The Liver in Biology and Disease 9780080459264, 9780762311248, 076231124X

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THE LIVER IN BIOLOGY AND DISEASE

PRINCIPLES OF MEDICAL BIOLOGY Series Editor: E. Edward Bittar

PRINCIPLES OF MEDICAL BIOLOGY VOLUME 15

THE LIVER IN BIOLOGY AND DISEASE EDITED BY

E. EDWARD BITTAR University of Wisconsin, Madison, WI, USA

2004

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© 2004 Elsevier Ltd. All rights reserved. This work is protected under copyright by Elsevier Ltd, and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may be sought directly from Elsevier’s Rights Department in Oxford, UK; phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. Requests may also be completed on-line via the Elsevier homepage (http://www.elsevier.com/locate/permissions). In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: (+1) (978) 7508400, fax: (+1) (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London W1P 0LP, UK; phone: (+44) 20 7631 5555; fax: (+44) 20 7631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of the Publisher is required for external resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier’s Rights Department, at the fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. First edition 2004 British Library Cataloguing in Publication Data A catalogue record is available from the British Library. ISBN: 0-7623-1124-X ISSN: 1569-2582 (Series) ∞  The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper). Printed in

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CONTENTS LIST OF CONTRIBUTORS

ix

PREFACE 1.

2.

3.

4.

5.

6.

7.

xiii

THE INTRAHEPATIC BILIARY TREE James M. Crawford

1

FUNCTIONAL HETEROGENEITY OF INTRAHEPATIC CHOLANGIOCYTES Gene D. LeSage, Shannon S. Glaser, Heather Francis, Jo Lynne Phinizy and Gianfranco Alpini

21

THE ACTIN CYTOSKELETON IN LIVER FUNCTION R. Brian Doctor and Matthew Nichols

49

MECHANISMS OF BILE FORMATION AND CHOLESTASIS M. Sawkat Anwer

81

THE ROLE OF BILE ACIDS IN THE MODULATION OF APOPTOSIS Cecília M. P. Rodrigues, Rui E. Castro and Clifford J. Steer

119

GROWTH FACTORS AND THE LIVER Clare Selden

147

CHEMOKINE AND CYTOKINE REGULATION OF LIVER INJURY Kenneth J. Simpson and Neil C. Henderson

167

v

vi

8.

9.

DRUG METABOLISM AND HEPATOTOXICITY J. Michael Tredger

207

ASSEMBLY AND SECRETION OF HEPATIC VERY-LOW-DENSITY LIPOPROTEIN Geoffrey Gibbons

229

10. BILIRUBIN METABOLISM Peter L. M. Jansen and E. Edward Bittar

257

11. CLINICAL BIOCHEMISTRY OF THE LIVER Neil McIntyre

291

12. ALCOHOLIC LIVER DISEASE S. F. Stewart and C. P. Day

317

13. FULMINANT HEPATIC FAILURE Watson Ng, Ian D. Norton and D. Brian Jones

361

14. PRIMARY BILIARY CIRRHOSIS James Neuberger

383

15. CHRONIC ACTIVE HEPATITIS Ian G. McFarlane

399

16. HEPATITIS B VIRUS F. Fred Poordad

427

17. CURRENT ISSUES IN HEPATITIS B VACCINES Jane N. Zuckerman and Arie J. Zuckerman

439

18. THE MOLECULAR VIROLOGY OF HEPATITIS C VIRUS Timothy L. Tellinghuisen and Charles M. Rice

455

vii

19. THE ROLE OF THE HEPATIC STELLATE CELL IN LIVER FIBROSIS Timothy J. Kendall and John P. Iredale

497

20. ORTHOTOPIC LIVER TRANSPLANTATION Gagandeep Singh, Pankaj Rajvanshi and Sanjeev Gupta

525

21. BIOLOGICAL PRINCIPLES AND NOVEL THERAPIES IN LIVER CELL TRANSPLANTATION Sanjeev Gupta, Mari Inada, Vinay Kumaran and Brigid Joseph

543

22. FLUID TRANSPORT IN THE GALLBLADDER Joar Svanvik and Bengt Nilsson

555

AUTHOR INDEX

577

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LIST OF CONTRIBUTORS Gianfranco Alpini

Department of Internal Medicine and Medical Physiology, Scott & White Hospital, The Texas A&M University System, Temple, TX, USA

M. Sawkat Anwer

Departments of Biomedical Sciences, Tufts University School of Veterinary Medicine, North Grafton, MA, USA

E. Edward Bittar

University of Wisconsin, Madison, WI, USA

Rui E. Castro

Centro de Patog´enese Molecular, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal

James M. Crawford

Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL, USA

C. P. Day

School of Clinical Medical Sciences (Hepatology) Medical School, Newcastle upon Tyne, UK

R. Brian Doctor

Division of Gastroenterology, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO, USA

Heather Francis

Department of Research & Education, Scott & White Hospital, Temple, TX, USA

Geoffrey Gibbons

Metabolic Research Laboratory, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, UK

Shannon S. Glaser

Division of Research & Education, Scott & White Hospital, Temple, TX, USA ix

x

Sanjeev Gupta

Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY, USA

Neil C. Henderson

MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, Scotland, UK

Mari Inada

Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY, USA

John P. Iredale

Division of Infection, Inflammation and Repair, University of Southampton General Hospital, Southampton, UK

Peter L. M. Jansen

Department of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam, The Netherlands

D. Brian Jones

Department of Gastroenterology and Hepatology, Concord Repatriation General Hospital, Sydney, Australia

Brigid Joseph

Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY, USA

Timothy J. Kendall

Division of Infection, Inflammation and Repair, University of Southampton General Hospital, Southampton, UK

Vinay Kumaran

Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY, USA

Gene D. LeSage

Department of Medicine, The University of Texas, Houston Medical School, Houston, TX, USA

Ian G. McFarlane

Institute of Liver Studies, King’s College Hospital, London, UK

Neil McIntyre

Department of Medicine, Royal Free Hospital and University College School of Medicine, London, UK

xi

James Neuberger

The Queen Elizabeth Hospital, Birmingham, UK

Watson Ng

Department of Gastroenterology and Hepatology, Royal Prince Alfred Hospital, Sydney, Australia

Matthew Nichols

Department of Gastroenterology, University of Colorado Health Sciences Center, Denver, CO, USA

Bengt Nilsson

Department of Surgery, University of G¨othenburg, G¨othenburg, Sweden

Ian D. Norton

Department of Gastroenterology and Hepatology, Concord Hospital, Concord, Australia

Jo Lynne Phinizy

Department of Research & Education, Scott & White Hospital, Temple, TX, USA

F. Fred Poordad

Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA, USA

Pankaj Rajvanshi

Division of Gastroenterology and Hepatology, University of Washington and Pacific Medical Center, Seattle, WA, USA

Charles M. Rice

Center for the Study of Hepatitis C, The Rockefeller University, New York, USA

Cec´ılia M. P. Rodrigues

Centro de Patog´enese Molecular, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal

Clare Selden

Centre for Hepatology, Royal Free Hospital and University College School of Medicine, London, UK

Kenneth J. Simpson

MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, Scotland, UK

xii

Gagandeep Singh

Division of Hepatobiliary-Pancreatic Surgery and Abdominal Organ Transplantation, University of Southern California, Keck School of Medicine, Los Angeles, CA, USA

Clifford J. Steer

Departments of Medicine and Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, MN, USA

S. F. Stewart

School of Clinical Medical Sciences (Hepatology) Medical School, Newcastle upon Tyne, UK

Joar Svanvik

Department of Surgery, University of Link¨oping, Link¨oping, Sweden

Timothy L. Tellinghuisen

Center for the Study of Hepatitis C, The Rockefeller University, New York, USA

J. Michael Tredger

Institute of Liver Studies, Guy’s King’s and St. Thomas’ School of Medicine, London, UK

Arie J. Zuckerman

WHO Collaborating Centre for Reference and Research in Viral Diseases, Royal Free Hospital and University College School of Medicine, London, UK

Jane N. Zuckerman

WHO Collaborating Centre for Reference, Research and Training in Travel Medicine, Royal Free Hospital and University College School of Medicine, London, UK

PREFACE In keeping with the spirit of its predecessors in the series entitled The Biological Basis of Medicine (Academic Press, London), followed by the series entitled Principles of Medical Biology, the present book represents an attempt at providing a set of essays and overviews for all readers seeking to understand the general principles and growing groundwork of molecular cell biology within the ambit of hepatology. Hepatology in this postgenomic era is advancing rapidly by leaps and bounds. A notable example of this is proteomics and informatics both of which are due primarily to progress in biotechnology. The striking fact is that the growth of biotechnology has been, and still is, spiral rather than gradual. It is thus safe to say that the future of hepatology rests with molecular cell biology and it cannot be studied separately from technique. It is abundantly clear that it would be wide of the mark to contemplate nowadays covering a large multi-discipline subject within the compass of a reference textbook. For there are serious disadvantages several of which need to be taken into account. First, producing a reference textbook is a slow process, thus making it most likely that a great deal of the information therein would be out of date by the time the book is published. Second, the price structure of textbooks is generally forbidding, and hence, unaffordable by most graduates and post-graduates. The same is true of numerous medical libraries whose budgets have been curtailed. And third, there is a widespread reluctance particularly among average students to use such textbooks because of the unending struggle they face, and because of the tedium they so frequently experience. This book, as it now appears, is hardly large and it contains 22 chapters written by recognized experts in their own field. The topics have been selected with care and are not only appropriate for experts and those seeking expertise but also for the novice and people wanting to become familiar with the fundamentals of molecular biology when blended with cell biology. The first chapter is by James M. Crawford whose pioneer studies of the biliary tract are widely recognized. He reviews the development and anatomy of the intrahepatic biliary tree, as well as the biology of cholangiocytes. As pointed out by him, the terminal architecture of the intrahepatic biliary tree comprises hemicircular canals of Hering linking the bile canaliculi between hepatocytes to the smallest complete channels of the biliary tree, that is, the ductules. xiii

xiv

PREFACE

Chapter 2 is an overview in which Gianfranco Alpini and his team summarize much of the work they have done thus far on cholangiocytes. They show that cholangiocyte heterogeneity in ductal secretion affords physiologic advantages: A correlation is drawn between cholangiocytic heterogeneity and non-transport proteins. They also elaborate on aspects of cholangiocytic apoptosis, bearing in mind that apoptosis is designed to rapidly remove unwanted and potentially dangerous cells (Rich). Chapter 3 is by R. Brian Doctor and Matthew Nichols who outline how actin and its associated proteins direct signaling and transport functions at the apical membrane of liver epithelial cells. The various actin-related diseases are touched upon. Chapter 4 by M. Sawkat Anwer concerns our present understanding of the mechanisms of transhepatic solute transport and bile formation. As is to be expected, he discusses how insight has been gained into the nature of the transport systems responsible for canalicular excretion of organic anions. Studies on patients with the Dubin-Johnson syndrome and on animal models such as mutant corridal sheep with chronic hyperbilirubinemia have shed new light on the question whether most of the transporters are members of the ABC superfamily. Chapter 5 by Cec´ılia M. P. Rodrigues, Rui E. Castro and Clifford J. Steer deals with the role of bile acids in the modulation of apoptosis. These investigators have recently shown that ursodeoxycholic acid and its conjugated derivative, tauroursodeoxycholic acid, play a unique role in modulating the apoptotic threshold in both hepatic and non-hepatic cells. Both act by blocking classic pathways and are able to appreciably activate survival pathways. As it turns out, tauroursodeoxycholic acid is neuro-protective in pharmacologic and transgenic animal models of Huntington’s disease, improves graft survival in Parkinsonian rats, and protects against neurological injury following acute brain ischemia and hemorrhagic stroke. Chapter 6 by Clare Selden is concerned with growth factors, and the paradigms of liver growth. Growth factor and cytokines are recognized as an important part of liver regeneration mechanisms. They contribute to the proliferation of the sub populations of the liver to maintain liver homeostasis after injury. Chapter 7 is by Kenneth J. Simpson and Neil C. Henderson who hold the view that our understanding of the immunological responses to liver damage is steadily increasing, but at the moment therapeutic options remain limited. Each of the different cellular constituents of the liver produce soluble protein mediators such as cytokines and chemokines. These mediators are able to bind to specific cell surface receptors, thus creating an autocrine loop of cellular activation. Numerous cytokines and chemokines play key roles in the pathogenesis of liver injury and repair.

Preface

xv

Chapter 8 by J. Michael Tredger is about drug metabolism and hepatotoxicity. A unifying theme throughout hepatotoxicity is its origin in an imbalance of intoxication over detoxication pathways. Where intoxication prevails, its products initiate one or more hepatotoxic cascades with adverse outcomes. Potential toxins that trigger sequences of key events mediating cell damage and dysfunction adds another layer of variable complexity which determines outcome. Chapter 9 is by Geoffrey Gibbons who documents what is currently known about molecular and cellular mechanisms involved in the production and secretion of very-low-density lipoprotein (VLDL). Changes in the secretion of VLDL are associated with physiological and nutritional transitions. The assembly of triacylglycerol (TAG)-rich lipoproteins by the intestine as chylomicrons, and by the liver as VLDL is an essential part of the process by which dietary and endogenously synthesized TAG become available for use or storage by extrahepatic tissues. The liver, however, makes a relatively small contribution to the body’s store of TAG. Chapter 10 by Peter L. M. Jansen and E. Edward Bittar presents an up-todate account of bilirubin biology, followed by accounts of neonatal jaundice and a number of syndromes in which the hallmark is jaundice. The recent finding that bilirubin is a more powerful antioxidant than glutathione and that it is cytoprotective are recognized as being of first importance with the proviso that the experimental results are reproducible in other laboratories, and are accurate. Chapter 11 by Neil McIntyre is titled Clinical Biochemistry of the Liver. It encompasses a small battery of liver function tests. These are aids to detect liver disease and are also used to monitor the course of the disease. Although some tests are of diagnostic value for certain conditions, the results may be difficult to interpret in the absence of liver biopsy, immunological tests and imaging techniques. Chapter 12 by S. F. Stewart and Christopher P. Day deals with the molecular aspects of alcoholic liver disease. The most important cause of liver disease in the world is alcohol abuse. Alcohol produces a wide range of clinico-pathologic syndromes in which acetylaldehyde is the initial toxic metabolite. This is followed by the development of an injurious “hypermetabolic state” that leads to the generation of reactive free radicals. Chapter 13 is by Watson Ng, Ian D. Norton and D. Brian Jones who review the topic of fulminant hepatic failure (FHP). In general, the term acute liver failure (ALF) has been used in the literature to describe rapid severe hepatocellular dysfunction in a previously normal liver. Fulminant hepatic failure is also similarly defined, except that FHF encephalopathy occurs within 8 weeks of the onset of symptoms of liver disease. The most common cause of ALF worldwide is viral hepatitis type B.

xvi

PREFACE

Chapter 14 is by James Neuberger who states that the etiology of primary biliary cirrhosis remains unknown. Its chief early feature is progressive destructive cholangitis accompanied by jaundice, hepatic fibrosis, portal hypertension, and finally, liver jaundice. Chapter 15 is by Ian G. McFarlane in which he draws our attention to the fact that the term chronic active hepatitis (CAH) is often used interchangeably with active chronic hepatitis (ACH). However, the histologic hallmark of CAH is piecemeal necrosis. The term non-A, non-B CAH was a catch-all category for patients with lupoid hepatitis and what was described as idiopathic or cryptogenic chronic liver disease. Until recently the terms CAH and CPH continued to dominate the field. Chapter 16 is by F. Fred Poordad who provides an up-to-date survey of hepatitis B virus (HBV) infection which is the most common chronic viral infection worldwide. There are seven genotypes and four sub types of this DNA virus. The genotypes A-G represent pathogenic differences, with C & D causing a more severe form of the disease that is less responsive to interferon therapy. Chapter 17 is about current issues in hepatitis B vaccines by Jane and Arie Zuckerman. Attention is largely focused on the emergence of hepatitis B surface antigen escape mutants, and the 5–15% of healthy people who are non-responders to the current vaccine. Chapter 18 concerning the molecular biology of hepatitis C virus is by Timothy L. Tellinghuisen and Charles M. Rice. They emphasize the point that about 70% of the patients infected with HCV remain persistently infected. HCV replication continues to occur in these patients often leading to serious liver disease and extra hepatic disorders, notably autoimmune diseases, cryoglobulinemia and nonHodgkin’s lymphoma. In addition, chronic HCV infection is associated with an increased risk of hepatocellular carcinoma. HCV associated liver disease is the leading indicator of liver transplantation. Chapter 19 is by Timothy J. Kendall and John P. Iredale who provide an overview of the role of the stellate cell in liver fibrosis. They state very emphatically that liver fibrosis is a bi-directional process with a large capacity for significant structural recovery from severe liver injury and fibrosis. The recovery phase involves a reduction in activated hepatic stellate cells (HSC) numbers and restoration of the original architecture. Crucial to this process of resolution is the interaction between HSCs and the extracellular matrix. Extracellular matrix signaling influences cell survival by modulating apoptosis and modifying levels of cell proliferation and differentiation. The HSCs present in the space of Disse supply most of the extracellular components in this space. Chapter 20 is by Gagandeep Singh, Pankaj Rajvanshi and Sanjeev Gupta who provide a survey of orthotopic liver transplantation. Liver transplantation has become a viable option because of the various improvements that have been made

Preface

xvii

in surgical techniques and immuno-suppression. A longstanding problem is the limited supply of donor organs. Efforts are being made to determine whether reseeding of the liver with hepatocytes helps recovery in patients with acute liver failure, and whether metabolic deficiency states can be ameliorated or corrected by transplanting healthy hepatocytes. Chapter 21 by Sanjeev Gupta, Mari Inada, Vinay Kumaran and Brigid Joseph deals with a variety of issues in which biological principles and novel therapies are sought by means of liver cell transplantation. Gupta and his colleagues consider the liver as affording paradigms with which insight can be gained into both stem cell biology. This is self-evident, considering the diversity of liver functions ranging from protein synthesis all the way to its regenerative capacity. Studies show that transplanted hepatocytes can proliferate indefinitely, repopulating the liver of recipient animals over a period of seven generations. This is analogous to the property of stem cells. Indeed, the liver is a source of stem/progenitor cells, in particular, during the fetal stage. Chapter 22 by Joar Svanvik and Bengt Nilsson is an up-to-date review of the state of the science in respect of gallbladder fluid transport. Bile reaching the gallbladder is concentrated by mucosal isotonic fluid absorption. Water movement is passive, and secondary to active Na transport. The epithelium is also capable of fluid secretion. The references present at the end of each chapter are reasonably ample, including papers published in 2003. I should add that I hold myself responsible for any glaring and major conceptual and data errors that may have crept in. Last but not least, I am deeply indebted to the contributing authors who in the face of many pressing tasks have managed to make this text possible. This goes without saying. I should also like to thank Ms. Jody Singh for her painstaking typing work and Joan Anuels and Hendrik van Leusen for their courtesy and assistance. E. Edward Bittar Editor

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1.

THE INTRAHEPATIC BILIARY TREE

James M. Crawford INTRODUCTION The biliary tree is the conduit between the hepatocellular parenchyma of the liver and the gut. Loss of patency of this conduit is incompatible with life, in the neonate or at any other time in life. The volume of the human adult intrahepatic biliary tree is estimated to be between 14–24 cm3 (about 1.2% of the liver volume), with an internal surface area of between 330–575 cm2 . The epithelial cells lining bile ducts and bile ductules represent only about 0.10% of the liver volume, but 3–5% of the total liver cellular population (Crawford, 2002). Fluid secreted by hepatocytes into the bile canalicular channels between hepatocytes, and thence, into the biliary tree is called bile. Bile is a lipid-rich fluid, whose major organic solutes are bile salts (sterol detergents, derivatives of cholesterol), phosphatidylcholine derived from the hepatocellular apical membrane, and cholesterol itself. Secretion of bile is the major route for elimination of cholesterol and other lipid-soluble or amphiphilic substances from the body; solutes that are insufficiently water soluble to be excreted in urine. The epithelial cells of the biliary tree are termed cholangiocytes. These cells are not just inert barriers to fluid but rather are dynamic secretory and absorptive cells, contributing up to 40% of the fluid volume secreted into bile. This chapter will review the development and anatomy of the intrahepatic biliary tree, and give consideration to the biology of cholangiocytes.

The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 1–20 © 2004 Published by Elsevier Ltd. ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15001-0

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JAMES M. CRAWFORD

The Hepatic Primordium The liver primordium buds off the ventral aspect of the embryonic foregut very early during development of the abdominal organs, at about 18 days of gestation when the human embryo is 2.5 mm in length. This primordium quickly lengthens and enlarges to form the hepatic diverticulum. Over the next few days, this endodermal sprout grows in a cranioventral fashion toward the septum transversum, a mesenchymal plate that incompletely separates the thoracic cavity from the abdominal cavity. The endodermal cells invade the mesoderm of the septum transversum as ramifying cords of cells, coincidental with the ingrowth of a sinusoidal vascular network from tributaries of the vitelline vein. The nascent liver is thus formed, and over the next three weeks of gestation grows rapidly and soon fills most of the abdominal cavity. The liver corpus separates from the septum transversum in the process, and the mesenchymal residua becomes the diaphragm. The extrahepatic biliary tree forms directly from the embryonic stalk of tissue off the foregut, and is substantively formed by week 16; the gallbladder and cystic duct are derived from a derivative diverticulum off the caudad portion of this stalk (Nakanuma et al., 1997). In contrast, while the architectural organization of the hepatic parenchyma and vasculature is well-established by week 16, development of the intrahepatic biliary tree continues throughout gestation and after birth. The intrahepatic biliary tree forms out of the interplay between the early hepatic endodermal cells, the hepatoblasts, and the mesenchyme of the primitive liver. The chronology for development of the biliary tree is given in Table 1.

Table 1. Chronology of Biliary Tract Development. 18 day embryo 22 day embryo 23 day embryo 3–8 week fetus 8–12 week fetus 12 week fetus to birth

Birth Birth to 4 weeks

liver bud develops on ventral endoderm of the foregut hepatic diverticulum protrudes into mesenchyme of septum transversum endodermal “cords” of hepatoblasts invade mesenchyme hepatic bile duct becomes patent up to hilus of liver “ductal plate” develops, from hilum outwards in centrifugal fashion remodeling of ductal plate into ductules and ducts within portal tract: 12 weeks: first generation (left and right hepatic ducts) 15 weeks: second generation 17–25 weeks: third generation 25 weeks: most ductal plates have become discontinuous 35 weeks: most portal tracts have a terminal bile duct peripheral portal tracts still lack terminal bile duct and retain discontinuous ductal plate maturation of intrahepatic biliary tree out to ≥ 7 generations

Source: From Crawford (2002).

The Intrahepatic Biliary Tree

3

It should be noted that the liver is the major hematopoietic organ in the embryo and fetus. Hematopoiesis (erythropoiesis and granulopoiesis) is intense and diffuse in the hepatic laminae between hepatoblasts and within portal tracts up to 24 weeks of gestation. After 25–28 weeks, the hematopoietic cells begin to form islands out of a previously diffuse distribution. By the 36th week, hematopoiesis exists only as scattered islands in the hepatic parenchyma. Little hematopoiesis is found in portal tracts after 32 weeks of gestation. In the discussion to follow, the microarchitecture of the liver is depicted in a clean and elegant form. The experienced observer will note that discerning epithelial architecture amidst the profound hematopoiesis in the early fetal liver can be a daunting prospect; the process is greatly assisted by the use of immunostains for cytokeratins (Desmet et al., 1989), as will be discussed.

Formation of the Intrahepatic Biliary Tree The original theory for development of the intrahepatic biliary tree was that it originates from the extrahepatic biliary tree at the porta hepatis and grows into the liver corpus in an infiltrative manner. More recent evidence clearly indicates that the intrahepatic bile ducts are derived from the endodermal cells already within the liver and their remodelling into the tubular anastomosing biliary tree of the adult (Tan & Moscoso, 1994). The challenge, then, is to understand how the intrahepatic and extrahepatic biliary trees develop in relation to one another. The buds of endodermal cells that extend from the hepatic diverticulum into the mesoderm of the septum transversum in the 3rd–5th weeks of gestation are termed hepatoblasts. These epithelial buds form anastomosing cords and plates that enmesh the sinusoidal vascular network from its origin off the vitelline vein tributaries to their effluence as a venous system draining into the nascent hepatic vein (Godlewski et al., 1997). Hepatoblasts throughout the liver corpus are bipotential, in that they are capable of maturing into either hepatocytes or bile duct epithelial cells when isolated and cultured in vitro. The key event in formation of the intrahepatic biliary tree occurs at the interface between the developing hepatic parenchyma and the mesenchyme of the portal tracts (Vijayan & Tan, 1997). Beginning around the 6th week of gestation in the hilar region of the liver, the hepatoblasts immediately adjacent to the portal tract mesenchyme flatten slightly and become a continuous layer of biliary-type cuboidal cells (see Fig. 1). This cellular layer is termed the ductal plate. The specification of hepatoblast differentiation into cells destined for the biliary tree, cholangiocytes, versus cells destined for the hepatic parenchyma, hepatocytes, appears to occur at the time of ductal plate formation. Ductal plate formation spreads centrifugally from the hilum toward the periphery of the liver, essentially

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JAMES M. CRAWFORD

Fig. 1. Depiction of the Ductal Plate. Note: The basic architecture of the primordial liver is depicted, beginning with (upper left) formation of the ductal plate around portal tracts which contain only a portal vein; (upper right) formation of a double layered ductal plate; (lower left) ingrowth of hepatic arteries and formation of crescentic swellings in the ductal plate; and (lower right) involution of the ductal plate with formation of mature tubular bile ducts adjacent to the hepatic arteries. (Modified from Roskams et al., 1998; Diagram by Aleta R. Crawford© 2003).

keeping up with the continued growth and enlargement of the organ. Following formation of the initial ductal plate layer, mesenchymal cells from the portal tract interpose between the ductal plate and the remaining hepatocellular parenchyma, and proliferate. In this manner the ductal plate becomes separated from the hepatocellular parenchyma around its circumference. The ductal plate duplicates, forming a second layer of biliary-type epithelium. This generates a double-layered plate that subsequently acquires a lumen to form a wreath of crescent-like lumenal structures around the portal vein (Desmet, 1992). Immunohistochemistry for cytokeratins has been the key methodology for identifying the ductal plate and monitoring its development. Undifferentiated hepatoblasts in the embryonic liver express cytokeratins 8, 18 and 19. The hepatoblasts immediately adjacent to the portal tract mesenchyme become more immunoreactive for CK-19 as the ductal plate is formed, while the hepatoblasts away from the ductal plate loose their CK-19 immunoreactivity, retaining only CK-8 and CK-18. CK-19 thus becomes a marker for biliary structures within the embryonic and fetal liver. By 20 weeks of gestation, immunoreactivity for CK-7 appears in epithelial cells of developing ducts near the hilum, and this

5

The Intrahepatic Biliary Tree

Table 2. Immunohistochemistry of the Intrahepatic Biliary Tree. Hepatoblasts

3–11 weeks

Ductal plate

6 weeks to birth

Hepatocytes

12 weeks to birth

Cholangiocytes

12 weeks to birth

CK-8, CK-18, CK-19 ␣-fetoprotein, albumin CK-7, CK-8, CK-18, CK-19 ␣-fetoprotein, albumin, ␣-1-antitrypsin ␥-glutamyltranspeptidase c-kit, CD-34 CK-8, CK-18 ␣-fetoprotein, albumin ␣-1-antitrypsin CK-7, CK-19, CK-20 ␥-glutamyltranspeptidase

Source: From Crawford (2002).

also progresses to the periphery. Immunoreactivity for CK-7 increases through term and reaches the adult level at 1 month after birth. Thus, normal adult hepatocytes express only cytokeratins 8 and 18, whereas intrahepatic bile ducts express cytokeratins 7 and 19. Faa et al. (1998) demonstrated that, in the rat, cytokeratin 20 may represent a late maturation marker for the fetal biliary tract, as it appears in bile duct epithelium one day prior to parturition. Confirming the concept of hilar-to-peripheral maturation of the intrahepatic biliary tree, CK-20 appeared in portal tracts of the hilar region, spreading outward from there. Notably, there is a tremendous increase in CK-20 positivity after birth, supporting the concept that maturation of the intrahepatic bile duct continues well after birth. The ductal plate and developing biliary epithelium also express hematopoietic stem cell markers such as c-kit, CD-34, and CD-33 (Blakolmer et al., 1995). Thus, there is a characteristic pattern of immunostaining for epithelial cells in the developing liver (Table 2; Tan et al., 1995). As noted, ductal plate remodelling into the mature tubular biliary tree starts at the porta hepatis, beginning between 11 and 13 weeks of gestation, and progresses toward the periphery of the liver (Koga, 1971). At first these tubular bile ducts are peripheral and have an ellipsoid or crescent-shaped lumen. With time, the duct cross sections become circular and are more centrally located in the portal tract mesenchyme (Kawarade et al., 2000). Remaining peripheral ductal plate structures regress. Through this orderly process of selection and deletion, the ductal plate is reorganized into the anastomosing system of longitudinally-oriented tubular bile ducts that mark the beginning of the mature architecture. Figure 2 demonstrates the histologic features of the ductal plate and its development into bile ducts. The mature biliary tree is supplied by a vascular plexus derived from the hepatic artery (Miyake et al., 1960). Development of arterial vessels and the peri-biliary

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Fig. 2. Histology of the Ductal Plate. Note: A maturing terminal bile duct is present at lower left. Arching from lower left to upper right along the parenchymal:portal tract interface is a layer of ductal plate cells. Mostly one cell thick, there is focal doublelayering upon approaching the terminal bile duct. The surrounding parenchyma (upper left) contains both hepatocytes and sinusoids filled with hematopoietic elements (extramedullary hematopoiesis). Hematoxylin and eosin stain, 100X.

plexus begins at the hilum and spreads to the periphery, mimicking the pattern of development of intrahepatic bile ducts. Indeed, ingrowth of hepatic arteries may serve as the final organizing event in the formation of the tubular bile ducts. In keeping with the post-partum maturation of the biliary system, the hepatic arterial system continues to proliferate and grow after birth, reaching an adult form only at 15 years of age. In the adult, approximately four arteries supply the largest intrahepatic bile ducts (Washington et al., 1997); out at the level of the terminal portal tracts there is a uniform 1:1 pairing of hepatic arteries and terminal bile ducts (Crawford et al., 1998).

ANATOMY The anatomy of the mature biliary tree can now be considered. The extrahepatic biliary tract consists of the common bile duct, cystic duct and gallbladder, and

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the common hepatic duct. Approximately 60% to 70% of the time, the common hepatic duct bifurcates into the right and left hepatic ducts before entering the liver. The predominant anatomic variation is absence of the right hepatic duct. Instead, posterior and anterior branches of bile ducts supplying the right portion of the liver arise from a hilar confluence with the left hepatic bile duct. This occurs in the form of a three-way branch point with the left hepatic bile duct, or variations of two-way confluences of the anterior or posterior branches with the left hepatic duct. Finally, while the common hepatic duct and its branches lie ventral to the portal vein system, the right posterior bile duct may wrap in an inferior/ventral or a superior/dorsal fashion around the right portal vein. The large intrahepatic bile ducts are defined as follows: right and left hepatic ducts (with origin just outside the liver corpus); segmental ducts (the first major branches of each hepatic duct: left medial and lateral, right anterior and posterior); and area ducts (the first major branches of each segmental duct: superior and inferior). The segmental bile ducts of the caudate lobe of the liver drain directly into the right or left hepatic duct or their major branches. These large ducts – right and left hepatic, segmental, and area – are grossly visible and are characterized by association with intrahepatic mucin-secreting peribiliary glands. Smaller biliary branches within the liver arise from non-dichotomous branching, in that radial trees of bile ducts do not divide symmetrically. As a result, there are considerable variations in the branching of the biliary tree within the liver as well as at its hilum. The finer branches of the biliary system are identifiable by light microscopy only, and are not associated with peribiliary glands. The most terminal branches are commonly called interlobular bile ducts, based on the concept that it is these branches which supply the lobules of the liver (see Fig. 3). Saxena et al. (1999) recommended calling these smallest branches terminal bile ducts, in part to recognize the fact that the microarchitectural units of the hepatic parenchyma go by many names other than “lobule,” and to refocus the terminology on the architecture of the biliary tree rather than on the hepatic parenchyma. However, within the lexicon of histopathology, “interlobular” remains the term in common usage. Ultimately, the terminology of the human intrahepatic biliary tree is based upon the size of the bile ducts, using the basement membrane as the point of reference: bile ductules are 800 ␮m in diameter. It is useful to consider how many terminal bile ducts are needed to supply the liver parenchyma. First, based on microanatomic study, it appears that one terminal bile duct is to be expected for every 2–3 mm3 of the liver. In an exhaustive analysis of the intrahepatic biliary tree of a single adult liver filled in a retrograde fashion by

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Fig. 3. The Normal Terminal Portal Tract, with a Hepatic Artery and Interlobular Bile Duct of Approximately Equal Diameter, and a Larger Diameter Portal Vein. Note: According to Crawford et al. (1998) there are on average two hepatic arteries, two interlobular bile ducts, and one portal vein per portal tract in the peripheral liver. The portal vein is absent about 30% of the time, hepatic arteries or bile ducts are absent only about 7% of the time. Hematoxylin & eosin stain, 100X.

contrast medium, Ludwig et al. (1998) demonstrated 10 orders of branching of the intrahepatic biliary tree in the adult human, 3 of which are external to the hepatic corpus and 7 of which are intrahepatic. Post-mortem cholangiograms in children have shown as many as 17 branch points in evaluable biliary “rays.” However, most identifiable ducts do not exhibit 16–17 branch points, but instead exhibit a Gaussian distribution of branches with a mode of 10. This matches the 10 orders of branching enumerated by Ludwig. However, Ludwig also alludes to smaller biliary radicles extending beyond the 10th order of branching. The fact that both Ludwig et al. (1998) and Landing and Wells (1991) inconsistently observe biliary branches beyond the 10th order may be due to the technical challenge of retrograde filling of the biliary tree – it is a dead-end compartment. However, on the basis of total liver volume and assuming: (1) that the volume of a liver microarchitectural unit supplied by a terminal bile duct is on the order of 2–3 mm3 ; and (2) that the biliary tree branches dichotomously, the biliary tree would require a consistent 17 orders of branches in the newborn liver, and between

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18–20 orders of branches in the adult liver. Beyond 20 branches, the biliary tree becomes prohibitively large; under 17 orders of branches, the liver parenchyma is undersupplied. However, as noted earlier, that the branching of the biliary tree is asymmetric and not dichotomous, so that one of any two branches may give rise to a lesser number of derivative branches. Variation in the final branching order is therefore to be expected. Regardless, the adult liver must be supplied by 400,000 to 500,000 terminal bile ducts, corresponding to the estimated 440,000 microarchitectural units (defined as “lobules” or otherwise) estimated to exist in the adult liver (Crawford, 2002).

Bile Ductules and the Canals of Hering In the current view, bile ductules are those channels branching off the terminal bile ducts that collect bile directly from the hepatocellular parenchyma via the canals of Hering. To the best of current knowledge, these structures are a single unit that drains bile from bile canaliculi within the hepatic parenchyma. However, the connection between intrahepatic biliary tree and parenchyma has long been the subject of study. In the stereoptic depiction of normal liver anatomy by Elias in 1949, the biliary tree was seen to drain the hepatic parenchyma via tubular structures, cholangioles, emerging from deep within the hepatic lobule. This concept has been propagated in liver textbooks with inconsistent fidelity in the ensuing years. Despite frequent comments that canals of Hering are inapparent by light microscopy, careful examination of the periportal parenchyma frequently permits identification of strings of cuboidal epithelial cells – partial sections of canals of Hering (see Fig. 4). Ultrastructural studies by Steiner and Carruthers in 1961 demonstrated that ductular channels were lined partially by hepatocytes and partially by bile ductular epithelial cells. This ultrastructural criterion has become the established definition of a canal of Hering, a term introduced by Hering a century ago. While the initial observation by Steiner and Carruthers pointed out that hemiductular structures extended within the hepatic parenchyma, over the ensuing 40 years these connections between biliary tree and parenchyma were generally viewed as occuring only at the interface between portal tract mesenchyme and parenchyma (although some authors allowed for an intralobular passage). In a light microscopy study of normal adult hyman liver anatomy, Crawford et al. (1998) found that isolated biliary ductular structures were actually readily observed in histological sections. Using a Masson-trichrome stain, about 1 intralobular bile ductule could be identified per portal tract. Simultaneously, Theise et al. (1999) found that staining of normal adult human liver sections with CK-19 revealed about 10 intralobular ductal systems per portal tract. More

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Fig. 4. The Periportal Hepatic Parenchyma, Showing Partial Strings of Cuboidal Epithelial Cells with Evidence of Branching. Note: These are partial sections through the canal of Hering, the most peripheral portion of the biliary tree. Hematoxylin & eosin stain, 400X.

importantly, superposition of up to 60 consecutive tissue sections stained for CK-19 demonstrated that the intralobular ductal systems all arose from terminal bile ducts, extending into the parenchyma for up to one third the distance to the terminal hepatic vein. In the normal liver intralobular branches were present but scattered; severe liver damage induced a massive proliferation and ramification of the intralobular ductal system. In all instances, the intralobular system marked by CK-19 immunostaining connected up to terminal bile ducts by bile ductules which traversed the portal tract mesenchyme. In no instances were intraparenchymal ductular “arcades” observed, as proposed by Landing and Wells (1991). Lastly, Ekataksin et al. (1996) has demonstrated that the bile ductules emerge from the terminal bile ducts accompanied by portal venous tributaries (termed septal veins). They propose that the local perfusion and drainage of hepatic parenchyma by vein/ductule, respectively, constitute a cholehepaton, the smallest architectural unit of the liver.

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Fig. 5. Anatomy of the Terminal Portal Tract, Containing an Interlobular Bile Duct from which Branches a Bile Ductule: Canal of Hering Unit. Note: The bile ductule is lined entirely by cholangiocytes resting on a basement membrane, and may end at the portal tract:parenchymal interface (intraportal bile ductule) or may extend into the parenchyma for a brief distance (intraparenchymal bile ductule). The canal of Hering is lined only partially by cholangiocytes on a basement membrane; the other hemicircumference is lined by hepatocytes (not shown). (Diagram by Aleta R. Crawford© 2003).

Taken collectively, these findings are interpreted to indicate that terminal bile ducts give rise to bile ductular branches, which traverse the portal tract mesenchyme to penetrate the hepatic parenchyma. Once within the parenchyma, the hemiductular structures penetrate into the lobule as the presumed canals of Hering. This interpretation does not exclude hemiductular structures rimming the circumference of the portal tract interface, and these can be observed ultrastructurally as well. The important concept, however, is that the bile ductule and canal of Hering constitute a single unit for drainage of bile from the hepatic parenchyma (see Fig. 5). Moreover, it is this unit which appears to contain the bipotential progenitor cells for reconstitution of the damaged adult liver, and serves as the site for influx and localization of intrahepatic and extrahepatic adult stem cells. From an embryological standpoint, the bile ductules are “tethers” that remain to drain bile from the hepatic parenchymal canals of Hering to the mature biliary tree. Peribiliary Glands The extrahepatic bile ducts and major intrahepatic bile ducts have peribiliary glands, nestled in the mesenchyme immediately adjacent to the duct lumena. In keeping with gland formation throughout the alimentary tract, the extrahepatic

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glands develop as microscopic diverticular outpouchings along the axis of the extrahepatic bile ducts. Nakanuma et al. (1997) report that the intrahepatic peribiliary glands also develop from the ductal plate epithelium. These glands first become recognizable at the hepatic hilum as small evaginations of the large bile ducts, which ramify and increase in number to form acinar structures surrounded by a relatively condensed mesenchyme by 40 weeks of gestation. After birth, the acini of the immature peribiliary glands continue to increase in number and organize, with full maturation complete at approximately 15 years of age (Terada & Nakanuma, 1993). Differentiation of these acinar structures into exocrine pancreas tissue also occurs during the infantile period and persists into adult life. This pancreatic differentiation appears to be a normal heterotopic event, and is not a metaplastic phenomenon.

