Figure 5), he died of progressive liver failure and lung metastases from HCC 9 mo after the treatment.

DISCUSSIONHCC is the most common malignant tumor of the

A

B

Figure 1 Ax ia l T1-w e i g h t e d m a g n e t i c resonance image of the head showing a huge mass in the left EAC with extension of the lesion into the mastoid (A), and axial CT of t h e t e m p o r a l b o n e demonstrating the mass occupying the left EAC (B).

Figure 2 Microscopically, the lesion in the external auditory canal is composed of proliferating atypical epithelial cells forming a solid and thick trabecular arrangement with necrosis, and a fibrous stroma (hematoxylin/eosin stain, x 400).

Figure 3 Angiography showing hypervascula-rity in the left external auditory canal supplied by the posterior auricular artery (the arrow indica-tes the tumor stain).

A

B

Figure 4 Metastatic hepatocellular carcinoma protruding from the left external auditory canal (A) and inserted and fixed catheter for the 192Ir remote afterloader system in the left external auditory canal after debulking surgery (B).

liver. Intrahepatic metastases occur early in the disease, and more than half of these tumors metastasize to extrahepatic sites[5]. Autopsy and surgical series have suggested the presence of metastases from HCC in the lungs (18.1%-49.2%), lymph nodes (26.5%-41.7%), bones (4.2%-16.3%), and adrenal glands (8.4%-15.4%)[6-8]. However, reports on metastases from distant primary malignancies to different sites in the ear including EAC, are uncommon. The temporal bones are among the more common sites of the ear to develop metastases from distant malignancies[9]. Gloria-Cruz et al[4] reviewed the autopsy records of 864 cases, and reported that 47 of 212 patients (22%) with primary non-disseminated neoplasms had metastatic diseases involving the temporal bones. The primary site of the majority of metastases to the temporal bones is the breast (21%), followed by the lungs (12%), kidneys, and prostate, and only three patients (6%) showed histologic features consistent with HCC[4]. No other study has reported HCC metastasis to the EAC. To the best of our knowledge, the present patient is the first case of HCC metastasis to the EAC.

To treat metastatic tumors including HCC of the EAC, curative surgical resection is sometimes impossible, radiation therapy and chemotherapy can be considered as palliative therapies. Although external irradiation was performed in the present case, it was not effective, and the tumor was continuously bleeding. Therefore, we attempted surgical debulking and 192Ir RALS as postoperative irradiation. As a result, fiberscopy after treatment confirmed the effectiveness and safety of this combined procedure. In general, side effects of conventional external irradiation to the auditory canal include many complications such as external canal stenosis, otitis media with effusion, cholesteatoma,

sensorineural hearing loss, vestibular impairment, facial nerve paralysis, and osteoradionecrosis. However, none of these complications was observed in this case.

192Ir RALS is the best choice of treatment to ensure decreased doses to other important organs such as the tympanic membrane, temporal bone, and carotid artery while providing a curable dose to a target volume in EAC tumors[10]. In addition, this treatment can be performed in a short period, without any invasive procedure, and may become a choice of modality for its efficacy and less severe side effects.

In conclusion, a rare patient with EAC metastasis from HCC can be treated with surgical debulking and high dose rate 192Ir RALS for postoperative intracavitary irradiation. This combined treatment can be used as a palliative modality for such patients.

REFERENCES1 Kim HS, Shin JW, Kim GY, Kim YM, Cha HJ, Jeong YK, Jeong

ID, Bang SJ, Kim do H, Park NH. Metastasis of hepatocellular carcinoma to the small bowel manifested by intussusception. World J Gastroenterol 2006; 12: 1969-1971

2 Nagai M, Yamada H, Kitamoto M, Ikeda J, Mori Y, Monzen Y, Fukuhara T. Facial nerve palsy due to temporal bone metastasis from hepatocellular carcinoma. J Gastroenterol Hepatol 2005; 20: 1131-1132

3 B r o w n N E , O ’ B r i e n D A , M e g e r i a n C A . M e t a s t a t i c hepatocellular carcinoma to the temporal bone in a post-liver transplant patient. Otolaryngol Head Neck Surg 2004; 130: 370-371

4 Gloria-Cruz TI, Schachern PA, Paparella MM, Adams GL, Fulton SE. Metastases to temporal bones from primary nonsystemic malignant neoplasms. Arch Otolaryngol Head Neck Surg 2000; 126: 209-214

5 Okada H , Kamino Y, Shimo M, Kitamura E, Katoh T, Nishimura H, Akimoto Y, Kaneda T, Hinata M, Yamamoto H. Metastatic hepatocellular carcinoma of the maxillary sinus: a rare autopsy case without lung metastasis and a review. Int J Oral Maxillofac Surg 2003; 32: 97-100

6 Kaczynski J, Hansson G, Wallerstedt S. Metastases in cases with hepatocellular carcinoma in relation to clinicopathologic features of the tumor. An autopsy study from a low endemic area. Acta Oncol 1995; 34: 43-48

7 Yuki K, Hirohashi S, Sakamoto M, Kanai T, Shimosato Y. Growth and spread of hepatocellular carcinoma. A review of 240 consecutive autopsy cases. Cancer 1990; 66: 2174-2179

8 Ho J, Wu PC, Kung TM. An autopsy study of hepatocellular carcinoma in Hong Kong. Pathology 1981; 13: 409-416

9 Hill BA, Kohut RI. Metastatic adenocarcinoma of the temporal bone. Arch Otolaryngol 1976; 102: 568-571

10 Uchida N, Kuroda S, Kushima T, Moriyama M, Sugimura K, Kawauchi H, Shirakawa R. Squamous cell carcinoma of the external auditory canal: two cases treated with high dose rate 192Ir remote afterloading system (RALS). Radiat Med 1999; 17: 443-446

S- Editor Liu Y L- Editor Wang XL E- Editor Ma WH

Figure 5 Fiberscopy of the external auditory canal 6 mo after treatment showing no evidence of residual tumor (the arrow indicates the left tympanic membrane).

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INTRODUCTIONPorto-pulmonary hypertension (PPHT) refers to the development of pulmonary arterial hypertension in the setting of portal hypertension with or without chronic liver disease and is defined by a mean pulmonary artery pressure > 25 mmHg in the presence of normal pulmo-capillary wedge pressure (< 15 mmHg)[1,2]. The pathogenesis of PPHT is under investigation, although histopathologic features are similar to those found in primary pulmonary hypertension[3]. A current study supports the hypothesis that pulmonary vasculature may be exposed to either cytokines or excess circulating vasoconstrictors, such as endothelin-1 (ET-1) produced by the diseased liver[4]. Although the vast majority of patients with PPHT are asymptomatic, dyspnea is the most frequent presenting symptom[5].

CASE REPORTA 39-year-old obese woman with Laennec’s cirrhosis (Child-Pugh B) was admitted to our hospital in January 2003 because of the recent onset of dyspnea (NYHA IV), dependent edema and abdominal pain. In 1991, she under-went surgical side to side portocaval shunt for refractory ascites and since then she has never complained of dys-pnea or edema. Physical examination showed platypnea, arterial blood gas analysis showed hypoxaemia (pO2: 63 mmHg) with orthodeoxia (pO2: 49 mmHg). On admis-sion, liver function tests were as follows: total bilirubin: 84.5 µmol/L, AST: 1.14 µkat/L, ALT: 0.55 µkat/L, GGT: 87 U/L, alkaline phosphatase: 4.2 nkat/L, serum albumin: 28 g/L, ammonia (as NH3): 88 µmol/L, prothrombin time 60%. Hepatic ultrasonography with Doppler imaging showed massive intra-shunt thrombosis, portal hyperten-sion (18 mmHg) and mild ascites. No gastroesophageal varices were found by digestive endoscopy. D-dimer level was 1.4 mg/L and antithrombin activity was 43%. The presence of both peripheral venous thrombosis and recur-rent microembolism was ruled out by Doppler ultrasonog-raphy and ventilation-perfusion lung scan. Computerized tomography confirmed recent intra-shunt thrombosis and showed also pleuro-pericardial effusions and ascites. Mark-ers of autoimmunity were negative.

Right atrial and ventricular enlargement with severe

CASE REPORT

Decompensated porto-pulmonary hypertension in a cirrhotic patient with thrombosis of portocaval shunt

Chiara Giannarelli, Antonio De Giorgi, Ferdinando De Negri, Franco Carmassi

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Chiara Giannarelli, Antonio De Giorgi, Ferdinando De Negri, Franco Carmassi, Department of Internal Medicine, University of Pisa, via Roma 67, Pisa 56126, ItalyCorrespondence to: Chiara Giannarelli, Department of Internal Medicine, University of Pisa, via Roma 67, Pisa 56126, Italy. c.giannarelli@virgilio.it Telephone: +39-50-992446 Fax: +39-50-553414Received: February 20, 2004 Revised: October 15, 2007

AbstractWe report a case of decompensated porto-pulmonary hypertension closely associated with the development of intra-portocaval shunt thrombosis. A woman with Laennec's c irrhosis was hospital ized because of severe dyspnea and edema. She underwent surgical portocaval anastomosis ten years ago. Imaging studies showed massive intra-shunt thrombosis, portal hypertension, ascites, pleuro-pericardial effusions and enlargement of right cardiac cavities. Cardiac catheterization allowed to rule out coronary and left-sided heart abnormalities and led to the diagnosis of pre-capillary pulmonary hypertension. Antithrombotic treatment with low molecular weight heparin was instituted. The management also included ACE inhibitors, spironolactone, low-salt diet and lactulose. The patient was discharged and three months later we observed the disappearance of edema, ascites and pleuro-pericardial effusions, a marked body weight reduction and improved dyspnea and liver function tests. A possible link between the development of intra-shunt thrombosis and clinical decompensation in our patient was hypothesized. In fact, it has been demonstrated that the increased portal pressure, caused by occlusion of portosystemic shunt, reduces renal plasma flow and increases systemic endothelin-1 concentration. In our patient the disappearance of edematous state and improved dyspnea observed after recanalization of the shunt strongly support this hypothesis.

© 2007 WJG. All rights reserved.