Biliary Epithelial Cells Moving from the periphery downstream, the lining cells of the biliary tree are as follows. By definition, canals of Hering appear in cross-section as hemichannels consisting of hepatocytes along one hemi-circumference and simple cuboidal epithelial cells along the other hemi-circumference (see Fig. 6). The hepatocytes exhibit their normal ultrastructural relationships to the basolateral sinusoidal space and to each other. The cuboidal epithelial cells are the most terminal biliary epithelial cells, resting for their part on a basally oriented basal membrane, and with a free apical membrane lining the luminal channel. These cells, like those downstream, are termed cholangiocytes. The cholangiocytes of the canals of Hering may be found within the parenchyma up to one third of the distance between portal tracts and the terminal hepatic vein, or within hemicanals at the very interface of the portal tract:parenchyma. Canals of Hering become bile ductules when the biliary epithelial cell lining is circumferential, resting upon a completely encircling basement membrane (Saxena et al., 1999). Bile ductules may be present within the parenchyma, or may be constituted only when the canal of Hering exits the parenchyma to enter the portal tract mesenchyme. Bile ductules are lined circumferentially by cholangiocytes and drain into the interlobular bile ducts present in terminal portal tracts. Interlobular bile ducts are lined by a continuous layer of tightly coupled cuboidal epithelial cells. The septal, area, segmental, and main hepatic bile ducts are lined by a progressively taller cuboidal epithelium, eventuating in hilar bile ducts which contain essentially a columnar epithelium. Cholangiocyte cell area increases from 8 to 80–100 ␮m2 over the length of the intrahepatic biliary tree, with a strong correlation between cholangiocyte area

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Fig. 6. Electron Micrograph of a Canal of Hering, Showing Low Cuboidal Epithelial Cells Abutting Hepatocytes to Form a Luminal Channel. Note: Lipid vesicles of varying sizes are present in the lumen, representing the phospholipid vesicles of bile. An endothelium-lined portal vein occupies the remainder of the image. (5000X, Courtesy of Donna Beer Stolz, University of Pittsburgh).

and external bile duct diameter (Glaser et al., 2003). In addition, the smallest interlobular bile ducts are lined by 4–5 cells only, whereas the largest bile ducts are lined by several hundred around their circumference (see Fig. 7). Throughout the intrahepatic biliary tree, cholangiocytes have a very characteristic ultrastructure (Marucci et al., 2003; see Fig. 8). The nuclei of the bile duct epithelial cells are basally located. The cells have a prominent Golgi complex located between the apical pole and the nucleus, numerous vesicles in the subapical region, scattered lysosomes, a few mitochondria, and abundant short luminal microvilli. Tight junctions and the underlying cytoskeleton are well-developed, providing an intact barrier between the biliary lumen and the basolateral space. The nucleus/cytoplasmic ratio of the smallest cholangiocytes is relatively high; the nucleus/cytoplasmic ratio becomes low within the largest bile

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ducts. Cholangiocytes exhibit heterogeneity in their function, with the smallest cholangiocytes showing minimal to no response to the secretory hormone secretin (owing to an absence of secretin receptors), and larger cholangiocytes showing both hormonal responsiveness and a more robust secretory physiology (Alpini et al., 1997a). The secretory function of cholangiocytes is well-characterized, as they generate a bicarbonate rich fluid constituting up to 40% of the bile volume (see Fig. 9). The smallest cholangiocytes in bile ductules most likely contribute little to fluid secretion; larger cholangiocytes are well-developed cells with considerable secretory capacity. These cholangiocytes increase their fluid secretion in response to a number of gastrointestinal hormones (secretin, gastrin, somatostatin, bombesin, and vasoactive intestinal peptide), peptides (endothelin-1), and neural

Fig. 7. Light Microscopy of a Portal Tract Containing a Terminal Bile Duct (A) and an Area Bile Duct (B). Note: Cholangiocytes in terminal bile ducts are roughly cuboidal, whereas in the larger bile ducts, they are taller and approach a columnar morphology. Hematoxylin & eosin, 400X (A) and 200X (B).

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Fig. 7. (Continued )

stimulation. As the dominant hormone, secretin interaction with its basolateral plasma membrane receptor activates membrane adenylyl cyclase, increasing cAMP levels, and hence, cAMP-dependent protein kinase A (PKA) activity. PKA phosphorylates the apical cystic fibrosis transmembrane regulator (CFTR), generating an efflux of chloride anion into bile. Action of the Cl− /HCO− 3 exchanger results in net secretion of bicarbonate into bile, with entrained sodium cation and water. While other secretory hormones and stimuli may activate other signaling pathways (e.g. via elevations in intracellular calcium levels), the net stimulatory effect is via the same CFTR and Cl− /HCO− 3 exchanger mechanism. Cholangiocytes also express aquaporin water channels in a regulated fashion, thereby regulating the amount of water that can follow the secretion of bicarbonate and sodium. Cholangiocytes also express the apical sodium-dependent bile acid transporter (ABAT), the same transporter responsible for ileal uptake of bile acids from the intestinal lumen (Alpini et al., 1997b). Bile salts entry into cholangiocytes from the

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Fig. 8. Electron Micrograph of a Cholangiocyte from a Terminal Bile Duct, Showing Cuboidal Cells Lying on a Delicate Basement Membrane, with Basally Located Nuclei, Apical Plasma Membranes Lightly Studded with Microvilli, and Tight Junctions Abutting Adjacent Cholangiocytes. (6000X, Courtesy of Donna Beer Stolz, University of Pittsburgh).

bile lumen enhances secretin-stimulated ductal secretion. This effect is greater for more hydrophobic bile salts such as taurocholate and taurolithocholate, and may help protect the biliary tree from the detergent activity of more hydrophobic bile salts. Secretion of bile salts across the cholangiocyte basolateral plasma membrane into the portal tract space also sets up a “cholehepatic shunt,” whereby bile salts are recirculated within the liver from the biliary tree back to hepatocytes. This occurs primarily for the more hydrophilic bile salts such as tauroursodeoxycholic acid, and is a good explanation for the marked choleresis that can occur during pharmacologic therapy with this agent. Through the peribiliary capillary plexus derived from the hepatic artery, the intrahepatic biliary tree is supplied primarily by the hepatic artery, with apparently little contribution from the portal venous circulation (Ekataksin & Wake, 1997). Hence, compromise to hepatic arterial circulation may lead to substantial ischemic

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Fig. 9. Biliary Transport Physiology of the Cholangiocyte. Note: The left diagram shows the relationship of upstream bile salt-secreting hepatocytes to both small cholangiocytes of bile ductules and the larger cholangiocytes of bile ducts. Small cholangiocytes lack secretin receptor, and respond to selected secretory stimuli with elevations in intracellular Ca++ . Through mechanisms which are not yet fully understood, this stimulates chloride secretion into bile by the cystic fibrosis transmembrane regulator (CFTR). The apical Cl/HCO− 3 exchanger takes up chloride and secretes bicarbonate. Sodium cation and water secretion follow; water secretion is facilitated by aquaporin channels in a regulated fashion. In addition to the receptors depicted for small cholangiocytes, larger cholangiocytes have a basolateral secretin receptor which, upon binding secretin, activates adenylyl cyclase, leading to elevated cAMP levels and activation of protein kinase A (PKA). This in turn stimulates CFTR. Somatostatin down-regulates adenylyl cyclase and the cAMP response to secretin. Larger cholangiocytes also take up bile acids from the biliary lumen via the apical Na+ -dependent bile acid transporter (ABAT). Intracellular bile acids enhance secretin stimulation of fluid secretion; they also are transported across the basolateral membrane by a basolateral transporter (I-ABST) to enter into a “cholehepatic” shunt for bile acid recirculation. This latter shunt enhances hepatocyte bile acid-dependent secretion as well. (from Crawford, 2003; diagram by Aleta R. Crawford© 2003).

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compromise to the cholangiocytes of the intrahepatic biliary tree. However, it should also be noted that, while mitotically dormant under normal conditions, cholangiocytes exhibit a marked capacity for replication. Hence, following injury to the biliary tree the epithelial lining may substantively heal if vascular blood flow is maintained. The smallest bile ductules also appear to constitute a bipotential regenerative compartment, whereby proliferation of these smallest cholangiocytes may generate not only new cholangiocytes, but also a population that may mature into hepatocytes. These cells serve as a reservoir for hepatic reconstitution not only following injury to the biliary tree but also following massive destruction of the hepatocellular parenchyma. The bile ductules also appear to be the compartment of entry for extrahepatically-derived stem cells, which are then capable of proliferating and maturing into cholangiocytes and hepatocytes (Sell, 2001). Hence, the cellular biology of cholangiocytes large and small has taken on major significance as we attempt to gain new insights into the pathobiology of hepatic disease.

SUMMARY Maturation of the intrahepatic biliary tree constitutes an elegant mechanism for maintaining patency of the biliary passages, whilst undergoing major structural reorganization throughout the second and third trimesters and into the postpartum period. As bile begins to flow around the 12th week of gestation, the physiologic events of bile formation can proceed without structural impediment. Throughout post-natal life, secretion of bile by hepatocytes is accompanied by secretion of a bicarbonate-rich fluid by the cholangiocytes lining the biliary tree, with cholangiocyte secretiono constituting up to 40% of bile volume. The final architecture of the intrahepatic biliary tree consists of: hemicircular canals of Hering linking the bile canaliculi between hepatocytes to the smallest complete channels of the biliary tree, bile ductules. The bile ductules serve as tethers between the hepatic parenchyma and the terminal twigs of the biliary tree, interlobular bile ducts. Interlobular bile ducts drain into septal bile ducts, and thence, into area bile ducts, segmental bile ducts, and finally, the main hepatic ducts.

REFERENCES Alpini, G., Glaser, S., Robertson, W. et al. (1997a). Bile acids stimulate proliferative and secretory events in large but not small cholangiocytes. American Journal of Physiology, 273, G518–G529.

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Alpini, G., Glaser, S. S., Redgers, R. et al. (1997b). Functional expression of the apical Na+ dependent bile acid transporter in large but not small rat cholangiocytes. Gastroenterology, 113, 1734–1740. Blakolmer, K., Jaskiewicz, K., Dunsford, H. A., & Robson, S. C. (1995). Hematopoietic stem cell markers are expressed by ductal plate and bile duct cells in developing human liver. Hepatology, 21, 1510–1516. Crawford, J. M. (2002). Development of the intrahepatic biliary tree. Seminars in Liver Disease, 22, 13–22. Crawford, J. M. (2003). Normal and abnormal development of the biliary tree. In: G. Alpini, D. Alvaro, G. LeSage & N. LaRusso (Eds), The Pathophysiology of Biliary Epithelia (pp. 13–39). Georgetown, TX: Landes Bioscience. Crawford, A. R., Lin, X. Z., & Crawford, J. M. (1998). The normal adult human liver biopsy: A quantitative reference standard. Hepatology, 28, 323–331. Desmet, V. J. (1992). Congenital diseases of intrahepatic bile ducts: Variations on the theme “ductal plate malformation”. Hepatology, 16, 1069–1083. Desmet, V. J., Van Eyken, P., & Sciot, R. (1989). Cytokeratins for probing cell lineage relationships in developing liver. Hepatology, 15, 125–135. Ekataksin, W., & Wake, K. (1997). New concepts in biliary and vascular anatomy of the liver. Progress in Liver Disease, 15, 1–29. Ekataksin, W., Zou, Z. Z., Wake, K., Chunhabundit, P., Somana, R., Nishida, J., & McCuskey, R. S. (1996). The hepatic microcirculatory subunits: An over-three-century-long-search for the missing link between an exocrine unit and an endocrine unit in mammalian liver lobules. In: P. M. Motta (Ed.), Recent Advances in Microscopy of Cells, Tissues and Organs (pp. 375–380). La Sapienza, Rome: University of Rome. Elias, H. (1949). A re-examination of the structure of the mammalian liver: II, The hepatic plate and its relation to the vascular and biliary systems. American Journal of Anatomy, 85, 379–456. Faa, G., Van Eyken, P., Roskams, T., Miyazaki, H., Serreli, S., Ambu, R., & Desmet, V. J. (1998). Expression of cytokeratin 20 in developing rat liver and in experimental models of ductular and oval cell proliferation. Journal of Hepatology, 29, 628–633. Glaser, S. S., Francis, H., Marzioni, M., Phinizy, J. L., LeSage, G., & Alpini, G. (2003). Functional heterogeneity of the intrahepatic biliary epithelium. In: G. Alpini, D. Alvaro, G. LeSage & N. LaRusso (Eds), The Pathophysiology of Biliary Epithelia (pp. 262–271). Georgetown, TX: Landes Bioscience. Godlewski, G., Gaubert-Cristol, R., Rouy, S., & Prudhomme, M. (1997). Liver development in the rat and in man during the embryonic period (Carnegie stages 11–23). Microscopy Research Technique, 39, 314–327. Kawarade, Y., Das, B. C., & Taoka, H. (2000). Anatomy of the hepatic hilar area: The plate system. Journal of Hepatobiliary and Pancreatic Surgery, 7, 580–586. Koga, A. (1971). Morphogenesisi of intrahepatic bile ducts of the human fetus. Light and electron microscopic study. Z Anat. Entwickl. Gesch., 135, 156–184. Landing, B. H., & Wells, T. R. (1991). Considerations of some architectural properties of the biliary tree and liver in childhood. In: C. R. Abramowsky, J. Bernstein & H. S. Rosenberg (Eds), Transplantation Pathology – Hepatic Morphogenesis. Perspectives in Pediatric Pathology (Vol. 14, pp. 122–142). Karger, S., Basel. Ludwig, J., Ritman, E. L., LaRusso, N. F., Sheedy, P. F., & Zumpe, G. (1998). Anatomy of the human biliary system studied by quantitative computer-aided three-dimensional imaging techniques. Hepatology, 27, 893–899.

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Marucci, L., Jezequel, A. M., & Benedetti, A. (2003). Ultra-structural analysis of the intrahepatic bile duct system. In: G. Alpini, D. Alvaro, G. LeSage & N. LaRusso (Eds), The Pathophysiology of Biliary Epithelia (pp. 63–71). Georgetown, TX: Landes Bioscience. Miyake, M., Okudaira, M., Sato, T., Kitagawa, M., & Hisauchi, T. (1960). The blood vessels of the liver. Nippon Byori Gakkai Kaishi. Transactions of the Society of Pathology of Japan, 49, 589–632. Nakanuma, Y., Hoso, M., Sanzen, T., & Sasaki, M. (1997). Microstructure and development of the normal and pathologic biliary tract in humans, including blood supply. Microscopy Research Technique, 38, 552–570. Roskams, T., Van Eyken, P., & Desmet, V. (1998). Human liver growth and development. In: A. Strain & A. M. Diehl (Eds), Liver Growth and Repair (pp. 541–557). London: Chapman & Hall. Saxena, R., Theise, N. D., & Crawford, J. M. (1999). Microanatomy of the human liver: Exploring the hidden interfaces. Hepatology, 30, 1339–1346. Sell, S. (2001). Heterogeneity and plasticity of hepatocyte lineage cells. Hepatology, 33, 738–750. Steiner, J. W., & Carruthers, J. S. (1961). Studies on the fine structure of the terminal branches of the biliary tree. 1. The morphology of the normal bile canaliculi, bile pre-ductules (ducts of Hering)and bile ductules. American Journal of Pathology, 38, 639–661. Tan, C. E. L., Chan, V. S. W., Yong, R. Y. Y., Vijayan, W. L., Tan, S. M. C., Fook Chong, S. M. C., Ho, J. M. S., & Cheng, H. H. (1995). Distortion in TGFb1 peptide immunolocalization in biliary atresia: Comparison with the normal pattern in the developing human intrahepatic bile duct system. Pathology International, 45, 815–824. Tan, C. E. L., & Moscoso, G. J. (1994). The developing human biliary system at the porta hepatis level between 11 and 25 weeks of gestation: A way to understanding biliary atresia. Part 2. Pathology International, 44, 600–610. Terada, T., & Nakanuma, Y. (1993). Development of human intrahepatic peribiliary glands. Histological, keratin immunohistochemical, and mucin histochemical analyses. Laboratory Investigation, 68, 261–269. Theise, N. D., Saxena, R., Portmann, B. C., Thung, S., Yee, H., Chiriboga, L., Kumar, A., & Crawford, J. M. (1999). Canals of Hering and hepatic stem cells in humans. Hepatology, 30, 1425–1433. Vijayan, V., & Tan, C. E. L. (1997). Developing biliary system in three dimensions. Anatomy Record, 249, 389–398. Washington, K., Clavien, P.-A., & Killenberg, P. (1997). Peribiliary vascular plexus in primary sclerosing cholangitis and primary biliary sclerosis. Human Pathology, 28, 791–795.

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FUNCTIONAL HETEROGENEITY OF INTRAHEPATIC CHOLANGIOCYTES

Gene D. LeSage, Shannon S. Glaser, Heather Francis, Jo Lynne Phinizy and Gianfranco Alpini INTRODUCTION The aim of this chapter is to summarize recent findings that support the concept that the biliary epithelium is morphologically and functionally heterogeneous. The knowledge of cholangiocyte functions is rapidly accumulating largely because of technical advances and more investigative work that has led to the recognition that cholangiocytes are almost always either primarily or secondarily involved in human liver diseases (Alpini et al., 2002; Roberts et al., 1997). The development and introduction of experimental models, the identification and characterization of transport systems, and their second messenger systems has enhanced our understanding of cholangiocyte pathobiology. In this overview we will describe the morphology of intrahepatic bile ducts and their blood supply in the liver. Next, we will summarize overall bile duct function, and then link the structural differences between large and small ducts with their functional differences. We also will review the physiological advantages of having regional distribution in secretion and absorption in the biliary tree. We also will describe the models for bile duct injury, and the mechanisms underlying restricted size-dependent bile duct injury. Correlations to human liver diseases in which

The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 21–48 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15002-2

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injury is restricted to limited ranges of bile duct sizes will be examined. This will be followed by a review of the repair of bile ducts by cholangiocyte proliferation, and mechanisms by which bile duct structure is restored. And finally, we will review the capacity of cholangiocytes lining small bile ducts to differentiate, and consider the role pluripotent liver cells play within small bile ducts or closely adjacent to them in response to liver injury.

MORPHOLOGY Only a resum´e needs to be given here since this subject is dealt with fully in Chapter 1. Biliary epithelial cells (cholangiocytes) line the intra- and extrahepatic bile ducts of the liver (Alpini et al., 2002; Kanno et al., 2000; Ludwig, 1987; Marzioni et al., 2002; Sasaki et al., 1967; Schaffner & Popper, 1961). Bile duct structure comprises an anastomosing network of small bile ducts, which coalesce into progressively larger ducts (Alpini et al., 2002; Kanno et al., 2000; Ludwig, 1987; Marzioni et al., 2002). As these ducts become larger, the lining cholangiocytes change from cuboidal to columnar cell morphology (Sasaki et al., 1967; Schaffner & Popper, 1961). Progressively, larger ducts merge to form the hepatic duct, and then the common bile duct, which drains into the intestinal tract (Kanno et al., 2000; Ludwig, 1987; Marzioni et al., 2002). When the three-dimensional formation of the intrahepatic bile duct is complete (Masyuk et al., 2001), it closely resembles a tree, with the common and hepatic ducts corresponding to the trunk, the intrahepatic bile ducts corresponding to the large branches and the small ducts corresponding to the smallest tree limbs (Masyuk et al., 2001). The structure of the bile duct system is well suited for its primary function, which is to produce and transfer bile from the liver to the intestinal tract (Alpini et al., 1988, 2002; Cho et al., 1995; Cho & Boyer, 1999).

BILE FORMATION AND COMPOSITION Bile is composed of bile acids, cholesterol, phospholipids, bile pigments and inorganic electrolytes (Alpini et al., 2002; Tietz et al., 1995). Bile secretion is initiated by the movement of these substances from hepatocytes into the bile canaliculi (Nathanson & Boyer, 1991) and during the transfer from hepatocytes to the intestine. Bile is subsequently modified in the bile ducts by cholangiocytes (Alpini et al., 1988, 2002; Cho et al., 1995; Cho & Boyer, 1999; Kanno et al., 2000; Marzioni et al., 2002; Tietz et al., 1995). Three primary steps lead to bile generation. The first is active transport of bile acids from blood into bile canaliculi

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(Nathanson & Boyer, 1991). And the second is a canalicular, bile acid-independent secretion representing 30–60% of basal bile flow (Nathanson & Boyer, 1991). The third step in bile formation is absorption and secretion of fluid and inorganic electrolytes by bile ducts (Alpini et al., 1988, 2002; Tietz et al., 1995). Ductal bile flow is primarily regulated by the hormone secretin functioning to produce a bicarbonate-rich bile secretion (Alpini et al., 1989, 2002; Alvaro et al., 1993, 1997) and represents 30–40% of basal bile flow in humans, and 10% in rats (Alpini et al., 1989). Secretin stimulates ductal bile flow by increasing intracellular cyclic adenosine monophosphate (cAMP) levels (Kato et al., 1992; LeSage et al., 1996, 1999), which promotes biliary HCO− 3 secretion (Alpini et al., 1988, 1989, 1996; Alvaro et al., 1993, 1997; LeSage et al., 1996; Tietz et al., 1995), by stimulating apical cystic fibrosis transmembrane regulator (CFTR) Cl− channels (Alpini, Glaser et al., 1997; Fitz et al., 1993), in addition to the Cl− /HCO− 3 exchanger (Alpini et al., 1996; Alvaro et al., 1993, 1997; LeSage et al., 1996; Strazzabosco et al., 1991). For a comprehensive treatment of this subject, see Chapter 4 by Anwer.

ABNORMALITIES OF BILE DUCT FUNCTION Abnormalities of bile duct structure-function, or gene expression are the primary cause of liver diseases that target biliary epithelium such as in cystic fibrosis (CF), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and the idiopathic ductopenia syndrome (Alpini et al., 2002). In the other forms of liver disease (e.g. cirrhosis, and chronic hepatitis C), bile ducts are secondarily involved as the result of cell proliferation caused by chronic liver inflammation (Alpini et al., 2002). Present understanding of the pathophysiology of cholangiocytes in these disorders is rather limited or not infrequently non-existent.

HETEROGENEITY IN OTHER EPITHELIA Heterogeneity of epithelial cell function, reactions to injury or capacity to differentiate may depend upon the cells’ position within the structure of the organ (Cohn et al., 1992; Katz & Jungermann, 1993; Nielsen et al., 1993). Examples include: (i) differences in water permeability and expression of aquaporins in the epithelial cells lining the distal and proximal tubules in the kidney (Nielsen et al., 1993); (ii) differing absorptive and secretory capacity in cells along the villous crypt axis in intestinal epithelium (Cohn et al., 1992); and (iii) differing transport and metabolic capacities of hepatocytes in the periportal and perivenular zones

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in the liver (Katz & Jungermann, 1993). Different functions in various regions inside the organ are considered as physiologic advantages as well illustrated by the dependency of the urine concentrating ability of the kidney on the heterogeneity of kidney epithelial cells, and aquaporin expression in different tubular segments (Nielsen et al., 1993). Epithelial heterogeneity is also reflected in the response to disease as evidenced in liver injury. Damage of hepatocytes is limited to certain zones of the liver lobule (Katz & Jungermann, 1993).

Heterogeneity of Cholangiocytes During the last ten years, our interest has centered on the heterogeneity of cholangiocyte secretory functions, bile duct reactions to injury, and cholangiocyte proliferation and differentiation (Alpini et al., 1996, 1997, 1998, 2001, 2002; LeSage et al., 1999, 2001). We have been able to show that in large intrahepatic bile ducts (exceeding 15 ␮m in diameter) compared to small intrahepatic bile ducts (less than 15 ␮m) there are: (i) clearly distinguishable secretory functions (Alpini et al., 1996, 1997); (ii) different capacities for differentiation and degrees (Alpini et al., 2001); (iii) varying sensitivity to injury (LeSage et al., 1999, 2001); and (iv) different proliferative capacities (Alpini et al., 1998; LeSage et al., 1999, 2001). The overall view of the model for cholangiocyte heterogeneity that we have developed based on experimental findings is shown in Fig. 1. As it turns out, the fundamental difference between large and small intrahepatic bile duct function is attributable to differences in gene expression (Alpini et al., 1996, 1998; Alpini, Glaser, Robertson, Phinizy et al., 1997; Alpini, Glaser, Robertson, Rodgers et al., 1997), with large ducts expressing the secretin receptor (Alpini et al., 1996, 1998; Alpini, Elias et al., 1997; Alpini, Glaser, Robertson, Rodgers et al., 1997; LeSage, Glaser et al., 1999; LeSage, Alvaro et al., 1999; LeSage et al., 2001), apical CFTR Cl− channels (Alpini et al., 1997) and Cl− /HCO− 3 exchanger (Alpini et al., 1996). This difference results in only large intrahepatic bile ducts secreting fluid in response to secretin (Alpini, Glaser, Robertson, Phinizy et al., 1996; Alpini, Glaser, Robertson, Rodgers et al., 1997; Alpini et al., 1997). The function of small ducts, in terms of secretion, remains to be determined. Increased sensitivity of large bile ducts to injury is due to the differential expression of drug metabolizing enzymes, and pro-apoptosis proteins in large and small ducts (LeSage, Alvaro et al., 1999; LeSage, Benedetti et al., 1999; LeSage et al., 2001). This difference results in primarily large bile duct damage in response to toxic injury from the administration of carbon tetrachloride (CCl4 ) or ␣-naphthylisothiocyanate (ANIT) (LeSage, Benedetti et al., 1999; LeSage, Glaser et al., 1999; LeSage et al., 2001). Differences in cholangiocyte

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Fig. 1. Overall View of the Model for Cholangiocyte Heterogeneity. Note: Large intrahepatic bile ducts (>15 ␮m in diameter) secrete a bicarbonate rich fluid, are lined by cholangiocytes, which are the only cells in the liver that express secretin receptors and are sensitive to injury from either toxins or drugs. Small intrahepatic bile ducts (800 ␮m), segmental ducts (400–800 ␮m), area ducts (300–400 ␮m), septal bile ducts (100–200 ␮m), interlobular ducts (15–100 ␮m), and bile ductules (15 ␮m in diameter) and cholangiocytes (>13 ␮m in diameter) in rats

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(Alpini et al., 1996, 1997). Previous studies using rats have shown that an isolated cholangiocyte population rich in large cholangiocytes (>13 ␮m in diameter) derives principally from large intrahepatic bile ducts (Alpini et al., 1996 (see Fig. 2, upper panel). In sharp contrast, an isolated cholangiocyte population rich in small cholangiocytes ( intracellular) and K+ (intracellular > extracellular). The electrochemical gradient of Na+ generated by Na+ -K+ -ATPase provides the driving force for secondary active (indirect ATP requirement) Na+ -coupled transport processes. The Na+ -H+ exchanger is

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responsible for a reversible and electroneutral exchange of extracellular Na+ for intracellular H+ . The Na+ -HCO3 − cotransporter facilitates hepatic uptake of HCO3 − . Both Na+ -H+ exchange and Na+ -HCO3 − cotransport are involved in the regulation of intracellular pH and cell volume (Anwer & Engelking, 1993; Strazzabosco & Boyer, 1996). The SO4 2− -OH− exchanger may be involved in hepatic uptake of SO4 2− , as well as organic anions, like oxalate and succinate. Two inorganic anion transport mechanisms, namely electroneutral Cl− -HCO3 − exchange and SO4 2+ -HCO3 − exchange, are identified at the canalicular membrane (Anwer, 1993; Trauner et al., 1999). SO4 2+ -HCO3 − exchange may be involved in canalicular excretion of sulfate and sulfate conjugated metabolites. A coupled operation of sinusoidal Na+ -H+ exchange and Na+ -HCO3 − cotransport and the canalicular Cl− -HCO3 − exchange may be responsible for biliary HCO3 − excretion (Anwer, 1993). Biliary excretion of inorganic ions can also take place as counterions. Thus, biliary excretion of organic anionic solutes, like bile acids, is associated with the excretion of primarily Na+ to maintain electroneutrality. Biliary excretion of inorganic ions is also facilitated by solvent drag and diffusion secondary to the movement of water induced by choleretic agents (see later).

Organic Anions The ability of the liver to efficiently transport organic anions was recognized as early as 1870. It is now well established that a number of organic anions are transported by the liver and concentrated in bile. Different transport processes are involved in efficient transcellular transport of endogenous (bile salts, bilirubin, glutathione) and exogenous (bromosulfopththalein, drugs) organic anions. At least three different transport mechanisms (Anwer, 1993; Trauner et al., 1999) are responsible for hepatic uptake of organic anions across the sinusoidal membrane: (a) Na+ -coupled cotransport; (b) Na+ -independent, carrier-mediated transport; and (c) passive non-ionic diffusion. The first two mechanisms are responsible for concentrative uptake. In general, conjugated bile acids (>80%), and to a lesser extent unconjugated cholate (80%; normal 3.5

Prothrombin time: International normalized ratio >6.5 Serum creatinine: >3.4 mg/dL

Disease etiology and clinical presentation can help to stratify patients into these two groups. For example, patients with FHF caused by hepatitis A have a relatively better prognosis than those with FHF of unknown etiology (O’Grady et al., 1989). Patients who develop stage 3 or 4 encephalopathy tend to do worse than those who reach only stage 1 or 2 (Trey & Davidson, 1970). However, these indicators do not allow accurate prediction of which patients will or will not require transplantation in order to survive. Clinical decision has been aided by the identification of prognostic markers. The most commonly used predictive model was developed at King’s College Hospital in London (Makin et al., 1995). The following variables had prognostic significance: disease etiology, age of patient, duration of jaundice, bilirubin, prothrombin time, arterial pH, and creatinine level (see Table 3). With the exception of patients with acetaminophen toxicity, the presence of any single adverse indicator was associated with a mortality rate of 80%; the presence of three adverse indicators was associated with a mortality rate of over 95%. For patients with acetaminopheninduced FHF, the presence of any one adverse prognostic indicator was associated with a mortality rate of at least 55%; severe acidosis was associated with a mortality rate of 95% (O’Grady et al., 1989, 1993). These mortality rates vastly exceed those associated with orthotopic liver transplantation. Therefore, liver transplantation should be considered in patients with any indicator of poor prognosis. In contrast, liver histology does not predict outcome (Hanam et al., 1995). Moreover, coagulopathy markedly increases the risk of biopsy, which must then be performed via the transjugular route. Other factors including plasma factor V levels and serum Gc protein concentration, do not appear to add significantly to assessment of outcome (Lee et al., 1995; Ostapowicz et al., 2000).

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MANAGEMENT Overview The mortality rate associated with FHF is very high, and management consists of intensive supportive care. These critically ill patients are best managed in an intensive care unit, ideally in a specialized liver unit (Williams & Gimson, 1991). Although intensive medical care enables some patients with FHF to survive long enough to allow for liver regeneration and recovery, the majority die without transplantation. A wide variety of therapies have been proposed and used for the specific treatment of FHF, including corticosteroids, prostaglandins, and exchange transfusions, but none of these have proved efficacious. Only the development of liver transplantation has allowed the salvage of patients with irreversible liver failure. Unfortunately, many patients do not undergo transplantation because of contraindications to transplantation or because of the unavailability of donor livers.

Initial Evaluation and Management The identification of the etiology of FHF is an important initial step of management. A small number of causes of FHF can be specifically treated. For example, acetaminophen toxicity can be treated with n-acetylcysteine (Harrison et al., 1990); herpes-induced fulminant hepatitis has been reported to respond to intravenous acyclovir (Klein et al., 1991) and emergency delivery for FHF caused by fatty liver of pregnancy. Assessment of the suitability and necessity for liver transplantation is critical in the early evaluation of patients with FHF. Urgent transfer to a liver transplant center is advisable for all potential liver transplantation candidates. Rapid clinical deterioration is not uncommon in patients with FHF, and transport may be dangerous at a later time. Initial laboratory work should include tests for: (1) determination of etiology (e.g. hepatitis virus serologic profiles, toxicology screening for acetaminophen and other drugs); and (2) assessment of the severity of liver failure (e.g. liver and renal function tests, blood glucose and arterial blood gas measurements).

Coagulopathy The management of coagulopathy in patients with FHF requires careful consideration. Prophylaxis against upper gastrointestinal hemorrhage is beneficial

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(Martin et al., 1993). Parental vitamin K to treat coagulopathy related to vitamin K deficiency is also reasonable. Unless active hemorrhage is present or an invasive procedure will be performed, the potential disadvantages and risks of plasma infusion outweigh its potential benefits. Replacing clotting factors in non-bleeding patients should be tempered by two issues: Most importantly, infusion of plasma can present a significant volume load which may precipitate respiratory failure from pulmonary edema, especially in renal insufficiency, and cerebral edema that may herald death. Second, infusion of agents such as plasma tends to normalize the prothrombin time, which is an important prognostic indicator, thereby reduce its accuracy. Moreover, empirical administration of fresh-frozen plasma has not been shown to improve clinical outcome.

Hypoglycemia Hypoglycemia commonly occurs in patients with FHF, which may occur abruptly. It is critical to monitor blood glucose levels frequently. Parenteral glucose administration often supports blood glucose levels adequately (e.g. a bolus of intravenous 50% dextrose followed by continuous dextrose solution).

Encephalopathy and Cerebral Edema The encephalopathy associated with FHF tends to be progressive unless liver failure is reversed. Sedative-hypnotic drugs should be avoided. Unlike chronic liver disease, lactulose is of no proven benefit. Reversible conditions associated with FHF that could contribute to altered mental status (e.g. hypoglycemia, hypoxemia) must be treated immediately. More difficult to diagnose and treat is cerebral edema and intracranial hypertension. Intracranial hypertension can be suspected non-invasively or detected directly. Non-invasive modalities such as physical examination and radiological imaging have important limitations. Impaired pupillary responses, posturing, or seizures, which may suggest the presence of intracranial hypertension, are not reliable, particularly when sedatives or neuromuscular blockade are used in mechanically ventilated patients. Computed tomographic (CT) scanning of the head is valuable for identifying mass lesions, intracranial hemorrhage, and evidence of brainstem herniation. Despite this, the correlation between radiographic CT evidence of cerebral edema and measured intracranial pressure is imperfect, ranging between 60 and 75% (Munoz et al., 1991).

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Intracranial pressure (ICP) monitoring to detect intracranial hypertension is rarely utilized because of risk of bleeding and infection (Blei et al., 1993). The goal of therapy is to maintain cerebral perfusion and to reduce intracranial pressure. Patients should be placed in a quiet environment with minimal stimulation to minimize elevation of intracranial pressure. Attempts should be made to keep patient from becoming agitated. Overhydration is a common problem that can elevate ICP dramatically. Therefore, fluid management is critical in these patients. In general, these patients are rarely filled to euvolamia to avoid precipitation of cerebral edema. Close monitoring of fluid status is absolutely critical and it often requires central venous catheter and/or pulmonary artery catheter placement. Other measures include elevation of the head of the bed to 45 degrees and avoidance of the head-down position (O’Grady et al., 1993). Hyperventilation to keep pCO2 less than 25 mmHg is another simple measure for reducing ICP (O’Grady et al., 1993). If this standard approach fails, osmotherapy is required. Osmotherapy with mannitol (0.5–1 g/kg) can be given as bolus intravenous injection and then on an as-needed basis to maintain plasma osmolality between 310 and 325 mosmol/kg. Osmotherapy is effective in about 60% of cases but requires preservation of renal function (or hemofiltration) (Caraceni & Vanthiel, 1995). It must be administered with care because of the added intravascular volume load that precedes diuresis. For patients with oliguric renal failure, fluid can be removed via continuous veno-venous hemofiltration. Uncontrolled data suggests that intravenous barbiturate thiopental has similar efficacy to mannitol for controlling intracranial pressure (Forbes et al., 1989). Thiopental has two relative advantages: Its onset of action is rapid, and it can be used in the presence of renal impairment. Its potential drawbacks are hypotension and, more important, masking of neurological indicators of recovery or deterioration. In general, it is reasonable to use mannitol as first-line therapy and to reserve barbiturates for patients with renal insufficiency or refractory intracranial hypertension. Corticosteroids are of no benefit (Canalese et al., 1982; Harrid et al., 1980) and should not be administered.

Infection Infection can be difficult to diagnose because signs such as hypothermia, hypotension, leucocytosis, and acidosis may reflect underlying liver failure. Therefore, surveillance cultures may be helpful. The use of prophylactic antibiotics is controversial. Prophylactic antibiotics may offset the development of infections

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that limit the applicability of liver transplantation, but may increase the risk of superinfection with resistant bacteria or fungi. A small randomized trial had shown a significant reduction in documented infections (from 61 to 32%), in comparison with those treated conservatively, and a modest (but statistically insignificant) improvement in survival (from 45 to 67%) (Rolando et al., 1993). In addition, enteral decontamination does not appear to alter clinical outcome in patients with FHF receiving prophylactic antibiotics (Rolando et al., 1996). A high level of suspicion of infection should be maintained and a low threshold for antibiotic administration should be adopted. If infection is suspected, the choice of antibiotics should be based on the spectrum of likely bacterial pathogens and local hospital antimicrobial sensitivities. A reasonable empirical regimen might include vancomycin and a third-generation cephalosporin. In view of the high risk of fungal infection (Rolando et al., 1991) it is not uncommon that a prophylactic antifungal agent may also be administered.

Multiple Organ Failure The ultimate goals of management of multiple organ failure in patients with liver failure are similar to those in patients with other causes of multiple organ failure, i.e. to optimize arterial pressure and tissue oxygenation. Ideally, the mean arterial pressure (MAP) should be maintained above 60 mm Hg. If MAP falls below this value, cerebral perfusion can drop precipitously (Munox et al., 1991). Hemodynamic monitoring with a central venous or pulmonary arterial catheter will be useful for determination of intravascular volume status. Blood or colloids should be given to correct hypotension secondary to intravascular volume depletion. Hypotension caused by reduced vascular resistance may be managed with alpha-adrenergic agonists. Although inotropic agents can be used to maintain MAP within a physiologic range, they have the potential drawbacks of causing further impairment of tissue oxygenation (Wendu et al., 1992). In small studies acetylcysteine and prostacyclin have been shown to improve tissue oxygenation without adverse effects on hemodynamics. The impact of these agents on patient outcome has not yet been investigated (Harrison et al., 1991). Endotracheal intubation and mechanical ventilation are frequently necessary for patients with FHF. Hypoxemia can result from respiratory depression related to coma or impaired gas exchange from ARDS or superimposed pneumonia. Renal failure can result from intravascular volume depletion (i.e. readily reversible) or other causes, such as acute tubular necrosis or hepatorenal syndrome. It is important to avoid nephrotoxic drugs, especially aminoglycosides and nonsteroidal anti-inflammatory agents, and care must be taken when contrast dye is

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used. Measurement of central venous (or pulmonary capillary wedge) pressure provides a guide to fluid therapy. Patients with FHF tolerate volume overload poorly and therefore early measurement of central venous or pulmonary arterial pressure in oligouric patients is preferable. If oliguria persists in the presence of adequate central filling pressure, continuous arteriovenous or venovenous hemofiltration should be initiated which is superior to intermittent hemodialysis because of hemdynamic instability in patients with FHF (Davenport et al., 1993). However, fluid replacement must be managed carefully. As previously mentioned, these patients tolerated fluid poorly and slight overhydration, sometimes even with euvolemia, cerebral edema can easily result.

Liver Transplantation Liver transplantation is the only effective management of patients with irreversible FHF. Before the liver transplantation era, fewer than 50% of patients survived. In contrast, survival rates since liver transplantation have been substantially higher: In the USA between 1987 and 1991, survival post orthotopic liver transplant for FHF was 60–70% (Detre et al., 1997). The decision to perform transplantation must balance the likelihood of spontaneous recovery with the risks of surgery and long-term immunosuppression. Furthermore, contraindications to transplantation – particularly irreversible brain damage, active extrahepatic infection, and multiple organ failure must be considered. The decision to place a patient on the waiting list for transplantation should be made promptly, because waiting times for donor organs under emergency conditions average two days or more in United States. A delay increases the likelihood of developing complications such as infection, multiple organ failure, and intracranial hypertension, which can preclude liver transplantation (Panwels et al., 1993). Auxiliary (temporary) liver transplantation may be a new therapeutic option for patients with fulminant hepatic failure (Hoofnagle et al., 1995). It involves placement of a graft either adjacent to the patient’s native liver (heterotopic) or in the hepatic bed after a portion of the native liver has been removed (orthotopic). The auxiliary liver supports the patient until the native liver regenerates. Theoretically, this may obviate the need for chronic immunosuppression. Moreover, a relatively smaller graft will be required; therefore, the majority of the donor graft can be utilized for standard orthotopic liver transplantation for another recipient.