Key words: Porto-pulmonary hypertension; Porto-caval shunt; Thrombosis

Giannarelli C, De Giorgi A, De Negri F, Carmassi F. De-compensated porto-pulmonary hypertension in a cirrhotic patient with thrombosis of portocaval shunt. World J Gastroenterol 2007; 13(47): 6439-6440

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tricuspidal regurgitation was also detected by echocardiog-raphy. Systolic pulmonary arterial pressure, as measured by Doppler analysis, was 50 mmHg. Cardiac catheterization showed no coronary and left-sided heart abnormalities. Hemodynamic parameters (Table 1) led to the diagnosis of moderate precapillary pulmonary hypertension. Anti-thrombotic treatment with low molecular weight heparin was instituted. The treatment also included ACE inhibitors, spironolactone, insulin, low-salt diet (2 gr pd) and lactu-lose. This management decreased the retention of sodium and water in the kidney from 12 mEq/d on admission to 186 mEq/d after 3 d of treatment, and improved edema, as demonstrated by natriuresis. The patient was discharged one month later with the above-mentioned prescription and, after three months of follow-up, physical examina-tion showed the disappearance of edema, marked weight reduction (20 kg) and improved dyspnea (NYHA II). Liver function tests were as follows: total bilirubin: 15.3 µmol/L, AST: 0.56 µkat/L, ALT: 0.47 µkat/L, GGT: 36 U/L, alkaline phosphatase: 1.85 nkat/L, serum albumin: 30 g/L, ammonia (as NH3): 76 µmol/L, prothrombin time 67%. Abdominal ultrasonography showed recanalization of porto-caval shunt, disappearance of ascites and right pleural effusions. No pericardial effusions were found by ecocardiography. Antithrombin activity was 56%, D-dimer 0.2 mg/L. Orthotopic liver transplantation was excluded

because of the high intra-operative mortality in patients with portopulmonary hypertension and the conflicting results reported in this condition[6]. Therefore, the patient was referred to a specialized center in order to start an appropriate vasodilatory therapy, which appears to be the only feasible treatment.

DISCUSSIONThis case report suggests a possible link between the development of intra-shunt thrombosis and clinical decompensation in our patient. In fact, a recent study showed that increased por ta l pressure caused by occlusion of porto-systemic shunt reduces renal plasma flow, leading to renal sodium and water retention, and increases systemic ET-1 concentration[4]. In our patient, disappearance of edema and improved dyspnea, observed after recanalization of the shunt, strongly support this hypothesis, although further experience with similar cases is needed.

REFERENCES1 Hadengue A, Benhayoun MK, Lebrec D, Benhamou JP.

Pulmonary hypertension complicating portal hypertension: prevalence and relation to splanchnic hemodynamics. Gastroenterology 1991; 100: 520-528

2 Simonneau G, Galiè N, Rubin LJ, Langleben D, Seeger W, Domenighetti G, Gibbs S, Lebrec D, Speich R, Beghetti M, Rich S, Fishman A. Clinical classification of pulmonary hypertension. J Am Coll Cardiol 2004; 43: 5S-12S

3 Passarella M, Fallon MB, Kawut SM. Portopulmonary hyper-tension. Clin Liver Dis 2006; 10: 653-663 x

4 Kapoor D , Redhead DN, Hayes PC, Webb DJ, Jalan R. Systemic and regional changes in plasma endothelin following transient increase in portal pressure. Liver Transpl 2003; 9: 32-39

5 Robalino BD, Moodie DS. Association between primary pulmonary hypertension and portal hypertension: analysis of its pathophysiology and clinical, laboratory and hemodynamic manifestations. J Am Coll Cardiol 1991; 17: 492-498

6 Tam NL, He XS. Clinical management of portopulmonary hypertension. Hepatobiliary Pancreat Dis Int 2007; 6: 464-469

S- Editor Liu Y L- Editor Wang XL E- Editor Ma WH

Table 1 Hemodinamic parameters during cardiac catheterization

Aortic pressure (mmHg) 103-76 (mean 85)Right atrial pressure (mmHg) 11 (mean)Right ventricular pressure (mmHg) 70-18 (mean 35)Pulmonary arterial pressure (mmHg) 70-30 (mean 43)Pulmonary capillary wedge pressure (mmHg) 10Cardiac output (l/min) 5.43Cardiac index (l/min per m2) 2.83Total pulmonary resistance (dyn•s/cm5) 632Pulmonary vascular resistance (dyn•s/cm5) 488Systemic vascular resistance (dyn•s/cm5) 1088

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Malignant fibrous histiocytoma presenting as hemoperitoneum mimicking hepatocellular carcinoma rupture

Hsin-Chi Chen, Chi-Jen Chen, Chin-Ming Jeng, Chan-Ming Yang

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CASE REPORT

Hsin-Chi Chen, Chan-Ming Yang, Department of Radiology, Cathay General Hospital, Hospital Sijhih Branch, No. 2, Lane 59, Jiancheng Road, Sijhih City, Taipei, Taiwan, China Chi-Jen Chen, Department of Radiology, E-Da Hospital, No. 1, E-Da Road, Jiau-Shu Tsuen, Yan-Chau Hsiang, Kaohsiung, Taiwan, ChinaChin-Ming Jeng, Department of Radiology, Cathay General Hospital, No. 280, Jen-Ai Road, Section 4, Taipei, Taiwan, ChinaCorrespondence to: Hsin-Chi Chen, Department of Radiology, Cathay General Hospital, Hospital Sijhih Branch, No. 2, Lane 59, Jiancheng Road, Sijhih City, Taipei, Taiwan, China. hsinchi.chen715@msa.hinet.netTelephone: +886-2-86463655 Fax: +886-2-87921723Received: June 14, 2007 Revised: October 10, 2007

AbstractMalignant fibrous histiocytoma (MFH) is a pleomorphic mesenchynal sarcoma. It is uncommonly arises primarily from the intra-peritoneal cavity. Primary peritoneal MFH with tumor bleeding and rupture is rare. We describe the imaging features of a 70-year-old patient presenting with ruptured hemorrhagic peritoneal MFH at subhepatic area, accompanied by massive hemoperitoneum, mimicking a ruptured pedunculated hepatocellular carcinoma. Computed tomography (CT) revealed a large heterogeneous enhanced subhepatic mass with adjacent liver, gallbladder and colon invasion. Tumor hemorrhage and rupture complicated with peritoneal seeding and massive bloody ascites were also detected. Angiography showed a hypervascular tumor fed by enlarged right hepatic arteries, cystic artery and omental branches of gastroepiploic artery. The patient underwent laparotomy for tumor resection, but the tumor recurred one month after operation. To our knowledge, the CT appearance of ruptured intraperitoneal MFH complicated by hemoperitoneum has not been previously described.

© 2007 WJG. All rights reserved.

Key words: Malignant fibrous histiocytoma; Peritoneum; Hemoperitoneum; Spontaneous rupture; Computed tomography

Chen HC, Chen CJ, Jeng CM, Yang CM. Malignant fibrous histiocytoma presenting as hemoperitoneum mimicking hepatocellular carcinoma rupture. World J Gastroenterol 2007; 13(47): 6441-6443

http://www.wjgnet.com/1007-9327/13/6441.asp

INTRODUCTIONMassive hemoperitoneum is an emergent life-threatening condition requiring prompt management. Tumor-associated hemoperitoneum is commonly related to hypervascular intra-abdominal visceral organ tumor rupture, but rarely related to hemorrhage of the hypervascular tumor of primary peritoneal origin. Malignant fibrous histiocytoma (MFH) is the most common sarcoma at late adult life. About 5%-10% of MFH lesions arise from the peritoneal cavity[1], and may develop various degrees of tumor bleeding[1-3]. Herein, we present a case of spontaneously bleeding peritoneal MFH located in the subhepatic area with direct liver invasion presenting as massive hemoperitoneum, mimicking a ruptured pedunculated hepatocellular carcinoma.

CASE REPORTA 70-year-old man was admitted to the emergency department because of intensifying abdominal pain of a 10-days’ duration with aggravation in the recent two days before admission. The patient also complained of body weight loss. Physical examination revealed a pallid face, orthopnea and a distended abdomen with a palpable tender mass in the right upper to middle quadrant of the abdomen. Pulse rate and blood pressure were normal. Mild anemia (hemoglobin of 130 g/L), leukocytosis (white blood cell count 16.83 × 109/L) and elevated C-reactive protein (247 mg/L) were also noted. Other laboratory data were unremarkable. Serological tests were negative for hepatitis B and positive for hepatitis C. The serum levels of tumor markers of alpha-fetoprotein, CEA and CA19-9 were within normal limits.

Abdominal sonography revealed a large mixed hyperechoic and hypoechoic mass at the hepatic right lower region with massive ascites. Abdominal paracentesis revealed bloody ascites, with an erythrocyte count of 100 000/mm3. Computed tomography (CT) depicted a large ill-defined hemorrhagic mass (approximately 7.5 cm × 8.6 cm × 12.4 cm in size) with heterogeneous enhancement involving segments Ⅴ and Ⅵ of liver with large extra-hepatic components in the peritoneal cavity accompanied with massive bloody ascites (Figure 1). The subhepatic mass also compressed and invaded the gallbladder and adjacent hepatic flexure of colon. Enlarged omental arteries were found surrounding the subhepatic mass (Figure 1). Tumor rupture with irregular tumor margin and peritoneal seeding were also present.

A presumptive diagnosis of pedunculated hepatocellular carcinoma rupture was made. Therefore, emergent angiography was performed and disclosed a large hypervascular tumor located at the right subhepatic region, supplied by enlarged right inferior hepatic arteries, cystic artery, and omental branches of gastroepiploic artery (Figure 2). Transarterial embolization with gelfoam pieces was performed to achieve hemostasis for these engorged feeding arteries.

Six days later, the patient underwent laparotomy for radical tumor excision. Intraoperative examination revealed a large bleeding peritoneal subhepatic tumor rupture with contiguous omentum, liver, gallbladder and colon invasion. Peritoneal seeding and 3 L bloody ascites were also found. Pathology of the excised specimen revealed a storiform-pleomorphic malignant fibrous histiocytoma with marked hemorrhage and necrosis.

One month after the operation, the patient was readmitted to the emergency department due to massive upper gastrointestinal bleeding. CT revealed multiple hepat ic and peri toneal metastases with duodenal involvement. Panendoscopy confirmed the duodenal invasion by the recurrent tumor causing internal bleeding. Despite intensive f luid supplementation and blood transfusion therapy, the patient died of hypovolemic shock 3 d later.

DISCUSSIONMFH is a pleomorphic sarcoma initially described by O’Brien and Stout[4]. It is the most common sarcoma in

late adulthood, with a peak incidence in the fifth and sixth decades. Men are affected twice as frequently as women. MFH is microscopically characterized by areas of spindle cells arranged in a storiform pattern, and pleomorphic areas with haphazardly arranged sheets of fibroblasts and histiocytes. This neoplasm has a variety of histologic subtypes, including storiform-pleomorphic, myxoid, giant cell, inflammatory and angiomatoid. MFH generally affects the extremities and retroperitoneum. Uncommon locations include the head and neck region, dura mater, brain, lung, heart, aorta, pancreas, liver, spleen, breast, intestine and mesentery[1,5,6]. The propensity of MFH for invasion of contiguous organs and local recurrence result in its poor prognosis.

The radiographic features of MFH are non-specific. On CT, FMH usually presents as a poorly demarcated or a well circumscribed mass, and exhibits peripheral solid enhanced component with intralesional hypodense areas of myxoid change, hemorrhage or necrosis. Although the vascularity of MFH is variable, the majority of lesions are moderately hypervascular with tumor supply derived from multiple surrounding vessels[1,7]. Because of the rich vascularity of the lesion, preoperative transarterial embolization may be considered to shrink the tumor and minimize intraoperative hemorrhage.

MFH can develop variable degrees of tumor hemo-rrhage either spontaneously or as a result of response to chemotherapy[1,8]. Approximately 5% MFHs will develop apparent intra-tumoral hemorrhage spontaneously, especially when MFH arises from the peritoneum or omentum[1]. The intratumoral hemorrhage can be so extensive as to obscure the underlying neoplasms and create a large cyst-like space that might be mistaken as a hematoma, abscess or cystic tumor. Previous reports described that intra-peritoneal MFHs situated at the mesovarium and gastrocolic ligament with marked intra-tumoral hemorrhage manifested as large cystic tumors, causing diagnostic confusion[2-3]. Nevertheless, these intra-peritoneal MFH lesions with extensive tumor hemorrhage were still confined to the tumor mass itself, rather than complicated with extra-tumoral bleeding into the peritoneal cavity. However, in the present case, the subhepatic peritoneal MFH not only experienced intra-tumoral hemorrhage but also developed tumor rupture with extra-tumoral bleeding into the peritoneal cavity, mimicking a ruptured hepatocellular carcinoma. Three previous cases of MFH causing extra-tumoral massive bleeding had

Figure 2 A: Arterial phase image of the celiac arteriogram shows hypertrophied right inferior hepatic arteries, cystic artery and omental branches of gastroepiploic artery (white arrows) supplying the hypervascular subhepatic mass; B: Venous phase shows prominent tumor stains in the peripheral portion of the huge subhepatic mass (black arrows).

Figure 1 A 70-year-old man with hemorrhagic peritoneal malignant fibrous histiocytoma located subhepatically and presenting as massive hemoperitoneum. A: Pre-enhanced computed tomography shows a high-density intra-lesional hemorrhage (black arrow) within the subhepatic tumor involving the anterior aspect of segment Ⅴ and Ⅵ of liver. Ascites are also noted; B: Post-enhanced CT shows heterogeneous enhancement of the subhepatic mass directly invading the gallbladder (black arrow); C: Post-enhanced CT study at a lower level shows enlarged omental branches (white arrows) of the gastroepiploic artery supplying the subhepatic tumor. A cleavage plane (black arrow) between the subhepatic mass and the liver tip parenchyma was identified, suggestive of an extra-hepatic origin of the mass; D: Post-enhanced CT study at the lower tumor level reveals the large subhepatic mass directly invades the adjacent hepatic flexure of colon (thick black arrow). Tumor rupture with irregular tumor margin (thin black arrow) is also evident.

I

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A B

C D

A B

been reported, all involving metastatic MFH lesions at the alimentary tract causing gastrointestinal bleeding[9-11]. A case of post-irradiation induced dermal MFH with spontaneous tumor hemorrhage, causing massive skin bleeding had also been reported[12]. Another case of primary aortic MFH presenting as aortic dissection was described as well[13]. However, to the best of our knowledge, intra-peritoneal MFH bleeding complicated with massive hemoperitoneum causing acute abdominal emergency has not been previously reported.

Tumor-associated hemoperitoneum is frequently related to rupture of underlying intra-abdominal hypervascular tumors. Hepatocellular carcinoma (HCC) rupture is the most common cause of tumor-associated hemoperitoneum in male patients of all ages in Asia and Africa, especially patients with large and peripherally located tumors. Compared to the incidence of intraperitoneal visceral organ tumor rupture causing hemoperitoneum, the occurrence of primary peritoneum-origin tumor bleeding presenting with hemoperitoneum is rare. A review of the literature found only one similar case report which described a hypervascular peritoneal hemangiopericytoma with spontaneous tumor bleeding causing a small amount of hemoperitoneum[14]. By contrast, in our present case, large amounts of bloody ascites were found, indicating that the MFH had tumor rupture with active tumor bleeding rather than minimal tumor bleeding only.

In the present case, CT showed that the vast majority of subhepatic tumor was situated in the extra-hepatic peritoneal cavity and the center of the mass was outside of the liver parenchyma. These findings suggest that the subhepatic mass may be a tumor of extra-hepatic origin rather than arising from the liver parenchyma. Furthermore, the subhepatic tumor was large enough not only to compress but also to directly invade the liver and gallbladder. However, the subhepatic tumor and the compressed liver tip parenchyma still exhibited negative “beak sign” on CT study, seen as an obtuse angle between the subhepatic mass and the compressed liver tip surface with partially preserved dull edges of the liver. These findings suggest that the subhepatic mass did not arise from the liver. Moreover, a cleavage plane between the subhepatic mass and the compressed lower-most liver tip parenchyma was also identified as negative “embedded organ sign” on CT scan, indicating the subhepatic mass was compressing the liver, but did not arise from the liver. In addition, CT also revealed enlarged omental arteries supplying the subhepatic mass, presenting as “prominent feeding artery sign”. This suggests the subhepatic mass may be a tumor of peritoneal omentum origin. Angiography also confirmed the subhepatic tumor was mainly supplied by multiple enlarged omental arteries, whereas the hepatic artery only accounted for a minority of tumor feeding. Advanced HCC with omentum invasion can develop extra-hepatic collateral vessels from the omental branches of the gastroepiploic artery, but HCC usually still preserves its dominant feeding arteries from the hepatic artery rather than from the extra-hepatic omental artery. The more prominent blood supply from

the omental arteries than the hepatic artery in the present case is an unusual phenomenon in HCC. Careful analysis of the CT imaging features of this case indicate that a subhepatically located bleeding peritoneal tumor with liver invasion should be taken into account as a possible cause of peri-hepatic tumor complicated with hemopertitoneum. Since MFH is the most common sarcoma in the elderly, primary peritoneal MFH, although rare, should be included among the causes of tumor-related hemoperitoneum. However, the radiog raphic features of MFH are indistinguishable from those of other peritoneal malignant neoplasms, such as leiomyosarcoma, gastrointestinal stromal tumor, fibrosarcoma or angiosarcoma.

In conclusion, MFH arising from the peritoneal subhepatic region is rare, but the potential exists for a hemorrhagic peritoneal sarcoma to directly invade adjacent organs, mimicking an exophytic hepatic tumor rupture. The possibility of MFH should be carefully considered when a patient presents with a hemorrhagic subhepatic tumor and no prior documented hepatitis episode and liver cirrhosis.

REFERENCES1 Weiss SW, Enzinger FM. Malignant fibrous histiocytoma: an

analysis of 200 cases. Cancer 1978; 41: 2250-22662 Ko SF, Ng SH, Lin JW, Lee TY. Cystic malignant fibrous

histiocytoma of the mesovarium. AJR Am J Roentgenol 2001; 176: 549-550

3 Huang CC, Ko SF, Ng SH, Lee TY, Wan YL, Lin JW, Chen WJ. Cystic malignant fibrous histiocytoma of the gastrocolic ligament. Br J Radiol 2001; 74: 651-653

4 O'Brien JE, Stout AP. Malignant fibrous xanthomas. Cancer 1964; 17: 1445-1455

5 Ros PR , Viamonte M, Rywlin AM. Malignant fibrous histiocytoma: mesenchymal tumor of ubiquitous origin. AJR Am J Roentgenol 1984; 142: 753-759

6 Bruneton JN , Drouillard J, Rogopoulos A, Laurent F, Normand F, Balu-Maestro C, Monticelli J. Extraretroperitoneal abdominal malignant fibrous histiocytoma. Gastrointest Radiol 1988; 13: 299-305

7 Goldman SM, Hartman DS, Weiss SW. The varied radiographic manifestations of retroperitoneal malignant f ibrous histiocytoma revealed through 27 cases. J Urol 1986; 135: 33-38

8 Panicek DM, Casper ES, Brennan MF, Hajdu SI, Heelan RT. Hemorrhage simulating tumor growth in malignant fibrous histiocytoma at MR imaging. Radiology 1991; 181: 398-400

9 Blaauwwiekel EE, Koopal S, Kreeftenberg HG. Metastasized malignant fibrous histiocytoma as a cause of gastrointestinal tract haemorrhage. Scand J Gastroenterol 1997; 32: 1272-1274

10 Adams HW , Adkins JR, Rehak EM. Malignant fibrous histiocytoma presenting as a bleeding gastric ulcer. Am J Gastroenterol 1983; 78: 212-213

11 Santoro MJ, Abdulian JD, Chase RL, Griffin RA, Solinger MR, Collen MJ. Malignant fibrous histiocytoma metastatic to the colon presenting as a lower gastrointestinal bleed. Am J Gastroenterol 1992; 87: 1051-1053