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EXPERIMENTAL THERAPY Advances in the therapy of FHF have been limited. Treatment strategies, such as charcoal hemoperfusion and prostaglandin E1, which showed early promise, have not been shown to be superior to standard care in analyses by randomized studies. Plasmapheresis and hepatectomy have been suggested as possible bridging strategies to transplantation, but prospective trials are yet to be performed. Three additional forms of therapy may provide a bridge to liver transplantation or to regeneration of the native liver with spontaneous recovery: bioartificial liver devices, hepatic xenotransplantation (use of non-human livers), and hepatocyte transplantation. However, all these experimental therapy are yet to be shown its efficacy and safety.

Liver Support Systems Liver support systems had been in development for over 30 years. They fall into two categories: non-cell-based systems and cell-based systems (Murray-Lyon et al., 1975; Stockmann et al., 2000). Non-cell-based systems include plasmapheresis and charcoal-based hemoadsorption. A charcoal-based, blood detoxification system is commercially available in United States. Though the system has demonstrated safety, survival benefit in treating either acute or chronic liver failure has not been shown (Ash, 1994; Ellis et al., 1999; Murray-Lyon et al., 1975). The molecular adsorbents recirculation system (MARS) is another non-cellbased system, available in Europe (Stange et al., 1999a, b, 2000). This system exposes patient ultrafiltrate to an albumin-rich solution across a membrane (Stange et al., 1993). The basic concept is that bilirubin and other albuminbound substances and toxins will move across a concentration gradient from the patient to a circulating albumin-rich (25%) solution. The ultrafiltrate is then passed through another compartment where conventional renal hemofiltration/dialysis will take place. Hence, this device will provide both liver and renal support. A recent study of 13 patients with chronic liver disease and with encephalopathy demonstrated that bilrubin, bile acids and creatinine, but not ammonia, improved with MARS. In addition, nine of 13 demonstrated improvement in liver and renal indices. Another study had used MARS in patients with hepatorenal syndrome (Stange, 2000). Mortality was 100% in the controlled group while it was 75% in the treated group (Stange, 1999b). In addition, both bilirubin and creatinine had improved in the treated group. However, these endpoints had not reached statistical significance.

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Cell-based systems involve incorporation of living hepatocytes into plasmapheresis or whole blood extracorporeal systems (Nyberg & Misra, 1998). These are also known as bioartificial liver support systems. Incorporated hepatocytes can be of either human or porcine origin. The extracorporeal liver assist device (ELAD) is one such system. It incorporates the hepatoblastoma-derived HepG24/C3A cell line. An initial small study showed promising result with significant survival benefit (Stockmann et al., 2000; Sussman et al., 1994). However, a subsequent controlled trial failed to reproduce a survival benefit (Ellis et al., 1996, 1999). Bioartificial extracorporeal liver support (BELS) and HepatAssist system are porcine-based system that had shown some promising result in small case series or small uncontrolled studies (Detry et al., 1999; Stevens et al., 2001). The full safety and benefit of these systems are yet to be defined. A detailed review of various bioartificial liver devices has been published (Allen et al., 2001).

SUMMARY The incidence of fulminant hepatic failure is poorly defined and there are a variety of causes, which varies among different geographic regions. On the whole, viral induced and toxin/drugs-related hepatitis are the most common causes. Apart from acetaminophen toxicity and pregnancy related liver disorders, most patients will require liver transplantation. Such patients should be recognized early and promptly transferred to a liver transplantation center. Patients must be managed in an intensive care unit with special attention to associated complications, in particular, volume overload, cerebral edema and infection. Regular assessment of the patient will be necessary. Therefore, early listing for liver transplantation should be considered for the appropriate patient since a delay might increase the likelihood to develop complications that may subsequently preclude the patient from liver transplantation.

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Harrison, P., Wender, J., & Gimson, A. (1991). Improvement during acetylcysteine of hemodynamics and oxygen transport in fulminant hepatic failure. N. Eng. J. Med., 324, 1852–1857. Hillenbrand, P., Parhoo, S. P., & Jedrychowski, A. (1974). Significance of intravascular coagulation and fibrinolysis in acute hepatic failure. Gut, 15, 83–88. Hoofnagle, J., Carithers, R., Sharpiro, C., & Ascher, N. (1995). Fulminant hepatic failure: Summary of a workshop. Hepatology, 21, 240–252. Hortnagel, H., Singer, E., & Leuz, K. (1984). Substance P is markedly increased in plasma of patient with hepatic coma. Lancet, 1, 480–482. Kim, W. R., Brown, S. B., Jr., Terrault, N. A., & El-Serag, H. (2002). Burden of liver disease in the United States: Summary of a workshop. Hepatology, 36, 227–242. Klein, N., Mabie, W., & Shaver, D. (1991). Herpes Simplex virus hepatitis in pregnancy: Two patients successfully treated with acyclovir. Gastroenterology, 100, 239–244. Larsen, F. S., Kundsen, G. M., & Hansen, B. A. (1997). Pathophysiological changes in cerebral circulation, oxidative metabolism and blood brain barrier in patients with acute liver failure. Tailored cerebral oxygen utilization. J. Hepatol., 27, 231–238. Lee, W. M. (1993). Acute liver failure. N. Eng. J. Med., 329, 1862–1866. Lee, W., Galbraith, R., & Watt, G. (1995). Predicting survival in fulminant hepatic failure using serum Gc protein concentration. Hepatology, 21, 101–105. Lidofshy, S. D. (1995). Fulminant hepatic failure. Crit. Care Clin., 11, 415–430. Makin, A. J., Wendon, J., & Williams, R. (1995). A 7-year experience of severe acetaminophen-induced hepatotoxicity (1987–1993). Gastroenterology, 109, 1907–1912. Martin, L., Booth, F., & Karlstadt, R. (1993). Continuous intravenous cimetidine decreases stressrelated upper gastrointestinal hemorrhage without promoting pneumonia. Crit. Care Med., 21, 19–30. Munoz, S., Maritz, M., & Martin, P. (1993). Relationship between cerebral perfusion pressure and systemic hemodynamics in fulminant hepatic failure. Transplant Proc., 25, 1776–1780. Munoz, S., Robinson, M., & Northrup, B. (1991). Elevated intracranial pressure and computerized tomography of the brain in fulminant hepatic failure. Hepatology, 13, 209–212. Murray-Lyon, I., Portmann, B., & Gazzard, B. (1975). Analysis of the cause of death in the treatment failures. In: R. Williams & I. Murray-Lyon (Eds), Artificial Liver Support (p. 242). Tunbridge. Wells, England: Pitman Medical. Nyberg, S., & Misra, S. (1998). Hepatocyte liver-assist system – a clinical update. Mayo Clin. Proc., 73, 765–768. O’Grady, J., Alexander, G., & Hayllar, K. (1989). Early indicators of prognosis in fulminant hepatic failure. Gastroenterology, 97, 439–445. O’Grady, J., Portmann, B., & Williams, R. (1993). Fulminant hepatic failure. In: L. Schiff & R. Schiff (Eds), Disease of Liver. Philadelphia: J. B. Lippinott. Ostapowicz, G., Fontana, R., & Nabarro, V. (2000). Use of liver transplantation in patients with acute liver failure. Hepatology, 2 (Part 2), 215A. Panwels, A., Mostefa-Kara, N., & Florent, C. (1993). Emergency liver transplantation for acute liver failure: Evaluation of London and Clichy criteria. J. Hepatol., 17, 124–128. Portmann, B., Talbot, I. C., & Day, D. W. (1975). Histopathological changes in liver following paracetamol overdose: Correlating clinical and biochemical parameters. J. Pathol., 117, 169–181. Rakela, J., Lange, S., Ludwig, J., & Baldus, W. (1985). Fulminant hepatitis: Mayo Clinic experience with 34 cases. Mayo Clin. Proc., 60, 289–292.

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Ring-Larsen, H., & Palazzo, U. (1981). Renal failure in fulminant hepatic failure and terminal cirrhosis: A comparison between incidence, types and prognosis. Gut, 22, 585–591. Riordan, S. M., & Williams, R. (1997). Treatment of hepatic encephalopathy. N. Eng. J. Med., 337, 473–479. Roberts, H. R., & Cederbann, A. L. (1972). The liver and blood coagulation: Physiology and pathology. Gastroenterology, 63, 297–320. Rolando, N., Gimson, A., & Wade, J. (1993). Prospective controlled trial of selective parental and enteral antimicrobial regimen in fulminant hepatic failure. Hepatology, 17, 196–201. Rolando, N., Harvey, F., & Braham, J. (1990). Prospective study of bacterial infection in acute liver failure: An analysis of fifty patients. Hepatology, 11, 49–53. Rolando, N., Harvey, F., & Braham, J. (1991). Fungal infection: A common, unrecognized complication of acute liver failure. J. Hepatol., 12, 1–9. Rolando, N., Wade, J., & Davalos, M. (2000). The systemic inflammatory response syndrome in acute liver failure. Hepatology, 32, 734–739. Rolando, N., Wade, J., & Stangon, A. (1996). Prospective study comparing the efficacy of prophylactic parental antimicrobials with or without enteral decontamination in patient with fulminant hepatic failure. Liver Transpl. Surg., 2, 8–12. Rubin, M. H., Weston, M. J., & Bullock, G. (1977). Abnormal platelet function and ultrastructure in fulminant hepatic failure. Q. J. Med., 46, 339–352. Saija, A., Princi, P., & Lanza, M. (1995). Systemic cytokine administration can affect blood-brain permeability in the rats. Life Sci., 56, 775–784. Samson, R., Trey, C., & Timme, A. (1967). Fulminating hepatitis with recurrent hypoglycemia and hemorrhage. Gastroenterology, 53, 291–293. Saunders, S. J., Hickmann, R., & Macdonald, R. (1972). The treatment of acute liver failure. In: H. Popper & F. Schaffer (Eds). Progress in Liver Disease (Vol. 4, pp. 333–353). New York: Grune & Stratton. Schafer, D. F., & Jones, E. A. (1982). Hepatic encephalopathy and the gamma-aminobutyric acid neurotransmitter system. Lancet, 1, 18–20. Schiodt, F., Atillasoy, E., & Shakil, A. (1999). Etiology and outcome from 295 patients with acute liver failure in the United States. Liver Transplant Surg., 5, 29–34. Schiodt, F., Rochling, F., Casey, D., & Lee, W. (1997). Acetaminophen toxicity in an urban county hospital. N. Eng. J. Med., 337, 1112–1117. Stange, J., Mitzner, S., & Risler, T. (1999). Molecular adsorbent recycling system (MARS): Clinical results of a new-membrane-based blood purification system for bioartificial liver support. Artif. Organs, 23, 319–330. Stange, J., Mitzner, S., & Klammt, S. (2000). Liver support by extracorporeal blood purification: A clinical observation. Liver Transpl., 6, 603–613. Stange, J., Ramlow, W., & Mitzner, S. (1993). Dialysis against a recycled albumin solution enables removal of albumin-bound toxins. Artif. Organs, 17, 809–813. Stevens, A., Han, B., & Baqerizo, A. (2001). An interim analysis of a phase II/III prospective randomized, multicenter, controlled trial of the Hepatassist bioartificial liver support system for the treatment of fulminant failure (abstract). Hepatology, 34, 299A. Stockmann, H., Hiematra, C., Marquet, R., & IJsermans, J. (2000). Extrocorporeal perfusion for the treatment of acute liver failure. Ann. Surg., 231, 460–464. Strauss, G., Adel Hausen, B., & Kirkegaard, P. (1997). Liver function, cerebral blood flow autoregulation and hepatic encephalopathy in fulminant hepatic failure. Hepatology, 25, 837–839.

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Sullivan, S., Chase, R., & Christofides, N. (1981). The gut hormone profile of fulminant hepatic failure. Am. J. Gastroenterol., 76, 338–341. Sussman, N., Gislason, G., Conlin, C., & Kelly, J. (1994). The Hepatix extracorporeal liver assist device: Initial clinical experience. Artif. Organs, 18, 390–396. Trewby, P. N., Warrens, R., & Contini, S. (1978). The incidence and pathophysiology of pulmonary oedema in fulminant hepatic failure. Gastroenterology, 74, 859–864. Trewby, P. N., & Williams, R. (1977). Pathophysiology of hypotension in patients with fulminant hepatic failure. Gut, 18, 1021–1026. Trey, C., & Davidson, L. S. (1970). The management of fulminant hepatic failure. In: H. Popper & F. Schaffner (Eds), Progress in Liver Disease (Vol. 4, pp. 282–292). New York: Grune & Stratton. Vale, J. A., & Proudfoot, A. T. (1995). Paracetamol (acetaminophen) poisoning. Lancet, 346, 547–552. Vento, S., Garofano, T., & Renzini, C. (1998). Fulminant Hepatitis associated with hepatitis A virus superinfection in patients with chronic hepatitis C. N. Eng. J. Med., 338, 286–290. Ware, A. T., & D’Agostino Awn Conises, B. (1971). Cerebral oedema, major complication of massive hepatic necrosis. Gastroenterology, 61, 877–883. Wendu, J., Harrison, P., & Keays, R. (1992). Effects of vasopressor agents and epoprostenil on systemic hemodynamics and oxygen transport in fulminant hepatic failure. Hepatology, 15, 1067–1071. Weston, M. J., Talbot, A. C., & Howath, P. J. N. (1976). Frequency of arrhythmia and other cardiac abnormalities in fulminant hepatic failure. Br. Heart J., 38, 1179–1188. Williams, R., & Gimson, A. E. (1991). Intensive liver care and management of acute liver failure. Dig. Dis. Sci., 36, 820–823. Wyke, R. J., Canalese, J. C., & Gimson, A. E. S. (1982). Bacteremia in patients with fulminant hepatic failure. Liver, 2, 45–52.

14.

PRIMARY BILIARY CIRRHOSIS

James Neuberger INTRODUCTION Primary Biliary Cirrhosis (PBC) is a disease of unknown etiology, characterized by progressive destruction of the middle-sized intrahepatic bile ducts. Although the name for the disease is inappropriate since many patients are diagnosed many years before the onset of cirrhosis, for historical reasons the name has remained and alternative names such as non-supportive destructive cholangitis and granulomatous cholangitis, while perhaps being more accurate, have not been accepted.

CLINICAL FEATURES The classical presentation of the disease is if a woman who, in her middle years, presents with lethargy and pruritus. However, in recent years, it has become clear that PBC covers a wider spectrum than previously appreciated: Pre-symptomatic stage. Anti-mitochondrial antibodies are detectable in the serum (as a consequence of routine screening or assessment for other conditions) but the patient is asymptomatic and both liver tests and liver histology may be normal. Most, but not all patients will become symptomatic at a median of 10–15 years. Inevitably, the prevalence of this stage is unknown. Asymptomatic stage. The patient has not of the symptoms attributed to PBC but has typical liver tests and liver histology shows the classical features of PBC. Indeed, up to half may have an established cirrhosis at this time. During the 5 years The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 383–398 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15014-9

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after diagnosis, 75% will develop symptoms and the median time to end-stage disease is 8–12 years. Symptomatic stage. This is the classical presentation, described above. As the disease progresses, jaundice develops and with the development of progressive hepatic fibrosis, symptoms of portal hypertension, such as ascites or variceal hemorrhage from esophageal or gastric varices occur. Jaundice progresses and liver failure ensues. The median time to end-stage disease (death or transplantation) is 7–10 years. End-stage disease. Some patients present with symptoms of portal hypertension, others present with jaundice. Ninety five percent of patients with PBC are female although the natural history in men and women is similar. The disease may be diagnosed at any age between 18 and old age. Unlike other presumed autoimmune diseases, the syndrome has not been described in children. The prevalence of the disease varies, with reported levels of up to 150/million in North America and Northern Europe. In contrast, the syndrome is rarely diagnosed in people from the Indian sub-continent (Table 1), but it is not clear whether this represents a true variation in disease prevalence or merely differences in diagnostic methods. In Olmstead country, Rochester in 1995, the age and sex adjusted prevalence per 100,000 was 65.4 for women and 12.1 for men. On examination, the patient may be asymptomatic. Pigmentation and xanthomata are often seen. The pigmentation is greatest in the temporal areas of the face but may occur in any part of the body. The cause of the pigmentation is not clear. Xanthelasma are seen around the eyes in particular and xanthoma in tendons or nerves may also occur; these are related to the associated hypercholesterolemia. Unlike many other causes of chronic liver disease, spider nevi are not usually present but the cutaneous stigmata of chronic liver disease, such as palmar erythema, leukonychia, clubbing of the nail bed, may all be present. The liver is Table 1. Prevalence of PBC. Cases/106 Victoria, Australia Ontario, Canada Sheffield, United Kingdom Malmo, Sweden Orebro, Sweden Umea, Sweden Newcastle, United Kingdom Source: From Watson et al. (1995).

19 22 54 92 128 151 154

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usually enlarged and remains enlarged during the course of the illness. The spleen also increases in size early in the disease. Other signs of hepatic decompensation may develop as the disease progresses. Biochemically the disease is characterized by “cholestatic liver tests”. That is, the serum alkaline phosphatase and Gamma Glutamyl Transpeptidase (GGT) and 5′ -nucleotidase are elevated early on in the course of the disease. As the disease progresses, the serum bilirubin starts to rise and markers of hepatic synthetic function such as serum albumin or clotting indices tend to fall. The classic immunological abnormalities of PBC is an elevation of the immunoglobulins, especially of IgM and to a lesser degree IgG, and the presence of non-specific autoantibodies for which the classical marker is the antimitochondrial antibody (AMA) (see below). The lipids may be elevated; in the early stages there may be striking elevation of the high-density lipoproteins (HDL) with more modest increases in the VLDL and LDL: as the disease progresses. HDL levels fall and LDL rises.

Natural History The natural history of PBC is one of slow progression. The median time from diagnosis to death or transplantation is about 15 years. The best guide to prognosis is the serum bilirubin. Once this reaches the level of 180 ␮mols/l, then the median survival (in the absence of transplantation) is about 18 months. Several specific models to predict survival have been developed (such as the Mayo Clinic Model and the European Prognostic model); these models correlate well with each other, and are helpful when applied to populations but the relatively wide confidence intervals reduces the applicability when applied to the individual patient. The development of cirrhosis, old age, and onset of ascites also herald a poor prognosis. As with any disease associated with cirrhosis, hepatocellular carcinoma may develop. Since male gender is an additional risk factor for the development of hepatocellular carcinoma, the number of patients with PBC developing hepatocellular carcinoma is relatively small since, as indicated above, the disease affects primarily females. PBC is associated with a variety of other diseases which are presumed to have an autoimmune basis; these include thyroid disease (both myxedema and thyrotoxicosis), arthritis, Addison’s disease, celiac disease, sclerodactyly, the CREST syncrome (chrondrocalcinosis, Raynaud’s phenomenon, esophageal abnormalities, sclerodactyly and telangiectasia), Raynaud’s syndrome, pulmonary fibrosis and glomerulonephritis. There may be an associated osteopenia, which exacerbates the natural bone loss that occurs in post-menopausal women. Malabsorption of the fat soluble vitamins (A, D, E and K) may occur either because of associated pancreatic insufficiency or because of steatorrhea consequent on

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Table 2. Histological Classification of PBC. Stage 1

Florid Duct Lesions Portal Hepatitis: Granulomas

Stage 2

Ductular Proliferation Periportal Hepatitis Ill-Defined Lymphoid Aggregates

Stage 3

Scarring and Fibrosis Peri-Portal Cholestasis

Stage 4

Cirrhosis Cholestasis

cholestasis; thus, maladsorption of vitamin D will be associated with an increased risk of osteomalacia.

LIVER HISTOLOGY The diagnosis of PBC is usually confirmed by histology. The classical hallmark of PBC is a granulomatous infiltration destroying the middle-sized intrahepatic bile ducts. The disease is histologically divided into four stages although features of all four stages may be present in the same liver (Table 2). The portal inflammatory infiltrate consists of lymphocytes (the major cell type), histiocytes, plasma cells, neutrophils and eosinophils. CD3 T cells are distributed throughout the parenchyma, whereas CD8 cells are present around the bile ducts; as the disease progresses, CD4 cells increase. Reports of Th1 and Th2 balance are conflicting.

VARIANTS OF PBC There are two variants of PBC: Autoimmune cholangitis. In autoimmune cholangitis, the patient has all the signs, symptoms and serological abnormalities of PBC but AMA are not detectable in the serum by immunofluorescence. In some cases, AMA can be detected by immunoblotting. Serum IgG and ANA are present in greater amounts than in classical PBC but his variant should be considered and treated as PBC. PBC and Autoimmune hepatitis (AIH) overlap. Here patients have signs, biochemical and histological features of both PBC and AIH and fulfill the international agreed criteria for the diagnosis of AIH, even though AMA are found and there is bile duct damage and granulomatous cholangitis on liver histology.

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There is controversy whether this represents true overlap of the two conditions or a variant of PBC; most recommend treatment with corticosteroids to control the portal inflammation.

BASIS FOR SYMPTOMS The two main symptoms related to PBC, neither of which is specific for the disease, are lethargy and pruritus.

Lethargy The lethargy associated with PBC may be so prolonged and profound that liver replacement may be the only effective therapy. While it is important to exclude treatable causes of lethargy (such as myxedema, Addison’s disease, hypercalcemia, depression or iatrogenic causes such as antihistamines, mistakenly given for treatment of pruritus), in most patients, there is no other identifiable precipitating factor. As yet no explanation for lethargy has been found. Data in rats, produced by Swain and Maric (1995), has shown that rats with cholestasis induced by bile-duct ligation may be associated with decreased release of corticotrophic releasing factor (CRH) by hypothalamus. Whether alterations of the hypothalamic-pituitaryadrenal axis is important in the lethargy of humans with lethargy associated with PBC remains to be shown. Recent data has associated lethargy with altered levels of manganese and manganese deposition in the globus pallidus (Forton et al., 2004).

Pruritus The pruritus used to be attributed to elevation of serum bile acids stimulating their fibers in the skin. This explanation was accepted despite the lack of correlation between the degree of elevation of bile acids and the presence or degree of pruritus and despite the observation that in many other forms of liver disease, bile acids are increased to a similar degree and yet the patient does not complain of pruritus. However, it was on this basis that the use of cholestryamine was introduced for the release of pruritus and there is little doubt the mainstay of therapy remains cholestyramine. It was assumed that this resin binds irreversibly bile acids in the lumen of the small bowel and so interrupting the enterohepatic circulation of bile acids. Other drugs which have been used with some effect for the management of pruritus include enzyme inducers such as phenobarbitone and rifampicin.

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Although there have been reports suggesting some benefit from these therapies, the improvement is relatively small and the mechanism of their effect is unclear. Alternative approaches which have been used, for which again there is no clearly understood mechanism, include plasmaphoresis, charcoal hemoperfusion or the use of liver support devices (such as the MARS machine) and external biliary diversion. These treatments, often effective, indicate that there is some factor or factors in serum and/or bile which are associated with the genesis of pruritus but fail to identify which factors these are. The use of ursodeoxycholic acid, a tertiary bile acid, has been used for the treatment of PBC; the relief of pruritus is inconsistent and the mechanism unknown. In the last few years, more interest has centered on the opiate system in the pathogenesis of pruritus. Thomas de Quincy in 1821 had already described his own experiences with intense pruritus as an opium addict. However, Bernstein and Swift (1979) reported a single case history that the Naloxone given subcutaneously resulted in relief of pruritus. Further studies of opiate antagonists have included Nalmefene and Naloxone. This work was extended by Bergasa and Jones (1995). They draw attention to the observation that opiates with agonist properties and opioid receptors have been implicated in the mediation of pruritus; such drugs include not only morphine but diamorphine, fentanyl, methadone and pethidine. They hypothesized that increased opiodurgic neurotransmission or neuromodulation in the CNS contributes to the pruritus of cholestasis. They advanced several lines of research to suggest this might indeed be the case. As indicated above, opiate drugs with agonist properties may induce pruritus. Oral administration of opiate antagonists, such as naltrexone, results in short term relief of pruritus in patients with chronic cholestasis. It is of interest that some of the side effects such as anorexia, nausea, insomnia and even hallucinations may be seen in patients with chronic cholestasis but in not normal subjects, suggesting there is an increased opiate antagonist induced reaction in patients with cholestasis. However, it still remains unclear what the mechanism underlying pruritus is, and what is the nature of the opioid antagonist. It is of interest that cholestyramine, very effective in some patients with cholestasis due, for example, to PBC, is relatively ineffective in treating pruritus associated with some other conditions such as cholestasis of pregnancy or drug induced cholestasis. It is possible, therefore, that the pruritus of cholestasis is multi-factorial.

IMMUNOLOGICAL ABNORMALITIES There are many, well-documented abnormalities of both the cellular and humoral system in patients with PBC. The extent to which these are secondary to

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the cholestasis remains uncertain. There is evidence of complement activation, as shown by increased levels of C3d. There are abnormalities of peripheral lymphocyte numbers and function; although findings are sometimes conflicting, it appears that, in the early stages of the disease, there are normal levels of CD4positive lymphocytes and increased CD8-positive cells; as the disease progresses there is a significant reduction of both lymphocyte subsets. Functionally, it can be shown that there is decreased suppressor T-cell function and functional impairment of lymphocyte function as evidenced by, for example, decreased production my lymphocytes of interferon-␥, tumor-necrosis factor on mitogen stimulation. As indicated elsewhere, there is increased ␥-globulinemia. There are increased circulating immune complexes, which correlate with the presence of arthralgia; some of these complexes contain antimitochondrial antibodies. Other serological markers of immune activation in patients with PBC include elevated levels of circulating ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular adhesion molecule-1) and ␤2-microglobulin. These analytes tend to rise with progression on the disease and thus may be markers of liver damage, rather than the cause. These changes are not specific to PBC. The X chromosome includes genes that are involved in immune tolerance; the recent demonstration that women with PBC have a significantly higher incidence of X monosomy than aged-matched controls; this haploinsufficiency could explain, at least in part, the high female preponderance (Invernizzi et al., 2004)

Antimitochondrial Antibodies Antimitochondrial antibodies were first described by Walker et al. (1965) using immunofluorescence. They showed that the serum of patients with PBC incubated with composite rat sections gave a characteristic staining which subsequent studies was shown to be localized to the mitochondria. This antibody, which is non-organ non-species specific, is characteristic of PBC and has been documented in 75–99% of patients with histological disease. A number of antimitochondrial antibodies have been defined and these have been labeled from M1 to M9 (Table 3). These may occur in many different situations including pseudolupus, some drug reactions but the classes specifically associated with PBC are M-2, M-4, M-8 and M-9. While some workers believe that these different antibody sub-types have different prognostic implications, this is uncertain. The M-2 antibody reacts with an antigen present on the inner mitochondrial membrane and is sensitive to trypsin. The antigen is found not only in mammalian liver, kidney, heart and other organs, but also in animals, including rat, rabbit and

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Table 3. Characterization of AMA Antibodies. AMA Type

Clinical Correlate

Organ Specificity

Mitochondrial Location

M1 M2 M3 M4 M5 M6 M7 M8 M9

Syphilis PBC Pseudolupus PBC and CAH Connective tissue disease Drug hepatitis Cardiomyopathy PBC PBC

– – – – – Liver Myocardium – –

Inner Inner Outer Outer Outer Outer Inner Outer Outer

Source: Adapted from Gershwin, Coppel and MacKay, Hepatology, 8, 147 (1980).

chicken, but also in yeast and bacteria. Initial studies showed that the AMA reacted with several different proteins on a Western blot, with apparent Mr of 36, 43–48, 50–56 and 70–75 kD. The dominant antigen was found to have a Mr of about 73 kD. The major antigen was subsequently defined by Gershwin and colleagues as the E2 component of pyruvate dehydrogenase complex. Subsequent studies showed that the M2 antigenic determinants were polypeptides of a group of respiratory enzymes termed the 2-oxo-acid dehydrogenase complexes (Table 4). These complexes are isolated from microbial and eukaryotic cells and three classes have been identified, all of which are involved in the oxidative decarboxylation of 2-oxo-acids. One is specific for PDH (pyruvate dehydrogenase complex). The second is specific for branch chain oxo-acids (BCOADC) and the third is specific for 2-oxo-glutarate (OGDC). Each complex has a similar structure Table 4. Reactions of AMA with M2 Auto Antigens. Frequency PDC E1´a sub unit E1ˆa sub unit E2 component Protein x

+ ± +++ ++++

OGDC E2 component

+++

BCOADC E2 component

++

Note: PDC: Pyruvate dehydrongenase complex; OGDC: 2-oxo-Glutarate dehydrogenase complex; BCOADG: Branched Chain 2-oxo-acid dehydrogenase complex.

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being composed of multiple copies of three basic enzymes: a substrate specific decarboxidase dehydrogenase (E1); a dihydrolipoamide acetyl transferase (E2) which is specific for each complex, and a dihydrolipoamide dehydrogenase (E3). There is also a small amount of a protein termed protein x which has a high affinity binding for E3 but whose structure remains unknown. Although all antibodies are present to all three proteins in the complex it seems that the immunodominant site of M2 antigens is the lipoyl domain of the E2 component of PCD; other antibodies may arise merely as a result of epitope spreading. These lipoyl domains are central to the activity of the complexes. What then is the role of these antibodies? As indicated in normal cells, these antigens are widely distributed and are present in all eukaryotic cells. They are neither organ nor species specific. These proteins are located in normal cells in the inner aspect of the mitochondrion, which itself is an intracellular structure. Although in vitro AMA functionally inhibit E2 activity, there is no evidence for such an effect in vivo. In common with other autoantigens and other autoimmune diseases such as in thyroid disease or diabetes mellitus, autoantibodies appear to be directed to general “housekeeping” enzymes. These antibodies are detectable not only in serum but also urine, bile and saliva; this suggests that AMA are found where there is secretory epithelium. It has also been suggested that PDCspecific dimeric AMA are taken up by the biliary epithelial cells and transcytosis could result in apoptosis and bile duct damage (Matsumura et al., 2004). There is, however, no evidence that the AMA are pathogenetic; immunization of a variety of animals with recombinant E2 results in the generation of AMA, but there is no evidence of bile duct damage. It has been shown that in patients with PBC there is aberrant expression of PBC on the membrane on bile ducts. This raises the possibility that such cells can become the target of immune response. Again, it remains unclear why biliary epithelial cells should have aberrant expression of this antigen. It is possible that this remains a cross reaction and equally it is uncertain how such an aberrant antigen leads to an immune response. Of interest, the E2 antigen in biliary epithelial cells appear to be resistant to apoptosis; although the extent of apoptosis of biliary epithelial cells in PBC is controversial, it is possible that apoptotic cells could release E2 and so allow exposure of the protein to the immune system.

Other PBC-specific Autoantibodies In addition to the characteristic antibodies to the enzymes of the two oxo-aciddehydrogenase complexes in the mitochondria, the serum of patients with PBC

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contains a number of PBC-specific autoantibodies: anti-Sp100 (anti-nuclear); antigp210 (nuclear pore complex), and antibodies to the nuclear membrane (antilamins). The significance of these antibodies remains uncertain; they may represent cross-reactions with epitopes of E2.

Other Autoantibodies Patients with PBC have several other circulating autoantibodies that are not specific for PBC; these include: anti-centromere; anti-nuclear; anti-platelet, and antilymphocyte antibodies. As indicated above, there is a close association between PBC and sclerodactyly. Anti-centromere antibody was first associated with patients with CRST syndrome and subsequent studies have shown that many patients with PBC and scleroderma have anti-centromere antibodies. Our own study showed CRST in 18% of 110 patients with PBC, whereas anticentromere antibody was detected in only 9%. The anti-Sp100 antibodies was again described in patients with PBC and is so known because the nuclear protein with which the antibody reacts gives a speckled appearance on immunofluorescence; it has an approximate molecular weight of 100 kD. There is no clinical subset characterized by patients with these antibodies. Anti-laminin antibodies are found in approximately one quarter of patients with PBC and show a ring-like staining of the nuclear limiting membrane. These antibodies react with proteins of molecular weight 60, 68 and 74 kD of the nuclear membrane. These antibodies are also found in the serum of patients with other autoimmune diseases.

HLA and PBC There are several reports of familial clustering of PBC and studies have shown a familial incidence of up to 2%. There have been many studies looking for an association between HLA phenotypes and genotypes and PBC. Many of the early studies used serological methods to assign HLA phenotype, and because of the inherent inaccuracies in such methods, the conclusions must remain uncertain. However, a number of studies have shown an association between PBC and the HLA phenotype DR8 but this association applies only for the minority of patients, suggesting that if there is a susceptibility allele, it lies some distance from the DR genes, possibly involving the class III region or the DP or DQ loci. Analysis of the DQB polymorphism showed that one allele, DQB1*0402, was more common in patients (11%) compared with controls (4%). Although this difference did not reach statistical significance, it is suggested there was an increased risk of 5.4 fold in

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patients who were DR8 DQB1 0401 positive. Other studies, for example, in Japan have shown different HLA susceptibility loci. The class II complement genes, have attracted some attention. Some have shown an increase in C4B2 (Briggs et al., 1987) allotypes and others have shown an increase in C4AQ0. Thus, if there is a susceptibility antigen, this is relatively weak.

HLA and the Liver The HLA system includes genes which are thought to play an important role in the immune response. The class I and class II molecules of the major histocompatibility complex (MHC) are highly polymorphic cell structures which are important in cell recognition and enabling the immune system to distinguish cell from non-cell. The class I molecules consist of a membrane bound glycoprotein which is non-covalently bound with ␤-2 microglobulin. Class I products usually bind intracellular peptides of 8–9 aminoacids which are involved primarily in recognition of antigens by CD8 T-lymphocytes, the majority of which are cytotoxic T-cells. Class II molecules are membrane bound heterodimers which consist of heavy and light glycoprotein chains. These are involved in activation of CD4 cells and class II molecules are able to present larger antigens of around 18 amino acids. There is considerable polymorphism of the class I and class II molecules due to amino acid substitutions. The distribution of HLA class I and class II antigens in the normal liver is shown in Table 5. Compared with the normal liver, in patients with PBC there is aberrant expression of HLA class II by biliary epithelial cells and increased class I and, to a lesser extent, class II expression by hepatocytes (Ballardini et al., 1984). The increased expression of class II antigens by biliary Table 5. MHC Expression in Human Liver. Sinusoidal Cells

BECs

Hepatocytes

Normal Class I Class II

+ +

+ −

− −

PBC Class I Class II

+ +

+ +

+ ±

Cholestasis (EHBO) Class I Class II

+ +

+ −

+ ±

Note: BEC: Biliary epithelial cell; EBHO: Extra hepatic biliary obstruction.

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Table 6. Characteristics of Professional and Opportunistic Antigen Presenting Cells in the Liver. Professional APCs

ICAM-1 ILFA-1 LFA-3 MHC Class I MHC Class II B7 AG Processing Cytokine Secretion

Others Portal DC

Kupffer cells

BEC

+ + + + + + + +

+ + + + + + (+) +

(+) − (+) + (+) − − (+)

Note: (+): Indicates positive after stimulation; DC: Dendritic cells; APC: Antigen presenting cells; AG: Antigen. Source: Courtesy of Dr. D. Adams.

epithelial cells raises the possibility that this would allow recognition of biliary antigens by the immune system and so set the scent for autoimmunity. However, this attractive hypothesis may not stand up to critical evaluation. First, the expression of aberrant class II antigen is patchy and may occur more commonly in late stage disease than early disease, suggesting that this aberrant expression is a consequence rather than a cause of the disease process. Second, as expression is patchy and many diseased bile ducts do not demonstrate increased class II expression. Third, examination of patient liver from those with cholestasis due to obstruction, for example, shows similar patterns of aberrant HLA class I and class II expression to those with PBC. Thus, it may be that this aberrant expression is a consequence of cholestasis rather than its cause. The underlying mechanism is unclear but may relate to the effect of pro-inflammatory agents such as TNF-␣ on inducing aberrant MHC expression. Furthermore, treatment such as ursodeoxycholic acid, which is associated with improvement in biochemistry, (see below) is associated with a reduction in the class II expression on hepatocytes; the effect on class II expression on biliary epithelial cells is uncertain and inconsistent. The biliary epithelial cells may not be able to present antigens, since they do not express the B7 antigen (Table 6).

TREATMENT In the absence of defined etiology it is difficult to find a treatment specific for the disease. The clinical trials have been hampered by the slow natural history of the

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disease; since the natural history from diagnosis to death may be well over 10 years, and clinical studies using death as an end-point may be difficult to perform. The increasing success of liver transplantation adds further difficulties to interpreting such studies since it cannot be assumed that all transplanted patients would have otherwise died. These considerations have led researchers to look for other endpoints but there remains a lack of consensus over agreed surrogate endpoints. Although serum bilirubin remains the best prognostic marker, alternations in prognostic markers do not necessarily imply alterations in the natural history. Overall, treatment with immunosuppressive therapy including cyclosporin, tacrolimus, and corticosteroids have been disappointing. Antifibrotics such as malotilate and colchicine have also failed to give convincing therapeutic benefit. A recent report raises a possible benefit from the use of Tamoxifen. In recent years, greatest interest has focused on the use of the tertiary bile acid ursodeoxycholic acid (UDCA). The therapeutic benefit was described initially by Leuschner et al. (1992) who showed that in patients with a variety of liver diseases, given the bile acid for therapy of gall stones, had significant improvement in liver function tests. Since that time there has been a number of both open and controlled studies. A number of conclusions can be drawn from these studies: the drug is very safe, gastro-intestinal symptoms being the only significant side effect. The drug is also well tolerated, and to date, no significant adverse features have been reported. There is a definite effect on improvement of serum biochemistry, a reduction in serum immunoglobulins and AMA titers and the peripheral eosinophilia but the effect on liver histology remains inconsistent. Several studies suggest that treatment with UDCA is associated with a 30% improvement in mortality. However, a recent Cochrane analysis has concluded that there is no convincing evidence for a therapeutic benefit on survival. Nonetheless, given the safety and the effectiveness on surrogate markers of prognosis, most clinicians offer the patients treatment at a dose of 10–15 mg/kg/day. The mechanism of action of UDCA is not fully understood. One of the main mechanisms is the effect of the UDCA on ileal hepatic bile acid transport system. UDCA reduces active ileal absorption of cholic and chenodeoxycholic acid with results in a reduction in the serum levels of these two bile acids. The bile acids are toxic and UDCA appear to be hepatoprotective. Secondly, UDCA is a choleretic and increases the Tm of bilirubin. Thirdly, UDCA has a significant effect on MHC expression in the liver, as indicated above, and there is now increasing evidence that UDCA may alter the immune system. UDCA also reduces the cytotoxic effects of more hydrophobic bile acids, and is anti-apoptotic. Of interest, treatment with UDCA appears to be associated with a reduction in colonic cancer in patients with PBC and with primary sclerosing cholangitis. Which, if any of these effects

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of UDCA is important in the therapeutic effect of the drug in patients with PBC remains uncertain at this time. At present, the only therapeutic modality which has been showing convincingly to alter prognosis in patients with PBC is orthotopic liver transplantation. Although it is likely that PBC recurs after transplantation, there is major improvement in the quality and length of life. It is only by further knowledge of the mechanisms of PBC that specific therapeutic intervention can be acquired.

CAUSES OF PBC There are no convincing animal models of PBC currently available. Initial reports of a PBC-like syndrome naturally occurring in rabbits has not been followed up. Attempts to induce the disease by immunizing SCID mice with lymphocytes from patients with PBC has not been disappointing. Several pieces of evidence are compatible with an infectious trigger; the clustering of cases and the familial occurrence (in the absence of known HLA associations), and the effect of migration on incidence are all compatible with this hypothesis. Thus, a study from Australia (Table 7) has suggested that immigrants to a country tend to develop the prevalence of their new residence. Furthermore, PBC recurs after liver transplantation (in up to 50% at 10 years) although the aberrant distribution of E2 on biliary epithelial cells in the allograft may be detected within weeks of transplantation. Other viral infections are associated with autoimmune phenomena (such as LKM antibodies in association with Hepatitis C viral infection). Hopf et al. (1989) and Stemorowicz et al. (1988) have suggested that there is a close relationship between PBC and enterobacteria. Indeed, these authors have shown that there is an increase in the R-forms of E. coli in patients with PBC but these observations have not been confirmed by other groups. Others have reported an increased incidence of antimitochondrial antibodies (albeit at low titer) in patients with recurrent urinary tract infections, and have suggested that a cross-reaction between E. coli might result in the disease (Butler et al., 1993). Nonetheless, these results have not been confirmed by other workers and, Table 7. Prevalence of PBC in Melbourne, Australia. Cases/106 Population Australian born population Immigrants from U.K. and Eire Immigrants from other ethnic groups Source: From Watson et al. (1995).

15 47 27

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in particular, these results have not been looked at in males with PBC where the prevalence of recurrent urinary tract infections is much lower than in females. Preliminary studies suggesting infection with Mycobacteria gordoni have not been confirmed by others. However, there are a few pointers suggesting that there may be an environmental trigger to the disease; there is clustering of cases which, in one instance, has been linked to water supply from a single reservoir but these findings have not been confirmed in a follow-up study. Other infectious agents implicated in the pathogenesis include Novosphingobium aomaticovorans and with Chlamydia. Other workers have suggested that there may be a retroviral trigger. Others have suggested that the disease may be triggered by pregnancy or drug ingestion; benoxaprofen and chlorpromazine have been implicated. It is difficult to distinguish whether the pregnancy or drugs have caused the syndrome or whether they have merely exacerbated the condition, and so leading to the diagnosis. Finally, it has been suggested that xenobiotics might trigger PBC; in particular, those agents (such as drugs or household detergents) that have the potential to form halogenated derivatives could trigger the disease. At present, however, none of these wide-ranging hypotheses has been substantiated.

CONCLUSIONS PBC remains an enigmatic disease but there is increasing knowledge of the natural history, which is that of slow progression. Clinically, the disease is associated with other autoimmune diseases. Serologically, the disease is associated with a variety of autoantibodies, but the presence of the anti-mitochondrial antibody is characteristic. These antibodies react with components of the 2-oxo acid dehydrogenase complex, of which the dihydrolipoamide acetyl transferase is the most reactive. However, the role of the antimitochondrial antibodies in the pathogenesis of the disease remains uncertain. Since the cause of the disease is uncertain, treatment is symptomatic. Specific treatment with the naturally occurring tertiary bile acid, ursodeoxycholic acid, is associated with some clinical and biochemical improvement, although its effect on the natural history remains to be proved. The only effective treatment for end-stage disease is liver replacement, but the disease may recur.

REFERENCES Ballardini, G., Mikrakian, R., Bianchi, F., Pisi, E., Doniach, D., & Bottazzo, F. (1984). Aberrant expression of HLA-Dr antigens on bile duct epithelium in primary biliary cirrhosis: Relevance to pathogenesis. Lancet, ii, 1009–1013.

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Bergasa, N. V., & Jones, E. A. (1995). The pruritus of cholestasis. Gastroenterology, 108, 1582–1588. Bernstein, J. E., & Swift, R. (1979). Relief of intractable pruritus with naloxone. Arch. Dermatol., 115, 1366–1367. Briggs, D. C., Donaldson, P. T., Hayes, P., Welsh, K. L., Williams, R., & Neuberger, J. (1987). A major histocompatibility complex Class III allotype C4B2 associated with primary biliary cirrhosis. Tissue Antigens, 29, 141–145. Butler, P., Valle, F., Hamilton-Miller, J. M. T., Brumfitt, W., Baum, H., & Burroughts, A. K. (1993). M2 mitochondrial antibodies and urinary rough mutant bacteria in patients with primary biliary cirrhosis and in patients with recurrent bacteriuria. J. Hepatol., 17, 408–414. Forton, D. M., Patel, N., Prince, M., Oatridge, A., Hamilton, G., Goldblatt, J., Allsopp, J. M., Hajnal, J. V., Thomas, H. C., Bassendine, M., Jones, D. E., & Taylor-Robinson, S. D. (2004). Fatigue and Primary Biliary Cirrhosis: Association of globus pallidus magnetisation transfer ratio measurements with fatigue severity and blood manganese levels. Gut, 54, 587–592. Gershwin, M. E., Coppel, R. L., & MacKay, I. R. (1980). Primary biliary cirrhosis and mitochondrial autoantigens – Insight from molecular biology. Hepatology, 8, 147–151. Hopf, U., Moller, B., Semerowicz, R., Rodloff, A., Lobeck, H., Freudenberg, M., Galanos, C., & Huhn, D. (1989). Escherichia coli rough (R) mutants in the gut and lipid A in the liver from patients with primary biliary cirrhosis (PBC). Lancet, 2, 1419–1422. Invernizzi, P., Miozzo, M., Battezzati, P. M., Bianchi, I., Grati, F. R., Simoni, G., Selmi, C., Watnik, M., Gershwin, M. E., & Podda, M. (2004). Frequency of monosomy X in women with primary biliary cirrhosis. Lancet, 363, 533–535. Leuschner, U., Guldutana, S., Imhof, M., & Leuschner, M. (1992). Ursodeoxycholic acid does not cure primary biliary cirrhosis but prolongs survival. Results of a 3–11 years’ study. Hepatology, 116, 192A. Matsumura, S., Van de Water, J., Leung, P., Odin, J. A., Yamamoto, K., Gores, G. J., Mostov, K., Ansari, A., Coppel, R. L., Shiratori, Y., & Gershwin, M. E. (2004). Caspase induction by IgA antimitochondrial antibody: IgA-mediated biliary injury in primary biliary cirrhosis. Hepatology, 39, 1415–1422. Stemorowicz, R., Hopf, U., Moller, B. et al. (1988). Are antimitochondrial antibodies in primary biliary cirrhosis induced by R (rough)-mutants of Enterobacteraceae? Lancet, ii, 1160–1170. Swain, M. G., & Maric, M. (1995). Detective corticotrophin releasing hormone mediated neuroendocrine and behavioral responses in cholestatis rats: Implications for cholestatic liver disease-related sickness behavior. Hepatology, 22, 1560–1564. Walker, J. G., Doniach, D., Roitt, I. M., & Sherlock, S. (1965). Serological tests in diagnosis of primary biliary cirrhosis. Lancet, 1, 827–831. Watson, R., Angus, R. W., Dewar, M., Gross, B., Jewell, R. B., & Smallwood, R. A. (1995). Low prevalence of PBC in Victoria, Australia. Gut, 36, 927–930.

15.

CHRONIC ACTIVE HEPATITIS

Ian G. McFarlane HISTORICAL PERSPECTIVE The term “chronic active (or aggressive) hepatitis” (CAH), often used interchangeably with “active chronic hepatitis,” owes its origins to World War II. Up to 10% of soldiers returning from the Mediterranean theatre who had contracted (presumed viral) hepatitis were found to be slow to recover and many continued to have signs and symptoms of liver disease long after the initial acute phase (Mackay & Tait, 1994; McFarlane & Williams, 1996). Other studies in the immediate post-war years documented persistence of clinically diagnosed acute viral hepatitis and noted progression to cirrhosis in a high proportion of cases. Saint and colleagues (1953) in Australia were the first to coin the term “active chronic hepatitis” to describe this aggressive form of protracted hepatitis. Prior to this, the main causes of cirrhosis had been considered to be excessive alcohol consumption, toxic liver damage (including iron and copper overload), and chronic biliary obstruction. An infectious agent as a possible etiologic factor had been only suspected. At about this time, reports were beginning to appear of cases of a severe form of fluctuating hepatitis that mainly affected young females, and which was associated with marked elevations in serum globulin concentrations, amenorrhea, hirsutism, acneiform rashes and spider angiomas. The first description of this syndrome is usually attributed to the Swedish physician Waldenstr¨om but there are several reports suggesting that the condition had been recognized earlier (Mackay & Tait, 1994; McFarlane & Williams, 1996). The post-war years were a period of intense interest in the concept of autoimmunity, and in diseases such as rheumatoid arthritis and systemic lupus erythematosus (SLE) which were beginning to be considered

The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 399–425 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15015-0

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to have a probable basis in loss of immunologic self-tolerance. It was noted that patients with this form of chronic hepatitis often had circulating lupus erythematosus (LE) cells – phagocytic leukocytes containing large Feulgen-positive cytoplasmic inclusions comprising degraded nuclear material complexed with antinuclear antibodies and complement components. Mackay and colleagues proposed that the syndrome should be termed “lupoid hepatitis,” although they were careful to distinguish it from SLE (Mackay et al., 1959). Nonetheless, the disease was still thought to have a viral etiology and throughout the 1950s and early 1960s most authorities did not discriminate between “lupoid” hepatitis and CAH, which was often termed “active chronic viral hepatitis.” Other terminology in use for “lupoid” hepatitis at that time included “juvenile cirrhosis” and “chronic plasma cell hepatitis.” The discovery in the mid-1960s of a marker of serum hepatitis, the “Australia antigen” (later to be recognized as part of the surface envelope of the hepatitis B virus and designated as HBsAg), made it possible to distinguish between patients with CAH with and without chronic hepatitis B. This led to adoption of the designations “HBsAg-positive” and “HBsAg-negative” as prefixes for CAH, with “lupoid hepatitis” often being relegated to the latter category along with all other forms of “non-B” chronic hepatitis. During this period, it was found that some patients with CAH responded to treatment with corticosteroids (vide infra) and for a time HBsAg-negative CAH was further sub-classified as “steroid-responsive” or “steroid non-responsive.” The morphological features of CAH were also becoming more clearly defined. The characteristic histologic picture was recognized to be a portal and periportal lymphocytic infiltration with disruption of the limiting parenchymal plate and what Popper (Popper et al., 1965) described as “piecemeal necrosis” of periportal hepatocytes (see Fig. 1). Popper was later to distinguish between this lymphocytic piecemeal necrosis and what he termed “biliary piecemeal necrosis” seen in patients with chronic cholestatic disorders (Popper, 1978), in which the inflammatory infiltrate includes vacuolated (“foamy”) macrophages often containing bilirubin. In this form also the hepatocytes frequently contain deposits of copper and copper-binding protein. By the late 1960s, with increasing use of percutaneous liver biopsy and biochemical liver tests as diagnostic tools, it became apparent that some patients with CAH had a milder, seemingly less aggressive, form of chronic hepatitis which was described as “chronic persistent hepatitis” (CPH). The latter was defined morphologically as a lymphocytic infiltrate in expanded portal tracts without disruption of the limiting plate or piecemeal necrosis. Recognition that CAH was heterogeneous in terms of both etiology and severity led Geall and colleagues (1968) to propose the all-encompassing term “chronic active liver disease”

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Fig. 1. Liver Biopsy Showing the Characteristic Histologic Features of Interface Hepatitis (Chronic Active Hepatitis). Note: The dense lymphoplasmacytic infiltrate in the portal tract on the left, spilling out into the surrounding parenchyma with disruption of the limiting plate (interface) and piecemeal necrosis of periportal hepatocytes, with little inflammation and no necrosis in the perivenular area on the right.

(CALD), which was adopted for a time. The year 1968 also saw the first formal attempt, by an international panel of liver pathologists (De Groote et al., 1968), to classify chronic hepatitis. The distinction between CAH and CPH was reinforced. Although this panel did not include piecemeal necrosis in their classification, it was regarded as an important morphological feature and later became the histologic hallmark of CAH (International Group, 1977). It was generally agreed that, other than in hepatitis A (vid´e infra) in which piecemeal necrosis is common but progression to chronicity does not occur, this feature was usually associated with transition to chronic hepatitis. A third category, “chronic lobular hepatitis” (CLH), was proposed by Popper and Schaffner (1971) to define patients with intralobular (intraacinar) pathologic changes (spotty necrosis and inflammation) similar to those seen in acute viral hepatitis, although this was not universally accepted (International Group, 1977). Popper himself later challenged the significance of piecemeal necrosis and suggested that the extent, type, duration and etiology of lobular changes were the main factors determining the outcome of chronic hepatitis (Popper, 1983). It must be noted, however, that lobular hepatitis is a histologic definition based on observations obtained from a two-dimensional microscopic view of a thin section

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of a liver biopsy. Account is not usually taken of the three-dimensional structure of the liver lobule, which would require examination of multiple serial sections of larger specimens. Thus, the focal intraacinar changes that characterise CLH might represent spill-over of a necroinflammatory infiltrate from an extensive periportal lesion lying above or below the plane of the histologic section. The importance of lobular changes in disease progression envisaged by Popper (1983) might therefore be related to severity of periportal piecemeal necrosis. This issue remains unresolved. Following the identification of the hepatitis A virus (HAV) in the early 1970s, it became apparent that HAV and HBV infections could not be implicated in many patients with acute or chronic liver disease of presumed viral etiology. This gave rise to the concept of “non-A, non-B” (NANB) hepatitis (Dienstag, 1983), and NANB-CAH or -CPH (or NANB-CALD) was used for many years as a catch-all category for patients with what was also described as “idiopathic” or “cryptogenic” chronic liver disease, often including those with “lupoid” hepatitis. Subsequent events have shown this to be a prudent precaution because, with the discovery of the hepatitis C virus (HCV) in 1989, it became clear that a significant proportion of such patients have chronic hepatitis C (Reesink, 1998). Nonetheless, the terms CAH and CPH continued to dominate the field until recently. Although these were entirely morphological definitions the implied differences in prognosis, and therefore in clinical management, led to their adoption as clinical entities.

DEVELOPMENT OF CURRENT NOMENCLATURE By the 1980s it had become clear that the defining morphological changes of CAH can be seen at various stages in a wide range of liver disorders (see Table 1), in many of which the diagnostic criteria for CAH are not fulfilled. But, somewhat surprisingly, rather than questioning the validity of CAH as a syndrome, this

Table 1. Some of the Liver Disorders in Which the Morphological Features of Interface Hepatitis (Formerly Chronic Active Hepatitis) May be Seen with Variable Frequency. Acute or chronic viral hepatitis Alcoholic liver disease Alpha-1-antitrypsin deficiency Autoimmune hepatitis

Drug-induced hepatitis Primary biliary cirrhosis Primary sclerosing cholangitis Wilson’s disease

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led to expansion of the list of diseases considered to be capable of causing it (Ludwig, 1993). The previously accepted view that CPH was associated with a good prognosis was also being challenged, because it was found that transitions can occur between patterns of clinical and histologic activity that affect outcome (Czaja, 1981). Furthermore, it was noted that the distinction between CAH and CPH could not be applied in many patients with NANB hepatitis (Dienes et al., 1982) and that CPH following treatment-induced remission of some forms of CAH did not preclude recrudescence of active disease (Czaja et al., 1981). Other criteria for CAH were also found to be untenable. A requirement for markedly elevated serum aminotransferases could not be upheld because it was recognized that these enzymes are poor correlates of histologic activity in chronic liver disease and that mild elevations of aminotransferases do not exclude severe disease (McFarlane, 2002). The temporal criterion for duration of disease of at least six months (to distinguish chronic from acute liver disease) also proved difficult to apply because it is often not possible to define the time of onset, and patients presenting with acute hepatitis who had clear evidence of chronic liver disease were being identified (Burgart et al., 1995; Nikias et al., 1994). The importance of lobular changes envisaged by Popper (1983) was also called into question by the observation that portal-portal or central-portal bridging necrosis (confluent necrosis) appears to be the determining factor in progression of CAH to cirrhosis (Cooksley et al., 1986). However, the suggestion by Cooksley et al. (1986) that the presence or absence of bridging necrosis should determine whether or not to institute appropriate therapy is probably not tenable, since the disease tends to fluctuate and the absence of bridging necrosis at one time point does not preclude its later development. The above observations prompted many hepatologists and hepatopathologists around the world to propose radical changes in the nomenclature of chronic liver diseases, with particular reference to CAH and CPH (Czaja, 1993; Desmet et al., 1994; Gerber, 1992; Johnson & McFarlane, 1993; Ludwig, 1993; Ludwig et al., 1995; Scheuer, 1995). By the mid-1990s a consensus view had emerged, the essentials of which are that: (1) Use of the terms “chronic active hepatitis (CAH)” and “chronic persistent hepatitis (CPH)” should be discontinued. (2) They should be replaced by precise morphological descriptions, graded for necroinflammatory activity and staged for degree of fibrosis. (3) The term “periportal hepatitis” or, preferably, “interface hepatitis” (with or without bridging necrosis) should be used to described the changes previously associated with CAH. (4) The term “piecemeal necrosis” may be retained.

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(5) Description of the changes previously associated with CPH should employ terms such as mild or moderate, portal or periportal hepatitis (as appropriate) without significant necrosis. (6) The above should be qualified by etiologic designations (e.g. autoimmune hepatitis, chronic hepatitis B, C, D, etc.), wherever possible and practicable. These recommendations have been generally accepted, and the terms CAH and CPH have now largely fallen into disuse. The recommendations are therefore observed in the following discussion of the chronic liver diseases which were formerly regarded as comprising the spectrum of conditions associated with CAH.

GENERAL FEATURES OF CHRONIC LIVER DISEASE Any discussion of chronic liver disease must take account of the wide range of signs and symptoms (many of which may not immediately suggest an underlying hepatic abnormality) that are common to most chronic liver disorders. The most frequent complaints are lethargy, often extreme fatigue, accompanied by feelings of general malaise. The cause of the fatigue is unknown but functional changes in the hypothalamic-pituitary-adrenal axis, altered neurotransmission, or disturbances of sleep patterns due to disease-associated complications (e.g. severe pruritus) or anxiety, have all been invoked (Cauch-Dudek et al., 1998; Jones, 1995). Other frequent symptoms include persistent or intermittent nausea, anorexia (and consequent weight loss), general abdominal discomfort (with or without pain), pruritus and/or skin rashes, arthralgia and/or myalgia, fluctuating low grade pyrexia, recurrent epistaxes, and menstrual irregularities in women. However, a significant proportion of patients seen in major referral centers today have either been identified through routine health screening programs and are entirely asymptomatic or their liver disease has been revealed during investigation of some other condition (Gordon, 1998; McFarlane, 2002). Jaundice is the most obvious sign of liver disease, but anicteric acute or chronic hepatitis is now well recognized. Today many patients diagnosed with chronic liver disorders have no history of jaundice while, in others, it is a relatively late feature associated with advanced disease. Other physical signs such as hepatomegaly, splenomegaly, ascites, peripheral edema, and cutaneous stigmata (e.g. spider angiomas) are common but vary in frequency and may be absent. Many patients already have cirrhosis at accession, indicating that they must have had their disease for some time before it manifest itself, and occasionally patients may even present with a hematemesis and/or melena due to bleeding from gastric or esophageal varices secondary to portal hypertension as the first sign of their underlying liver disorder.

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Biochemical liver test abnormalities in chronic liver disease are also very variable and do not reliably reflect the severity of tissue damage, but the overall pattern may suggest the cause of the disorder. It is customary to classify these abnormalities as either a “cholestatic” pattern (elevated serum alkaline phosphatase and gammaglutamyl transferase, and in some cases also bilirubin, out of proportion to any elevation of aminotransferases) which is usually indicative of a biliary disorder, or a “hepatitic” pattern (elevated serum aminotransferases with or without an elevation in bilirubin, but with normal or only moderately raised alkaline phosphatase and gammaglutamyl transferase) which is more suggestive of parenchymal liver damage. Abnormalities in prothrombin time and other markers of coagulopathy usually reflect decreased synthetic function of the liver associated with parenchymal damage. Low serum albumin concentrations also reflect diminished synthetic function but, in chronic liver disorders, are generally a later feature of advanced disease. Abnormalities in other biochemical parameters may provide clues to etiology (vid´e infra).

DISEASES ASSOCIATED WITH INTERFACE HEPATITIS (FORMERLY CAH) Autoimmune Hepatitis An international panel has defined autoimmune hepatitis (AIH) as: “an unresolving, predominantly periportal hepatitis, usually with hypergammaglobulinemia and tissue autoantibodies, which is responsive to immunosuppressive therapy in most cases” (Ludwig et al., 1995). This broadly corresponds to the original descriptions of “lupoid” hepatitis (vid´e supra). It is a disease of unknown etiology but is presumed to have a basis in aberrant autoreactivity underlying progressive destruction of the hepatic parenchyma, often leading to cirrhosis. There appears to be a genetic predisposition for susceptibility to AIH (Czaja & Donaldson, 2000) and it is therefore regarded as a priori chronic. It is a relatively rare condition but it is important to make the diagnosis because it is one of the few chronic liver diseases that can usually be very successfully controlled by drug therapy, and failure to institute appropriate treatment can have serious consequences (Heneghan & McFarlane, 2002). Nonetheless, a true cure is rarely (if ever) achieved, ergo it is “unresolving.” Diagnosis AIH should be suspected in any patient with an unexplained hepatitic illness associated with hypergammaglobulinemia. It can present at any age and affects both sexes, although the large majority of patients are above forty years of age and

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females predominate (4:1) (Alvarez et al., 1999). Diagnosis requires the finding of a suggestive pattern of clinical and laboratory abnormalities together with careful exclusion of other liver disorders that are associated with similar abnormalities (see Table 1). There are no specific signs or symptoms (other than those of acute or chronic liver disease generally), but suspicion should be heightened if there is a skin rash, oligomenorrhea in females, or a history of other autoimmune conditions (thyroid disease, diabetes, rheumatoid arthritis, vitiligo) in the patient or the family. In about 50% of cases, onset is insidious, with symptoms or signs that fluctuate with a periodicity of a few weeks to many months. A further 30% of patients present with an acute hepatitis that can mimic acute viral hepatitis clinically. Two thirds of these symptomatic patients will be jaundiced or report prior icteric episodes. In the remaining 20% of patients with AIH, the disease is “asymptomatic” – in the sense that there are initially no obvious signs or symptoms of liver disease (Alvarez et al., 1999; Gordon, 1998; McFarlane, 2002). The serum biochemical liver test abnormalities show a typically hepatitic pattern. However, the aminotransferase activities and bilirubin concentrations vary widely and do not reliably reflect severity of disease. Thus, low values do not necessarily indicate mild or inactive disease nor mitigate against a diagnosis of AIH (McFarlane, 2002). The hypergammaglobulinemia in AIH is due to a selective increase in the immunoglobulin G (IgG) fraction. Approximately 80% of patients have significant serum titers of antinuclear (ANA) or anti-smooth muscle (SMA) autoantibodies, or both, and about 3–4% overall have liver-kidney microsomal antibodies (anti-LKM1) in their sera. Most of the remaining 20% have one or more of a range of other autoantibodies, including perinuclear antineutrophil nuclear antibodies (pANNA) (Terjung & Worman, 2001), thyroid antibodies or rheumatoid factor (even in patients with normal thyroid function or without clinically significant rheumatic disease), and several other autoantibodies that are more specifically related to the liver but for which tests are not yet widely available (Alvarez et al., 1999). Although there have been occasional reports of antimitochondrial antibodies (AMA) in the sera of apparently genuine cases of AIH (Gregorio et al., 1997a), in view of the strong association of these autoantibodies with primary biliary cirrhosis (vide infra), it is generally recommended that AMA-positive patients should not be considered to have AIH (Alvarez et al., 1999). Several attempts have been made to classify AIH according to patients’ autoantibody profiles, but this is controversial (McFarlane, 1998). Nevertheless, it has become the convention to classify the disease broadly into two main sub-divisions: Type 1 (ANA and/or SMA positive) and Type 2 (anti-LKM1 positive). Type 1 may present at any age, whereas Type 2 is mainly confined to children and young adults. Clinically, there is little difference between Type 1

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and Type 2 in young people (Gregorio et al., 1997b) but there is evidence to suggest that they may represent two pathogenetically distinct groups of patients (McFarlane, 2002). AIH is associated with an increased frequency of inheritance of the human leukocyte antigen (HLA) haplotype A1-B8-DR3 and particularly with the DR3 and DR4 allotypes. These markers are not diagnostic in themselves, because they are also increased in frequency in other autoimmune disorders and occur in healthy individuals, but their presence adds weight to the diagnosis. They are also potentially useful as prognostic indicators. The DR3 and DR4 allotypes are independent risk factors for AIH and are associated with different clinical expressions of the disease (Czaja & Donaldson, 2000). The DR3 allotype tends to be associated with a younger age at onset, but DR3-positive patients of any age usually have more severe disease which is more difficult to control with immunosuppressive therapy and generally have a less favourable outcome, while DR4-positive patients usually present at an older age (>40 years) with generally milder disease and show a more rapid and complete response to treatment (McFarlane, 1998, 2002). A diagnosis of AIH should not be made without histologic examination of a liver biopsy, if at all practicable (Alvarez et al., 1999). The morphological features are those of interface hepatitis with a predominantly lymphoplasmacytic necroinflammatory infiltrate. In severe cases, a lobular hepatitis with bridging necrosis and formation of liver cell rosettes is often seen. As noted above, these changes are not pathognomonic of AIH but they reinforce the diagnosis. Patients with cholangiolitic changes reminiscent of biliary diseases (vide infra) should not be classified as having AIH. Other changes, such as lymphoid aggregates, bile ductule replication, or deposits of copper or iron may sometimes be present and do not necessarily mitigate against AIH unless they are particularly prominent (Alvarez et al., 1999). Treatment and Prognosis Standard therapy with oral corticosteroids (prednisone or prednisolone at approximately 0.5 mg/kg/day) induces remission in 80–95% of cases (Heneghan & McFarlane, 2002). If azathioprine is added, the dose of corticosteroids required to maintain remission can be substantially reduced (2.5–7.5 mg/day) or, in many cases, may be withdrawn and remission can be sustained with azathioprine alone. Although most patients respond to standard therapy, there is considerable variation in response and the doses of the two drugs required to induce and maintain remission therefore need to be titrated for each patient individually. Most leading authorities agree that, once remission is induced and the maintenance doses are established, patients should continue on the maintenance regimen for at least one

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year before any further changes in treatment are contemplated. Certainly, early withdrawal of treatment is associated with a high rate of relapse (Kanzler et al., 2001) and many patients require some form of immunosuppressive therapy in the longer term, perhaps for the rest of their lives (Johnson et al., 1995). Several other immunosuppressive drugs and other agents have been tried for treating AIH, mainly selectively as alternative therapy in the small proportion of cases that do not respond satisfactorily to steroids and azathioprine (Heneghan & McFarlane, 2002). Experience with these alternative therapies is still limited and none has yet been shown to offer significant advantages over standard therapy for the majority of cases. Very little is known about the true natural history of AIH because, since the advent of effective therapy thirty years ago, most patients are treated at an early stage – which radically alters the course of the disease. Prior to that, it was considered to be a particularly aggressive disorder which rapidly progressed to cirrhosis, and up to 80% of untreated patients died within five years of diagnosis (Heneghan & McFarlane, 2002). In part, this high mortality may have been related to diagnosis of the condition only in patients with severe AIH, with those with milder disease going unrecognised. Today the 10 year mortality for carefully managed patients is less than 10% (even if cirrhosis has developed), and reports from several specialist centers suggest that survival is not significantly different from that of the normal population matched for age and gender (Kanzler et al., 2001; Roberts et al., 1996; Schvarz et al., 1993). Furthermore, there is evidence to suggest that careful attention to maintenance of remission can lead to significant regression of hepatic fibrosis (Cotler et al., 2001; Dufour et al., 1997; Schvarz et al., 1993). For patients who are refractory to standard therapy and for those with end stage disease, liver transplantation is a viable treatment option. However, it seems that AIH recurs in about 30% of cases within three years (Manns & Bahr, 2000). Hepatocellular carcinoma, which is an important late sequel in other forms of chronic liver disease, is very rare in AIH (even in patients with long-standing cirrhosis) unless there is a superimposed chronic hepatitis virus infection (Ryder et al., 1995).

Chronic Hepatitis B and D The hepatitis B (HBV) and D (Delta) viruses are transmitted parenterally. The latter is a defective RNA pathogen which is wholly dependent on HBV and is acquired either as a superinfection in an individual already carrying HBV or as a coinfection with the initial HBV inoculum (Negro & Rizzetto, 1995). Worldwide there are about 350 million people chronically infected with HBV, the large majority of

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whom acquired the virus perinatally, but expanding screening and vaccination programmes are beginning to reduce the incidence of new infections. It is generally thought that about 5% of adults infected with HBV develop chronic infections but a long-term follow-up study of a large number of soldiers infected through contaminated yellow fever vaccine during World War II suggests that the proportion may be lower (Norman et al., 1993). Delta virus infections are most commonly seen in intravenous drug users, but the incidence has declined markedly in recent years (Gaeta et al., 2000). The spectrum of liver disease in subjects chronically infected with HBV varies widely. Many carriers will remain “healthy,” with normal biochemical liver tests and minimal changes on liver biopsy, while others will have a severe interface hepatitis with lobular changes which rapidly progresses to cirrhosis. It was this observation that mainly led to the earlier development of the concept of CPH and CAH as two distinct syndromes with different prognoses. It is now recognized that the HBV carriage state is a dynamic condition which is strongly influenced by fluctuations in the interaction between viral activity and the host immune response to the virus, and that transitions occur between “CAH” and “CPH” – which probably represent the two extremes of a continuous spectrum of liver disease. Patients with concomitant Delta virus (HDV) infections usually have more severe disease (Negro & Rizzetto, 1995). Diagnosis Diagnosis of chronic hepatitis B usually depends on the finding of persistence of hepatitis B surface antigen (HBsAg) and the immunoglobulin M isotype of antibodies (IgM-HBcAb) against the HBV “core” antigen (HBc) in the blood (Badur & Akgun, 2001). Early in the course of the disease, patients will usually also be seropositive for the HBV “e” antigen (HBeAg), which is the soluble form of HBc. This phase may or may not be associated with significant liver damage manifest by elevated serum aminotransferase activities, but patients will be highly infectious. After some time, which may extend to several years, most individuals will seroconvert and develop antibodies (HBeAb) to HBeAg. Seroconversion is often immediately preceded by a “flare” of the aminotransferases. Hepatic inflammation then usually decreases and biochemical liver tests tend to return towards normal but the patient often remains HBsAg positive. The development of HBeAb is considered to herald a decrease or cessation of active viral replication and, consequently, to be associated with a reduction in the potential to transmit the virus. This pattern of events is, however, not invariable. It is possible for active viral replication to continue for some time after seroconversion has occurred, as evidenced by persistence of HBV genomic material (HBV-DNA) in the blood

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or the liver – measurement of which is therefore the most reliable indicator of continuing infection. Indeed, HBV-DNA can be detected in the serum, peripheral blood mononuclear cells or livers of some individuals several decades after disappearance of the usual markers of ongoing active infection (Blackberg & Kidd-Ljunggren, 2000; Br´echot et al., 2001). The clinical and biochemical features of chronic hepatitis B depend on the stage and severity of liver damage. As with other forms of chronic liver disease, there are no specific signs or symptoms and biochemical liver test abnormalities can vary widely. While patients with abnormal serum aminotransferase activities will usually have some degree of interface hepatitis, normal values do not necessarily indicate histologic inactivity. The histologic picture of interface hepatitis is indistinguishable from that shown in see Fig. 1, although there may also be lobular necroinflammation and hepatocytes containing large amounts of HBsAg show a typical “ground glass” appearance in liver sections stained with hematoxylin and eosin (Ludwig, 1992). Concomitant Delta virus infection is generally diagnosed by testing for serum antibodies against a specific viral protein, the Delta antigen (HDAg). It is customary to test for total anti-HDAg, i.e. IgG, IgM and IgA class antibodies together, but identification of the individual isotypes may provide important information about the nature of the infection (Negro & Rizzetto, 1995). Thus, the finding of IgG anti-HDAg alone indicates recovery from a previous infection, while persisting high titers of IgM anti-HDAg are indicative of chronic infection, and IgA anti-HDAg seems to be associated with active liver necroinflammation (McFarlane et al., 1991). Two other serum markers of HDV infection, HDAg itself and the viral genomic material (HDV-RNA), the presence of which indicate viremia, can be helpful in diagnosis (Negro & Rizzetto, 1995). However, for technical reasons, tests for HDAg and HDV-RNA are not routinely employed by most diagnostic laboratories. Delta virus superinfection is usually associated with an exacerbation of the underlying HBV-related liver disease. HDV suppresses replication of HBV and superinfections are accompanied by a marked decline in titers, and even disappearance, of the various serum markers of HBV infection (HBsAg, IgM-HBcAb, HBeAg, HBV-DNA). This can give a false impression of spontaneous clearance of HBV when, in fact, superinfections often lead to chronic Delta hepatitis which has a worse prognosis (Negro & Rizzetto, 1995). Treatment and Prognosis The question of whether or not to treat patients with chronic hepatitis B, and if so how, has been the subject of much debate. Prognosis is related to the duration and severity of liver damage but is unpredictable and depends also on whether there

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are concomitant infections with HDV or other viruses, which are associated with more severe disease. Most patients with interface hepatitis and cirrhosis remain relatively stable for many years before decompensating, with development of ascites, jaundice, hepatic encephalopathy or variceal bleeding, but there is also a definite risk for development of hepatocellular carcinoma (HCC). Whether HBV inherently has oncogenic potential is still uncertain. In most cases of HCC in chronic hepatitis B, the malignancy has developed against a background of long-standing cirrhosis. But cirrhosis is itself an independent risk factor for HCC (Fattovich et al., 1995; Zaman et al., 1985). The Delta virus does not appear to play a pathogenetic role in development of HCC (Fattovich et al., 1995). Over the years, a wide variety of immunomodulatory and antiviral therapies have been tried for treating chronic hepatitis B but none has so far proved effective for complete virus eradication in the majority of patients. As in AIH, corticosteroids reduce the hepatic parenchymal necroinflammatory activity in patients with interface hepatitis. However, steroids are considered deleterious in chronic hepatitis B because they may suppress the host anti-viral response and they enhance replication of the virus, as evidenced by exacerbation of disease when treatment is stopped (Hoofnagle et al., 1986). The two agents that have been most widely used for treating chronic hepatitis B are interferon-␣ (IFN) and the nucleoside analogue lamivudine (Malik & Lee, 1999). With either therapy, a satisfactory response (in terms of disappearance of serum HBV-DNA and/or HBeAg/HBeAb seroconversion) is obtained in only about one third of patients, and neither treatment seems to have a marked effect in patients with chronic Delta hepatitis (Lau et al., 1999). A recent report suggests that the response rate might be increased by using a combination of both agents (Schalm et al., 2000). However, each is associated with significant problems (Malik & Lee, 1999). The drawbacks of IFN include the need for parenteral administration and its side-effect profile. With lamivudine there is a major problem of drug resistance after one or two years of treatment. This is due to the development of mutations affecting the catalytic site (YMDD) of the virus’ reverse transcriptase by which it replicates itself and of which lamivudine is a potent inhibitor. These YMDD mutations involve substitution of valine (V) or isoleucine (I) for methionine (M) to yield YVDD or YIDD variants which are resistant to lamivudine and allow viral replication to proceed, sometimes resulting in acute exacerbations of chronic hepatitis B (Liaw et al., 1999). Liver transplantation is currently the only option for patients with advanced disease, but re-infection of the graft is almost universal. This is because the virus is harbored (and replicates) at numerous extrahepatic sites (Mason et al., 1993), and is very difficult to eradicate completely peri-operatively. Continuous passive immunoprophylaxis with HBsAb (HBIG) does, however, help to suppress viral (including HDV) replication post-transplantation (Shouval & Samuel, 2000).

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Chronic Hepatitis C Following the discovery of the hepatitis C virus (HCV) in 1989, it soon became apparent that this agent is the major cause of chronic “NANB” hepatitis worldwide (Reesink, 1998). The virus is transmitted parenterally. Maternal/fetal, sexual, or other close-contact transmission does occur but, overall, the risk seems to be much lower than for HBV (Seef & Hoofnagle, 2002). However, in marked contrast to HBV, up to 85% of adults exposed to HCV become chronically infected and, with the success of screening and vaccination programmes for HBV, HCV is rapidly becoming the main cause of chronic hepatitis around the world. Chronic hepatitis C is usually a slow, indolent disease and the majority of patients seem to remain entirely asymptomatic for many years. It is a fluctuating condition, both clinically and virologically, and there is wide variation in outcome between infected individuals (Seef & Hoofnagle, 2002). In some cases the infection seems not to lead to significant liver disease even after more than 20 years (Barrett et al., 2001) but, overall, about 20–30% of infected individuals will eventually develop cirrhosis within that timeframe. Compounding factors such as concomitant infections with other viruses or heavy alcohol consumption are usually associated with more severe and more rapidly progressive disease. Six distinct genotypes of the virus have been identified, which vary in geographical distribution and with severity of liver disease and responses to treatment (Reesink, 1998; Seef & Hoofnagle, 2002). Diagnosis In symptomatic cases, the clinical features differ little from those of other chronic liver diseases except with respect to the extrahepatic manifestations (purpura, Raynaud’s phenomenon, glomerulonephritis) of cryoglobulinemia (which occurs frequently but is often asymptomatic). The virus has also been associated with a number of other extrahepatic disorders including thyroiditis, polyarteritis nodosa, porphyria cutanea tarda, and Sj¨ogren’s syndrome (Seef & Hoofnagle, 2002). Diagnosis usually relies initially on the finding of a positive serum test for antibodies (anti-HCV) against the virus. These are not neutralising antibodies because they occur in conjunction with viral genomic material (HCV-RNA) in the blood (which is indicative of active infection), although they can persist after clearance of the virus. Seropositivity for anti-HCV will therefore often, but not always, indicate ongoing infection. By comparison with other viral infections, the level of viraemia in chronic hepatitis C is low and demonstration of active infection requires detection of HCV-RNA in serum by sensitive polymerase chain reaction (PCR) techniques. Seronegativity for anti-HCV and HCV-RNA does not, however, necessarily exclude ongoing infection. The virus is harboured both in the liver and

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at extrahepatic sites, notably the peripheral blood mononuclear cells (PBMCs), and it is possible for patients to be seronegative for both of these markers but to still have HCV-RNA detectable in their livers and/or PBMCs (Koskinas et al., 1995; Pereira et al., 1995; Ryder et al., 1995; Saleh et al., 1994; Schmidt et al., 1997). Some 20–40% of patients with chronic hepatitis C have circulating autoantibodies reminiscent of AIH (mainly ANA, SMA and anti-LKM1, albeit usually at low titers), which can present a diagnostic problem in differentiating between the two diseases and previously raised the question of whether perhaps AIH was related to HCV infection. This issue has now largely been resolved and it seems clear that the two diseases are distinct (Reesink, 1998), although very occasionally patients with AIH may also have HCV infection (Ryder et al., 1995). The clinical management of such patients is challenging. Serum biochemical liver tests are not reliable indices of disease activity in chronic hepatitis C. Other than in patients with severe and/or advanced disease, most parameters (such as bilirubin, alkaline phosphatase, albumin) are within the normal range. Elevations in serum aminotransferases are often mild and fluctuate. Although patients with raised aminotransferases tend to have significant liver damage, this is not always the case (Healey et al., 1995). Conversely, patients with persistently normal aminotransferases quite frequently have moderate (occasionally severe) hepatic necroinflammation (Healey et al., 1995; Puoti et al., 1997). Thus, without a liver biopsy it can be difficult to distinguish between “healthy” carriers of the virus and those with underlying liver damage. The histologic findings in chronic hepatitis C range from virtually normal liver in a small proportion of cases, through mild reactive hepatitis to moderate interface hepatitis. The latter is indistinguishable from that seen in AIH or chronic hepatitis B (see Fig. 1) but tends to be less severe and there are almost always additional morphological changes which, although not pathognomonic, point to the diagnosis. The latter include focal lobular necroinflammation, lymphoid aggregates, biliary changes, and micro- or macro-vesicular steatosis (Gerber, 1995). Treatment and Prognosis A detailed discussion of the treatment of chronic hepatitis C is beyond the scope of this chapter. Suffice it to say that several immunomodulatory and antiviral drug regimens have been tried. However, assessment of efficacy of therapy in chronic hepatitis C is difficult because, as noted above: (1) serum aminotransferases are unreliable indices of disease activity; and (2) seronegativity for HCV-RNA does not preclude persistence of the virus in the liver or at extrahepatic sites. Histologic assessment is the most reliable method but is limited with respect both to the possibility of sampling error and to the frequency with which it can be performed. Furthermore, due to the fluctuating nature of the condition, changes in some or all of

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the parameters may occur in untreated patients – including apparently spontaneous clearance of HCV-RNA from serum, liver and PBMCs (Saleh et al., 1994). Thus, it is sometimes impossible to say with certainty whether a “response” is due to the treatment or is simply part of the natural history of the infection. As with chronic hepatitis B, corticosteroids reduce the hepatic necroinflammatory activity but may be deleterious because they appear to enhance viral replication and there is a rebound of serum aminotransferase activities when treatment is stopped. Interferon-alpha (IFN) is the treatment that has been most widely used to date. Overall, between 30 and 50% of patients show some response to IFN in terms of reduction or normalization of serum aminotransferases, reduction or clearance of serum HCV-RNA, and/or improvement of hepatic fibrosis, but probably in less than 20% is this sustained after stopping treatment. However, recent studies using IFN in combination with the antiviral agent ribavirin (which on its own seems to have little effect on chronic HCV infection) are providing more promising results (Seef & Hoofnagle, 2002). The outcome of chronic hepatitis C is very variable. Most patients will have some degree of hepatic inflammation and fibrosis and, even in those with mild histological changes and persistently normal serum aminotransferases, cirrhosis can develop after many years (Yano et al., 1996). Nonetheless, because the interval between HCV infection and development of significant liver disease is very long, this slow progression means that it will not have a major impact on life expectancy in most cases. In the relatively small proportion (about 20%) of patients who develop more severe disease (with interface hepatitis), cirrhosis and its complications develop more rapidly. In these cases, there is a high risk of development of hepatocellular carcinoma (HCC). However, as with HBV, it is not known whether HCV is directly oncogenic and the available evidence suggests that HCC in chronic hepatitis C is related to development of cirrhosis.