12 Yamamoto Y, Arata J, Yonezawa S. Angiomatoid malignant fibrous histiocytoma associated with marked bleeding arising in chronic radiodermatitis. Arch Dermatol 1985; 121: 275-276

13 Chen WJ, Chen CL, Liau CS, Chu SH, Lee YT. Primary malignant fibrous histiocytoma of the aorta associated with aortic dissection. Chest 1991; 99: 1049-1050

14 Crusco F, Chiodi M, Pugliese F, Mosca S, Fischer MJ, Lupattelli L. Benign omental hemangiopericytoma presenting with hemoperitoneum: radiologic findings. AJR Am J Roentgenol 2005; 184: S67-S69

S- Editor Ma N L- Editor Ma JY E- Editor Yin DH

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Eosinophilic enteritis presenting as a rare cause for ileo-ileal intussusception

Ashok Yadavrao Kshirsagar, Sunil Vitthalrao Jagtap, Rajkamal Prabhudayal Kanojiya, Yogesh Bhupal Langade, Sanjay Laxman Shinde, Niraj Shekhar

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CASE REPORT

Ashok Yadavrao Kshirsagar, Sunil Vitthalrao Jagtap, Rajkamal Prabhudayal Kanojiya, Yogesh Bhupal Langade, Sanjay Laxman Shinde, Niraj Shekhar, Department of Surgery, Department of Pathology, Krishna Institute of Medical Sciences & Deemed University, Dhebewadi Road, Malkapur, Karad 415110, Maharashtra, IndiaCorrespondence to: Dr. Ashok Yadavrao Kshirsagar, Department of Surgery, Krishna Institute of Medical Sciences & Deemed University, Dhebewadi Road, Malkapur, Karad 415110, Maharashtra, India. drayk@indiatimes.comTelephone: +91-9422400435 Fax: +91-21-64242170Received: June 15, 2007 Revised: October 10, 2007

AbstractEosinophilic enteritis, a relatively rare entity, usually involves gastric antrum or proximal small bowel. Our case is rarer in its involvement of the distal small bowel and presents unusually as intussusception. The disease if diagnosed in the initial stages responds well to medical treatment but if associated with complications or misdiagnosed, surgical modality is the treatment of choice. In our case, the patient presented with acute intestinal obstruction due to intussusception and emergency laparotomy with i leoi leal anastomosis was done. Histopathology confirmed the diagnosis as eosinophilic enteritis. This case with such a presentation is discussed here.

© 2007 WJG. All rights reserved.

Key words: Eosinophilic enteritis; Intussusception; Intestinal obstruction; Eosinophils; Ileoileal

Kshirsagar AY, Jagtap SV, Kanojiya RP, Langade YB, Shinde SL, Shekhar N. Eosinophilic enteritis presenting as a rare cause for ileo-ileal intussusception. World J Gastroenterol 2007; 13(47): 6444-6445

http://www.wjgnet.com/1007-9327/13/6444.asp

INTRODUCTIONEosinophilic enteritis is a rare, poorly understood condition presenting with a bizarre spectrum of unexplained symptoms mimicking any other acute abdominal conditions. Diagnosis initially is based on exclusion of other abdominal

conditions though ultimately histopathology is definitive. The majority of patients respond to medical treatment but if associated with complications like obstruction, perforation, intussusception etc., surgical intervention is necessary. We present a case of eosinophilic enteritis of the distal ileum with intussusception.

CASE REPORTA 40-year-old man was admitted to the emergency department of our hospital for severe colicky upper abdominal pain and vomiting. He had a history of mild colicky abdominal pain a month ago but no history of food sensitization, allergic disease, asthma, parasitic infestations or any abdominal surgery. Physical examination revealed generalized distention with tenderness all over the abdomen. Per rectal examination was unremarkable. Blood investigations revealed 117 g/L hemoglobin, 8700/mm3 white blood cells with 67% neutrophils, 36% lymphocytes and 3% eosinophils. The rest of haematological investigations were within normal limits. Abdominal radiograph revealed multiple air-fluid levels suggestive of small bowel obstruction. Ultrasonography revealed intussusception of the distal small bowel. Emergency laparotomy revealed ileo-ileal intussusception with gangrenous changes, about 15 cm proximal to the ileo-caecal junction. Resection with ileo-ileal anastomosis was done.

Histopathology revealed the surrounding mucosa and submucosa. Muscular layers were heavily infiltrated by eosinophils (50-60/Hpf) along with polymorphonuclear cells, lymphocytes and few plasma cells. Intestinal wall was thickened with areas of necrosis suggestive of eosinophilic enteritis with gangrene. No evidence of malignancy was found. The patient was asymptomatic, his follow-up serum immunoglobulin E levels were within normal limits, and repeated peripheral smears did not show eosinophilia(Figures 1-3).

DISCUSSIONEosinophilic gastroenteritis is one of the rare conditions and its etiology is poorly understood. It usually involves the gastric antrum and proximal small bowel, but rarely involves the distal gut. In about 85% of cases it is associated with eosinophilia. Classification of eosinophilic enteritis is based upon the presence of eosinophilia

Kshirsagar AY et al . Eosinophilic enteritis presenting as intussusception 6445

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or eosinopenia[1]. Clinical features depend upon the most prominent layer of visceral wall involvement by eosinophils, i.e., mucosal, muscular or serosal[2-6]. Involvement of muscularis results in obstructive symptoms and serosal involvement produces ascites. Evaluation of immunoglobulin E levels may help to detect an allergic cause. Despite all clinical factors, definitive diagnosis can only be made by histopathology confirming eosinophilic involvement of the affected area. In cases of intussusception, malignancy should always be ruled out[7].

At present, laparoscopic full thickness biopsy can be definitive in suspected cases[8]. Corticosteroid therapy is the mainstay of medical treatment. Long term follow-up is required as there are always chances of recurrence[9].

Figure 1 Ileo-ileal intussusception with a 10-cm ileal segment showing blackish brown discoloration.

Figure 2 Microphotograph of ileum showing severe infiltration by eosinophils in all coats (HE, × 40).

Figure 3 Microphotograph of ileum muscle coat showing severe eosinophilic infiltration (HE, × 100).

ACKNOWLEDGMENTSThe authors thank Dr. HR Tata, Head of the Department of Surgery and Medical Director, for allowing us to publish the data and Mrs. MC Deshingkar for her secretariat support in preparing this paper.

REFERENCES1 O'Neill T. Eosinophilic granuloma of the gastro-intestinal

tract. Case report and suggested classification. Br J Surg 1970; 57: 704-708

2 Lee CM, Changchien CS, Chen PC, Lin DY, Sheen IS, Wang CS, Tai DI, Sheen-Chen SM, Chen WJ, Wu CS. Eosinophilic gastroenteritis: 10 years experience. Am J Gastroenterol 1993; 88: 70-74

3 Talley NJ, Shorter RG, Phillips SF, Zinsmeister AR. Eosinophilic gastroenteritis: a clinicopathological study of patients with disease of the mucosa, muscle layer, and subserosal tissues. Gut 1990; 31: 54-58

4 Lee M, Hodges WG, Huggins TL, Lee EL. Eosinophilic gastroenteritis. South Med J 1996; 89: 189-194

5 Klein NC , Hargrove RL, Sleisenger MH, Jeffries GH. Eosinophilic gastroenteritis. Medicine (Baltimore) 1970; 49: 299-319

6 Kraft SC, Kirsner JB. Immunology in gastroenterology. 4th ed. Philadelphia: WB Saunders, 1985: 4487-4524

7 Corman ML. Carcinoma of colon. 3rd ed. Philadelphia: JB Lippincott Co, 1993: 487-595

8 Edelman DS. Eosinophilic enteritis. A case for diagnostic laparoscopy. Surg Endosc 1998; 12: 987-989

9 Naylor AR. Eosinophilic gastroenteritis. Scott Med J 1990; 35: 163-165

S- Editor Ma N L- Editor Wang XL E- Editor Liu Y

CASE REPORTA 56-year-old woman with mild right upper quadrant pain for 2 d was admitted to our hospital for acute onset of severe upper abdominal pain radiating to the back accompanied with vomiting for 12 h. On physical examination, she had no fever, the sclera was icteric, and there was tenderness on the right upper quadrant of the abdomen and the epigastric area with no rigidity and rebound pain. Her amylase and lipase levels were 2066 U/L and 1980 U/L, respectively. Her liver function tests were as follows: 5 mg/dL direct bilirubin, 303 IU/L gamma glutamyl transpeptidase, 135 IU/L alkaline phosphatase, 127 mg/dL glucose, 12 g/dL blood urea nitrogen, 170 IU/L lactate dehydrogenase, 8.6 mg/dL calcium, 3.8 g/dL albumin, 66 mmHg arterial Po2, 6800/μL white blood cells, 35% hematocrit , and 12 g/dL hemoglobin. She underwent selective noncomplicated LC 6 mo prior to this episode of acute pancreatitis.

Plain abdominal X-rays showed three endoclips in the right upper quadrant, two of them were close to each other and one was located inferiomedially to the others. Abdominal ultrasonography (US) revealed that the common bile duct was dilated to 12 mm in diameter and the pancreas was swollen. The distal common bile duct could not be evaluated adequately by US.

The patient was diagnosed as mild biliary acute pancreatitis based on the modified Imrie criteria. After medical treatment was started, ERCP performed on the second day of admittance, demonstrated a stone (12 mm in diameter) and an imbedded surgical clip at the distal part of common bile duct which was dilated to 13 mm in diameter (Figure 1A). A sphincterotomy of 15mm was performed. The extraction balloon catheter was easily burst when the stone was extracted. The stone was removed via a Dormia basket catheter (Figure 2A and B).

Her symptoms improved on the first day after ERCP and the serum amylase level decreased to 250 IU/L. Her amylase levels and other biochemical parameters were normal on d 2 after ERCP and she was discharged from the hospital without any complications. There was no clinical or biochemical abnormality attributable to the biliary system or pancreas during the 1-year follow-up period after ERCP.