Alcoholic Liver Disease The majority of individuals who consume excessive amounts of alcohol have, at worst, relatively benign hepatic steatosis which usually resolves with abstinence, but a small proportion develop the much more serious conditions of alcoholic hepatitis and/or “active” cirrhosis, sometimes with features of interface hepatitis (Crapper et al., 1983). There are, however, several morphological changes that distinguish these cases from other conditions associated with interface hepatitis. In contrast to AIH and chronic viral hepatitis, the inflammatory infiltrate contains a high proportion of polymorphonuclear leukocytes. Additionally, in most cases there is marked hepatocellular steatosis and lipogranulomas may be seen, alcoholic

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(Mallory’s) hyalin is typically present, and there is usually perivenular necrosis and often swelling of mitochondria (Ishak et al., 1991). Diagnosis usually depends on a clear history of excessive alcohol intake. Apart from the histologic changes there are no laboratory tests that are of sufficient specificity for diagnosis, but a number of abnormalities may be suggestive (McFarlane et al., 2000). Thus, the plasma gammaglutamyl transferase activity is usually increased out of proportion to other biochemical liver test abnormalities, the ratio of aspartate:alanine aminotransferase activity is usually greater than 2:1, and the serum cholesterol concentration is often increased. Haematological changes include increased erythrocyte sedimentation rate and mean corpuscular volume (often with a marked macrocytosis). Plasma vitamin B12 and folate concentrations tend to be decreased, while urinary coproporphyrins are usually increased. There may be hypergammaglobulinemia but, in contrast to AIH, this is due mainly to increased IgA concentrations. Autoantibodies may occasionally be present but usually only at low titer (McFarlane, 2000). Thus, the differential diagnosis from AIH is generally not a problem when account is taken of all of these parameters.

Alpha-1-Antitrypsin Deficiency Alpha-1-antitrypsin (AAT) is a protease inhibitor which occurs normally in serum. Deficiency of AAT is associated with pulmonary emphysema and often with liver disease, in which interface hepatitis may be a feature (Primhak & Tanner, 2001). The latter can be distinguished from interface hepatitis in other forms of liver disease by the finding of eosinophilic globules in hepatocytes that show positive staining with the periodic acid/Schiff reagent but are diastase resistant, and which represent accumulation of AAT within the cells. The concentration of AAT in the blood is determined by two alleles on chromosome 14, inherited in an autosomal co-dominant mode, which are identified phenotypically (Pi) by the electrophoretic mobility of the expressed protein. The commonest is the M phenotype, while deficiency states are most frequently associated with inheritance of the F, S or (particularly) Z phenotypes. Liver disease is seen most often in PiZZ homozygotes but also occurs in heterozygotes (FZ, MZ or SZ) (Primhak & Tanner, 2001). Diagnosis of liver disease due to AAT deficiency is usually straightforward. In addition to the morphological changes described above, the findings of a markedly reduced serum AAT concentration together with the PiZZ or other phenotypic combinations associated with deficiency are diagnostic. Abnormalities of serum biochemical liver tests vary with the underlying liver pathology but, in patients with interface hepatitis, show

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the typically hepatitic pattern. Autoantibodies are very rarely a feature. Outcome depends on severity of the liver disease (greatest in PiZZ homozygotes) and its duration. An increased risk of hepatocellular carcinoma has been reported but, overall, the outcome from the liver point of view is generally favourable (Sveger & Eriksson, 1995).

Drug-Induced Liver Disease The list of drugs and other chemical agents that can cause predictable or idiosyncratic reactions affecting the liver is exceedingly long and continues to grow (Zimmerman, 2000). Some drugs, such as the laxative oxyphenisatin (use of which has been discontinued), the antibiotic minocyline, and the non-steroidal anti-inflammatory agent diclofenac, can idiosyncratically induce hepatic disease with interface hepatitis, hypergammaglobulinemia and/or circulating autoantibodies that can be virtually indistinguishable from AIH (Gough et al., 1996; Lawrenson et al., 2000). Withdrawal of the drug usually results in complete resolution. However, the diagnosis in such cases may be missed if the drug is not suspected as a cause and fatalities have be recorded when an offending drug has not been withdrawn.

Biliary Diseases Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis are the two biliary disorders in which interface hepatitis is most often seen. The frequency of this feature seems to vary with the stage of the underlying biliary disease, tending to be more frequent in the early stages. Well organized lymphoid aggregates or granulomas surrounding damaged bile ducts are the classic histologic finding in PBC, whereas “onion-skin” fibrosis in portal tracts is characteristic of PSC. In both conditions, biochemical liver tests show a cholestatic pattern but serum aminotransferases may also be raised, especially in those with interface hepatitis. PBC is usually associated with elevated serum IgM concentrations (cf. AIH and alcoholic liver disease) and with circulating antimitochondrial autoantibodies, which are virtually pathognomonic, but 20–40% of PBC patients may also have antinuclear antibodies (Courvalin & Worman, 1997; Leung et al., 1997; Szostecki et al., 1997). Diagnosis of PSC can be more difficult, particularly in the early stages and in children (in whom the condition has been termed “autoimmune sclerosing cholangitis”) (Gregorio et al., 2001). Suggestive biliary changes may not be evident in liver biopsies, and hypergammaglobulinemia with

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circulating autoantibodies is not uncommon (Boberg et al., 1996; Gregorio et al., 2001). In these cases, the underlying biliary disease may be revealed only by cholangiography. Cases of PBC or PSC with features that overlap with those of AIH are generally grouped under the heading of “autoimmune cholangitis” or “autoimmune cholangiopathy” (Ben-Ari & Czaja, 2001). Many such patients, as well as others with PBC or PSC, will show some response to corticosteroids but the biliary lesions tend to progress (Ben-Ari & Czaja, 2001; McNair et al., 1998). Recent evidence suggests that ursodeoxycholic acid treatment may delay progression of PBC (Corpechot et al., 2002) but otherwise there is not yet any particularly effective treatment for either PBC or PSC, and liver transplantation is the only option for end stage disease.

Wilson’s Disease Wilson’s disease is an autosomal recessive disorder of copper transport characterized by the abnormal and toxic accumulation of copper in a number of organs, particularly the brain and liver, which usually manifests itself before 30 years of age in homozygotes (Schilsky & Sternlieb, 1999). There are no morphological features that are specific to Wilson’s disease. Steatosis, Mallory bodies, cuprinosis and increased copper-associated protein deposition are often seen, but failure to demonstrate these changes does not exclude the diagnosis. Between 5 and 10% present with histological features of interface hepatitis and, especially in children, there may also be hypergammaglobulinemia and circulating autoantibodies. It is essential to differentiate these cases from AIH because failure to promptly institute the appropriate therapy for either condition can lead to development of fulminant hepatic failure (and/or, in Wilson’s disease, neurological complications), with potentially catastrophic consequences (Johnson & Williams, 1991). The gene for Wilson’s disease, designated ATP7B, is located on chromosome 13 but the multiplicity of disease-specific mutations presents difficulties for genetic screening, and diagnosis still relies on a combination of clinicopathological findings (Schilsky & Sternlieb, 1999). Typically, the serum ceruloplasmin and total copper concentrations are very low, while free serum copper, 24-hour urinary copper excretion and total liver copper are high. These abnormalities can also occur in other liver disorders but when seen in conjunction with the presence of corneal copper deposits (Kayser-Fleischer rings) are virtually diagnostic of Wilson’s disease (Schilsky & Sternlieb, 1999). In the absence of Kayser-Fleischer rings, the diagnosis can be confirmed by measurement of urinary copper excretion following D-penicillamine challenge (Martins da Costa et al., 1992). Copper

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chelation with D-penicillamine or trientine therapy is very effective in most cases, although there are reports that corticosteroids are also effective in those with interface hepatitis (Johnson & Williams, 1991).

PATHOGENESIS OF INTERFACE HEPATITIS Although microscopically all hepatocytes appear similar, there is in fact marked functional heterogeneity across the liver lobule (Jungermann & Kietzmann, 2000). Blood entering the lobule via the portal tracts perfuses the hepatocytes unidirectionally. As the blood flows towards its exit via the central vein, solutes are exchanged with the liver cells in a sequential manner. Thus, the composition of the blood in the perivenular zone is quite different from that entering the periportal area. For example, periportal blood is rich in oxygen and ammonia. Oxidative metabolism and conversion of ammonia to urea occurs mainly in the periportal zone. Consumption of oxygen during the metabolic processes in this area leads to a fall in oxygen tension (of about 50%) as the blood flows through the lobule (Jungermann & Kietzmann, 2000). Ammonia concentration also decreases, with a concomitant increase in urea. Processes that do not require high oxygen tension, such as the metabolism of drugs and other xenobiotics, are performed mainly (or often exclusively) by the perivenular hepatocytes. Although all hepatocytes carry the genetic information required to perform all of these functions, metabolic zonation means that many genes encoding enzymes and other proteins involved in the various processes are switched on or off (sometimes constitutively) in cells in different parts of the lobule. Also, since many of the metabolic processes are mediated via receptors on the surfaces of the cells, there is variability in cell-surface expression of different receptors across the lobule. Direct, chemically-induced liver injury caused by drugs and other xenobiotics is typically associated with perivenular hepatocyte necrosis. This pattern of liver damage is thought to be due to toxic metabolites produced during the preferential metabolism of these chemical compounds by cells in this area of the lobule. In contrast, little is known about the mechanisms underlying the development of periportal hepatocellular necrosis, or interface hepatitis. In viral hepatitis, liver damage is thought to be due mainly to a host immune response against virus-infected cells (Chisari & Ferrari, 1995). However, this alone cannot explain periportal necroinflammation in patients with viral hepatitis, because virus-infected cells are not particularly concentrated in the periportal area but are scattered throughout the liver lobule (including areas where there is no apparent inflammation or necrosis) (Ballardini et al., 1995; Chisari & Ferrari, 1995; Ludwig, 1992; Nouri-Aria et al., 1995).

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It is possible that the development of interface hepatitis may be related to differences in oxygen tension across the liver lobules and zonal metabolic effects, or to cytokine gene expression and polymorphisms that may result in localized differences in cytokine production (Cookson et al., 1999; Simpson et al., 1997; Treichel et al., 1994), which may render periportal hepatocytes more susceptible to injury. However, there is little direct evidence to support this. An alternative hypothesis is that autoimmunity may have a role to play. It has long been recognized that there is an autoimmune component in several of the diseases in which features of interface hepatitis can be seen (McFarlane, 1991). Patients with AIH have autoantibodies against the hepatic asialoglycoprotein receptor (ASGPR), titers of which correlate with histological severity of interface hepatitis (McFarlane, 1996; Treichel & Meyer zum Buschenfelde, 1998). This receptor, which has been shown to be an important target autoantigen in AIH, participates in the binding and endocytosis of glycoproteins bearing terminal galactose residues and is unique to hepatocytes. Although it is present in all hepatocytes, there is evidence for selective differential cell surface expression of ASGPR in certain areas of the liver lobule. Precisely which hepatocytes display the receptor is the subject of some debate, but studies to investigate this in vivo under fairly physiological conditions have suggested that it is preferentially expressed at high density on periportal hepatocytes (McFarlane et al., 1990). The available evidence suggests that, in AIH, liver damage involves antibody-dependent cellular cytotoxic (ADCC) reactions in which autoantibodies against hepatocellular surface components cooperate with a non-T (K) lymphocyte subpopulation in cell lysis or inducing apoptosis, and that direct T cell cytotoxicity does not appear to play a major role (McFarlane, 1999). Thus, ADCC reactions against the ASGPR (or any other liver-specific autoantigen which is preferentially displayed on the surfaces of periportal hepatocytes in vivo) might account for the histological picture of interface hepatitis. Anti-ASGPR autoantibodies are also found with variable frequency in most of the other liver disorders in which interface hepatitis can be seen, including PBC, PSC, and chronic viral hepatitis (McFarlane, 1996; Treichel & Meyer zum Buschenfelde, 1998). However, extensive studies of the possible contribution of autoimmune mechanisms to hepatocellular damage in these conditions have so far been undertaken only for chronic hepatitis B. Direct T cell cytotoxic reactions against virus-infected hepatocytes are undoubtedly involved in chronic hepatitis B (Chisari & Ferrari, 1995) but, as in AIH, patients have circulating anti-ASGPR autoantibodies at titers that correlate with severity of interface hepatitis and they also show ADCC reactions in vitro against uninfected hepatocytes (McFarlane, 1991). A similar autoreactive mechanism to that proposed for AIH might therefore contribute to liver damage, particularly interface hepatitis, in chronic hepatitis B virus (HBV) infection. Support for this hypothesis has come from

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studies in woodchucks infected with woodchuck hepatitis virus (WHV) (Diao & Michalak, 1997; Diao et al., 1998). This is a well established model of human HBV infection, in which development of chronic hepatitis (similar to that seen with HBV), and often autoantibodies, is seen in a proportion of animals following WHV infection. In particular, woodchucks infected with WHV develop anti-ASGPR autoantibodies which are capable of inducing complement-mediated cytolysis of uninfected woodchuck hepatocytes in vitro (Diao & Michalak, 1997; Diao et al., 1998). Nonetheless, the evidence remains circumstantial and it is still not clear what are the precise mechanisms leading to development of interface hepatitis in patients with the various liver diseases associated with these histologic changes.

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HEPATITIS B VIRUS

F. Fred Poordad INTRODUCTION Hepatitis B Virus (HBV) and C (HCV) infections are two of the most common chronic viral infections in the world affecting roughly 600 million people worldwide. With over two billion individuals infected with HBV and approximately 400 million chronically infected, and hence, chronic carriers, it is the most common human hepatotropic viral infection (WHO data). It is responsible for a large number of liver-related deaths particularly in Africa and Asia, and is an ongoing epidemiologic problem, in spite of the availability of an effective vaccine. HCV, though, less common worldwide, is more prevalent in developed countries compared to HB. It is also a leading cause of liver related morbidity and mortality including liver carcinoma. While there is no primary preventive vaccine against it, there is an effective therapy (see Chapters 17 and 18). Treatment options for both of these viruses will continue to expand over the next decade and will undoubtedly become more complex. The aim of this chapter is to survey the molecular virology, natural history and treatment options for HBV. In 1947, MacCallum introduced the terms hepatitis A and hepatitis B so as to classify infections (epidemic) and serum hepatitis. Six years later the World Health Organization Committee on Viral Hepatitis adopted both terms. Subsequent to the discovery of Australian antigen (Au) in an Australian aborigine, it became apparent that Au antigen could only be found in the sera of patients infected with type B hepatitis. The antigen is today referred to as hepatitis B surface antigen (HBsAg). A second milestone in discovery was the work of Kaplan and his colleagues demonstrating that the virus-like particles present in the sera of patients infected with type B hepatitis is associated with DNA-dependent DNA polymerase. The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 427–438 © 2004 Published by Elsevier Ltd. ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15016-2

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Robinson and Greenman took this work one step farther by demonstrating that the DNA polymerase is situated in the core of the particle. Thus, the accepted view is that DNA-dependent DNA-polymerase is surrounded by the core particle (capsid), the surface coat of which constitutes an outer shell composed of protein (HBs). In other words, an outer shell surrounds the inner shell composed of HBc protein which constitutes the core particle. The HB virus is spheroidal in shape and approximately 42 nm in diameter. Upon attaching to a cell membrane, for example, that of a hepatocyte, the virus is transported into the cell interior, and then, into the nucleus. With viral DNA and its DNA polymerase in the nucleoplasm, the virus reproduces itself. Copies of the virus and excess antigen are released by the hepatocyte to the exterior, thereby reaching neighboring cells and the bloodstream. Mistakes in copying viral DNA may be made during reproduction, and hence, different strains (and mutants) may occur. A week or so after infection, it is possible to detect HBV in the bloodstream using the PCR method. However, this is not the case with core protein (HBcAg). The standard test involves the assessment of hepatitis B surface protein (HBsAg) which is produced in quantities larger than those required by the virus to reproduce. The presence of HBsAg in the bloodstream for periods in excess of 6 months is interpreted as indicative of chronic infection. Very little is known about “e” antigen (HBeAg), a peptide that is present in the blood when HBV is reproducing. Antibodies against the “e” antigen are found a few weeks after HBeAg disappears. This is considered as a good prognostic sign. The first detectable antibody following infection is HBcAb, whereas HBsAb is the last. Antibodies predominatly of type IgG against HBc appear in the bloodstream about two months after infection. They are not detectable after vaccination.

MOLECULAR VIROLOGY AND THE IMMUNE RESPONSE HBV is a DNA virus belonging to the Hepadnaviridae family, and is similar genetically to viruses that infect primates, squirrels, woodchucks and ducks (Ganem & Schneider, 2001). There are seven genotypes and four subtypes of HBV, with various insertions or deletions of nucleotides defining the genotypes, but with less than 10% variation overall between them (Kann & Gerlich, 1998). The genotypes (A–G) have pathogenic differences, with C and D causing more severe disease generally, and being less responsive to interferon therapy (Kao et al., 2002). The HBV genome consists of two linear DNA strands partially overlapping and double stranded, circular and 3200 nucleotides in length. The minus strand is open at the 5′ end where the DNA polymerase is bound and has four open reading frames

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(ORFs): Pre-S/S, Pre-C/C, Pol, and X(1). The ORFs overlap and multiple internal AUG codons allow for synthesis of sets of proteins from each ORF. Pre-core and core mutations are relatively common, especially in some endemic areas. HbeAg synthesis is governed by both the pre-core region and the X ORF, which houses the core promoter. Mutations in both regions are associated with changes in eAg production. The most common of these is the precore stop codon mutation at nucleotide position 1896 which completely stops eAg synthesis (Hadziyannis & Vassilopoulos, 2001). This mutation is not commonly seen in genotypes A and F and some strains of C. While this mutation was initially thought to be associated with more virulent disease, it is now seen increasingly in asymptomatic individuals. Core gene mutations are common, and can be seen in those with precore mutants, especially during the elimination phase of HBV, when escape mutations occur that make it difficult for cytotoxic T-cells to recognize HBV. In addition to the X gene mutations that can affect eAg, X mutations in hepatocellular carcinoma patients have implicated this area with carcinogenesis (Sirma et al., 1999). In the case of Pol mutations, they have largely been the product of pressure from nucleoside/nucleotide analog therapies. The best studied is lamivudine resistance which occurs in the YMDD locus in the Pol gene, and can result in the predominance of this strain after several weeks or months and can lead to worsening liver disease (Liaw et al., 1999). HBV is generally noncytopathic to the hepatocyte. Transmission occurs through parenteral routes, and incubation can be about one to six months, but generally within a few weeks. Only a third of patients develop overt signs of disease, with fewer than 20% having a serum sickness-like presentation with malaise, fever, and arthralgias followed by jaundice and right upper abdominal discomfort. The jaundice may persist for several weeks, along with fatigue and malaise. The aminotransferases (ALT) which are liver enzymes that are often relied upon by investigators, are generally elevated as with other acute hepatitides, and the bilirubin elevation follows this with profound jaundice in some patients. These values all normalize as sAg is lost in the recovery phase. In immune competent adults, fewer than 5% fail to clear virus spontaneously, whereas up to half of those infected between ages 1–5 may develop chronicity, and over 90% of those infected perinatally (Tassopopoulos et al., 1987). Interestingly, however, true viral eradication may never be possible, even in those with apparent antibody formation, since HBV DNA can be detected using sensitive PCR assays. Fortunately, fulminant liver failure occurs in less than 0.5% of cases, and is thought to be due to massive necrosis through immunologically mediated mechanisms. While the precore mutant strains had been thought to be often the cause of fulminant failure, this alone is unlikely to explain why some patients show this form of the

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Table 1. Terminology of HBV Infections. Terms

Description

Acute hepatitis B

Clinical or subclinical infection resulting in HBsAg and anti-core IgM + state with detectable HBV DNA Surface Ag loss, anti-HBc IgM loss, anti-HBc IgG+, +/− anti-HBs, DNAHBsAg+, ongoing necroinflammation with elevated ALT, DNA+, eAg + or − strain HBsAg+, HBeAg−, anti-HBe+, negative DNA, anti-HBc IgG+, normal ALT HBsAg+, DNA+, anti-HBc IgM and IgG+, HBeAg loss and anti-HBe +, DNA − Reappearance of eAg in anti-HBe + individual, DNA+/−

Recovery or convalescence from acute HBV Chronic HBV HBV Carrier (Asymptomatic) HBV Reactivation of Carrier state HBV e antigen seroconversion HBV e antigen reversion

disease. Table 1 defines the clinical terminology associated with HBV, and Fig. 1 reveals the typical serologic patterns in acute and chronic infections. Immunologic factors and inflammation that occur as a consequence of immune stimulation, result in a very high incidence of hepatocellular carcinoma

Fig. 1. Serologic Patterns in Acute and Chronic HBV Infection.

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(HCC). Half of HBV-related HCC cases occur in patients without cirrhosis, in contradistinction to hepatitis C, where cirrhosis almost always precedes HCC development (McMahon, 1997). HBV DNA sequences are found in HCC cells and is known to suppress p53 gene function through the X gene product, and activates multiple transcription factors including NF-␬B. The regeneration that occurs in the liver is also thought to lead to oxidative stress and oxygen free radical formation, resulting in DNA mutations and oncogenesis (Rehermann, 2003).

EPIDEMIOLOGY AND NATURAL HISTORY OF HEPATITIS B The significance of HBV as a world health issue is particularly striking in Asia, sub-Sahara Africa and parts of the South Pacific. Lesser affected areas but with still large numbers affected are the Middle East, and some indigenous populations in Alaska, Canada and Australia. Over 400 million individuals worldwide are chronically infected, but one third of the world’s 6 billion inhabitants have serologic evidence of past or present infection. At least one million die yearly of HBV related illness and this may be an underestimate (Mast et al., 1999). In the United States, roughly 1.2 million people are infected chronically, and there is a 5% lifetime risk for Americans to be exposed to hepatitis B. The modes of parenteral transmission of the highly infectious HBV include percutaneous, sexual and vertical/perinatal routes. The durability of HBV outside the body for more than one week allows for spread via contaminated inanimate objects. While immune competent adults rarely go on to have chronic infection, the majority of those infected under the age of 5, and especially neonates, develop chronicity. In areas with less than 2% prevalence of HBV, sexual contacts and drug use by injection are the main modes of spread, whereas in high prevalence areas, household contact and perinatal transmission are the biggest risks (Bernier et al., 1982). Nosocomial infections are a significant risk in developing countries as well. In these highly endemic areas, 90% of the population has had HBV exposure, and over 8% of the population is chronically infected. Immune suppressed individuals are more at risk of developing chronic infection as demonstrated in the HIV population (Bodsworth et al., 1989). Table 2 outlines who should be screened for hepatitis B. Given the various serologic patterns that can occur with HBV, there are some commonalities based on geography. In Asia, the majority of infections are perinatal, and hence due to immune tolerance, HBeAg is positive for many decades, but ALT is often normal in spite of elevated DNA levels (Lok & Lai, 1988). ALT may become elevated in later years. In Africa, the Mediterranean and Alaska, childhood transmission is common, and immune tolerance is not seen; hence ALT is elevated

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Table 2. Individuals to Screen for Hepatitis B. Pregnant women Household and sexual contacts of infected individuals Immune incompetence, any cause HIV-infected HCV-infected Dialysis patients Homosexual men High risk sexual contacts Any history of injection drug use Persons born in endemic areas

but HBeAg is more likely to seroconvert in early adulthood (Ruiz-Moreno et al., 1999). Adulthood infection is most common in developed countries through sexual spread and most will clear virus spontaneously. A large Alaskan prospective study followed 1,536 adults and children with HBV infection and found at 12 years that spontaneous HBeAg clearance occurred in 45% at five years, 80% at 10 years (McMahon et al., 2001). Although seroconversion is generally durable, up to one-fifth may have acute flares of hepatitis with or without reversion to their previous serologic status, and over time may develop insidious cirrhosis as a result of this process (Davis et al., 1984). Precore and core promoter mutations allow for HBeAg negative chronic HBV with elevated DNA and ongoing necroinflammation and fibrosis. This is prevalent in the Mediterranean regions and Asia, and is most commonly seen with HBV genotype D, and rarely with genotype A, seen mostly in Western countries (Lindh et al., 1997). As already mentioned, the most common precore mutation is a stop codon at position 1896, while the most common promoter mutations, A1762 T and G 1764 A, lead to decreased production of HBeAg (Buckwold et al., 1996). sSpontaneous clearance of HBsAg is rare and occurs in less than 1% of carriers yearly. Even in these individuals, sporadic serum DNA by polymerase chain reaction (PCR) assays is detectable, but likely not clinically significant. This may, however, predispose to development of hepatocellular carcinoma (HCC), since half of all HCCs associated with HBV occur in non-cirrhotic carriers (Liaw et al., 1991). Cirrhosis may develop at a rate of 3% per year, and is associated with the presence of HBeAg, older age and duration of disease, and ongoing necroinflammation as suggested by elevated ALT. While 5 year survival in the compensated cirrhotic is excellent at 71%, once decompensation occurs, survival falls to 14% at 5 years (De Jongh et al., 1992). Hepatitis D, or delta virus, is not commonly seen in Western countries, but rather in the Mediterranean. Superinfection of a chronically infected individual leads to

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chronic infection with both. There is a higher risk of cirrhosis, decompensation and HCC in these individuals.

DIAGNOSIS AND FOLLOW UP OF HBV Evaluation of HBV includes serologic evaluation and assessment for other viruses. HCV and HIV should be checked in all patients that have potential risk factors, along with HDV. Hepatitis A antibody screening should be done to determine who needs vaccination in areas where there is a high prevalence rate. In individuals that are not to undergo therapy, liver chemistries should be monitored every three months if stable, or more frequently in those with known advanced disease. Liver biopsy should be considered in every hepatitis B patient to assess degree of inflammation and fibrosis. Although quantification of DNA levels above 10 × 5 copies/mL have historically been used to define chronic infection (Lok et al., 2001), PCR assays of DNA can now detect below 10 × 3 copies/mL, and these assays have large supplanted the bDNA testing previously used. Transmission counseling is necessary to prevent spread of virus. Household and close contacts should be vaccinated after having serologic tests for HBsAg and anti-HBs. While individuals with higher DNA levels are more infectious, all carriers are potentially infectious to others through close contact. The issue of HCC surveillance is one that has an incomplete literature to base strong recommendations upon. The standard of care in many Western countries is to screen cirrhotics every six months with ultrasound (US) and ␣-fetoprotein (AFP). This, however, is not possible in all parts of the world. There has been only one randomized trial assessing this, and the follow up period was too short to allow conclusions to be drawn. Furthermore, there is no real consensus on how to follow non-cirrhotic HBV carriers, since they too are at risk for HCC. AFP alone is not very sensitive, but when followed over time it is more useful (McMahon et al., 2000). Other tumor markers have not been extensively studied, though des␥-carboxy prothrombin (DCP) and AFP may yield higher sensitivity for detecting HCC. Practice based studies using AFP and U.S. in combination suggest that screening every six months is a reasonably sensitive strategy to detect small HCC.

TREATMENT Treatment of HBV has undergone rapid change over the past several years and with the advent of newer nucleoside/nucleotide analog agents, this evolution will continue. The primary goal of any therapy is to suppress replication of virus, and

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ideally to clear virus. The hope with this strategy is to also normalize biochemical and histologic parameters. There are currently three FDA approved agents which are indicated for chronic HBV, usually for patients with an abnormal ALT. Response rates in HBeAg negative and positive patients is different. In HBeAg positive individuals, loss of eAg, and ultimately conversion to anti-HBe positivity is the ultimate measure of response. Clearance of HBsAg, or true clearance of virus, rarely occurs with short term nucleoside analogs and occurs in fewer than 10% of those treated with interferon as well. High ALT and low HBV DNA is a positive predictor of response to interferon. Table 3 illustrates the response rates to interferon, lamivudine and adefovir in HBeAg positive patients (Dienstag et al., 1999; Schalm et al., 2000). Data on the newer pegylated interferons is lacking, but early reports show promise that efficacy may be higher than standard interferon (Cooksley et al., 2003). In those with HBV/HDV co-infection, high dose interferon three times a week for one year has proven more effective than therapy with lamivudine (Lau et al., 1999). No data is available using adefovir for this population. Lamivudine ((-) enantiomer of 2′ 3′ dideoxy-3′ -thiacytidine) results in premature chain termination and inhibits HBV DNA synthesis. Extending lamivudine therapy beyond one year improves seroconversion rates of HBeAg to roughly 50% at five years in those who have not developed the YMDD mutation, which occurs in the majority of those treated beyond three years (Leung et al., 2001). Table 3. Comparison of HBV Therapies in eAg Positive Individuals. Interferon –24 wk (24–48 wk in eAg-) DNA loss eAg loss eAb conversion sAg loss

37% (70 in eAg neg) 33% 8% Lamivudine (52 wk)

DNA loss eAg loss eAg conversion sAg loss

44% (70 in eAg neg) 32% 18% 50 IU/l. A lower and asymptomatic infection rate of 0.8 per 100 person years was observed after immunization of health care workers in nephrology units who had antibody titres of < 50 IU/l (Courouce et al., 1988).

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The titer of vaccine induced anti-HBs declines, often rapidly, during the months and years following immunization. The highest anti-HBs titers are generally observed one month after booster vaccination followed by rapid decline during the next 12 months and thereafter more slowly (see, for example, Ambrosch et al., 1987; Gibas et al., 1988; Hilleman, 1984; Jilg et al., 1984; Nommensen et al., 1989; Wismans et al., 1989 and others). Mathematical models were designed and an equation was derived consisting of several exponential terms with different half-life periods. It is considered by some researchers that the decline of antiHBs concentration in an immunized subject can be predicted accurately by such antibody kinetics and preliminary recommendations before the next booster have been made (Ambrosch et al., 1987; Fagan et al., 1987a, b; Jilg et al., 1984; Nommensen et al., 1989 and others). If the minimum protective level is accepted at 10 IU/l, which is being debated, consideration should be given to the diversity of the individual immune response and the decrease in levels of anti-HBs as well as possible errors in quantitative anti-HBs determinations, then it would be reasonable to define a level of > 10 IU/l and < 100 IU/l as an indication for booster immunization. It has been demonstrated that a booster inoculation results in a rapid increase in anti-HBs titres within 4 days (Jilg et al., 1988). However, even this time delay might permit infection of hepatocytes (Nommensen et al., 1989). Several options are therefore under consideration for maintaining protective immunity against hepatitis B infection:  Relying upon immunological memory to protect against clinical infection and its complications (Centers for Disease Control, 1991, and reviewed in Tilzey, 1995), a view which is supported by in vitro studies showing immunological memory for HBsAg in B cell derived from vaccinated subjects who have lost their anti-HBs but not in B cells from non-responders (van Hattum et al., 1991), and, indeed, one cannot recall what has never been memorized (McIntyre, 1995).  Providing booster vaccination to all vaccinated subjects at regular intervals without determination of anti-HBs. This option is not supported by a number of investigators because non-responders must be detected (McIntyre, 1995; Tedder et al., 1993) and because while an anti-HBs titre of about 10 IU/l may in theory be protective, this level is not protective from a laboratory point of view since many serum samples may give non-specific reactions at this antibody level (Tedder et al., 1993; Westmoreland et al., 1990).  Testing anti-HBs levels one month after the first booster and administering the next booster before the minimum protective level is reached, which is the preferred option. A protective level of 100 IU/l seems to be appropriate. There are studies that hepatitis B vaccine provides a high degree of protection against clinical symptomatic disease in immunocompetent persons despite

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declining levels of anti-HBs. These studies encouraged the Immunization Practices Advisory Committee of the United States, the National Advisory Committee on Immunization of Canada and the European Consensus Group (2000) to recommend that routine booster immunization against hepatitis B is not required. Caution, however, dictates that those at high risk of exposure, such as cardio-thoracic surgeons and gynaecologists would be prudent to maintain a titre of 100 IU/l of anti-HBs by booster inoculations, more so in the absence of an appropriate international antibody reference preparation. Breakthrough infections have been reported and, whereas long term follow-up of children and adults indicated that protection is attained for at least nine years after immunization against chronic hepatitis B infection, even though anti-HBs levels may have become low or declined below detectable levels (reviewed by the European Consensus Group, 2000), brief periods of viremia may not have been detected because of infrequent testing. Longer follow-up studies of immunized subjects is required to guide policy, as is well illustrated by a study carried out in Gambian children (Whittle et al., 2002), who found, by a cross-sectional study of hepatitis B infection in children in The Gambia, that the efficacy of hepatitis B vaccination against chronic carriage of HBV 14 years after immunization was 94%, and the efficacy against infection was 80% and lower (65%) in those vaccinated at the age of 15–19 years. Further and longer follow-up studies of immunized subjects are therefore required to guide policy. An early placebo-controlled study was carried out with a plasma-derived vaccine in an HBV “high-risk” setting in 353 staff, patients on maintenance hemodialysis and their relatives in France in 1975 (Maupas et al., 1979). Follow-up of 73 patients and 191 staff showed that vaccinated subjects who did not respond to the vaccine by developing anti-HBs were infected at the same rate as the unvaccinated controls i.e. nearly 50% as indicated either by anti-HBc production alone (5%), transient antigenemia (15%) or prolonged antigenemia (25%). Many of the subjects who developed infection within 2 months of immunization were patients, who tend to mount a delayed or slow anti-HBs response, and were likely to be incubating the infection. Thirteen staff members (6.8%) were non-responders and nine became infected with HBV within 4–12 months after the first inoculation. It should be noted that interpretation of parts of the report is difficult. Other studies referred to above (Courouce et al., 1988; Coursaget et al., 1986; Hadler et al., 1986; Stevens et al., 1984; Taylor & Stevens, 1988 and others) have shown that the risk of HBV infection increases as anti-HBs levels decline to 10 IU/l in responders. There are few reports concerning non-responders. Nevertheless, the initial efficacy trials of the plasma-derived hepatitis B vaccine (produced by Merck, Sharp & Dohme in the USA) provide evidence of the continuing susceptibility of persons who receive a complete course of vaccine but develop less than 10 IU/l of

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anti-HBs. For example, the study conducted by Szmuness et al. (1981) revealed that 7 of 21 (33%) of vaccinated non-responder male homosexuals became infected during an 18 month period of surveillance. That compared with 92 of 426 (22%) placebo recipients infected during the same period. The evaluation in another study of long-term protection by hepatitis B vaccine for 7–9 years revealed 36 HBV infections among 139 male homosexuals who had no detectable anti-HBs after three doses of vaccine (Hadler et al., 1991). In an earlier trial, the same investigators noted that HBV infection occurred in 55 vaccinated subjects with a poor antibody response, and two became carriers of HBV both of whom were nonresponders (Hadler et al., 1986). In another study there were four “vaccine failures” among 15 babies born to “high risk” mothers; one infant non-responder became infected after the age of 10 months and one poor responder became infected at the age of 6.5 months and remained e antigen positive for five months of the follow-up (Flower & Tanner, 1988). There are apparently no reports of a cohort of healthy non-responders to vaccination who have been surveyed systematically for a sufficient number of person-years to estimate closely susceptibility to infection. It is proposed to followup by serological surveillance the 86 participants in the Hepacare vaccine over a period of several years.

Non-responders and Silent Infection A brief report (Lou et al., 1992) noted that 6.4% of 214 subjects in China who were immunized with the Merck, Sharp & Dohme hepatitis B vaccine and 12.5% of 96 subjects who received a locally produced vaccine did not respond. Hepatitis B virus DNA was detected by PCR in over 60% of the non-responders in each group, suggesting that non-responsiveness to hepatitis B vaccine may be due to immunotolerance or immunosuppression induced by latent HBV infection. Other reports suggested that HBV e antigen can cause immunotolerance and chronic HBV infection (Brunetto et al., 1991), and that HBV itself may cause immunotolerance by infecting directly T and B lymphocytes resulting in viral persistence (Oldstone, 1989) or through different mechanisms triggered by viral infection leading to imbalance in immunoregulation (Paller & Mallory, 1991).

CONCLUSIONS Systematic vaccination of individuals at risk of exposure to hepatitis B virus remains the principal method for controlling this important infection. The

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development of a universal strategy for immunization against hepatitis B is essential if a significant reduction in the world reservoir of 350 million carriers is to be attained. Epidemiological monitoring of hepatitis B surface antigen mutants is essential if the blood supply is to be protected. The development of third generation vaccines incorporating pre-S1 and pre-S2 epitopes may overcome nonresponsiveness to the current vaccines, may provide a mechanism for preventing the emergence of vaccine-associated mutants and may provide enhanced immunogenicity.

REFERENCES Alberti, A., Cavalletto, D., Pontisso, P., Chemello, L., Tagariello, G., & Belussi, F. (1988). Antibody response to pre-S2 and hepatitis B virus induced liver damage. Lancet, i, 1421–1424. Alper, C. A., Kruskall, M. S., Marcus-Bagley, D., Craven, D. E., Katz, A. J., Brink, S. J., Dienstag, J. L., Awdeh, Z., & Yunis, E. J. (1989). Genetic prediction of nonresponse to hepatitis B vaccine. N. Engl. J. Med., 321, 708–712. Ambrosch, F., Frisch-Niggemeyer, W., Kremsner, P., Kunz, Ch., Andre, F., Safary, A., & Wiedermann, G. (1987). Persistence of vaccine-induced antibodies to hepatitis B surface antigen and the need for booster vaccination in adult subjects. Postgrad. Med. J., 63(Suppl. 2), 129–135. Arif, M., Mitchison, N. A., & Zuckerman, A. J. (1988). Genetics of non-responders to hepatitis B surface antigen and possible ways of circumventing “nonresponse”. In: A. J. Zuckerman (Ed.), Viral Hepatitis and Liver Disease (pp. 714–716). New York: Alan R. Liss. Banatvala, J. E., Boxall, E., Heptonstall, J., & Zuckerman, A. J. (1991). Proposal drafted in London, December. Carman, W. F., Zanetti, A. R., Karayiannis, P., Waters, J., Manzillo, G., Tanzi, E., Zuckerman, A. J., & Thomas, H. C. (1990). Vaccine induced escape mutant of hepatitis B virus. Lancet, 336, 325–329. Centers for Disease Control (1987). Update on hepatitis B prevention. Morb. Mort. Wkly Report, 36, 353–366. Centers for Disease Control (1991). Hepatitis B virus: A comprehensive strategy for eliminating transmission in the United States through universal childhood vaccination: Recommendations of the Immunization Practices Advisory Committee (ACIP). Morb. Mort. Wkly Report, 40(RR-13), 1–19. Clements, M. L., Miskovsky, E., Davidson, M., Cupps, T., Kumwenda, N., Sandman, L. A., West, D., Hesley, T., Ioli, V., Miller, W., Calandra, G., & Krugman, S. (1994). Effect of age on the immunogenicity of yeast recombinant hepatitis B vaccines containing surface antigen (S) or pre-S2+S antigens. J. Inf. Dis., 170, 510–516. Courouce, A.-M., Laplanche, A., Benhamou, E., & Jungers, P. (1988). Long-term efficacy of hepatitis B vaccine in healthy adults. In: A. J. Zuckerman (Ed.), Viral Hepatitis and Liver Disease (pp. 1002–1005). New York: Alan R. Liss. Coursaget, P., Yvonnet, B., Chotard, J., Sarr, M., Vincelot, P., N’Doye, R., Diop-Mar, I., & Chiron, J. P. (1986). Seven-year study of hepatitis B vaccine efficacy in infants from an endemic area (Senegal). Lancet, 2, 1143–1145.

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Dienstag, J. L., Werner, B. G., Polk, F., Snydman, D. R., Craven, D. E., Platt, R., Crumpacker, C. S., Ouellet-Hellstrom, R., & Grady, G. F. (1984). Hepatitis B vaccine in health care personnel: Safety, immunogenicity, and indicators of efficacy. Ann. Int. Med., 101, 34–40. Ferrari, C., Cavalli, A., Penna, A., Valli, A., Bertoletti, A., Pedretti, G., Pilli, M., Vitali, P., Neri, T. M., Giuberti, T., & Fiaccadori, F. (1992). Fine specificity of the human T-cell response to the hepatitis B virus preS1 antigen. Gastroenterology, 103, 255–263. Francis, D. P., Hadler, S. C., Thompson, S. E., Maynard, J. E., Ostrow, D. G., Altman, N., Braff, E. H., O’Malley, P., Hawkins, D., Judson, F. N., Penley, K., Nylund, T., Christie, G., Meyers, F., Moore, J. N., Gardner, A., Doto, I. L., Miller, J. H., Reynolds, G. H., Murphy, B. L., Schable, C. A., Clark, B. T., Curran, J. W., & Redeker, A. G. (1982). The prevention of hepatitis B with vaccine. Ann. Int. Med., 97, 362–366. Francois, G., Kew, M., van Damme, P., Mphahlele, M. J., & Meheus, A. (2001). Mutant Hepatitis B viruses: A matter of academic interest only or a problem with far-reaching implications? Vaccine, 3799–3815. Gerlich, W. H., Deepen, R., Heermann, K. H., Krone, B., Lu, X. Y., Seifer, M., & Thomssen, R. (1990). Protective potential of hepatitis B virus antigens other than the S gene protein. Vaccine, 8, S63–S68. Gibas, A., Watkins, E., Hinkle, C., & Dienstag, J. (1988). Long term persistence of protective antibody after hepatitis B vaccination of healthy adults. In: A. J. Zuckerman (Ed.), Viral Hepatitis and Liver Disease (pp. 998–1001). New York: Alan R. Liss. Goldwater, P. N. (1994). Randomized comparative trial of interferon-alpha vs. placebo in hepatitis B vaccine non-responders and hyporesponders. Vaccine, 12, 410–414. Grob, P. J., Joller-Jemelka, H. I., Binswanger, U., Zaruba, K., Descoeudres, C., & Fernex, M. (1984). Inteferon as an adjuvant for hepatitis B vaccination in non- and low-responder populations. Europ. J. Clin. Microbiology, 3, 195–198. Hadler, S. C., Francis, D. P., Maynard, J. E., Thompson, S. E., Judson, F. N., Echenberg, D. F., Ostrow, D. G., O’Malley, P. M., Penley, K. A., Altman, N. L., Braff, E., Shipman, G. F., Coleman, P. J., & Mandel, E. J. (1986). Long-term immunogenicity and efficacy of hepatitis B vaccine in homosexual men. N. Engl. J. Med., 315, 209–214. Hadler, S. C., Coleman, P. J., O’Malley, P., Judson, F. N., & Altman, N. (1991). Evaluation of long-term protection by hepatitis B vaccine for seven to nine years in homosexual men. In: F. B. Hollinger, S. B. Lemon & H. S. Margolis (Eds), Viral Hepatitis and Liver Disease (pp. 766–768). Baltimore: Williams & Wilkins. Hilleman, M. R. (1984). Immunologic prevention of human hepatitis. Persp. Biol. Med., 27, 543–557. Hsu, H. Y., Chang, M. H., Liaw, S. H., Ni, Y. H., & Chen, H. L. (1999). Changes of hepatitis B surface antigen variants in carrier children before and after universal vaccination in Taiwan. Hepatology, 30, 1312–1317. Jilg, W., Schmidt, M., Deinhardt, F., & Zachoval, R. (1984). Hepatitis B vaccination: How long does protection last? Lancet, 2, 458. Jilg, W., Schmidt, M., & Deinhardt, F. (1988). Immune response to hepatitis B revaccination. J. Med. Virology, 24, 377–384. Jungers, P., Devillier, P., Salomon, H., Cerisier, J. E., & Courouce, A. M. (1994). Randomised placebocontrolled trial of recombinant interleukin-2 in chronic uraemic patients who are non-responders to hepatitis B vaccine. Lancet, 344, 856–857. Klinkert, M., Theilmann, L., Pfaff, E., & Schaller, H. (1986). Pre-S antigens and antibodies early in the course of acute hepatitis B virus infection. J. Virology, 58, 522–525.

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Kruskall, M. S., Alper, C. A., Awdeh, Z., Yunis, E. J., & Marcus-Bagley, D. (1992). The immune response to hepatitis B vaccine in humans: Inheritance patterns in families. J. Exp. Med., 175, 495–502. Marescot, M. R., Budkowska, A., Pillot, J., & Debre, P. (1989). HLA linked immune response to S and pre-S2 gene products in hepatitis B vaccination. Tissue Antigens, 33, 495–500. McDermott, A. B., Cohen, S. B. A., Zuckerman, J. N., & Madrigal, J. A. (1999). Human leukocyte antigens influence the immune response to a pre-SIS hepatitis B vaccine. Vaccine, 17, 330–339. McIntyre, P. J. (1995). Hepatitis B vaccination follow-up. Lancet, 345, 1575. Melappioni, M., Baldassari, M., Saldini, S., Radicioni, R., & Panichi, N. (1992). Use of immunomodulators (Thymopentin) in hepatitis B vaccine in elderly patients undergoing chronic hemodialysis. Nephron, 61, 358–359. Meuer, S. C., Dumann, H., Meyer zum Buschenfelde, K.-H., & Kohler, H. (1989). Low dose interleukin2 induces systemic immune responses against HBsAg in immunodeficient non-responders to hepatitis B vaccination. Lancet, 1, 15–17. Milich, D. R. (1991). Immune response to hepatitis B virus proteins: Relevance of the murine model. Sem. Liver Dis., 11, 93–112. Milich, D. R., McLachlan, A., Chisari, F. V., Kent, S. B., & Thornton, G. B. (1986). Immune response to the pre-S(1) region of hepatitis B surface antigen (HBsAg): A pre-S(1)-specific T cell response can bypass nonresponsiveness to the pre-S(2) and S regions of the HBsAg. J. Imm., 137, 315–322. Milich, D. R., McNamara, N. K., McLachlan, A., Thornton, G. B., & Chisari, F. V. (1985a). Distinct H-2 linked regulation of T-cell responses to the pre-S and S regions of the same hepatitis B surface polypeptide allows circumvention of nonresponsiveness to the S region. Proc. Nat. Acad. Sci. USA, 82, 8168–8172. Milich, D. R., Thornton, G. B., Neurath, A. R., Kent, S. B., Michel, M.-L., Tiollais, P., & Chisari, F. V. (1985b). Enhanced immunogenicity of the pre-S region of hepatitis B surface antigen. Science, 228, 1195–1199. Nainen, O. V., Khristova, M. L., Byun, K. S., Xia, G., Taylor, P. E., Stevens, C. E., & Margolis, H. S. (2002). Genetic variation of hepatitis B surface antigen coding region among infants with chronic hepatitis B virus infection. J. Med. Virology, 68, 319–327. Nommensen, F. E., Go, S. T., & MacLaren, D. M. (1989). Half-life of HBs antibody after hepatitis B vaccination: An aid to timing of booster vaccination. Lancet, 2, 847–849. Ogata, N., Miller, R. G., Ishak, K. G., Zanetti, A. R., & Purcell, R. H. (1994). Genetic and biological characterization of two hepatitis B virus variants: A precore mutant implicated in fulminant hepatitis and a surface mutant resistant to immunoprophylaxis. In: K. Nishioka, H. Suzuki, S. Mishiro & T. Oda (Eds), Viral Hepatitis and Liver Disease (pp. 238–242). Tokyo: SpringerVerlag. Oon, C.-J., Lim, G.-K., Ye, Z., Goh, K.-T., Tan, K.-L., Yo, S.-L., Hopes, E., Harrison, T. J., & Zuckerman, A. J. (1995). Molecular epidemiology of hepatitis B virus variants in Singapore. Vaccine, 13, 699–702. Oon, C.-J., Tan, K.-L., Harrison, T. J., & Zuckerman, A. J. (1996). Natural history of hepatitis B surface antigen mutants in children. Lancet, 348, 1524. Stevens, C. E., Taylor, P. E., Tong, M. J., Toy, P. T., & Vyas, G. N. (1984). Hepatitis B vaccine: an overview. In: G. N. Vyas, J. L. Dienstag & J. H. Hoofnagle (Eds), Viral Hepatitis and Liver Disease (pp. 275–291). Orlando: Grune and Stratton. Suzuki, H., Iino, S., Shiraki, K., Akahane, Y., Okamoto, H., Domoto, K., & Mishiro, S. (1994). Safety and efficacy of a recombinant yeast-derived pre-S2+S-containing hepatitis B vaccine (TGP943): Phase 1, 2 and 3 clinical testing. Vaccine, 12, 1090–1095.

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Szmuness, W., Stevens, C. E., Zang, E. A., Harley, E. J., & Kellner, A. (1981). A controlled clinical trial of the efficacy of the hepatitis B vaccine (Heptavax B). A Final Report. Hepatology, 1, 377–385. Taylor, P. E., & Stevens, C. E. (1988). Persistence of antibody to hepatitis B surface antigen after vaccination with hepatitis B vaccine. In: A. J. Zuckerman (Ed.), Viral Hepatitis and Liver Disease (pp. 995–997). New York: Alan R. Liss. Tedder, R. S., Zuckerman, M. A., & Brink, N. (1993). Hepatitis B vaccination: Non-responders must be detected. Lancet, 307, 732. Tilzey, A. J. (1995). Hepatitis B vaccine boosting: The debate continues. Lancet, 345, 1000–1001. van Hattum, J., Maikoe, T., Poel, J., & de Gast, G. C. (1991). In vitro anti-HBsproduction by individual B cells of responders to hepatitis B vaccine who subsequently lost antibody. In: B. F. Hollinger, S. M. Lemon & H. Margolis (Eds), Viral Hepatitis and Liver Disease (pp. 774–776). Baltimore: Williams and Wilkins. Westmoreland, D., Player, V., Heap, D. C., & Hammond, A. (1990). Immunization against hepatitis B – what can we expect? Epidem. Infection, 104, 499–509. Whittle, H., Jaffar, S., Wansborough, M., Mendy, M., Dumpis, U., Collinson, A., & Hall, A. (2002). Observational study of vaccine efficacy 14 years after trial of hepatitis B vaccination in Gambian children. Brit. Med. J., 325, 569–572. Wilson, J. N., Nokes, D. J., & Carman, W. F. (1999). The predicted pattern of emergence of vaccineresistant hepatitis B: A cause for concern? Vaccine, 17, 973–978. Wismans, P., van Hattum, J., Mudde, G. C., Endeman, H. J., Poel, J., & de Gast, G. C. (1989). Is booster injection with hepatitis B vaccine necessary in healthy responders? A study of the immune response. J. Hepatology, 8, 236–240. Wood, R. C., MacDonald, K. L., White, K. E., Hedberg, C. W., Hanson, M., & Osterholm, M. T. (1993). Risk factors for lack of detectable antibody response following hepatitis B vaccination of Minnesota health care workers. J. Am. Med.l Ass., 270, 2935–2939. Yap, I., Guan, R., & Chan, S. H. (1995). Study on the comparative immunogenicity of a recombinant DNA hepatitis B vaccine containing pre-S components of the HBV coat protein with non pre-S containing vaccines. J. Gastroenter. Hepatology, 10, 51–55. Zanetti, A. R., Tanzi, E., Manzillo, G., Maio, O., Sbreglia, C., Caporaso, N., Thomas, H., & Zuckerman, A. J. (1988). Hepatitis B variant in Europe. Lancet, 2, 1132–1133. Zaruba, K., Rastorfer, M., Grob, P. J., Joller-Jemelka, H., & Bolla, K. (1983). Thymopentin as adjuvant in non-responders or hyporesponders to hepatitis B vaccination. Lancet, 2, 1245. Zuckerman, A. J. (2000). Effect of hepatitis B virus mutants on efficacy of vaccination. Lancet, 355, 1382–1384. Zuckerman, A. J., Harrison, T. J., & Oon, C.-J. (1994). Mutations in the S region of hepatitis B virus. Lancet, 343, 737–738. Zuckerman, J. N., & Zuckerman, A. J. (2002). Recombinant hepatitis B triple antigen vaccine: HepacareTM. Exp. Rev. Vaccine, 1, 141–144.

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THE MOLECULAR VIROLOGY OF HEPATITIS C VIRUS

Timothy L. Tellinghuisen and Charles M. Rice INTRODUCTION Hepatitis C virus (HCV) infection is a significant public health problem of international scope. Estimates from an epidemiologic study by the World Health Organization (WHO) in 1997 place the number of HCV infected individuals at approximately 170 million, representing nearly 3% of the world’s population (Anonymous, 1997). It is important to note that the HCV infection is five times more prevalent than that of the human immunodeficiency virus (HIV), underscoring the pandemic nature of HCV infection. More recent data from the National Health and Nutrition Examination Survey (NHANES) on HCV infection in the United States indicate 3.9 million Americans have been exposed to HCV (for a summary of the NHANES report see Kim, 2002). The natural course of HCV infection has two distinct virological outcomes, acute infection with subsequent viral clearance, and viral persistence leading to chronic infection. Acute HCV infection is largely asymptomatic and rarely diagnosed. Unfortunately, only 30% of patients are capable of naturally clearing and acute HCV infection, with the vast majority remaining persistently infected (Alter et al., 1992; Alter & Seeff, 2000). The NHANES data places the number of persistently infected individuals in the United States at approximately 2.7 million. HCV replication can occur for decades in these patients, often leading to serious liver disease and a variety of extra hepatic disorders, including autoimmune disorders, cryoglobulinemia, and non-Hodgkin’s lymphoma. The most common hepatic manifestations of a persistent infection are chronic hepatitis and a progressive cirrhosis. Persistent HCV infection has also The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 455–495 © 2004 Published by Elsevier Ltd. ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15018-6

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been linked to an increased risk of hepatocellular carcinoma (for review see Block et al., 2003). Currently, HCV associated liver disease is the leading indicator of liver transplantation in the United States (Fishman et al., 1996). The NHANES data estimated the direct healthcare cost of hepatitis C infection in the United States at more than 1 billion dollars per year in 1998, with predictions of dramatic increases in future years (Kim, 2002). The approved treatment for HCV infection, a combination therapy of pegylated interferon-␣ and ribavirin, is of limited efficacy and is often poorly tolerated by patients (Heathcote et al., 2000; Zeuzem et al., 2000). The efficacy of drug therapy correlates with the genotype of HCV present in the infected individual. There are currently six recognized HCV genotypes, and a number of more closely related subtypes (Bukh et al., 1995). Sequence variability between genotypes is considerable, with the most distantly related genotypes differing by up to 30%. Rates of sustained virologic response of therapy are as high as 80% for genotypes 2 and 3, and as low as 40% with genotype 1 (Chander et al., 2002). The molecular mechanism of variations in drug efficacy for the different HCV genotypes is not clear. In addition to genotypic variations, HCV is present as numerous closely related quasi-species in the infected individual. The diversity of these quasi-species, combined with the high mutation rate of RNA virus replication, greatly complicates the specific targeting of HCV RNA and proteins (Pawlotsky, 2003). Vaccine development has been equally problematic, and despite significant effort, no effective HCV vaccine exists (Lechmann & Liang, 2000). Clearly, much work is needed in the development of effective anti-HCV therapeutics, and understanding of the molecular virology of HCV is of paramount importance to this process.

OVERVIEW OF HCV BIOLOGY The beginning of hepatitis C virus molecular virology heralds to the late 1980s with the identification of HCV as the causative agent for what was termed non-A non-B hepatitis (Choo et al., 1989). The cDNA clone generated in this landmark work has provided the basis for the classification and molecular dissection of HCV (Choo et al., 1991). Infectious consensus clones of a variety of HCV genotypes have been generated (Beard et al., 1999; Bukh et al., 1998; Kolykhalov et al., 1997; Yanagi et al., 1997, 1998, 1999a). Initial examination of these HCV sequences led to the classification of this virus as a member of the diverse Flaviviridae family of enveloped, positive strand RNA viruses. HCV represents the sole member of the Hepacivirus genus within this family (Lindenbach & Rice, 2001). It is worth noting that the Flaviviridae family also contains the genera Flavivirus and Pestivirus,

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which contain numerous important human and animal pathogens, respectively. The HCV genome consists of an RNA molecule of approximately 9.6 kb containing a single open reading frame (ORF) flanked by large, highly structured 5′ and 3′ non-translated regions (NTRs). The viral RNA lacks both a 5′ cap structure and a 3′ poly(A) tail. Viral proteins are translated as a polyprotein via an internal ribosome entry site (IRES) located within the 5′ NTR. The organization of the polyprotein is similar to that of the other members of the Flaviviridae family, with structural proteins located at the 5′ end of the genome, and non-structural proteins downstream. The ten HCV proteins are organized in the polyprotein in the order: NH2 - C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (see Fig. 1) (Grakoui et al., 1993b). The polyprotein processing is complex and involves both host and viral proteinase activities to carry out the numerous co- and posttranslational cleavage events in the maturation of the viral proteins (Grakoui et al., 1993a). An additional HCV protein generated from an overlapping reading frame in the core (C) protein coding sequence, designated ARFP (alternate reading

Fig. 1. Organization of the HCV Genome and Polyprotein Processing. Note: The HCV genome consists of a single, positive sense RNA molecule flanked by structured 5′ and 3′ non-translated regions (NTRs). The overall organization of the HCV polyprotein is similar to other Flaviviridae, with a single large open reading frame (ORF) with structural proteins (shaded in grey) at the amino terminal end, and non-structural proteins (shaded in white) located downstream. The proteins are organized in the polyprotein in the order; NH2-C-E1E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. The coding sequence of the putative alternate reading frame protein (ARFP), or frame shift protein (F), is indicated with a dark grey box containing the letter F. The locations of known enzymatic activities of the HCV nonstructural proteins have been indicated, including the NS2-NS3 autocatalytic proteinase, the NS3 serine proteinase, the NS3 helicase, and the NS5B RNA dependent RNA polymerase. The cleavage sites utilized in the complex polyprotein processing mechanism have been indicated. Black circles represent the cleavages mediated by the host cell signal peptidase within the structural proteins. The open circle indicates the putative cleavage by signal peptide peptidase involved in generating the mature C protein. The open arrow indicates the site of autocatalytic cleavage by the NS2-NS3 proteinase. The proteolytic cleavage sites processed by the NS3 serine proteinase activity are indicated by black arrows.

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Fig. 2. Membrane Topology of the HCV Proteins. Note: The membrane topology and membrane association of the processed forms of the HCV proteins is shown in relation to the lumen of the endoplasmic reticulum (ER) and the cytoplasm. The location of amino (N) and carboxy (C) termini of the proteins are indicated. The immature and mature forms of the C protein are shown, with the signal peptide peptides maturation cleavage site shown (SPP). The trans membrane spanning anchor of E1 is colored in black to indicate the reorganization of this sequence required for the correct topological insertion of E2. NS3 is shown associated with NS4A, which is believed to localize this protein to membranes, The unusual horizontal membrane topology of the amino terminal helix of NS5A is shown.

frame protein) or F (frame shift protein), has been proposed. The membrane topology of the mature HCV proteins is shown in Fig. 2. The structural proteins, C, E1, and E2 are cleaved from the polyprotein by the endoplasmic reticulum (ER) signal peptidases, and following maturation, most likely serve as components for the assembly of progeny virions. By analogy to other members of the Flaviviridae, assembly of HCV most likely occurs on ER derived vesicles with budding of virions into internal membrane compartments and subsequent cellular exit via the ER trafficking system. The function of the small hydrophobic p7 protein, located at the polyprotein junction between the structural and non-structural proteins, is only beginning to be unraveled. The HCV non-structural proteins, NS2 through NS5B, are thought to comprise the viral replicase complex. The proteolytic processing of these proteins requires two distinct viral proteases. The NS2 protein, together with the amino terminal region of the NS3 protein, constitutes the NS2–3 proteinase that catalyzes the autocatalytic removal of NS2 from the polyprotein. Following this cleavage, NS2 has no known additional function, and is dispensable for subsequent steps in RNA replication. Once released from NS2, the amino terminal domain of the NS3

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proteins serves as a distinct proteinase for the cleavage of all downstream sites in the polyprotein. The carboxyl-terminal region of NS3 is an NTPase/RNA helicase. The NS4A protein serves as a cofactor/enhancer for the proteolytic activities of NS3. The function of the hydrophobic integral membrane protein NS4B is unknown, but this protein appears to interact with the viral replicase and play a role in the reorganization of cellular membranes, presumably to a conformation is amenable to HCV replication. NS5A is a hydrophilic membrane associated phosphoprotein of unknown function. The NS5B protein comprises the RNA dependent RNA polymerase activity. Viral RNA replication is believed to occur in association with peri-nuclear membranes of ER origin, as has been observed for

Fig. 3. Steps in the HCV Lifecycle. Note: A general overview of the steps of the HCV lifecycle. Following binding of the extracellular virion to the host cell receptors(s) and endocytosis of the virion-receptor complex, the virus penetrates the host cell membrane vesicle via the pH dependent glycoprotein fusion activity. Once release into the cytoplasm, the nucleocapsid disassembles (uncoating) and releases the HCV genomic RNA. The input RNA serves as a template for translation of the polyprotein. Once translation and processing of the polyprotein has occurred, the HCV replicase complex assembles in association with ER derived membranes and generates progeny RNA via a minus strand replicative intermediate. These progeny RNA are then packaged into nucleocapsid structures. Nucleocapsids associate with the mature glycoprotein heterodimers and budding into internal membrane vesicles occurs. Following budding, the virions mature and exit the cell via the host vesicle trafficking system. The figure presented is a general schematic, and it should be noted that these processes are dynamic with numerous overlaps and interactions likely between the steps shown.

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other members of the Flaviviridae. An overview of the HCV lifecycle is presented in Fig. 3. Considerable progress has been made in determining the properties of the HCV proteins and RNA. The complex interactions between these macromolecules required for the processes of RNA replication and virion biogenesis is an area of active research and is not fully understood. The aim of this chapter is to present a brief overview of the systems used for the study of HCV and examine the properties of the HCV RNA and proteins as they relate to the numerous processes of virus replication. It is nearly impossible to provide a detailed discussion the vast body of HCV research articles, so a sample of current and classic literature has been selected for review with the intent of providing the reader a general understanding of HCV molecular virology. This chapter is by no means complete, and wherever possible references for more comprehensive review articles covering specific aspects of HCV virology have been provided.

SYSTEMS FOR THE STUDY OF HEPATITIS C VIRUS Perhaps the most significant difficulty in HCV research has been establishing robust systems for the study of HCV replication. This topic has been recently reviewed (Grakoui et al., 2001; Lanford & Bigger, 2002; Pietschmann et al., 2003). Humans and chimpanzees represent the only known animals capable of being infected with HCV. The use of human samples is complicated by the quantity of research material obtainable and the variability in these samples arising from natural infections outside of a controlled laboratory environment. The use of chimpanzees has alleviated some of the problems associated with human samples by allowing inoculation with defined molecular clones of HCV under controlled laboratory conditions, however both cost and ethical issues limit the number and type of experiments that can be performed using this system. Reviews on the use of the chimpanzee in HCV research are available (Lanford et al., 2001a). Although they have been instrumental in defining the natural course of HCV infection as well as addressing the complex interactions of HCV with the immune response, both the chimpanzee model and available human samples lack the tractability and availability needed for understanding the complete molecular details of HCV replication. The development of a small animal laboratory model for HCV has been difficult. Aside from an isolated report of HCV replication in tree shrews (Xie et al., 1998), attempts at obtaining HCV replication in small laboratory animals have been unsuccessful. A clever small animal model system using immunodeficient

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mouse hybrids (SCID/Alb-uPA) has been recently described (Mercer et al., 2001). Following the elimination of the native murine hepatocytes and the delivery of human hepatocytes up to half of the liver mass can be repopulated with human cells. These animals can be inoculated with infected human sera and develop persistent HCV viremia. Although an important breakthrough, the complexities of this system and the requirement of immunodeficient mice make it far from an ideal small animal model for studying HCV. The ability of the closely related flavivirus, GBV-B to replicate in tamarins and tamarin hepatocytes in culture has led to recent interest in this virus as a model for HCV (Bukh et al., 1999; Lanford et al., 2001b). The study of the properties of HCV ex vivo has been limited to the use of a variety of surrogate expression systems, which although capable of producing HCV proteins, fail to allow for RNA replication. Efficient cell culture HCV RNA replication systems based on replicon technology have recently become available, allowing the molecular dissection of RNA replication (Blight et al., 2000, 2003; Guo et al., 2001; Ikeda et al., 2002; Lohmann et al., 1999, 2001; Yi et al., 2003). In the replicon system, bicistronic RNAs containing the HCV non-structural proteins under translational control of an IRES and a selectable marker under the control of a second IRES are generated with HCV 5′ and 3′ NTR sequences. In the human hepatoma cell line Huh7, these RNA molecules express HCV non-structural proteins, which then replicate the viral RNA. RNA replication is monitored by either real-time quantitative PCR analysis of HCV RNA, or by monitoring the expression of a reporter gene. Although the initial replicon system was extremely inefficient, a large number of cell culture adaptive mutations have been described that greatly enhance RNA replication (Blight et al., 2000; Krieger et al., 2001; Lohmann et al., 2001). Interestingly, the combination of these adaptive mutations in the same RNA is often deleterious, suggesting multiple mechanisms of adaptation exist (Lohmann et al., 2003). It is important to note that adaptive mutations appear to be a cell culture specific phenomenon, and these changes are debilitating to RNAs in chimpanzee infections (Bukh et al., 2002). The generation of an adapted cell line for replicons has been described, suggesting the importance of host factors (Blight et al., 2002). The generation of genome length HCV replicons has been described, but despite the presence of the HCV structural proteins, these systems fail to generate infectious virus particles (Blight et al., 2002, 2003; Ikeda et al., 2002; Pietschmann et al., 2002). Recently, HCV replicons have been adapted to non-hepatic human epithelial cells and a murine hepatoma cell line, thereby eliminating the previous limitation of replicons to a single cell type (Zhu et al., 2003). A number of reviews detailing the development and use of the HCV replicon system have been published (Pietschmann & Bartenschlager, 2001; Randall & Rice, 2002).

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HCV RNA, IRES AND NTRS As mentioned in the introduction, the HCV genome consists of a single positive sense, non-capped and non-polyadenylated, RNA molecule of approximately 9.6 kb flanked by large, highly structured 5′ and 3′ NTRs. These regions represent areas of considerable sequence conservation among all HCV isolates, suggesting an important role in virus biology. The 5′ HCV NTR is a 341 nucleotide element consisting of 4 highly structured domains, designated I though IV. Domains II, III, IV, and a portion of the coding sequence of the C protein comprise the viral IRES, a structure required for the cap-independent translation of the HCV polyprotein. The IRES contains two large stem loop structures and an RNA pseudoknot. A number of the smaller elements of domain III have been visualized in NMR and x-ray structures (Collier et al., 2002; Kieft et al., 2002; Lukavsky et al., 2000), and the entire 5′ NTR has been extensively mapped by structure probing. A cryo-electron microscopy image reconstruction (cryo-EM) of the HCV IRES complexed with the 40S ribosomal subunit has been determined, indicating that the IRES is capable of altering the conformation of the ribosomal subunit through a mechanism requiring domain II of the IRES (Spahn et al., 2001). It is believed that this conformational change in the IRES allows HCV to bypass the necessity for the typical canonical translation factors. For a more detailed review on the function of the HCV IRES in translation, the reader is directed to (Hellen et al., 1999; Rijnbrand & Lemon, 2000). The HCV IRES has garnered significant interest as a target for anti-viral therapeutics (Jubin, 2003). Domain I, the most 5′ element of the 5′ NTR, forms a stable stem loop structure. Deletion of this stem loop positively affects translation, although this region is not required for IRES activity (Honda et al., 1996; Rijnbrand et al., 1995; Yoo et al., 1992). More recently, this region has been shown to be important for RNA replication. Deletion of the 5′ terminal 40 nucleotides of the HCV RNA, thereby disrupting this element, abolished RNA replication in the replicon system, while only moderately affecting translation (Friebe et al., 2001). Generation of artificial RNAs containing the first 125 nucleotides of the HCV 5’ NTR has shown this region is sufficient for HCV specific RNA replication, suggesting that domain I and a portion of the HCV IRES, are essential for replication (Friebe et al., 2001). The 3′ NTR consists of a short variable region, a polyuridine/polypyrimide tract of approximately 40 nucleotides, and a conserved 98 nucleotide region (designated the 3′ X region) containing a stable stem loop structure at the extreme 3′ end of the genomic RNA (Kolykhalov et al., 1996). Structural probing of the 3′ end of the RNA has partially confirmed the predicted secondary structure (Blight & Rice, 1997). The 3′ NTR is essential for HCV RNA replication in cell

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culture and required for infection of chimpanzees (Friebe & Bartenschlager, 2002; Kolykhalov et al., 2000; Yanagi et al., 1999b). Recent experiments have shown the 98 nucleotide 3′ X region is essential for RNA replication but plays little role in translation or RNA stability (Friebe & Bartenschlager, 2002). Deletion of the hypervariable region is debilitating to HCV replicons, but is not absolutely required for RNA replication. The polyuridine/polypyrimidine tract is essential for replication, but portions of this region can be replaced with polyuridine homopolymers. The 3′ NTR is believed to be the site of initiation of viral RNA synthesis.

HCV Structural Proteins Core The HCV core (C) protein lies at the amino terminus of the viral polyprotein and is the site of initiation of viral translation. The early translation of C is thought to be cytoplasmic, with a redistribution of the nascent polypeptide to the ER following the translation of the C/E1 junction, which functions as an internal signal sequence for ER insertion. Once this sequence has been inserted, the remainder of the HCV polyprotein can be translated and processed in association with the ER. Cleavage of the C/E1 junction by the ER resident signal peptidase generates a 191 amino acid form of C which is inserted in the ER membrane based on the retention of the C/E1 junction signal sequence (Santolini et al., 1994). A second processing event within the ER membrane, presumably by signal peptide peptidase, removes the C/E1 signal sequence peptide and generates what is believed to be the mature 179 amino acid form of the C protein (McLauchlan et al., 2002). The majority of this form of the C protein remains associated with the ER, despite the removal of the carboxy terminal membrane anchor peptide, although other sub cellular localizations of the C have been observed (discussed below). The mature C protein is a small, hydrophilic protein that is believed to be the sole protein component of the HCV nucleocapsid. The binding of C to the HCV 5′ NTR has been observed, and this has been proposed to be a potential RNA packaging signal for nucleocapsid assembly (Hwang et al., 1995; Tanaka et al., 2000). This 5′ NTR interaction has also been shown to alter translation of the HCV polyprotein, possibly serving as a mechanism to regulate the switch between translation, replication, and virion assembly (Shimoike et al., 1999; Zhang et al., 2002). The non-specific nucleic acid binding activity of C has also been reported, possibly representing the non-specific charge neutralization of RNA required for nucleocapsid assembly (Hwang et al., 1995; Santolini et al., 1994). C has been shown to make homotypic interactions via a tryptophan rich sequence in the amino terminal portion of the protein,

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and these interactions have been proposed to be steps in the assembly of the HCV nucleocapsid (Matsumoto et al., 1996; Nolandt et al., 1997). C protein has been shown to be modified by tissue transglutaminase, resulting in the cross-linking of C proteins into a stable dimeric form that may play a role in the assembly process (Lu et al., 2001). Additionally, interactions of C with the 60S ribosomal subunit have been described, possibly mediating the disassembly of the nucleocapsid during virus entry (Santolini, Migliaccio & La Monica, 1994). The amino terminal region of C has also been shown to contain a number of cryptic nuclear localization signals, although these findings are controversial (Chang et al., 1994). The sub cellular localization of C is complex, with protein found mainly associated with the ER and lipid droplets, although nuclear localization of C, presumably via one or more of the putative nuclear localization signals, has been described (reviewed in McLauchlan, 2000). The localization of C to ER associated complexes containing the HCV structural proteins, non-structural proteins, and presumably RNA is believed to be the most relevant localization observed for HCV replication and virion production (Egger et al., 2002). The association of C with cytoplasmic lipid droplets has been observed, and this interaction may play a role in HCV pathogenesis. The regions of the C protein that are responsible for this association have been mapped to the carboxy terminal hydrophobic region of the protein, and these sequences bear a resemblance to plant olesin, a lipid binding protein (Hope et al., 2002). Additionally, C has been shown to bind to apolipoprotein II, possibly mediating lipid interactions (Perlemuter et al., 2002; Sabile et al., 1999; Shi et al., 2002). The interaction of C with lipid vesicles has been implicated in HCV related steatosis (Moriya et al., 1997). Transgenic mice expressing C develop steatosis (Moriya et al., 1997; Perlemuter et al., 2002) and liver cancer (Moriya et al., 1998), although the later observation appears to be mouse strain specific. The C protein has been shown to lead to a reduction in microsomal triglyceride transfer protein activity, leading to defects in the assembly and secretion of very low-density lipoproteins (Perlemuter et al., 2002). The C protein may play a role in lipid metabolism, lipid reorganization and trafficking, but the functional significance of these observations to HCV replication remain unclear. As mentioned previously, the C protein has been proposed to localize to the nucleus via several cryptic nuclear localization signals, although efficient localization requires artificial constructs lacking the hydrophobic carboxy terminal region of C (Chang et al., 1994; Liu et al., 1997; Lo et al., 1995; Ravaggi et al., 1994; Suzuki et al., 1995; Yasui et al., 1998). The nuclear localization of C is a significant area of debate. A large number of transcriptional regulatory activities have been proposed for the nuclear form of C. In addition to the role

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of C in nuclear transcriptional regulation, the cytoplasmic form of C has been proposed to interact with numerous cellular signaling cascades, suggesting links to transcription, apoptosis, carcinogenesis, and evasion of the host immune system. Much of the data regarding both the nuclear and cytoplasmic roles of C in gene expression are controversial and contradictory, and in most cases, it is unclear if these interactions occur in the course of a normal infection. The reader is directed to (McLauchlan, 2000) for an extensive review on these activities of C.

F Protein(s) or ARFP(s) One of the most surprising observations in recent HCV research is the presence of multiple overlapping reading frames in the core protein coding sequence that give rise to what has been called the frame shift (F) or alternate reading frame protein (ARFP) (Varaklioti et al., 2002; Xu et al., 2001). The majority of HCV isolates have been shown to contain an open reading frame in the −2/+1 frame that overlap the core protein coding sequence. Analysis of non-synonymous codon usage in the core protein coding sequence has indicated an unusual conservation of codons, presumably to maintain the integrity of the ARFP ORF. The translation of the ARFP ORF via a ribosomal frame-shifting event can generate a protein of up to 180 amino acids, although the exact size and composition of the ARFP is not clear. The generation of the ARFP requires only codons 8−14 of the core protein-coding sequence, a region that has been designated the HCV type I frame shift sequence (Xu et al., 2001). The frame shift junction that generates ARFP is believed to be located at codon 11 within this sequence. A double stem-loop structure located downstream of the frame shift signal has been shown to enhance frame shifting in the presence of the puromycin (Choi et al., 2003). More recent data suggests the generation of an additional 1.5 kDa ARFP using the −1/+2 frame of the core protein (Choi et al., 2003). This smaller ARFP is largely uncharacterized. Immunoflourescence studies suggest the larger ARFP, like many of the other HCV proteins, is localized to ER or ER derived membranes (Xu et al., 2003). Pulse chase experiments reveal a surprisingly short, 10-minute half-life of ARFP in Huh7 cells (Xu et al., 2003). The ARFP is most likely degraded by the proteosome complex, the final resting place of many misfolded proteins, as proteosome inhibitors seem to stabilize the ARFP (Xu, 2003). It is easy to dismiss the generation of the ARFP as an artifact of the in vitro translation and over expression systems used in the description of this phenomenon, save the presence of antibodies directed against ARFP sequences observed in infected HCV patient sera. The presence of these anti-ARFP antibodies in patient sera suggests this protein is generated in the natural

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course of an HCV infection (Varaklioti et al., 2002; Xu et al., 2001). The function of the ARFP(s) remain to be elucidated.