DISCUSSIONAlthough the long term behavior of metal endoclips placed during LC is not clear, it is a well known-phenomenon

CASE REPORT

Migrated endoclip and stone formation after cholecystectomy: A new danger of acute pancreatitis

Kemal Dolay, Halil Alis, Aliye Soylu, Gulum Altaca, Ersan Aygun

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Kemal Dolay, Halil Alis, Aliye Soylu, Gulum Altaca, Ersan Aygun, Department of General Surgery1 and Gastroenterology 2, Endoscopy Unit, Bakırkoy Research and Training Hospital, Bakirkoy, Istanbul, Turkey Correspondence to: Kemal Dolay, MD, Bakırköy Eğitim ve Araştırma Hastanesi Genel Cerrahi Kliniği Endoskopi Ünitesi Istanbul, Turkey. dolayk@yahoo.comTelephone: +90-212-6011909 Fax: +90-212-5713278Received: August 5, 2007 Revised: September 28, 2007

AbstractEndoclip migration into the common bile duct following laparoscopic cholecystectomy (LC) is an extremely rare complication. Migrated endoclip into the common bile duct can cause obstruction, serve as a nidus for stone formation, and cause cholangitis. We report a case of obstructive jaundice and acute biliary pancreatitis due to choledocholithiasis caused by a migrated endoclip 6 mo after LC. The patient underwent early endoscopic retrog-rade cholangiopancreatography (ERCP) with endoscopic sphincterotomy and stone extraction.

© 2007 WJG. All rights reserved.

Key words: Laparoscopic cholecystectomy; Endoclip mig-ration; Biliary pancreatitis; Endoscopic retrograde cho-langiopancreatography

Dolay K, Alis H, Soylu A, Altaca G, Aygun E. Migrated endoclip and stone formation after cholecystectomy: A new danger of acute pancreatitis. World J Gastroenterol 2007; 13(47): 6446-6448

http://www.wjgnet.com/1007-9327/13/6446.asp

INTRODUCTIONLaparoscopic cholecytectomy (LC) has become the standard approach in the treatment of benign gallbladder disease. The widespread use of LC does not increase the total incidence of postoperative complications[1]. Endoclip migration into the common bile duct was first reported by Raul et al[2] in 1992 as an extremely rare complication. To our knowledge, this is the first case of a migrated clip eventually resulting in acute biliary pancreatitis after LC, although choledocholithiasis due to endoclip migration into the common bile duct has been reported[1].

Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6446-6448www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

that the remained foreign materials in abdominal cavity tend to migrate towards the organs with lumens. Since stone formation within the common bile duct due to silk suture material after open cholecystectomy (OC) was first described in 1987, various foreign bodies causing nidus formation in bile ducts have been reported[3,4]. Ban et al[3] have divided biliary tract foreign bodies into three categories: operative residuals, penetrating missiles, and ingested items.

The first cases of choledocholithiasis due to endoclip migration into the common bile duct after OC and LC were reported by Walker et al[5] in 1979 and by Raoul et al[2] in 1992, respectively.

The true pathogenesis of migration of endoclips into the common bile duct is unclear. According to the first hypothesis, cystic duct remains patent due to ineffective clipping resulting in bilioma with bile leakage[2,6-8]. This bilioma leads to necrosis in the stump of the cystic duct by inducing chronic inflammation. The liberated clip in the necrotized cystic duct stump migrates into the common bile duct by eroding it mechanically. Presentation of the bile leakage might be subclinical, self-limited or clinically overt, which may explain why migrations occur in the early postoperative period with bile leakage.

According to the second hypothesis, the clip migrates into the ductal system by eroding the bile duct due to the local inflammation around the endoclip[6,9-12]. Short cystic duct or cystic artery, clip placement close to the common bile duct, manipulations after clipping (uncontrolled aspi-ration and irrigation, retraction of porta hepatis, etc.), local suppurative complications and ischemia on the ductal wall (excessive dissection) are risk factors for clip migration.

There have been rare repor ts concerning the migration of clips into the common bile duct resulting in complications other than stone formation, such as cholangitis, obstruction and stenosis of the common bile duct[6,11,13]. Cetta et al[14] reported a case of transient acute pancreatitis in the absence of associated stones 15 d after LC, probably due to spontaneous passage of an endoclip through the common bile duct. The case presented here is unique since stone formation and eventually acute pancreatitis occurred due to clip migration. It was reported that the time of endoclips to migrate into the bile ducts after LC is between 11 d and 6 years[2,6,7,15,16].

Absorbable clips and ultrasonic dissection without clipping in LC can prevent occurrence of clip migration

Dolay K et al. Acute pancreatitis due to choledocholithiasis caused by a migrated endoclip 6447

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and its complications[13,17]. However, these methods have not yet been used commonly by surgeons probably due to the complication and high costs of their applications. Bile duct stones associated with migration of clips after LC are usually extracted during ERCP[2,8,14]. However, when ERCP fails or in cases with additional pathology, open surgery or percutaneous transhepatic cholangioscopy can be performed[8,18].

The role of early ERCP and endoscopic sphincte-rotomy (ES) in the treatment of acute biliary pancreatitis is still controversial[19]. At present, early endoscopic intervention is suggested in patients with acute biliary pancreatitis when criteria for severity are met and/or there are coexistent cholangitis, jaundice, dilated common bile duct, smoldering or deteriorating clinical course[19]. In this case, we preferred early ERCP due to the presence of jaundice and dilated common bile duct.

In conclusion, endoclips after LC may migrate into the common bile duct leading to stone formation, obstruction, cholangitis, stenosis and even pancreatitis. Care should be taken during clip application, and clips should be completely squeezed by the clip applicator and unnecessary manipulations after clipping should be avoided.

REFERENCES1 Keus F , de Jong JA, Gooszen HG, van Laarhoven CJ.

Laparoscopic versus open cholecystectomy for patients with symptomatic cholecystolithiasis. Cochrane Database Syst Rev 2006; 4: CD006231

2 Raoul JL, Bretagne JF, Siproudhis L, Heresbach D, Campion JP, Gosselin M. Cystic duct clip migration into the common bile duct: a complication of laparoscopic cholecystectomy treated by endoscopic biliary sphincterotomy. Gastrointest Endosc 1992; 38: 608-611

3 Ban JL, Hirose FM, Benfield JR. Foreign bodies of the biliary

Figure 1 ERCP demo-nstrating a stone (12 mm in diameter) and an imbedded surgical clip at the distal part of the common bile duct.

Figure 2 Placement of balloon catheter (A) and (B) Dormia basket catheter to extract the stone above the common bile duct after endoscopic sphincterotomy.

A

B

tract: report of two patients and a review of the literature. Ann Surg 1972; 176: 102-107

4 Homans J. VIII. Gall-Stones formed around Silk Sutures Twenty Months after Recovery from Cholecystotomy. Ann Surg 1897; 26: 114-116

5 Walker WE, Avant GR, Reynolds VH. Cholangitis with a silver lining. Arch Surg 1979; 114: 214-215

6 Arnaud JP, Bergamaschi R. Migration and slipping of metal clips after celioscopic cholecystectomy. Surg Laparosc Endosc 1993; 3: 487-488

7 Matsuura T, Kanisawa Y, Sato T, Saito T, Hirata K. Migration of “endo-clips” into common bile-duct after laparoscopic cholecystectomy. Lancet 1992; 340: 306

8 Tsumura H, Ichikawa T, Kagawa T, Nishihara M, Yoshikawa K, Yamamoto G. Failure of endoscopic removal of common bile duct stones due to endo-clip migration following laparoscopic cholecystectomy. J Hepatobiliary Pancreat Surg 2002; 9: 274-277

9 Hansen KA , Wood R . An unusua l compl ica t ion o f laparoscopic cholecystectomy. Endoscopy 1994; 26: 322-323

10 Mansoa A, Martins A, Brito E Melo M, Coito P. Surgical clips as a nidus for stone formation in the common bile duct. Surg Endosc 2000; 14: 1189

11 Matsumoto H, Ikeda E, Mitsunaga S, Naitoh M, Furutani S, Nawa S. Choledochal stenosis and lithiasis caused by penetration and migration of surgical metal clips. J

Hepatobiliary Pancreat Surg 2000; 7: 603-60512 Yoshizumi T, Ikeda T, Shimizu T, Ohta S, Nagata S, Sonoda T,

Sugimachi K. Clip migration causes choledocholithiasis after laparoscopic cholecystectomy. Surg Endosc 2000; 14: 1188

13 Allan A, Cooper MJ, Leaper DJ. A new absorbable ligating clip for use in cholecystectomy. J R Coll Surg Edinb 1984; 29: 53-54

14 Cetta F, Lombardo F, Baldi C, Cariati A. Clip migration within the common duct after laparoscopic cholecystectomy: a case of transient acute pancreatitis in the absence of associated stones. Endoscopy 1997; 29: S59-S60

15 Birkett DH. Spilled cells, spilled clips, spilled stones. New problems or old challenges. Surg Endosc 1995; 9: 269-271

16 Sato T, Denno R, Yuyama Y, Matsuura T, Kanisawa Y, Hirata K. Unusual complications caused by endo-clip migration following a laparoscopic cholecystectomy: report of a case. Surg Today 1994; 24: 360-362

17 Westervelt J. Clipless cholecystectomy: broadening the role of the harmonic scalpel. JSLS 2004; 8: 283-285

18 Hai S, Tanaka H, Kubo S, Takemura S, Kanazawa A, Tanaka S, Hirohashi K. Choledocholithiasis caused by migration of a surgical clip into the biliary tract following laparoscopic cholecystectomy. Surg Endosc 2003; 17: 2028-2031

19 Toouli J, Brooke-Smith M, Bassi C, Carr-Locke D, Telford J, Freeny P, Imrie C, Tandon R. Guidelines for the management of acute pancreatitis. J Gastroenterol Hepatol 2002; 17 Suppl: S15-S39

S- Editor Liu Y L- Editor Wang XL E- Editor Ma WH

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WJG from 1998 to 2007 are covered by PubMed (Figure 1).The data from PubMed also show that the average

monthly hits to WJG articles linked on PubMed has been increasing every year since WJG started being covered by PubMed in Apr, 2004. Since that time, the total number of hits has been 344 499 in 2004, 47 110 in 2005, 66 305 in 2006, and 67 760 in 2007, with a milestone being reached in 2006 when WJG was recovered by SCI.