E1 and E2 E1 and E2 represent the HCV viral glycoproteins and are presumably the virion components required for receptor binding and fusion with the cellular membranes. E1 and E2 are type I trans membrane proteins with large amino terminal ectodomains facing the lumen of the ER. The ectodomain of E1 consists of 160 amino acids, and this region of E2 is considerable larger, consisting of 334 residues. Both proteins contain small (approximately 30 residue) trans membrane spanning anchors (TM) located at their carboxy termini. The requirement of both ectodomains to be within the ER lumen, combined with the location of the TMs, necessitates a complex interaction of the proteins with the ER translocation machinery in which the TM of E1 must be repositioned to allow for the correct topology of E2 (Cocquerel et al., 2002). In addition to their role in anchoring the glycoproteins, the TMs of E1 and E2 are involved in the formation of noncovalent E1-E2 heterodimers (Cocquerel et al., 1998; Michalak et al., 1997; Selby et al., 1994). A number of reports have also demonstrated the formation of large disulfide linked aggregates of E1 and E2 (Dubuisson et al., 1994; Grakoui et al., 1993b). The relevant disulfide bond formations are believed to be solely intramolecular. Interactions between the ectodomains of E1 and E2 are important in stability and processing of the proteins (Cocquerel et al., 2001; Patel et al., 2001), and a recent publication has demonstrated the importance of C in this process (Merola et al., 2001). Both E1 and E2 have been shown to interact with the ER chaperones BiP, calnexin, calreticulin, and the enzyme protein disulfide isomerase at various stages in the maturation process (Choukhi et al., 1998). E1 and E2 are heavily modified with complex N-linked glycosylation, containing 5 and 11 such modifications, respectively. The membrane insertion, folding, disulfide bond formation, glycosylation, and oligomerization of the envelope proteins are complex events that have been reviewed in detail elsewhere (Op De Beeck et al., 2001). A structural model of E2 has recently been proposed based on the solved structure of the related flavivirus tick borne encephalitis virus E protein, and the organization of the proteins is believed to be similar (Yagnik et al., 2000). Little is known about the structure of E1, or the structure of the mature dimeric glycoproteins. The search for the cellular receptor for HCV binding and entry has a long history, beginning with demonstration of the binding of a soluble form of E2

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to cells and potential receptor molecules, and eventually leading to the use of artificial HCV like particles, including artificial membrane vesicles containing the HCV glycoproteins, virus like particles, and virus pseudotypes (reviewed in Flint et al., 2001). Current experimental data has implicated tetraspanin CD81, low density lipoprotein receptor, scavenger receptor class B type I, dendritic cellspecific intracellular adhesion molecule 3 grabbing nonintergrin (DC-SIGN), the related molecule DC-SIGNR, liver/lymph node specific intracellular adhesion molecule 3 grabbing integrin (L-SIGN), and heparan sulfate as potential HCV receptor molecules (Barth et al., 2003; Bartosch et al., 2003; Flint et al., 1999a; Gardner et al., 2003; Pohlmann et al., 2003; Scarselli et al., 2002). Many of these interactions can be blocked by anti-E2 neutralizing antibodies, suggesting the specific nature of the observed interactions. An emerging body of evidence suggests that a number of these molecules are required in a “receptor complex” for productive binding and entry of HCV pseudotypes, and that no one molecule is the HCV receptor. Little is known about the interactions of the glycoproteins involved in membrane fusion, although pseudotype virus infection studies indicate this is a pH dependent mechanism (Hsu et al., 2003; Meyer et al., 2000). A 26 amino acid region of E1 is similar to other viral fusion peptides, but the demonstration of this sequence as a functional fusion peptide has not been performed (Flint et al., 1999b). It is important to note that psuedotyped viruses may not completely represent the properties of the HCV glycoproteins as they exist in a native virus particle. Nonetheless, the ability to generate psuedotypes virions bearing the HCV glycoproteins is an exciting and powerful tool, and when used with appropriate experimental controls, is quite useful for characterizing the early steps in HCV infection. The reader is directed to a recent review on pseudotypes and the study of HCV entry (Castet, 2003). Undoubtedly, the generation of a system capable of producing infectious HCV virions will greatly aid in the understanding of the mechanisms of receptor binding and fusion. An unusual and surprising finding for the E2 protein is the reported interaction and inhibition the activity of the double stranded RNA dependent protein kinase (PKR) (Taylor et al., 1999). Interactions between E2 and PKR have been observed in cells over expressing E2, and similar interactions have been observed using an in vitro binding system (Taylor et al., 2001). It is unclear how the ectodomain of E2, located in the ER lumen, would interact with the cytoplasmic PKR protein. A newer report indicates that the glycosylated form of E2 in the ER does not interact with PKR, but rather a novel, non-glycosylated cytoplasmic E2 mediates this interaction (Pavio et al., 2002). How exactly this cytoplasmic form of E2 is generated in the normal course of polyprotein translation and processing is difficult to envision. A recent publication has demonstrated that no correlation

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exists between the presence of the E2-PKR interaction sequence of E2 and response to interferon therapy (Abid et al., 2000). In addition to the proposed role in PKR signaling, E2 has been shown to induce the unfolded protein response signal cascade of the ER (Liberman et al., 1999).

p7 Protein The p7 protein is a small, 63 amino acid protein found at the junction of the structural and non-structural proteins in the HCV polyprotein. p7 is an integral membrane protein that crosses the ER membrane twice, leaving the amino and carboxy termini of the protein on the same membrane surface, most likely the ER luminal facet (Carrere-Kremer et al., 2002). The transmembrane spanning regions of p7 have been modeled as ␣-helices, and spatial conservation of residues suggests these transmembrane regions are involved in specific helix-helix interactions (Carrere-Kremer et al., 2002). The orientation of p7 places the E2/p7 and p7/NS2 cleavage sites within the ER lumen, where they are likely cleaved by signal peptidase. Unlike the other HCV polyprotein processing events, the cleavage of both termini of the p7 protein is inefficient, with intermediates of E2-p7 and E2p7-NS2 readily observable. This delayed cleavage of p7 has led to speculation of a regulatory role of p7 processing in virion assembly and downstream protein processing. The p7 protein is dispensable for RNA replication in HCV replicon systems (Blight et al., 2000; Lohmann et al., 1999). Evidence of a possible role for p7 in virion morphogenesis can be found in studies on the analogous p7 protein of the pestiviruses. Although not associated with mature virions, the pestivirus p7 protein is required for the production of infectious progeny (Harada et al., 2000). The p7 protein can be supplied in trans in these systems, suggesting more than a simple protein topology and processing role for p7. Immunoflouresence studies on HCV p7 sub-cellular localization indicate that p7, in addition to the aforementioned ER localization, is also found on internal vesicles and the cell surface, suggesting a role in modulation of cellular vesicular trafficking for progeny virion maturation and release (Carrere-Kremer et al., 2002). Recent data has proposed another role for p7, that of a viroporin ion channel (Griffin et al., 2003). Cross-linking studies suggest p7 forms discrete hexameric structures with a ring-like appearance (Griffin et al., 2003). In artificial membrane/p7 peptide systems, this protein allows ion flux across membranes that can be blocked with a variety of ion channel blockers (Griffin et al., 2003; Pavlovic et al., 2003). The function of p7 as a viroporin in the course of a natural infection has been postulated to be involved in virion morphogenesis. As with the other HCV proteins that have been proposed to be involved in morphogenesis, p7 will likely remain a mystery until a system for

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generating infectious HCV virions that is amenable to molecular techniques is developed.

HCV VIRIONS AND HCV ASSEMBLY The determination of the structure and composition of the HCV virion is one of the most daunting tasks in HCV research. Presumably, the virion is composed of the C protein complexed with RNA in a nucleocapsid structure, enveloped by a host derived lipid bilayer containing the E1 and E2 glycoproteins. Experiments demonstrating the composition of the virion are greatly complicated by the inability to culture HCV in a tractable laboratory system, and little is therefore known about virus particles. Filtration experiments of infected materials suggest the virus particle is between 30 and 80 nm (Bradley et al., 1985; He et al., 1987; Yuasa et al., 1991). Density gradient centrifugation of infected chimpanzee serum suggests a buoyant density of 1.03−1.1 g/ml, consistent with an enveloped virus (Carrick et al., 1992; Hijikata et al., 1993b). It should be noted that considerable variation in density of HCV particles have been observed, presumably through interaction with immunoglobulins and lowdensity lipoproteins (Andre et al., 2002; Carrick et al., 1992; Hijikata et al., 1993b; Thomssen et al., 1992, 1993; Watson et al., 1996). Stripping the membranes off of HCV particles with chloroform or detergents releases what is believed to be the nucleocapsid (buoyant density of 1.17−1.25 g/ml) (Hijikata et al., 1993b; Kanto et al., 1994; Miyamoto et al., 1992). Nucelocapsids have been directly observed in the cytoplasm of infected human hepatocytes in at least one report (Falcon et al., 2003). A system for the in vitro assembly of nucleocapsids has been developed using purified C protein (Kunkel et al., 2001). Preliminary in vitro assembly data suggests that C protein undergoes conformation changes during oligomerization and nucleocapsid assembly. Additionally, in vitro assembled nucleocapsids are RNAse sensitive, suggesting a structural role for nucleic acid in maintenance of the nucleocapsid structure. The mechanism of nucleocapsid and virion formation in infected cells is unknown. A single report has demonstrated a weak interaction between C and E1 during immunoprecipitation, hinting at an interaction involved in budding (Lo et al., 1996). Interactions of the C protein with the HCV 5′ NTR may represent an early step in nucleocapsid assembly (Hwang et al., 1995; Tanaka et al., 2000). The aforementioned heterodimerization of the glycoproteins is believed to be a requirement for virion assembly. Complete HCV virions have been directly visualized in pooled human plasma and both chimpanzee and human liver samples by electron microscopy (De Vos et al., 2002; Falcon et al., 2003; Prince et al., 1996; Takahashi et al., 1992). These particles appear to be enveloped

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structures with a diameter of approximately 50−60 nm. A recent publication has observed these particles in both cytoplasmic membrane vesicles and the ER of infected human hepatocytes (Falcon et al., 2003). This correlates well with the hypothesis that HCV assembly is similar to that observed for other members of the Flavivirdae, with budding into internal membranes and release via host vesicle trafficking system. A number of reports using surrogate viruses to express the HCV structural proteins, thereby generating what are referred to as virus like particles (VLPs) have been published (Baumert et al., 1998; Ezelle et al., 2002). The VLPs generated by these systems appear to have a similar size and morphology to the native HCV virions. These VLPs, in addition to their use in potential vaccine development and receptor binding studies, may provide important insights into the composition of the native HCV virion (Baumert et al., 1999). Additional data about the structure of the HCV virion may be gleaned from the recent cryo-EM image reconstruction of the flavivirus Dengue, although it is not clear how similar this particle is to the HCV virion (Kuhn et al., 2002). The major impasse in understanding the HCV virion is the lack of a robust system for the generation of particles.

HCV Non-Structural Proteins NS2 The NS2 protein is an integral membrane protein of approximately 23 kD. The membrane topology of NS2, although not completely understood, is an area of active research with current models suggesting four transmembrane spanning ␣-helices with the amino and carboxy termini of the protein located in the ER lumen. Although the topology of the p7 protein places the amino terminus of NS2 in the ER lumen, the protein appears to associate with membranes when expressed alone, due to the presence of at least 2 internal signal sequences (Yamaga et al., 2002). The localization of the carboxy terminus of NS2 in the ER lumen by glycosylation site mapping is somewhat controversial, as the NS2/NS3 cleavage site must be located on the cytoplasmic face of the ER membrane to generate a cytoplasmically localized NS3 protein (Yamaga et al., 2002). The carboxy terminus of NS2 has been proposed to insert into the ER membrane after the cleavage of the NS2/NS3 junction. The cleavage of the NS2/NS3 junction is performed in a co-translational manner by the NS2–3 autoproteinase activity composed of approximately half of the NS2 protein and the amino terminal 180 amino acids of NS3 (Grakoui et al., 1993a; Hijikata et al., 1993a). This cleavage event marks the only known function of the NS2 protein, which is dispensable for RNA replication in cell culture (Blight et al., 2000;

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Lohmann et al., 1999). It is important to note that the NS2–3 autoproteinase activity is distinct from the serine proteinase activity of the amino-terminal region of NS3, despite the physical overlap of these activities in the NS3 protein sequence. The NS2–3 protease activity is stimulated by addition of microsomal membranes, detergent and zinc in cell free translation extracts (Grakoui et al., 1993a; Hijikata et al., 1993a; Pieroni et al., 1997; Santolini et al., 1995). Although the stimulatory nature of zinc, and the conversely inhibitory activity of EDTA on the NS2–3 proteinase suggest a metalloproteinase activity, the presence of a structural zinc atom in the amino terminal region of NS3 required for the NS2–3 and NS3 proteinase activities complicates this interpretation. Site-directed mutagenesis implicates amino acids His 143 and Cys 184 of NS2 in the NS2–3 proteinase activity, with current models placing these two residues as the catalytic diad of a thiol protease (Neddermann et al., 1997). The NS2–3 proteinase has been shown to require, presumably through a direct protein-protein interaction, the host cell chaperone protein hsp90 for proper cis-cleavage activity (Waxman et al., 2001). A cellular J-domain protein has been shown to be involved in the NS2–3 protein cleavage in a pestivirus, and similar interactions have been proposed for HCV (Rinck et al., 2001). In pestiviruses the processing of the NS2/NS3 junction is often incomplete, and the production of cleaved NS3 has been correlated with pathogenesis (discussed in Lindenbach & Rice, 2001). Additionally, processing in the NS2/NS3 region has been shown to be involved in virion morphogenesis (Kummerer & Rice, 2002). The cleavage of the NS2/NS3 junction appears to be complete in HCV. The possible link between NS2/NS3 processing and HCV pathogenesis or virion morphogenesis have not yet been addressed. NS3 and NS4A The NS3 protein is a large (approximately 70 kD) multifunctional enzyme comprised of two domains; a serine protease domain at the amino terminus (independent of the NS2–3 proteinase activity), and an NTPase/helicase domain at the carboxy terminus. The NS3 serine protease activity is modulated by the small (54 amino acid) NS4A protein (Failla et al., 1994; Pang et al., 2002), and NS4A plays an important structural role in the serine protease (reviewed in (De Francesco & Steinkuhler, 2000)). In addition NS4A is also responsible for localizing the cytoplasmic NS3 to perinuclear ER membranes via an amino terminal hydrophobic region (W¨olk et al., 2000). For these reasons NS3 and NS4A are usually considered as a complex. NS3–4A represents the best characterized of the HCV proteins, with numerous crystal structures of the NS3 protease domain alone and complexed with NS4A, the NS3 NTPase/helicase domain with and without nucleic acid, and the full length NS3 protein complexed with the NS4A protein are available (Cho et al., 1998;

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Kim et al., 1996, 1998; Love et al., 1996a; Yan et al., 1998; Yao et al., 1997, 1999). The intricacies of these crystal structures are beyond the scope of this review, and the reader is thereby directed to (De Francesco et al., 2003; De Francesco & Steinkuhler, 2000; Kwong et al., 1999) for a review of these structures. The amino terminal serine proteinase activity of NS3–4A catalyzes the cis cleavage of the NS3/NS4A junction, as well as the downstream trans cleavages of the NS4A/4B, NS4B/NS5A, and NS5A/NS5B junctions. The NS3 serine protease has a overall fold reminiscent of chymotrypsin, with two six stranded squashed ␤-barrel sub-domains forming an active site cleft/substrate binding pocket between the sub-domain interface. The function of NS4A in the NS3–4A protease activity becomes clear when the structures of the protease domain with and without NS4A are compared (Kim et al., 1996; Love et al., 1996b; Yan et al., 1998). NS4A forms a ␤-strand that interacts with the amino terminal residues of NS3 to generate a two strand antiparallel ␤-sheet that is important in orienting the active site catalytic triad. A structural zinc atom is also important for the proper folding and activity of the protease domain of NS3 (reviewed in De Francesco et al., 1998). Some of the residues responsible for coordinating this zinc atom lie within the loop connecting the two ␤ barrels and therefore may affect the geometry of the active site that lies between the barrels. These features are indicated in the crystal structure of the protease domain complexed with an NS4A peptide presented in Fig. 4A (Yan et al., 1998). The association of NS3–4A protease with trans substrates can be inferred by the crystal structures of this complex with peptide mimetic drugs (Di Marco et al., 2000). Insights into the cis cleavage of the NS3/4A site can be gleaned from the crystal structure of a recombinant single chain NS3NS4A protein construct that generates the 14 residues of NS4A responsible for interaction with the NS3 protease domain as an amino terminal extension of the complete NS3 protein (Yao et al., 1999). In this structure the carboxy terminus of NS3 lies adjacent to the NS3 active site, presumably in a conformation similar to that seen in cis cleavage of NS3-NS4A (Fig. 4C) (Yao et al., 1999). What rearrangements of the carboxy terminus of NS3 render the active site accessible for subsequent trans cleavages remains to be demonstrated. The crystal structure of this NS3-NS4A construct indicates the true multi-domain nature of NS3, with the protease and NTPase/helicase domains clearly separated by a flexible loop region. Nevertheless, some interdomain contacts exist, most notably the interaction of the protease domain in generating a portion of the nucleic acid binding site on the helicase domain and the more compact subdomain contacts within the helicase induced by the protease domain. The NTPase/helicase region of NS3 is similar to members of helicase superfamily 2. This domain of NS3 is comprised of three subdomains, two ␤-␣-␤ subdomains and a third helical subdomain (Cho et al., 1998;

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Fig. 4. Overview of HCV NS3 and NS5B Structures. Note: Panel A. The crystal structure of the NS3 protease domain complexed with a peptide corresponding to a portion of NS4A (Love et al., 1996b). The active site cleft between the two ␤-barrel subdomains is designated. The structural zinc atom required for protease activity is shown (Zn) coordinated by the loop structure connecting the two subdomains. The amino (N) and carboxy (C) termini of the NS4A peptide are shown. Panel B. Crystal structure of the helicase domain of NS3 complexed with a short synthetic nucleic acid (Yao et al., 1997). The locations of the 3 subdomains of NS3 are indicated, as is the location of the bound nucleic acid. Panel C. The crystal structure the entire NS3 protein and a portion of NS4A (Yan et al., 1998). The well separated protease and helicase domains are shown. The position of the carboxy terminus of NS3, adjacent to the protease domain active site, is labeled (C). Panel D. The crystal structure of the NS5B RNA dependent RNA polymerase (Lesburg et al., 1999). The classic palm, fingers, thumb domain organization of the polymerase is shown. The active site (AS) region of the polymerase is designated. Note that the active site is completely enclosed by interactions between the finger and thumb domains.

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Kim et al., 1998; Yao et al., 1999). The crystal structure of the helicase domain of NS3 complexed with nucleic acid is shown in Fig. 4B (Kim et al., 1998). The ␤-␣-␤ subdomains are arranged with a central hydrophobic core of ␤-sheets and flanking ␣-helices. The two ␤-␣-␤ subdomains are structurally similar, each containing a 6 stranded parallel ␤ sheet, with the exceptions of the presence of a single antiparallel ␤-strand in subdomain I and two anti-parallel ␤-strands in subdomain II and the arrangement of the flanking helices of each subdomain. Subdomain I contains the NTPase activity, with subdomain II involved in RNA binding. The subdomains are arranged in configuration similar to the shape of the letter Y, with clefts between subdomains that contain the conserved helicase motifs (between subdomains I and II) and RNA binding site (between subdomain III and I and II). Both the helicase domain alone and full length NS3 have in vitro helicase activity, although the activity of the full length protein is enhanced compared to the helicase domain alone, likely due to the aforementioned role of the protease domain in the generation of the RNA binding site (Gallinari et al., 1998; Howe et al., 1999; Kumar et al., 1997; Urvil et al., 1997). The NS3 helicase activity unwinds double stranded RNA and DNA, as well as RNA/DNA heteropolymers in a 3′ –5′ orientation in the presence of ATP and the appropriate divalent cations, Mn++ and Mg++ (Tai et al., 1996). The NTPase/helicase activities of NS3 show what appear to be a complex regulation, presumably via cross-talk between subdomains (Levin et al., 2003). The regulation of this enzyme in the complex process of genome replication is undoubtedly regulated in an intricate manner, but the exact nature of this regulation remains unclear. The NS3 protein has recently been shown to bind to the poly(U/C) tract located in the 3′ NTR of the HCV genome, and it is an attractive hypothesis that this protein is involved in unwinding structured RNAs during replication or unpairing of plus and minus strand replicative intermediates (Banerjee & Dasgupta, 2001). The binding of NS3 to the HCV RNA polymerase further suggests a direct role in the manipulation of RNA during replication (Piccininni et al., 2002). The NS3 and NS3–4A proteins have further been demonstrated to interact with and modulate the phosphorylation of the NS5A protein, a putative component of the replicase (Koch & Bartenschlager, 1999; Neddermann et al., 1999). The NS3 NTPase and helicase activities are absolutely required for RNA replication in HCV and the related flavivurses and pestiviruses, yet the actual role of these activities in RNA replication is undefined (Gu et al., 2000; Matusan et al., 2001). Nevertheless, the absolute requirement for both the NS3 protease and NTPase/helicase activities for replication has led to the development of exciting new anti-viral drugs targeting NS3 (De Francesco et al., 2003). NS3 has been proposed to interact with a large number of cellular proteins with a myriad of effects, but the relevance of these interactions to HCV replication

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remain to be demonstrated. The majority of the proposed interactions for NS3 involve alteration of normal cellular signaling pathways, a feature that has been proposed for many of the HCV proteins. Sequences in the NS3 protein resemble the consensus target sequences and autophosphorylation sequences of both PKA and PKC (Borowski et al., 1996, 1997, 1999a, 1999c, 2000). NS3 has been shown to interact with the catalytic subunit of PKA and with PKC, thereby inhibiting the activity of these kinases by both blocking their interaction with normal cellular targets and preventing the relocalization of these proteins upon activation (Borowski et al., 1996, 1997, 1999a, 1999c, 2000). The function of these interactions in an infected cell is not clear. The NS3 protease domain appears to be weakly oncogenic in cell culture, possibly through an observed interaction with the p53 tumor suppressor (Ishido et al., 1997; Ishido & Hotta, 1998; Sakamuro et al., 1995). NS3 can interact with several histone proteins in vitro via an internal histone binding sequence, possibly allowing the modification of host cell transcription, although it is unclear how a cytoplasmic protein can interact with nuclear histone proteins (Borowski et al., 1999b). Recent publications demonstrate the covalent modification of NS3 by arginine methyltransferase 1, possibly modifying the interaction of NS3 with other proteins (Rho et al., 2001). NS4B NS4B is probably the least well characterized of the HCV proteins. NS4B is small hydrophobic integral membrane protein that co-translationaly associates with ER membranes via an internal signal sequence. The protein has been predicted to cross the ER membrane between four and six times, resulting in the orientation of the amino and carboxy termini in the cytosol (H¨ugle et al., 2001). Despite the large number of predicted membrane spanning regions and relative hydrophobicity of the protein, the bulk of NS4B appears to be on the cytoplasmic face of the ER membrane (H¨ugle et al., 2001). Immunoflouresence studies indicate NS4B is associated with ER or ER-like membranes, and when expressed alone this protein alters the ER into convoluted vesicular structures referred to as membranous webs (Egger et al., 2002; H¨ugle et al., 2001). Further characterization of these membranous webs has shown the presence of all of the HCV non-structural and structural proteins as well as replicating HCV RNA within these structures (Gosert et al., 2003). The membranous web has therefore been proposed to be the site of HCV RNA replication and possibly the site of the early stages of virion assembly. An additional role of NS4B has been proposed. NS4B protein may have oncogenic properties, based on transformation of cells expressing NS4B in conjunction with Ha-ras, but this observation has yet to be linked to HCV replication or pathogenesis (Park et al., 2000).

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NS5A NS5A is a large (56–58 kDa), hydrophilic protein of unknown function. The ability of adaptive mutations in NS5A to greatly stimulate HCV replication in cell culture, and the association of this protein with other members of the putative replicase suggests NS5A plays an important role in RNA replication (Blight et al., 2000; Lohmann et al., 2001). NS5A is associated with ER derived membranes via an amino-terminal amphipathic ␣-helix that has been proposed to be partially buried in one leaflet of the cellular membrane (Brass et al., 2002). Deletion of this helix leads to a diffuse cytoplasmic localization of NS5A and is lethal for RNA replication. Although NS5A has been clearly shown to be an ER associated protein in cells with actively replicating HCV replicons, numerous publications suggest an alternate nuclear localization. The presence of a cryptic nuclear localization signal in the interior of NS5A has been proposed (Ide et al., 1996). The exposure of this nuclear localization signal by a caspase mediated cleavage in apoptotic cells has been observed to allow the nuclear localization of NS5A, where is has been proposed to function as a PKA-regulated transcription factor (Goh et al., 2001; Satoh et al., 2000). The nuclear localization of NS5A and its function as a transcription factor are areas of significant controversy in the HCV community. The NS5A protein exists in multiple phosphorylation states, designated p56 (basal) and p58 (hyper) based on their migration on SDS-PAGE gels. The majority of phosphorylation occurs on serine residues, although some threonine phosphorylation has been observed (Kaneko et al., 1994; Reed et al., 1997). A number of phosphorylation sites have been mapped for 1a and 1b genotype NS5A sequences (Katze et al., 2000; Reed & Rice, 1999). The hyper phosphorylation of NS5A appears to require the presence of other HCV non-structural proteins in cis (Asabe et al., 1997). NS5A appears to be directly associated with the cellular kinase(s) responsible for its phosphorylation ( Reed et al., 1997; Tanji et al., 1995). A number of kinases have been proposed to be responsible for NS5A phosphorylation (Arima et al., 2001; Kim et al., 1999), but inhibitor studies suggest that a yet to be identified enzyme of the CMGC group (an abbreviation reflecting the best characterized members; CDK, MAPK, GSK3, CKII) of kinases is responsible (Reed et al., 1997). The same kinase activity is believed to phosphorylate the NS5A and NS5 proteins of pestiviruses and flaviviruses, respectively (Reed et al., 1998). Much of the work to date regarding the characterization and identification of the NS5A associated kinase activity has been performed in surrogate expression systems, and although the kinase activity appears to be evolutionarily conserved in yeast, insect and mammalian cells, the consequences of phosphorylation have not been examined in the context of HCV RNA replication. The NS5A phosphorylation state varies in a number of adaptive NS5A mutations in HCV replicons (Blight et al., 2000), but the link between NS5A phosphorylation and replication is unknown.

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The phosphorylation of NS5A, and its direct association with a cellular kinase or kinases, has led to the investigation of interactions between NS5A and cellular signal transduction pathways. The interactions observed are too numerous and convoluted to discuss in detail herein, and the reader is directed to (Reyes, 2002) for a review. The vast majority of these publications rely on the over expression of NS5A in the absence of a functional replicase, thereby complicating the interpretation of the data in the context of HCV replication. In addition, many of these studies are contradictory, with NS5A activating and inhibiting some of the same cellular pathways in different experimental systems. Another area of active research, and active debate, in the HCV community is the interaction of NS5A protein with PKR and the IFN/chemokine systems. The NS5A protein contains a short sequence in the central region of the protein referred to as the interferon sensitivity determining region (ISDR), named for the weak association of hypermutation in this region with response to IFN therapy for patients infected with HCV genotype 1b (Enomoto et al., 1996). Surprisingly, deletion of the ISDR does not affect the IFN sensitivity of HCV replicons (Lohmann et al., 2001). NS5A has been shown to bind PKR, via the ISDR, and inhibit the IFN induced activity of PKR on downstream targets, most notably eIF2␣, thereby preventing the antiviral effects of IFN (Gale et al., 1997, 1998, 1999; Gale & Katze, 1998; Gale, Korth & Katze, 1998; Pawlotsky et al., 1998; Pawlotsky, 1999). Another interaction of NS5A with the IFN response has been observed with the ability of NS5A to induce interleukin 8, leading to the inhibition of the antiviral effects of IFN (Pflugheber et al., 2002). The intricacies of the interaction of NS5A with PKR and IFN have been reviewed elsewhere (Reyes, 2002; Tan & Katze, 2001). NS5B The NS5B protein comprises the viral RNA dependent RNA polymerase activity (RdRp) required for the generation of a minus strand complimentary genome templates, and the subsequent synthesis of progeny plus strand genomic RNAs from this replicative intermediate. NS5B was initially predicted to function as an RNA polymerase based on the presence of the conserved GDD motif common to the active site of other polymerases (Choo et al., 1989). Mutation of this GDD motif abolishes infectivity of HCV transcripts in chimpanzees and blocks RNA replication in the replicon system (Blight et al., 2000; Kolykhalov et al., 2000; Lohmann et al., 1999). NS5B is a large (68 kD) hydrophilic protein that is found associated with ER derived membranes (Ivashkina et al., 2002; Schmidt-Mende et al., 2001). The association of NS5B with membranes has been determined require a hydrophobic 21 amino acid sequence at the carboxy terminus of the protein that has been proposed to form an ␣-helix (Ivashkina

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et al., 2002). The insertion of this sequence into membranes is believed to be posttranslational, making NS5B a member of the tail-anchored class of membrane proteins (Ivashkina et al., 2002). Deletion of this sequence leads to a predominantly nuclear localization of the polymerase (Ivashkina et al., 2002). Removal of this sequence in heterologous expression systems has allowed to generation of a soluble form of NS5B that retains enzymatic activity in vitro (Ferrari et al., 1999; Lohmann et al., 1997). A number of crystal structures of the soluble form of NS5B have been generated (Ago et al., 1999; Bressanelli et al., 1999, 2002; Lesburg et al., 1999; O’Farrell et al., 2003). These structures have been reviewed elsewhere in great detail (De Francesco et al., 2003; Hagedorn et al., 1999). The overall fold of NS5B is similar to that of other single chain polymerases, with a classic right hand topology containing distinct palm, finger and thumb subdomians (see Fig. 4D). Extensive interactions exist between the finger and thumb subdomains, thereby restricting movement of these domains relative to each other, resulting in a fully enclosed active site capable of binding nucleotides without further conformational changes (Lesburg et al., 1999). It is therefore believed that the structures of NS5B represent a polymerase during initiation events, and further conformational changes that have yet to be observed are required for elongation. Another unique feature of the NS5B polymerase is the presence of a ␤-hairpin in the thumb subdomain that is located close to the enzyme active site. This loop has been shown to restrict access to the active site and is believed to play a role in the initiation of RNA synthesis (Bressanelli et al., 2002; Hong et al., 2001). The thumb subdomain also contains an allosteric regulatory site that has been shown to bind GTP (Bressanelli et al., 2002). Recent structures of NS5B complexed with nonnucleoside inhibitors suggest the importance of the region of the thumb subdomain near this allosteric site in conformational changes required for the transition from initiation to elongation (O’Farrell et al., 2003). NS5B has been an important target for the development of antiviral drugs (see De Francesco, 2003 for review). The enzymatic activity of NS5B has been extensively studied (see De Francesco et al., 1996; Hagedorn et al., 1999; Lohmann et al., 2000 for review). NS5B is capable of the extension of both RNA and DNA primers in vitro using a variety of templates (Al et al., 1997, 1998; Behrens et al., 1996; Lohmann et al., 1997; Yamashita et al., 1998). Additionally, NS5B is capable of the synthesis of the entire HCV genome in vitro via a copy back method in which the 3′ end of the genome serves as an artificial primer for the extension of the nascent RNA (Behrens et al., 1996; Lohmann et al., 1997). The polymerase is also capable of de novo synthesis of RNA in the absence of any primer, and this mechanism of action is widely accepted as the relevant mechanism of action for NS5B (Luo et al., 2000; Zhong et al., 2000). A recent crystal structure of NS5B with bound nucleotides strongly supports the de novo synthesis of HCV RNA, as this structure is similar to the structure of the de

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novo initiation complex of the bacteriophage phi 6 polymerase (Bressanelli et al., 2002). An oligomerization of the HCV polymerase has been observed that may be important in cooperative activity of the enzyme (Qin et al., 2002; Wang et al., 2002). NS5B has been shown to directly interact with NS3, NS4A, and NS5A, and some of these interactions modify the enzymatic activity of NS5B (Ishido et al., 1998; Shirota et al., 2002). It is exciting to speculate these contacts mimic interactions in the functional HCV replicase.

HCV GENOME REPLICATION AND THE HCV REPLICASE The general mechanism of HCV RNA replication is believed to involve de novo initiation of RNA synthesis at the 3′ end of the genome followed by extension in the 5′ to 3′ direction to generate a minus strand complimentary genome template, and the subsequent synthesis of progeny plus strand genomic RNAs from this minus strand replicative intermediate. Examination of RNA copy number from cells bearing HCV replicons suggest 50–5,000 genomic RNAs are present per cell (Blight et al., 2000; Lohmann et al., 1999). The ratio of plus strand to minus strand RNAs in these systems is approximately 5–10:1. The mechanism for regulating the ratio of positive and negative strand RNA synthesis is unknown. Similar numbers have been determined from the examination of infected hepatocytes (Lanford et al., 1995). Replication is believed to occur in association with ER membranes and require the activity of the viral polymerase, helicase, and a mixture of the other nonstructural and structural proteins. Numerous host factors are likely to be involved in this process as well. The RNA replication complex has recently been observed in Huh7 cells containing HCV replicons, and this structure has been termed the membranous web (Gosert et al., 2003). The membranous web has been shown to contain all of the HCV structural and non-structural proteins as well as actively replicating RNA (Gosert et al., 2003). This structure is similar in appearance to the sponge-like inclusions seen in infected chimpanzee hepatocytes, and is most likely a modification of the host ER membrane (Pfeifer et al., 1980). The interaction of all of the HCV proteins with ER membranes has been described (see Dubuisson et al., 2002, for review). Recent biochemical assays have described several novel homo- and heterotypic interactions among HCV non-structural proteins that may be involved in the replicase (Dimitrova et al., 2003). Cell culture adaptive mutations that increase RNA replication efficiency in replicons have been observed in all of the non-structural proteins, suggesting the entire non-structural region is important for replication (Blight et al., 2000; Lohmann et al., 1999). The incompatibility seen when combining adaptive mutations in the same RNA suggests these mutations

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affect multiple phenomena to generate increased HCV RNA replication (Lohmann et al., 2003), although the mechanism of action of is not clear. Adapted cell lines that allow increased HCV replication suggest the importance of host cell factors in the replicase (Blight et al., 2002). Numerous host factors have been proposed to play a role in replication, but none of these have been validated (Tellinghuisen & Rice, 2002). Despite all of these observations, little is known about what constitutes a functional HCV replicase. Advances in the efficiency of HCV replicon systems in the past few years, yielding a tractable system for reverse genetics, will likely aid the understanding of HCV replication in future years (Blight et al., 2000, 2003). The recent development of a system allowing replication of HCV RNA in cell lysates generated from replicon bearing Huh7 cells may be a valuable tool in defining the relevant components of the HCV replicase, as has been performed for poliovirus (Ali et al., 2002; Hardy et al., 2003). Unfortunately, our understanding HCV replication and the HCV replicase are in their infancy, but this is an active area of research, and significant progress is expected in future years.

CONCLUSIONS In just 14 short years since the initial discovery of HCV as an infectious agent, significant progress has been made in understanding the molecular virology of this important pathogen, despite the numerous experimental difficulties associated with HCV research. A number of powerful experimental systems for the study of HCV have been developed, and through the use of these tools the biology of HCV has slowly started to emerge. The processing, localization, membrane association/topology, and putative functions of the majority of the HCV proteins have been determined. Considerable characterization of the HCV IRES and the viral NTRs has been performed. The site of HCV replication in cells bearing replicons has been observed. Considerable enzymatic characterization of HCV proteins with known activities has been performed. Molecular structures of half of the non-structural proteins have been determined to atomic resolution. Additional structural characterization of portions of other HCV proteins and the viral RNA has been performed. The structural and biochemical data generated to date has served to further the development of anti-HCV therapeutics, with many new potential pharmacological agents in development. Literally hundreds of interactions of the various HCV proteins and RNA with host cell proteins have been described, with effects attributed to many important cellular processes. The structure and properties of the HCV virion and the interactions of the virion components in assembly, receptor binding, penetration, and disassembly have only begun to be characterized, but newly described systems for studying these processes will likely

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lead to a greater understanding of HCV morphogenesis and infection. Despite the amazing progress made in understanding HCV, many questions remain to be answered. It is perhaps the greatest challenge in HCV research to distill the information in hand, as well as future observations, into concise mechanisms to describe the molecular virology of HCV.

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THE ROLE OF THE HEPATIC STELLATE CELL IN LIVER FIBROSIS

Timothy J. Kendall and John P. Iredale INTRODUCTION Hepatic fibrosis is the generic wound healing response of the liver which occurs as a consequence of a variety of injurious stimuli. Iterative or chronic injury mediated by viruses, autoimmune diseases and toxins such as alcohol all lead to a common histopathological outcome. In fibrosis the normal liver architecture becomes increasingly effaced. With continued damage the gross architectural distortion which characterizes cirrhosis develops (see Fig. 1). Fibrosis is characterized by qualitative and quantitative abnormalities of extracellular matrix. Increased amounts of fibrillar matrix, usually found only in the liver capsule and around large portal tracts, is present throughout the entire liver parenchyma, although the distribution may differ, depending on the exact nature of the injury. Cirrhosis can be considered the extreme end of the fibrotic spectrum, being characterized by the complete loss of normal liver architecture with nodules of regenerating hepatocytes surrounded by thick fibrous bands. The key effector cell co-ordinating the development of liver fibrosis has been shown to be the hepatic stellate cell (HSC) (Alcolado et al., 1997; Friedman, 1993), previously known as the Ito cell or lipocyte. As a consequence of liver injury, the HSC undergoes a phenotypic change called “activation” (Reeves & Friedman, 2002). The HSC is normally present as a small rounded quiescent cell but after activation becomes a myofibroblast-like cell (Sato et al., 2003). The activated HSC The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 497–523 © 2004 Published by Elsevier Ltd. ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15019-8

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Fig. 1. Histological Development and Recovery from Liver Fibrosis. Note: Damage to the normal liver (A, H&E stain) results in inflammation (B) and activation of hepatic stellate cells (staining positively ␣ smooth muscle actin), culminating in the development of fibrosis (C, after 4 weeks CCl4 , Sirius red staining) and ultimately in cirrhosis (D, after 12 weeks CCl4 , Sirius red staining). Withdrawal of the injurious stimulus may allow remodelling of the fibrillar matrix, leaving an attenuated cirrhosis (E, after 12 weeks CCl4 + 168 days recovery, Sirius red staining). Spontaneous resolution of fibrosis after removal of injury results in a return to near normal architecture (F, after 4 weeks CCl4 + 28 days recovery, Sirius red staining). It is unknown whether attenuated cirrhosis can undergo further remodelling with complete architectural resolution. Adapted from Iredale (2003), with permission from the author and BMJ Publishing Group.

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expresses a different range of genes that have a net profibrotic effect and produces the characteristic fibrotic extracellular matrix. Whilst contributing greatly to the change in the extracellular environment in fibrotic liver, HSCs are themselves influenced by the signals provided by this specialized environment. Extracellular matrix functions as a mechanical framework anchoring cells, and also enabling migration in a directional fashion. The underlying extracellular matrix of any given cell greatly influences the phenotype and range of genes expressed by interaction between extracellular matrix components and cell surface receptors, including integrins. Signals provided by extracellular matrix components can influence cell survival by promoting or preventing apoptosis, and modifying proliferative potential, in addition to influencing differentiation. Additionally, extracellular matrix acts to sequester and present or prevent exposure of soluble factors to cells, also influencing cell behavior and survival. Liver fibrosis is not an immutable entity, steadfastly replacing the normal architecture. Rather, it is a bi-directional process with a large capacity for significant architectural recovery from severe injury and fibrosis, and on the basis of current evidence, limited recovery from cirrhosis, once the profibrotic injury has ceased or been removed (Fig. 1) (Iredale, 2003; Iredale et al., 1998). There are two major features evident during the recovery process; a reduction in activated HSC numbers and a remodeling of fibrotic extracellular matrix with restoration of the original architecture. The interactions between HSCs and the extracellular matrix are critical to this process.

THE MICRO-ARCHITECTURE OF THE HEPATIC SINUSOID The liver is arranged microscopically into a sinusoidal pattern. Plates of hepatocytes, the epithelium, are separated from the endothelial cells lining the sinusoid by the space of Disse. Branches of the hepatic artery, hepatic portal vein and a bile duct form the portal tracts at one end of the sinusoids, and a tributary of the hepatic vein is at the other. The endothelial cells form a sheet with numerous fenestrations. The fenestrations are of two types, small (0.1␮m diameter) and large (up to 1 ␮m diameter). The small fenestrations are intracellular and the larger ones are thought to be intercellular. The fenestrations may be capable of changing in size in response to factors such as alcohol (Arias, 1990). Experimental evidence suggests that the composition of the extracellular matrix within the space of Disse is necessary for maintaining this fenestrated phenotype. Culture of sinusoidal endothelial cells in the absence of this complex cell-matrix contact results in loss

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of the fenestrations, whereas culture on the basement membrane side of human amnion allows the fenestrations to persist (McGuire et al., 1992). There is a free connection between the environment of the Space of Disse and the sinusoidal lumen since there is no membrane over the fenestrations, nor is there a “classical” basement membrane (in humans), although a loose matrix of basement membrane components can be demonstrated (see below). This allows the fenestrations to freely filter the sinusoidal blood so that solutes within the blood can easily diffuse into contact with the plates of hepatocytes but larger particles are unable to do so. The space of Disse separates the sinusoidal endothelial cells from the plates of hepatocytes. The sinusoidal surface of hepatocytes is covered by numerous microvilli, facilitating exchange of substances between the hepatocytes and the extravascular filtrate. The extracellular matrix present within the space of Disse does not act as a diffusion barrier. It is a low density matrix with both an interstitial and a basement membrane like component. There is a small amount of fibrillar collagens I, III and V, and microfibrillar collagen VI in addition to basement membrane-like collagens IV and XVIII, and non-collagenous components such as decorin, fibronectin, tenascin, laminin, heparan sulfate proteoglycans and others (Abdel-Aziz et al., 1991). The proteoglycan components function to regulate matrix assembly and the architectural construction. Some proteoglycans molecules have glycosaminoglycans side-chains, allowing them to interact with collagens and glycoproteins. There are differences in the components composing the extracellular matrix in the space of Disse according to the distance from the portal tracts (MartinezHernandez & Amenta, 1993). In the periportal areas laminin, heparan sulfate and collagen IV predominate but in the perivenular regions there is more collagen III and dermatan sulfate. Interstitial-type extracellular matrix, composed of fibrillar collagens and less basement membrane type components, is found under physiological circumstances around portal tracts, central veins and the liver capsule. The true basement membrane of the endothelium of bile ducts and blood vessels in these areas is an exception to this. The specialized environment of the space of Disse contains quiescent hepatic stellate cells (HSCs). HSCs are responsible for producing the majority of extracellular components present in the space of Disse (Abdel-Aziz et al., 1991), and the specialized matrix is responsible for maintaining the quiescent non-proliferative phenotype. The matrix also has a role in maintaining the normal physiological function of the other cell types it has contact with, namely Kupffer cells, the sinusoidal endothelial cells and hepatocytes (Kim et al., 1997). Hepatocytes have receptors on their cell surfaces to recognize matrix components such as fibronectin, laminin and collagen type IV (Clement et al., 1990; Forsberg et al., 1990).