The above data clearly indicate that the quality of articles published in WJG has been increasing, and attracting more widespread attention at the same time.

S- Editor Liu Y L- Editor Walker C E- Editor Ma WH

NEWS

Number of monthly hits to WJG articles linked to PubMed surpasses 70 000

Yan Jiang

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Yan Jiang, World Journal of Gastroenterology, The WJG Press and Beijing Baishideng BioMed Scientific Co., Ltd., Beijing 100025, ChinaCorrespondence to: Yan Jiang, Assistant Editor, World Journal of Gastroenterology, The WJG Press and Beijing Baishideng BioMed Scientific Co., Ltd., Room 903, Building D, Ocean International Center, No.62 Dongsihuan Zhonglu, Chaoyang District, Beijing 100025, China. submission@wjgnet.comTelephone: +86-10-85381892 Fax: +86-10-85381893Received: October 17, 2007 Revised: October 28, 2007

© 2007 WJG. All rights reserved.

Jiang Y. Number of monthly hits to WJG articles linked to PubMed surpasses 70 000. World J Gastroenterol 2007; 13(47): 6449

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The latest data from LinkOut Team NCBI/US NLM show that the number of hits to World Journal of Gastroenterology (WJG) articles linked to LinkOut of PubMed reached 2 254 327 during the period of April, 2004, to September, 2007; further, the number was 72 532 for the month of September, 2007, which was 7344 hits higher than that of the same period in 2006.

The MEDLINE database, as one of most important components of PubMed, a web searching system managed by NCBI of America, provides statistical data for life science related journals. All 6200 articles published on

Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6449www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

Figure 1 All 6200 articles published on WJG from 1998 to 2007 have been indexed by PubMed.

Items 1-5 of 6200

1: Patel NM, Tatar EL.

Unusual colonoscopy finding: Taenia saginata proglottid.World J Gastroenterol. 2007 Nov 7;13(41):5540-1. PMID: 17907306 [PubMed - in process]

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ACKNOWLEDGMENTS

Acknowledgments to Reviewers of World Journal of Gastroenterology

Many reviewers have contributed their expertise and time to the peer review, a critical process to ensure the quality of World Journal of Gastroenterology. The editors and authors of the articles submitted to the journal are grateful to the following reviewers for evaluating the articles (including those published in this issue and those rejected for this issue) during the last editing time period.

Hitoshi Asakura, Director, Emeritus ProfessorInternational Medical Information Center, Shinanomachi Renga Bldg.35, Shinanomachi, Shinjukuku, Tokyo 160-0016, Japan

Stefano Bellentani, ProfessorFondo Studio Malattie Fegato-ONLUS, Sezione di Campogalliano, Via R. Luxemburg, 29/N, 41011 Campogalliano (MO), Italy

Filip Braet, Associate ProfessorAustralian Key Centre for Microscopy and Microanalysis, Madsen Building (F09), The University of Sydney, Sydney NSW 2006, Australia

Giuseppe Chiarioni, PhDGastroenterological Rehabilitation Division of the University of Verona, Valeggio sul Mincio Hospital, Azienda Ospedale di Valeggio s/M, Valeggio s/M 37067, Italy

Mark G Clemens, PhD, ProfessorDepartment of Biology, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223, United States

Garcia-Compean Diego, MD, ProfessorFaculty of Medicine, University Hospital, Department of Gastroenterology, Autonomous University of Nuevo Leon, Ave Madero y Gonzalitos, 64700 Monterrey, NL, Mexico

Emad M El-Omar, ProfessorDepartment of Medicine & Therapeutics, Aberdeen AB25 2ZD, United Kingdom

Francesco Feo, ProfessorDipartimento di Scienze Biomediche, Sezione di Patologia Sperimentale e Oncologia, Università di Sassari, Via P, Manzella 4, 07100 Sassari, Italy

Hongjin Huang, PhDCelera Diagnostics, 1401 Harbor Bay Parkway, Alameda, California 94502, United States

Michael P Jones, MD, FACP, FACG, Associate ProfessorNorthwestern University Feinberg School of Medicine, Division of Gastroenterology, Suite 1400, 676 North St Clair Street, Chicago, IL 60611-2951, United States

Leonidas G Koniaris, ProfessorAlan Livingstone Chair in Surgical Oncology, 3550 Sylvester Comprehensive Cancer Center (310T), 1475 NW 12th Ave., Miami, FL 33136, United States

Javier San MartinGastroenterology and Endoscopy, Sanatorio Cantegril, Av. Roosevelt y P 13, Punta del Este 20100, Uruguay

James Neuberger, ProfessorLiver Unit, Queen Elizabeth Hospital, Birmingham B15 2TH, United Kingdom

Valerio Nobili, PhDLiver Unit, Research Institute, Bambino Gesù Children’s Hospital, S. Onofrio 4 Square, 00165 Rome, Italy

Samuel Babafemi Olaleye, PhDDepartment of Physiology, University of Ibadan, Ibadan, Nigeria, Ibadan 022, Nigeria

Raffaele Pezzilli, MDDepartment of Internal Medicine and Gastroenterology, Sant’Orsola-Malpighi Hospital, Via Massarenti, 9, Bologna 40138, Italy

D Mark Pritchard, PhD, FRCPGastroenterology of University of Liverpool, 5th Floor UCD Building, Daulby St, Liverpool L69 3GA, United Kingdom

Richard A Rippe, PhDDepartment of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7038, United States

Mitsuo Shimada, ProfessorDepartment of Digestive and Pediatric Surgery, Tokushima University, Kuramoto 3-18-15, Tokushima 770-8503, Japan

Tadashi Shimoyama, MDHirosaki University, 5 Zaifu-cho, Hirosaki 036-8562, Japan

Seng-Lai Tan, Principal ScientistEli Lilly and Company, Indianapolis 46285, United States

Wei Tang, MD, EngD, Assistant ProfessorH-B-P Surgery Division, Artificial Organ and Transplantation Division, Department of surgery, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan

Hitoshi Togashi, Associate ProfessorDepartment of Gastroenterology, Course of Internal Medicine and Therapeutics, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan

Yvan Vandenplas, ProfessorDepartment of Pediatrics, AZ-VUB, Laarbeeklaan 101, Brussels 1090, Belgium

Vincent W Yang, Professor and Director201 Whitehead Research Building, 615 Michael Street, Atlanta, GA 30322, United States

Weimin Ye, ProfessorDepartment of Medical Epidemiology and Biostatistics, Karolinska Institutet, Box 281, SE 171 77, Stockholm, Sweden

Hiroshi Yoshida, MDFirst Department of Surgery, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan

Michael E Zenilman, MDClarence and Mary Dennis Professor and Chairman, Department of Surgery, SUNY Downstate Medical Center, Box 40, 450 Clarkson Avenue, Brooklyn, NY 11202, United States

Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6450www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

Events Calendar 2007-2009Meeting Falk Research Workshop: Morphogenesis and Cancerogenesisof the Liver 25-26 January 2007Goettingen symposia@falkfoundation.de

Meeting Canadian Digestive Diseases Week (CDDW)16-20 February 2007Banff-ABcagoffice@cag-acg.org www.cag-acg.org/cddw/cddw2007.htm

Meeting Inflammatory Bowel Diseases 20071-3 March 2007Innsbruck ibd2007@come-innsbruck.atwww.come-innsbruck.at/events/ibd2007/default.htm

Meeting Falk Symposium 158:Intestinal Inflammation and Colorectal Cancer 23-24 March 2007 Sevilla symposia@falkfoundation.de

Meeting BSG Annual Meeting 26-29 March 2007Glasgow www.bsg.org.uk

Meeting 42nd Annual Meeting of theEuropean Association for the Study of the Liver 11-15 April 2007 Barcelonaeasl2007@easl.ch www.easl.ch/liver-meeting

Meeting SAGES 2007 Annual Meeting -part of Surgical Spring Week18-22 April 2007 Paris Hotel and Casino, Las Vegas, Nevadawww.sages.org/07program/index.php

Meeting Falk Symposium 159: IBD 2007-Achievements in Research and Clinical Practice 4-5 May 2007Istanbul symposia@falkfoundation.de

Meeting European Society for Paediatric Gastroenterology, Hepatology and Nutrition Congress 20079-12 May 2007 Barcelona espghan2007@colloquium.fr

Meeting Gastrointestinal Endoscopy Best Practices: Today and Tomorrow, ASGE Annual Postgraduate Courseat DDW 23-24 May 2007 Washington-DCtkoral@asge.org

Meeting ESGAR 2007 18th AnnualMeeting and Postgraduate Course12-15 June 2007 Lisbonfca@netvisao.pt

Meeting Falk Symposium 160:Pathogenesis and Clinical Practice in Gastroenterology 15-16 June 2007 Portorozsymposia@falkfoundation.de

Meeting ILTS 13th Annual International Congress 20-23 June 2007 Rio De Janeiro www.ilts.org

Meeting 9th World Congress on Gastrointestinal Cancer 27-30 June 2007 Barcelona meetings@imedex.com

Meeting 15th International Congress of the European Association for Endoscopic Surgery 4-7 July 2007 Athens info@eaes-eur.org www.congresses.eaes-eur.org

Meeting 39th Meeting of the European Pancreatic Club 4-7 July 2007 Newcastle www.e-p-c2007.com

Republic of meeting ISNM2007The 21st International Symposium on Neurogastroenterology and Motility 2-5 September 2007Jeju Islandisnm2007@intercom.co.kr www.isnm2007.org/00main/main.htm

Meeting ⅩⅩth International Workshop on Heliobacter and related bacteria in cronic degistive inflammation 20-22 September 2007Istanbulwww.heliobacter.org

Meeting European Society of Coloproctology (ESCP) 2nd Annual Meeting 26-29 September 2007 Malta info@escp.eu.comwww.escp.eu.com/index.php

Meetings

Global Collaboration forGastroenterologyFor the first time in the history of gastroenterology, an international conference will take place which joins together the forces of four pre-eminent organisations: Gastro 2009, UEGW/WCOG London. The United European Gastroenterology Federation (UEGF) and the World Gastroenterology Organisation (WGO), together with the World Organisation of Digestive Endoscopy (OMED) and the British Society of Gastroenterology (BSG), are jointly organising a landmark meeting in London from November 21-25, 2009. This collaboration will ensure the perfect balance of basic science and clinical practice, will cover all disciplines in gastroenterology (endoscopy, digestive oncology, n u t r i t i o n , d i g e s t i v e s u r g e r y , hepatology, gastroenterology) and ensure a truly global context; all presented in the exciting setting of the city of London. Attendance is expected to reach record heights as participants are provided with a compact “all-in-one” programme merging the best of several GI meetings. Faculty and participants from all corners of the earth will merge to provide a truly global environment conducive to the exchange of ideas and the forming of friendships and collaborations.

18th World Congress of the International Association of Surgeons, Gastroenterologists and Oncologists 8-11 October 2008Istanbul

Meeting Falk Workshop: Mechanisms of Intestinal Inflammation 10 October 2007Dresdensymposia@falkfoundation.de

Meeting Falk Symposium 161: Future Perspectives in Gastroenterology 11-12 October 2007 Dresdensymposia@falkfoundation.de

American College of Gastroenterology Annual Scientific Meeting 12-17 October 2007 Philadelphia

Meeting Falk Symposium 162: Liver Cirrhosis-From Pathophysiology to Disease Management 13-14 October 2007Dresdensymposia@falkfoundation.de

Meeting APDW 2007-Asian Pacific Digestive Disease Week 2007 15-18 October 2007Kobeapdw@convention.co.jp www.apdw2007.org

15th United European Gastroenterology Week, UEGW 27-31 October 2007 Paris

Meeting The Liver Meeting®2007-57th Annual Meeting of the American Association for the Study of Liver Diseases 2-6 November 2007 Boston-MAwww.aasld.org

Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6451www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

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Instructions to authorsGENERAL INFORMATIONWorld Journal of Gastroenterology (WJG, World J Gastroenterol ISSN 1007-9327 CN 14-1219/R) is a weekly journal of more than 48 000 circulation, published on the 7th, 14th, 21st and 28th of every month.

Original Research, Clinical Trials, Reviews, Comments, and Case Reports in esophageal cancer, gastric cancer, colon cancer, liver cancer, viral liver diseases, etc., from all over the world are welcome on the condition that they have not been published previously and have not been submitted simultaneously elsewhere.

Indexed and abstracted inCurrent Contents®/Clinical Medicine, Science Citation Index Expanded (also known as SciSearch®) and Journal Citation Reports/Science Edition, Index Medicus, MEDLINE and PubMed, Chemical Abstracts, EMBASE/Excerpta Medica, Abstracts Journals, Nature Clinical Practice Gastroenterology and Hepatology, CAB Abstracts and Global Health. ISI JCR 2003-2000 IF: 3.318, 2.532, 1.445 and 0.993.

Published byThe WJG Press

SUBMISSION OF MANUSCRIPTSManuscripts should be typed double-spaced on A4 (297 mm × 210 mm) white paper with outer margins of 2.5 cm. Number all pages consecutively, and start each of the following sections on a new page: Title Page , Abstract, Introduction, Materials and Methods, Results, Discussion, acknowledgements, References, Tables, Figures and Figure Legends. Neither the editors nor the Publisher is responsible for the opinions expressed by contributors. Manuscripts formally accepted for publication become the permanent property of The WJG Press, and may not be reproduced by any means, in whole or in part without the written permission of both the authors and the Publisher. We reserve the right to put onto our website and copy-edit accepted manuscripts. Authors should also follow the guidelines for the care and use of laboratory animals of their institution or national animal welfare committee.

Authors should retain one copy of the text, tables, photographs and illustrations, as rejected manuscripts will not be returned to the author(s) and the editors will not be responsible for the loss or damage to photographs and illustrations in mailing process.

Online submissionsOnline submissions are strongly advised. Manuscripts should be submitted through the Online Submission System at: http://www.wjgnet.com/index.jsp. Authors are highly recommended to consult the ONLINE INSTRUCTIONS TO AUTHORS (http://www.wjgnet.com/wjg/help/instructions.jsp) before attempting to submit online. Authors encountering problems with the Online Submission System may send an email you describing the problem to wjg@wjgnet.com for assistance. If you submit your manuscript online, do not make a postal contribution. A repeated online submission for the same manuscript is strictly prohibited.

Postal submissionSend 3 duplicate hard copies of the full-text manuscript typed double-spaced on A4 (297 mm × 210 mm) white paper together with any original photographs or illustrations and a 3.5 inch computer diskette or CD-ROM containing an electronic copy of the manuscript including all the figures, graphs and tables in native Microsoft Word format or *.rtf format to:

Editorial Office World Journal of GastroenterologyEditorial Department: Apartment 1066, Yishou Garden,58 North Langxinzhuang Road,PO Box 2345, Beijing 100023, ChinaE-mail: wjg@wjgnet.comhttp: //www.wjgnet.comTelephone: +86-10-85381892Fax: +86-10-85381893

MANUSCRIPT PREPARATIONAll contributions should be written in English. All articles must be submitted using a word-processing software. All submissions must be typed in 1.5

line spacing and in word size 12 with ample margins. The letter font is Tahoma. For authors from China, one copy of the Chinese translation of the manuscript is also required (excluding references). Style should conform to our house format. Required information for each of the manuscript sections is as follows:

Title pageFull manuscript title, running title, all author(s) name(s), affiliations, institution(s) and/or department(s) where the work was accomplished, disclosure of any financial support for the research, and the name, full address, telephone and fax numbers and email address of the corresponding author should be included. Titles should be concise and informative (removing all unnecessary words), emphasize what is new, and avoid abbreviations. A short running title of less than 40 letters should be provided. List the author(s)’ name(s) as follows: initial and/or first name, middle name or initial(s) and full family name.

AbstractAn informative, structured abstract of no more than 350 words should accompany each manuscript. Abstracts for original contributions should be structured into the following sections: AIM: Only the purpose should be included. METHODS: The materials, techniques, instruments and equipments, and the experimental procedures should be included. RESULTS: The observatory and experimental results, including data, effects, outcome, etc. should be included. Authors should present P value where necessary, and the significant data should accompany. CONCLUSION: Accurate view and the value of the results should be included.

The format of structured abstracts is at: http://www.wjgnet.com/wjg/help/11.doc

Key wordsPlease list 5-10 key words that could reflect content of the study mainly from Index Medicus.

TextFor most article types, the main text should be structured into the following sections: INTRODUCTION, MATERIALS AND METHODS, RESULTS and DISCUSSION, and should include in appropriate Figures and Tables. Data should be presented in the body text or in Figures and Tables, but not in both.

IllustrationsFigures should be numbered as 1, 2, 3 and so on, and mentioned clearly in the main text. Provide a brief title for each figure on a separate page. No detailed legend should be involved under the figures. This part should be added into the text where the figures are applicable. Digital images: black and white photographs should be scanned and saved in TIFF format at a resolution of 300 dpi; color images should be saved as CMYK (print files) but not as RGB (screen-viewing files). Place each photograph in a separate file. Print images: supply images of size no smaller than 126 mm × 85 mm printed on smooth surface paper; label the image by writing the Figure number and orientation using an arrow. Photomicrographs: indicate the original magnification and stain in the legend. Digital Drawings: supply files in EPS if created by freehand and illustrator, or TIFF from photoshops. EPS files must be accompanied by a version in native file format for editing purposes. Existing line drawings should be scanned at a resolution of 1200 dpi and as close as possible to the size where they will appear when printed. Please use uniform legends for the same subjects. For example: Figure 1 Pathological changes of atrophic gastritis after treatment. A: ...; B: ...; C: ...; D: ...; E: ...; F: ...; G: ...

TablesThree-line tables should be numbered as 1, 2, 3 and so on, and mentioned clearly in the main text. Provide a brief title for each table. No detailed legend should be included under the tables. This part should be added into the text where the tables are applicable. The information should complement but not duplicate that contained in the text. Use one horizontal line under the title, a second under the column heads, and a third below the Table, above any footnotes. Vertical and italic lines should be omitted.

Notes in tables and illustrationsData that are not statistically significant should not be noted. aP<0.05, bP<0.01 should be noted (P>0.05 should not be noted). If there are other series of P values, cP<0.05 and dP<0.01 are used. Third series of P values can be expressed as eP<0.05 and fP<0.01. Other notes in tables or under

Online Submissions: wjg.wjgnet.com World J Gastroenterol 2007 December 21; 13(47): 6452-6454www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327wjg@wjgnet.com © 2007 WJG. All rights reserved.

illustrations should be expressed as 1F, 2F, 3F; or some other symbols with a superscript (Arabic numerals) in the upper left corner. In a multi-curve illustration, each curve should be labeled with ●, ○, ■, □, ▲, △, etc. in a certain sequence.

AcknowledgmentsBrief acknowledgments of persons who have made genuine contributions to the manuscripts and who endorse the data and conclusions are included. Authors are responsible for obtaining written permission to use any copyrighted text and/or illustrations.

REFERENCESCoding systemThe author should code the references according the citation order in text in Arabic numerals, put references codes in square brackets, superscript it at the end of citation content or the author name of the citation. For those citation content as the narrate part, the coding number and square brackets should be typeset normally. For example, Crohn’s disease (CD) is associated with increased intestinal permeability[1,2]. If references are directly cited in the text, they would be put together with the text, for example, from references [19,22-24], we know that... When the authors code the references, please ensure that the order in text is the same as in reference part and also insure the spelling accuracy of the first author’s name. Do not code the same citation twice.

PMID requirementPMID roots in the abstract serial number indexed by PubMed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed). The author should supply the PMID for journal citation. For those references that have not been indexed by PubMed, a printed copy of the first page of the full reference should be submitted. The accuracy of the information of the journal citations is very important. Through reference testing system, the authors and editor could check the authors name, title, journal title, publication date, volume number, start page, and end page. We will interlink all references with PubMed in ASP file so that the readers can read the abstract of the citations online immediately.

Style for journal referencesAuthors: the first author should be typed in bold-faced letter. The surname of all authors should be typed with the initial letter capitalized and followed by their name in abbreviation (For example, Lian-Sheng Ma is abbreviated as Ma LS, Bo-Rong Pan as Pan BR). Title of the cited article and italicized journal title (Journal title should be in its abbreviation form as shown in PubMed), publication date, volume number (in black), start page, and end page [PMID: 11819634] Note: The author should test the references through reference testing system (http://www.wjgnet.com/cgi-bin/index.pl)

Style for book referencesAuthors: the first author should be typed in bold-faced letter. The surname of all authors should be typed with the initial letter capitalized and followed by their name in abbreviation (For example, Lian-Sheng Ma is abbreviated as Ma LS, Bo-Rong Pan as Pan BR) Book title. Publication number. Publication place: Publication press, Year: start page and end page.

FormatJournals English journal article (list all authors and include the PMID where applicable)1 Grover VP, Dresner MA, Forton DM, Counsell S, Larkman DJ, Patel N,

Thomas HC, Taylor-Robinson SD. Current and future applications of magnetic resonance imaging and spectroscopy of the brain in hepatic encephalopathy. World J Gastroenterol 2006; 12: 2969-2978 [PMID: 16718775]

Chinese journal article (list all authors and include the PMID where applicable)2 Lin GZ, Wang XZ, Wang P, Lin J, Yang FD. Immunologic effect of

Jianpi Yishen decoction in treatment of Pixu-diarrhoea. Shijie Huaren Xiaohua Zazhi 1999; 7: 285-287

In press3 Tian D, Araki H, Stahl E, Bergelson J, Kreitman M. Signature of

balancing selection in Arabidopsis. Proc Natl Acad Sci U S A 2006; In press

Organization as author4 Diabetes Prevention Program Research Group. Hypertension,

insulin, and proinsulin in participants with impaired glucose tolerance. Hypertension 2002; 40: 679-686 [PMID: 12411462]

Both personal authors and an organization as author 5 Vallancien G, Emberton M, Harving N, van Moorselaar RJ; Alf-One

Study Group. Sexual dysfunction in 1, 274 European men suffering from lower urinary tract symptoms. J Urol 2003; 169: 2257-2261 [PMID: 12771764]

No author given6 21st century heart solution may have a sting in the tail. BMJ 2002; 325:

184 [PMID: 12142303]Volume with supplement7 Geraud G, Spierings EL, Keywood C. Tolerability and safety of

frovatriptan with short- and long-term use for treatment of migraine and in comparison with sumatriptan. Headache 2002; 42 Suppl 2: S93-99 [PMID: 12028325]

Issue with no volume8 Banit DM, Kaufer H, Hartford JM. Intraoperative frozen section

analysis in revision total joint arthroplasty. Clin Orthop Relat Res 2002; (401): 230-238 [PMID: 12151900]

No volume or issue9 Outreach: bringing HIV-positive individuals into care. HRSA Careaction

2002; 1-6 [PMID: 12154804]Books Personal author(s)10 Sherlock S, Dooley J. Diseases of the liver and billiary system. 9th ed.

Oxford: Blackwell Sci Pub, 1993: 258-296Chapter in a book (list all authors)11 Lam SK. Academic investigator’s perspectives of medical treatment for

peptic ulcer. In: Swabb EA, Azabo S. Ulcer disease: investigation and basis for therapy. New York: Marcel Dekker, 1991: 431-450

Author(s) and editor(s)12 Breedlove GK, Schorfheide AM. Adolescent pregnancy. 2nd ed.

Wieczorek RR, editor. White Plains (NY): March of Dimes Education Services, 2001: 20-34

Conference proceedings13 Harnden P, Joffe JK, Jones WG, editors. Germ cell tumours V.

Proceedings of the 5th Germ Cell Tumour Conference; 2001 Sep 13-15; Leeds, UK. New York: Springer, 2002: 30-56

Conference paper14 Christensen S, Oppacher F. An analysis of Koza's computational effort

statistic for genetic programming. In: Foster JA, Lutton E, Miller J, Ryan C, Tettamanzi AG, editors. Genetic programming. EuroGP 2002: Proceedings of the 5th European Conference on Genetic Programming; 2002 Apr 3-5; Kinsdale, Ireland. Berlin: Springer, 2002: 182-191

Electronic journal (list all authors) Morse SS. Factors in the emergence of infectious diseases. Emerg

Infect Dis serial online, 1995-01-03, cited 1996-06-05; 1(1): 24 screens. Available from: URL: http//www.cdc.gov/ncidod/EID/eid.htm

Patent (list all authors)16 Pagedas AC, inventor; Ancel Surgical R&D Inc., assignee. Flexible

endoscopic grasping and cutting device and positioning tool assembly. United States patent US 20020103498. 2002 Aug 1

Inappropriate referencesAuthors should always cite references that are relevant to their article, and avoid any inappropriate references. Inappropriate references include those that are linked with a hyphen and the difference between the two numbers at two sides of the hyphen is more than 5. For example, [1-6], [2-14] and [1, 3, 4-10, 22] are all considered as inappropriate references. Authors should not cite their own unrelated published articles.

Statistical dataPresent as mean ± SD or mean ± SE.

Statistical expressionExpress t test as t (in italics), F test as F (in italics), chi square test as χ2 (in Greek), related coefficient as r (in italics), degree of freedom as γ (in Greek), sample number as n (in italics), and probability as P (in italics).

UnitsUse SI units. For example: body mass, m (B) = 78 kg; blood pressure, p (B) = 16.2/12.3 kPa; incubation time, t (incubation) = 96 h, blood glucose concentration, c (glucose) 6.4 ± 2.1 mmol/L; blood CEA mass concentration, p (CEA) = 8.6 24.5 µg/L; CO2 volume fraction, 50 mL/L CO2 not 5% CO2; likewise for 40 g/L formaldehyde, not 10% formalin; and mass fraction, 8 ng/g, etc. Arabic numerals such as 23, 243, 641 should be read 23 243 641.

The format about how to accurately write common units and quantum is at: http://www.wjgnet.com/wjg/help/15.doc

Instructions to authors 6453

AbbreviationsStandard abbreviations should be defined in the abstract and on first mention in the text. In general, terms should not be abbreviated unless they are used repeatedly and the abbreviation is helpful to the reader. Permissible abbreviations are listed in Units, Symbols and Abbreviations: A Guide for Biological and Medical Editors and Authors (Ed. Baron DN, 1988) published by The Royal Society of Medicine, London. Certain commonly used abbreviations, such as DNA, RNA, HIV, LD50, PCR, HBV, ECG, WBC, RBC, CT, ESR, CSF, IgG, ELISA, PBS, ATP, EDTA, mAb, can be used directly without further mention.

ItalicsQuantities: t time or temperature, c concentration, A area, l length, m mass, V volume.Genotypes: gyrA, arg 1, c myc, c fos, etc.Restriction enzymes: EcoRI, HindI, BamHI, Kbo I, Kpn I, etc.Biology: H pylori, E coli, etc.

SUBMISSION OF THE REVISED MANUSCRIPTS AFTER ACCEPTEDPlease revise your article according to the revision policies of WJG. The revised version including manuscript and high-resolution image figures (if any) should be copied on a floppy or compact disk. Author should send the revised manuscript, along with printed high-resolution color or black and white photos, copyright transfer letter, the final check list for authors, and responses to reviewers by a courier (such as EMS) (submission of revised manuscript by e-mail or on the WJG Editorial Office Online System is NOT available at present).

Language evaluation The language of a manuscript will be graded before sending for revision. (1) Grade A: priority publishing; (2) Grade B: minor language polishing; (3) Grade C: a great deal of language polishing; (4) Grade D: rejected. The revised articles should be in grade B or grade A.

Copyright assignment formPlease downloaded CAF from http://www.wjgnet.com/wjg/help/9.doc. We certify that the material contained in this manuscript:Ms:

Title:

is original, except when appropriately referenced to other sources, and that written permission has been granted by any existing copyright holders. We agree to transfer to WJG all rights of our manuscript, including: (1) all copyright ownership in all print and electronic formats; (2) the right to grant permission to republish or reprint the stated material in whole or in part, with or without a fee; (3) the right to print copies for free distribution or sale; (4) the right to republish the stated material in a collection of articles or in any other format. We also agree that our article be put on the Internet.Criteria for authorship: The WJG requests and publishes information about contributions of each author named to the submitted study. Authorship credit should be based on (1)direct participation in the study, including substantial contributions to conception and design of study, or acquisition of data, or analysis and interpretation of data; (2) manuscript writing, including drafting the article, or revising it critically for important intellectual content; (3)supportive work, including statistical analysis of data, or acquisition of funding, or administration, technology and materials support, or supervision, or supportive contributions. Authors should meet at least one of the three conditions. The WJG does not publish co-first authors and co-corresponding authors.

We hereby assign copyright transfer to WJG if this paper is accepted.Author Name in full (Full names should be provided, with first name

first, followed by middle names and family name at the last, eg, Eamonn MM Quigley). Handwritten names are not accepted.

Author Name in abbreviation (Family name is put first in full, followed by middle names and first name in abbreviation with first letter in capital, eg, Quigley EMM). Handwritten names are not accepted.

1 Full Name: Abbreviation Name: Signed: Date:

2 Full Name: Abbreviation Name: Signed: Date:

3 Full Name: Abbreviation Name: Signed: Date:

4 Full Name: Abbreviation Name: Signed: Date:

5 Full Name: Abbreviation Name: Signed: Date:

6 Full Name: Abbreviation Name: Signed: Date:

7 Full Name: Abbreviation Name: Signed: Date:

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Final check list for authorsThe format is at: http://www.wjgnet.com/wjg/help/13.doc

Responses to reviewersPlease revise your article according to the comments/suggestions of reviewers. The format for responses to the reviewers’ comments is at: http://www.wjgnet.com/wjg/help/10.doc

Proof of financial supportFor paper supported by a foundation, authors should provide a copy of the document and serial number of the foundation.

Publication feeAuthors of accepted articles must pay publication fee.EDITORIAL and LETTERS TO THE EDITOR are free of charge.

6454 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol December 21, 2007 Volume 13 Number 47