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The architecture of the liver and its extracellular matrix is assessed in liver biopsies by simple microscopy of H & E stained sections and reticulin and orcein staining. This allows the extent, age and distribution of fibrosis to be assessed. Staining with Sirius red allows the quantitative assessment of the distribution of extracellular matrix components, and is used particularly as a research tool. Individual components of the extracellular matrix can be localized in normal and diseased liver by immunohistochemical techniques, and quantitative assessment can be made of the relative amounts of specific mRNAs and proteins in tissue samples by PCR or Western blotting, respectively.

THE QUIESCENT HEPATIC STELLATE CELL Hepatic stellate cells account for 5–8% of cells within normal liver. Their embryonic derivation has been the subject of some debate. The traditional view is that HSCs are derived from mesenchymal cells of the septum transversum. Hepatocytes and cells of the bile duct system derive from endodermal cells of the hepatic diverticulum (Zajicek, 1991) and sinusoidal endothelial cells also derive from the septum transversum. However, it has been suggested that HSCs may have a common precursor with hepatocytes based on the embryonic expression of putative lineage markers (Kiassov et al., 1995; Vassy et al., 1993). More recently, it has been shown that HSCs express a number of neuronal markers including glial fibrillary acidic protein (GFAP) (Neubauer et al., 1996), p75 (Trim et al., 2000), synaptophysin (Cassiman et al., 1999), and N-CAM (Knittel et al., 1996; Nakatani et al., 1996), and also have dendritic processes similar to those of astrocytes rather than fibroblasts. This has led to speculation that HSCs may be neuroectodermal cells. Quiescent HSCs in normal liver reside within the space of Disse. They are rounded cells with numerous cytoplasmic processes. These processes allow a single HSC to make contact with a number of hepatocytes, endothelial cells and other HSCs. The cytoplasm of quiescent HSCs contains characteristic lipid droplets rich in vitamin A (Sato et al., 2003). These droplets produce rapidly fading luminescence when excited by fluorescent light of 328 nm wavelength. Under normal conditions, HSCs are non-proliferative, as assessed by tritiated thymidine or bromodeoxyuridine incorporation. Quiescent HSCs have an important role in vitamin A homeostasis (Blomhoff & Wake, 1991). Retinoid in the diet is esterified by small intestinal epithelial cells and enters the body within chylomicrons. Chylomicrons containing retinol esters are taken up by hepatocytes. Within hepatocytes the esters are hydrolysed back into retinol. Retinol can then either enter the circulation or be transferred to HSCs. Ninety percent of the body’s retinol stores are in the liver (Blaner & Olson,

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1994), and 75% of this is within HSCs (Blomhoff et al., 1985a, b). Once in HSCs retinol is again esterified, and the esterified forms are stored. These stores can be mobilized as necessary. The interaction of HSCs with the extracellular matrix is also of critical importance (Gaca et al., 2003; Sohara et al., 2002). The communication of HSCs and other cell types with extracellular matrix components is mediated by integrins (Imai & Senoo, 1998). These membrane-bound molecules connect the extracellular matrix to the cytoskeleton of the cells involved, particularly the ␣1 ␤1 , ␣2 ␤1 , ␣v ␤3 and ␣3 ␤1 integrins (Pinzani et al., 1998; Sato et al., 1998). Quiescent HSCs contribute heavily toward the formation of the extracellular matrix found within the space of Disse in normal liver. They express fibrillar collagen type III, basement membrane type IV and laminin (Abdel-Aziz et al., 1991). Contributions to this environment are also made by other cell types exposed to it, particularly endothelial cells and hepatocytes (Du et al., 1999). Hepatocytes may produce a small amount of collagen and proteoglycans (MacSween et al., 2002). The normal homeostasis of extracellular matrix involves both production and degradation of all components. The turnover rates for the various molecules vary. HSCs play an important part in facilitating matrix degradation as part of this process. mRNA transcripts for matrix metalloproteinases 1, 3 and 13 (MMP-1, -3, -13) have been identified in freshly isolated quiescent HSCs (Herbst et al., 1997; Iredale et al., 1996; Knittel et al., 1999). Inhibitors of these MMPs, tissue inhibitors of metalloproteinase -1 and -2 (TIMP-1 and -2), are not strongly expressed in quiescent HSCs. The expression of all of these genes, involved in the production of proteins that contribute to the extracellular matrix or its turnover, is crucially altered after HSC activation to produce a profibrotic activated HSC phenotype (Benyon & Arthur, 2001).

HEPATIC STELLATE CELL ACTIVATION AND THE ACTIVATED PHENOTYPE Hepatic stellate cell activation represents a key step in the final common pathway that results from acute and chronic liver injury (Reeves & Friedman, 2002). Phenotypically, HSCs undergo a range of fundamental changes. They adopt a myofibroblast-like phenotype and shed the stored retinoids. In addition, a number of contractile proteins are expressed, including, characteristically, ␣ smooth muscle actin (Sato et al., 2003). The activated HSC expresses a range of different receptors on its cell surface, allowing it to respond in differing ways from the quiescent phenotype, and reflecting its role as a wound healing myofibroblast. For example, activated HSCs express PDGF receptors, facilitating a proliferative response on

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exposure to PDGF (Kinnman et al., 2001). IL-10 expression has been demonstrated in activated HSCs in vitro and in vivo, and IL-10 receptor mRNA expression was induced by injury in vivo and culture activated HSCs (Mathurin et al., 2002). The process of activation follows a predictable course both at the molecular and cellular level. The nuclear events underlying this change are complex and have been extensively reviewed elsewhere (Mann & Smart, 2002). The activation of HSCs is accompanied by proliferation, accounting for the greatly increased numbers of activated HSCs observed in fibrotic livers when compared with the numbers of quiescent HSCs seen in normal livers. The kinetics of HSC proliferation during activation have been examined. Using an in vivo model of liver fibrosis, HSC proliferation, determined by BrdU incorporation, was observed to occur after 24–48 hours after bile duct ligation (Kinnman et al., 2001). This behavior coincided with the expression of PDGFR-␤ protein, a receptor with a well described mitogenic effect. Using primary cultures of isolated HSCs, the phenomenon of activation and proliferation can be mimicked by culture on plastic in the presence of serum. In this model, activation is associated with a burst of proliferative activity after which cell turnover decreases, but to a level above that seen in quiescent cells (Suzuki et al., 2001). In contrast, the production of procollagen I and III increased steadily during this time period. The establishment of such in vivo and in vitro models have greatly facilitated our investigation and understanding of the cell and molecular events underlying hepatic fibrogenesis. The factors driving HSC proliferation during activation have been studied. The most potent HSC mitogen is platelet-derived growth factor (PDGF) (Failli et al., 1995; Pinzani, 2002). This has been detected in the liver in fibrosis, and HSCs express PDGF receptors (Wong et al., 1994). The addition of PDGF to cultures of activated HSCs proliferation, while PDGF receptor tyrosine kinase inhibitors are highly effective at reducing HSC proliferation (Kinnman et al., 2001). Other molecules present in the context of liver fibrosis induce HSC proliferation. During activation there is increased expression of proteinase-activated receptor1 and -2 mRNA (Gaca et al., 2002). Agonists for these receptors, thrombin and tryptase, respectively, induced proliferation when added to cultures of activated HSCs. The addition of retinoic acid acts to reduce activation, measured by the ␣ smooth muscle actin production, and reduce proliferation, assessed by bromodeoxyuridine incorporation (Chi et al., 2003). The progression through the cell cycle that is necessary to allow cell division is intimately linked to apoptosis. A cell can be forced to undergo apoptosis by the action of the key molecules pRB and p53 (Morris, 2002; Vermeulen et al., 2003). This occurs, for example, when irreparable DNA damage is identified in the dividing cell. Adenovirus-mediated transfer of p53 or pRB to HSCs induces increased apoptosis and decreased proliferation (Abriss et al., 2003). The balance between

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cell proliferation and apoptosis is the determinant of overall population number, and is significant in both the increase in HSC numbers seen during the development of fibrosis and the decrease seen during recovery. Most cells, apart from stem cells, are unable, under physiological conditions, to multiply indefinitely. The limited replicative potential, therefore, results in cells adopting a state of retirement called replicative senescence (Bree et al., 2002; Lundberg et al., 2000). This occurs due to the shortening of telomeres that occurs with each cell division, since non-stem cells do not have the ability to replicate telomeric material (Newbold, 2002). Proliferative populations such as activated HSCs will obviously be susceptible to this phenomenon, although it may be more significant in vitro and not of major pathophysiological consequence. To study this, early passage HSCs and HSCs expressing human telomere reverse transcriptase (hTERT) were compared with senescent activated HSCs. Senescent HSCs showed increased synthesis of inflammation and stress-related proteins but decreased extracellular matrix synthesis (Schnabl et al., 2003). Senescent cells had a greatly increased apoptotic rate, assessed by TUNEL staining. Telomerase-deficient mice lacking the essential telomerase RNA gene develop accelerated cirrhosis with pro-fibrotic stimuli. The delivery of the gene by adenoviral vectors alleviated the cirrhotic pathology (Rudolph et al., 2000). In other organs the wound healing myofibroblast expresses angiotensin II which promotes inflammation and fibrosis (Weber, 1997). Active angiotensin II is produced from angiotensin I by the action of angiotensin converting enzyme (ACE). Angiotensin I itself is produced from angiotensinogen by active renin. Angiotensin II has a strong pressor effect, and inhibition of its action by ACE inhibition or receptor blockade is a widely used strategy in the treatment of hypertension. However, angiotensin II also has a number of other actions at sub-pressor doses at a cellular level, for example in promoting myocardial fibrosis (Brilla, 2000), possibly via regulation of TGF-␤1. In quiescent HSCs there is limited expression of renin, angiotensinogen and ACE, and no active angiotensin II production (Bataller et al., 2003a). In activated HSCs, in vivo and in vitro, there is expression of active renin and ACE, and angiotensin II is secreted (Paizis et al., 2002). Angiotensin II protein has been localized to the cytoplasm of activated HSCs (Bataller et al., 2003a). The degree of angiotensin II production is increased in the presence of soluble factors such as PDGF, EGF, endothelin-1 and thrombin (Bataller et al., 2003a). When angiotensin II is infused into rats at both pressor and sub-pressor doses there is liver injury, demonstrated by a rise in liver enzymes, increased NF␬B and AP-1 DNA binding activity, and increased c-Jun N-terminal kinase activity, nuclear events associated with HSC activation (Bataller et al., 2003b). There is also an increase in nitric oxide synthase (NOS) and inducible cyclo-oxygenase. Histologically, there is portal inflammation, an increase in activated HSCs and a slight increase in collagen deposition. Cultured activated HSCs have been shown by

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binding studies to express angiotensin type I receptors (AT-1) (Wei et al., 2001), and to express the equivalent mRNA (Paizis et al., 2001, 2002). The addition of angiotensin II to cultures causes an increase in [Ca2+ ]i , increases the rate of activation, causes HSC proliferation (Bataller et al., 2000; Zhang et al., 2003), and increases the expression of collagen and TGF-␤ mRNA. These findings are inhibited by AT-I receptor antagonsists. The addition of angiotensin II to HSC cultures also accelerates activation. Using in vivo models of liver fibrosis, carbon tetrachloride-induced (Wei et al., 2001), bile duct ligation (Paizis et al., 2002) and pig serum induced (Yoshiji et al., 2001), it has been demonstrated that there is increased expression of ACE and AT-I receptors, especially in the fibrotic areas. Immunohistochemical analysis has shown that this is in ␣ smooth muscle actin expressing cells. Given these results, it is perhaps not surprising that the blockade of the renin-angiotensin system has been shown to reduce fibrogenesis. Using a range of either ACE inhibitors or AT-I receptor blockers during experimental liver injury has shown a decrease in the degree of fibrosis, on histological grounds, and a reduction in other markers or mediators of fibrosis, for example AT-I, TGF-␤1 and collagen mRNA (Jonsson et al., 2001; Kurikawa et al., 2003; Ohishi et al., 2001; Wei et al., 2000; Yoshiji et al., 2001, 2002a).

THE PRODUCTION OF FIBROTIC MATRIX Activated HSCs secrete excess amounts of fibrillar collagens I and III that predominate in the matrix of fibrotic liver, in addition to a range of other matrix components. As discussed previously, there is an increase in all matrix components and collagen types in fibrotic liver but with the production of the fibrillar collagens being increased disproportionately and deposited in unfamiliar sites. The major cellular source of this is the activated HSC (Alcolado et al., 1997; Friedman, 1993). Using the model of HSC activation in vitro by culture on tissue culture plastic, increased levels of mRNAs coding for pro-collagens I, III and IV have been identified in activated HSCs (Du et al., 1999). Transcripts and protein of noncollagenous matrix components such as laminin, fibronectin, elastin, and tenascin have also been identified (Abdel-Aziz et al., 1991; Kanta et al., 2002; Odenthal et al., 1993; Ramadori et al., 1991; Van Eyken et al., 1992). Dystroglycan, a membrane component of the dystrophin-glycoprotein transmembrane complex needed for the spatial organization of laminin is upregulated in activated HSCs compared with their quiescent counterparts (Bedossa et al., 2002). Other cell types present in fibrotic liver also produce matrix components, but at much lower levels than activated HSCs. For example, the endothelial cells of the sinusoid and small septal blood vessels show procollagen IV mRNA expression

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and type IV collagen expression in experimentally induced rat liver fibrosis (Du et al., 1999).

EXTRACELLULAR MATRIX DEGRADATION – TIMPS AND MMPS The accumulation of matrix in liver fibrosis is a result of a reduction in the normal matrix degradation pathways as well as increased production, resulting in a net increase. Matrix metalloproteinases (MMPs) are a group of enzymes with a physiological role in normal matrix homeostasis, and a pathological role in the development of liver fibrosis (Arthur, 2000; Benyon & Arthur, 2001). There are a wide range of substrates susceptible to the action of these enzymes, including both collagenous and non-collagenous matrix components. The activity of the MMPs is not purely regulated by the rate of their transcription and translation but also heavily dependent on post-translational processing. MMPs are transcribed and translated initially as pro-enzymes, requiring the cleavage of a prodomain that functions to inhibit the catalytic domain. This is achieved in vivo in a number of different ways, depending, in part, on the exact MMP in question. Natural inhibitors of MMPs are found in vivo, specifically the tissue inhibitors of matrix metalloproteinase (TIMP) family. The general inhibitor ␣2 -macroglobulin also inhibits MMP activity in a non-specific manner. The MMPs can be broadly grouped together based on their substrate specificity although there is a degree of overlap using this system. Those MMPs that are able to degrade mature interstitial collagen (Types I, II, III, and X) by cleaving ␣ chains of a specific Gly-Ile/Leu site, are said to have interstitial collagenase activity. MMP-1 (Interstitial collagenase), -8 (Neutrophil collagenase), -13 (Collagenase3) and -14 (MT1-MMP) (Ohuchi et al., 1997) have this ability, although their individual affinities for the various substrates varies. The gelatinases, gelatinase-A (MMP-2) and gelatinase-B (MMP-9), are enzymes that degrade denatured interstitial collagen (i.e. gelatin) in addition to other non-collagenous matrix components and MMP-2 may also have interstitial collagenase activity (Aimes & Quigley, 1995). The membrane-type MMP (MT-MMPs) are a subgroup that have a transmembrane domain or anchor to glycosyl phosphatidyl inositol (GPI), thus rendering them membrane bound. They have a broad substrate range, some with interstitial collagenase activity, as well as being able to act upon a variety of non-collagenous and gelatin substrates. Importantly, certain MT-MMPs have a role in the activation of pro-MMPs. Activation of pro-MMPs is achieved in vivo in a number of different ways. MMP-3 (stromelysin) activates a number of other pro-MMPs. MMP-3 can be activated by plasmin and a number of enzymes related to inflammation such as

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tryptase from mast cells (Gruber et al., 1989) and neutrophil elastase (Okada & Nakanishi, 1989). This means that inflammation can produce widespread MMP activation and, thus, matrix destruction. The stromelysins and MMP-1 and -8 are activated by plasmin, which is itself produced from plasminogen by the action of the plasminogen activators uPA or tPA, a step that is inhibited by plasminogen activator inhibitor-1 (PAI-1) (Irigoyen et al., 1999). The full activation of MMP1 requires the action of MMP-3 in addition to plasmin (Suzuki et al., 1990). Gelatinase A is activated by MT1-MMP at the cell surface (Murphy et al., 1999), a process which requires TIMP-2 to form the activating complex (Sato et al., 1996). Natural inhibitors of MMP activity are present within the liver under normal conditions and their expression changes during the development of liver fibrosis with the net outcome facilitating excess matrix deposition (McCrudden & Iredale, 2000). ␣2 -macroglobulin is a large serum proteinase inhibitor that inhibits all MMPs in a general manner (Enghild et al., 1989). Within the liver ␣2 macroglobulin is produced by hepatocytes and HSCs (Andus et al., 1987). However, in vivo probably the most quantitatively significant inhibitors of MMPs are the TIMPs (McCrudden & Iredale, 2000). There are four TIMPs described (TIMPs 1–4) and all are able to inhibit all MMPs, but with differing affinities (Gomez et al., 1997). The only exception to this is that TIMP-1 is unable to inhibit MT1-MMP, MT2-MMP, and MT5-MMP (Will et al., 1996). They bind non-covalently and reversibly to the active site of the MMPs. The TIMPs are often produced by the same cell types that produce the MMPs themselves, suggesting that autocrine and paracrine regulation of matrix turnover is significant. Both TIMP and MMP expression can be influenced by soluble factors in a complex manner, increasing or decreasing expression of either type of molecule. For example, TGF-␤1 induces TIMP-1 and inhibits TIMP-2 expression (Zafarullah et al., 1996) but also inhibits MMP-3 and -1. TIMPs also appear to function as survival factors, preventing certain cells from undergoing apoptosis, another autocrine and paracrine effect.

TIMP AND MMP EXPRESSION BY ACTIVATED HSCS IN FIBROTIC LIVER In vivo and in vitro experimentation has helped to delineate the role of HSC expression of MMPs and TIMPs in the development of liver fibrosis. Much work has utilized an in vitro method of activation of HSCs by culture on tissue culture plastic. This produces a fully activated phenotype; the retinoids are shed early in this process, and there is expression of ␣-smooth muscle actin and pro-collagen 1 after 14 days of primary culture, and this phenotype persists with subsequent passaged cultures.

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The study of cultured rat HSCs has demonstrated that HSCs express MMPs and TIMPs in a sequence during the course of activation. During the first three days of primary culture, HSCs have a predominantly matrix-degrading pattern of gene expression, expressing MMP-3 (Vyas et al., 1995), MMP-13 (Iredale et al., 1996) (in rats) but not TIMP-1 or -2. It has been suggested that this is an acute phase response that could contribute to tissue damage seen with an acute liver injury. HSCs in early primary culture also express elements of the plasminogen activating system that is necessary for MMP-1 activation (Leyland et al., 1996). Similar results were obtained with cultured human HSCs except that the main interstitial collagenase expressed is MMP-1 (Iredale et al., 1995). After the initial stages of activation, and persisting in fully activated cells, MMP1 or -13 expression is markedly reduced, and TIMP-1 and -2 expression increases and is maintained (Benyon et al., 1996; Iredale et al., 1995, 1996). This pattern of expression favors the accumulation of fibrillar collagen-rich matrix. There is also increased expression of MMP-2 (Gel A) (Benyon et al., 1999) and MT1-MMP. Plasma-membrane enriched fractions from hepatocytes and conditioned medium from pure cultures together promote activation of MMP-2 (Theret et al., 1997). The combination of gel A, MT1-MMP and TIMP-2 allows the activation of gel A. This means that the activated HSC has the capacity to degrade matrix rich in collagen IV that is found in normal liver and in the matrix within the space of Disse. In culture, the activation of MMP-2 was enhanced in the presence of collagen I, but not laminin (Preaux et al., 1999). This effect was blocked by antibodies against ␣2 ␤1 integrins (Theret et al., 1999) and demonstrates that HSCs are responsive to the extracellular matrix in which they reside. MMP-2 was shown to be expressed at maximal levels corresponding to the peak time of HSC proliferation, and antisense oligonucleotides to pro-gelatinase A or experimental MMP inhibitors reduced the proliferation of cultured HSCs by over half (Benyon et al., 1999). However, although there is still potential gelatinase activity allowing the degradation of such substrates as collagen IV, the overall amount of all matrix components including collagen IV, is increased in fibrotic liver. It may be that the gelatinase activity is restricted to the sinusoidal areas where fibrillar matrix is deposited, and other regions see increased deposition. Indeed, in tissue culture experiments where the inhibitory effect of TIMP is removed by chromatography, the activity of HSC produced MMP-2 increases 20 fold, indicating that activated HSCs mediate a profound inhibition of matrix degradation. The inhibition of HSC TIMP-1 expression by antisense TIMP-1 recombinant plasmids increases collagenase activity in the media of cultured HSCs and inhibits accumulation of type I and III fibrillar collagens in vitro. This illustrates the complex nature of the interactions between matrix, TIMPs, and MMPs in relation to hepatic stellate cells (Liu et al., 2003a).

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Much of this work has been supported by in vivo studies, both in animal models and studying diseased human liver. Increased TIMP-1 and -2 mRNA and protein expression has been described in the fibrotic livers of patients with sclerosing cholangitis, primary biliary cirrhosis, biliary atresia and autoimmune chronic active hepatitis (Benyon et al., 1996). During the development and progression of chronic liver fibrosis in animal models, the levels of MMP-13 or -1 expression does not change (Milani et al., 1994) but there is a marked increase in the expression of TIMPs -1 and -2 (Iredale et al., 1996; Herbst et al., 1997). In situ hybridization studies have localized the expression of these, mainly to hepatic stellate cells. This increased expression of TIMP-1 has been shown to occur prior to the increase in pro-collagen I expression and collagen I deposition (Iredale et al., 1996), implying that fibrotic matrix is deposited in a microenvironment where the degradative capacity has already been reduced. The importance of TIMP-1 in the development of liver fibrosis has been further highlighted by studies using mice overexpressing human TIMP-1. Overexpression of TIMP-1 in the absence of liver injury did not produce any liver fibrosis, as adjudged by hepatic collagen accumulation (Yoshiji et al., 2000). There was also no difference between ␣-smooth muscle actin mRNA expression in wild-type and TIMP-1 overexpressers, implying that TIMP-1 by itself did not produce any HSC activation. However, after experimental liver injury with carbon tetrachloride the over-expressing group had a seven fold increase in liver fibrosis measured by densitometric analysis and hydroxyproline content. Collagen I and IV accumulation was also markedly increased. The production of active MMP-2 and MT1-MMP demonstrated in vitro has also been borne out by in vivo studies (Kossakowska et al., 1998; Milani et al., 1994; Watanabe et al., 2001). In chronic liver fibrosis, there is a prolonged increase in pro-MMP-2 expression, and increased active MMP-2 (Takahara et al., 1995, 1997). The production of pro-MMP-2 and MT1-MMP has been localised in sections of diseased liver to HSCs (Watanabe et al., 2001). TIMPs are also able to influence other cellular behavior. They have been shown to affect apoptosis and proliferation in various cell types. For example, TIMP-1 protects human breast epithelial cells against apoptosis (Li et al., 1999; Liu et al., 2003b), an effect independent of the MMP inhibiting property of the molecule. However, in human T lymphocytes, TIMP-2 is pro-apoptotic (Lim et al., 1999), and acts as a growth factor for mesenchymal cells in rat kidney (Barasch et al., 1999). In liver fibrosis, TIMP-1 mediates its development and persistence not merely by inhibiting MMP activity but also by enhancing activated HSC survival, acting as an autocrine and paracrine survival factor. TIMP-1 protein was shown to inhibit apoptosis induced by cycloheximide, serum deprivation or nerve growth factor in cultured rat HSCs, and in human cultured HSCs by cycloheximide, and serum deprivation, based on the recognition of apoptotic morphology and TUNEL

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staining (Murphy et al., 2002). The autocrine and paracrine role of TIMP-1 in HSC cultures was established by the demonstration that the addition of a TIMP-1 neutralizing antibody to activated HSC cultures in normal growth media increased the rate of apoptosis. In HSCs, this anti-apoptotic effect of TIMP-1 was dependent on the MMP-inhibiting capacity since a mutated form of TIMP-1, whose MMPinhibiting domain was defective but was otherwise normal, did not produce the same anti-apoptotic results. Moreover, a synthetic MMP-inhibitor was shown to protect HSCs from apoptosis (Murphy et al., 2002).

INTERACTIONS BETWEEN HSCS AND EXTRACELLULAR MATRIX HSCs are critical in both extracellular matrix deposition and degradation. However, the extracellular matrix itself is able to exert a wide-ranging influence over HSC behavior, phenotype and survival. HSCs studied in an in vitro Boyden chamber allowed the effect of the putative microenvironment on HSC behavior to be studied, particularly the migratory capacity. Addition of the soluble factor TGF-␤, a molecule with a known profibrotic role, upregulated MMP-2 activity and increased the migratory capacity of activated HSCs (Yang et al., 2003). The increased migration was inhibited by the addition of MMP-2 and -9 inhibitors, and by antibodies that blocked ␣1 and ␣2 integrins. Additionally, collagen I alone induced HSC migration. This demonstrates that MMP-2 activity may be required to facilitate migration of HSCs, and that the interstitial collagens that predominate in fibrosis also promote migration. Rearrangement of newly produced actin filaments occurred during the activation process. The most effective single component of matrix for HSC migration is laminin (Amakawa & Endo, 2002). HSC activation has been shown to be influenced by the fibrotic matrix. When activated HSCs were removed from tissue culture plastic and replated onto matrigel, a synthetic basement-membrane like product, the proliferation index fell by > two-thirds, and type I pro-collagen and ␣-smooth muscle actin mRNA expression fell to undetectable levels (Gaca et al., 2003). In the control experiment, the cells were replated directly back onto plastic and the activated phenotype was maintained. If the cells were plated onto collagen I instead, the state of activation also persisted. HSCs replated on matrigel are also able to re-acquire lipid droplets (Sohara et al., 2002). This suggests that there is notable phenotypic plasticity, with HSCs able to change between quiescent and activated phenotype depending on their environment. Currently, there are no experimental tools available to determine the significance of phenotypic reversion in liver fibrosis in vivo.

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The importance of collagen I in maintaining the activated state was demonstrated using a mouse strain bearing a mutated collagen gene that confers resistance to degradation by collagenase. During spontaneous recovery from carbon tetrachloride induced liver fibrosis markers for fibrillar collagen cross-linking remained at the level that occurred at peak fibrosis (Issa et al., 2003). Markers of activated HSCs also remained high. The control wild-type mice showed the expected significant reduction in hydroxyproline content, and HSC markers fell to normal levels. This demonstrates that the microenvironment is not only formed by HSCs but is also critical in maintaining their phenotype. Generally, extracellular matrix components transmit signals to cells via transmembrane receptors. The most important group of this type is the integrins. The integrins are dimeric transmembrane receptors with an ␣ and ␤ subunit. A number of different subunits have been identified within the liver (Imai & Senoo, 1998), and in HSCs. As an example of their importance, antibodies toward ␣2 ␤1 integrins blocked the collagen I induced MMP-2 activation in cultured HSCs (Theret et al., 1999). The integrin ␣8 ␤1 has also been shown to be expressed by activated HSCs in carbon tetrachloride induced liver fibrosis (Levine et al., 2000). When cell contact is lost completely the normal behavior is severely compromised. HSCs in complete suspension stop FAK tyrosine phosphorylation after 12 hours, and the usual PDGF induced Ras-GTP loading no longer occurs (Carloni et al., 2002). The proliferative response to PDGF is also markedly reduced.

THE INFLUENCE OF SOLUBLE FACTORS ON THE HSC PHENOTYPE Extracellular matrix does not only influence cell behavior by physical interaction. The matrix also acts as a reservoir and presenter of a legion of soluble factors that influence cells (Taipale & Keski-Oja, 1997). Binding to extracellular matrix components can protect some soluble factors from degradation, and conversely by being bound to matrix can limit the availability of others to interact with cells (Raines et al., 1992). The proteoglycans function as an important growth factor binding component via interaction of their protein core or glycosaminoglycan side chains. Hepatic stellate cells are responsive to soluble factors, with their proliferation, differentiation or activation state, and migration all modifiable. For example, platelet-derived growth factor-AA (PDGF-AA), a potent HSC mitogen, is present in the context of liver injury and fibrosis (Kinnman et al., 2001; Wong et al., 1994). It interacts with heparan sulphate in order to allow correct interaction with the membrane-bound receptor (Andersson et al., 1994). A variety of collagens

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found in normal and cirrhotic liver have been shown to bind PDGF isoforms (Somasundaram & Schuppan, 1996). TGF-␤1 is the classically described profibrotic growth factor. TGF-␤1 mRNA is present in normal liver mainly in Kupffer cells, with small amounts in HSCs and endothelial cells (De Bleser et al., 1997). Much lower levels of TGF-␤2 and TGF-␤3 mRNA are present. It is found at increased levels in fibrotic livers, and activated HSCs are an important cellular source. In fibrotic liver, TGF-␤1 mRNA is expressed strongly in all sinusoidal cells, 12 times more in HSCs and six times more in endothelial cells but with no increase seen in Kupffer cells (De Bleser et al., 1997). Increased levels of TGF-␤1 mRNA have also been demonstrated in fibrotic human liver and in the bile duct ligation model of liver fibrosis (Roulot et al., 1999). The addition of TGF-␤1 to cultures of HSCs causes increased matrix production and affects TIMP and MMP to enhance the fibrotic phenotype (Knittel et al., 1999; Schaefer et al., 2003). In TGF-␤1 knock-out mice, the fibrotic response to liver injury is markedly reduced. Adenovirus mediated transfection of antisense mRNA to the coding sequence of TGF-␤1 suppresses the synthesis of TGF-␤1 in HSC cultures and reduces their fibrotic activity (Arias et al., 2002). When TGF-␤1 activity is artificially reduced in animal models of liver fibrosis there is a reduction in the extent of fibrosis achieved. The addition of a soluble TGF-␤ type II receptor that inhibits cellular type II receptor binding, at the time of, or 4 days after, bile duct ligation produces less collagen mRNA and protein (George et al., 1999). A similar result was obtained using an adenoviral vector to locally express truncated type II TGF-␤ receptor in a dimethylnitrosamine (DMN) induced model of liver fibrosis (Qi et al., 1999). Latent TGF-␤1 is activated by the action of plasmin. The addition of a protease inhibitor that blocks this process produced decreased active TGF-␤ and blocked HSC activation (Okuno et al., 2001). In vivo this protease inhibitor also reduces the degree of liver fibrosis and HSC activation. Using a double transgenic mouse with a tetracycline regulated gene expression system, TGF-␤1 levels could be increased by 10–30 times controllably. Cyclical increases in TGF-␤1 induced intermediate fibrosis and increased the number of activated HSCs. High rates of hepatocyte apoptosis were also observed. This fibrosis was reversible when the TGF-␤1 levels were allowed to return to, and remain at, normal (Ueberham et al., 2003). There is also an interaction between the loss of retinol from quiescent HSCs and TGF-␤1.9,13-di-cis-retinoic acid is a metabolite of retinol that is found in HSC cultures as they lose their lipid droplets during activation, and is also found in increased amounts in a pig-serum induced model of liver fibrosis (Okuno et al., 1999). This isomer enhances cellular plasminogen activator and plasmin, and produces more active TGF-␤1 (Okuno et al., 1997). The addition of it to cultured HSCs produced increased TGF-␤-dependent collagen synthesis via RAR␣ receptors, and also accelerates fibrosis in the pig-serum model (Okuno et al., 1997, 1999).

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HSCS AND THE RECOVERY FROM LIVER FIBROSIS Liver fibrosis is not irreversible. Recent work has demonstrated a great capacity for resolution and remodeling of fibrotic matrix if the pro-fibrotic injury is withdrawn (Iredale et al., 1998). Studies using rodent models of recovery have delineated this process. In both bile duct ligation and carbon tetrachloride induced rat liver fibrosis (Iredale et al., 1998; Yoshiji et al., 2002b) there is spontaneous recovery of the normal liver architecture after reanastomosis of the bile duct (Abdel-Aziz et al., 1990; Issa et al., 2001) or cessation of carbon tetrachloride administration (Iredale et al., 1998; Watanabe et al., 2001), respectively (see Fig. 1). During this recovery period the liver architecture is remodeled, with resorption of fibrotic matrix and reconstitution of the normal architecture. The number of activated HSCs is reduced dramatically during this period. A significant part of this loss of activated HSCs results from stellate cell apoptosis. Apoptosis, or programmed cell death, can broadly be considered to be triggered in two different ways. A cell will default to the apoptotic sequence if there is a loss of normal constitutive survival signals; alternatively, apoptosis will result if an proapoptotic signal is present. Survival signals can be in the form of cell-cell contact, cell interaction with the local extracellular matrix, or provided by soluble factors such as growth factors or cytokines. The process of apoptosis requires energy from the dying cell, and, critically, does not result in inflammation and bystander damage to the surrounding tissues. Apoptosis is the physiological mechanism used to delete unwanted cell populations, for example during embryonic development or in the context of an immune or inflammatory response. During apoptosis, cells undergo a number of characteristic cellular and molecular changes, associated with the activation of a cascade of enzymes called caspases. A number of these enzymes cleave chromosomes between histones, thus producing fragments of DNA that are multiples of 200 bp. The cells also undergo a series of morphological changes visible under the microscope, with nuclear condensation and cytoplasmic blebbing, producing small apoptotic bodies containing the remnants of the cell. During this process the cytoplasmic membrane remains intact. This allows the identification of apoptotic cells and related activity on morphological grounds, or by the measurement of the activity of caspases. A number of factors have been shown to influence activated HSC survival with respect to apoptosis. Some soluble factors, such as insulin-like growth factor I (IGF-1), prevent cultured activated HSCs from undergoing apoptosis triggered by the removal of growth factor-containing serum (Issa et al., 2001). Other soluble factors, such as nerve growth factor (NGF) are proapoptotic (Trim et al., 2000). Stellate cell survival is also influenced by cell-matrix interactions (Gaca et al., 2003) and appears to be promoted by contact with collagen I.

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With the reduction in the numbers of activated stellate cells in recovering liver, there is an alteration in the levels of gene products that are important to develop or maintain the fibrotic phenotype. In recovering liver the expression of TIMP-1 and 2 decreases to pre-fibrotic levels. There is, accordingly, an increase in collagenase activity, but without any increase in MMP-13 expression (Iredale et al., 1998). The source of the interstitial collagenase in these models is currently the subject of debate. As HSC numbers decline rapidly during recovery, they may not be the source. An alternative source of MMP-13 in this setting may be Kupffer cells (KC) or macrophages, which have been shown to produce both MMP-13 and -9 in rats (Hironaka et al., 2000). When rats with established thioacetamide induced cirrhosis were transfected with adenovirus expressing MMP-1, the degree of fibrosis was dramatically reduced, the number of activated HSCs was decreased and there was increased hepatocyte proliferation (Iimuro et al., 2003). The importance of the reduction in TIMP-1 production during recovery to allow an increase in interstitial collagenase activity to mediate architectural restitution has been further illustrated using transgenic mouse models. Studies using a transgenic mouse strain that overexpresses TIMP-1 have shown that, during spontaneous recovery from carbon tetrachloride-induced liver fibrosis, the overexpression of TIMP-1 greatly reduces the degree of recovery at the same time points as wild-type controls (Yoshiji et al., 2002b). Moreover, the number of apoptotic stellate cells was greatly reduced. Some interstitial collagenase activity during the recovery period may be due to MT1-MMP and MMP-2 acting together. It has been shown that when hepatic myofibroblasts are triggered to undergo apoptosis, there was increased pro-MMP2 activation but not any associated pro-MMP-2 mRNA expression (Preaux et al., 2002). This activation was inhibited by TIMP-2 but not by TIMP-1. MT1-MMP protein expression was increased. These findings suggest a mechanism linking HSC apoptosis with increased interstitial collagenase activity, the two events characterizing recovery from fibrosis.

SUMMARY AND CONCLUSIONS Liver fibrosis is a bi-directional process induced by a wide variety of injurious stimuli. The key step is the activation and proliferation of the hepatic stellate cell. The activated HSC phenotype results in a pattern of gene expression which favors the development of pathological fibrosis as part of the wound healing response. Activated HSCs produce high levels of TIMPs 1 an 2, potent inhibitors of matrix metalloproteinases, including those with interstitial collagenase activity. This establishes a microenvironment where the degradation of secreted fibrillar

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collagen I and III is reduced. This changes the composition of the space of Disse that usually contains basement membrane-type extracellular matrix, and resulting in its replacement with matrix rich in interstitial collagens. Critical alterations in the phenotype of other sinusoidal cells occurs by virtue of altered cell-matrix interactions, namely the sinusoidal endothelial cells, hepatocytes and the hepatic stellate cells themselves. Matrix metalloproteinase production is still present during liver fibrosis, but its activity is held in check by the concurrently secreted TIMPs. Liver fibrosis is reversible, and recovery is characterized by the parallel processes of reduction of activated HSC numbers by apoptosis and remodeling of fibrotic matrix occur. Again, there is a complex set of interactions between matrix components, TIMPs, MMPs and HSCs. Fundamentally, understanding the cellular and molecular processes in the development of and recovery from liver fibrosis will lead to the development of anti-fibrotic and pro-resolution drugs (Iredale, 2003).

ACKNOWLEDGMENTS JPI gratefully acknowledges the grant support of the MRC (UK), the Wellcome Trust and the Children’s Liver Disease Foundation (UK).

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Suzuki, K., Enghild, J. J., Morodomi, T., Salvesen, G., & Nagase, H. (1990). Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin). Biochemistry, 29, 10261– 10270. Taipale, J., & Keski-Oja, J. (1997). Growth factors in the extracellular matrix. FASEB J., 11, 51–59. Takahara, T., Furui, K., Funaki, J., Nakayama, Y., Itoh, H., Miyabayashi, C., Sato, H., Seiki, M., Ooshima, A., & Watanabe, A. (1995). Increased expression of matrix metalloproteinase-II in experimental liver fibrosis in rats. Hepatology, 21, 787–795. Takahara, T., Furui, K., Yata, Y., Jin, B., Zhang, L. P., Nambu, S., Sato, H., Seiki, M., & Watanabe, A. (1997). Dual expression of matrix metalloproteinase-2 and membrane-type 1-matrix metalloproteinase in fibrotic human livers. Hepatology, 26, 1521–1529. Theret, N., Lehti, K., Musso, O., & Clement, B. (1999). MMP2 activation by collagen I and concanavalin A in cultured human hepatic stellate cells. Hepatology, 30, 462–468. Theret, N., Musso, O., L’Helgoualc’h, A., & Clement, B. (1997). Activation of matrix metalloproteinase-2 from hepatic stellate cells requires interactions with hepatocytes. Am. J. Pathol., 150, 51–58. Trim, N., Morgan, S., Evans, M., Issa, R., Fine, D., Afford, S., Wilkins, B., & Iredale, J. (2000). Hepatic stellate cells express the low affinity nerve growth factor receptor p75 and undergo apoptosis in response to nerve growth factor stimulation. Am. J. Pathol., 156, 1235–1243. Ueberham, E., Low, R., Ueberham, U., Schonig, K., Bujard, H., & Gebhardt, R. (2003). Conditional tetracycline-regulated expression of TGF-beta1 in liver of transgenic mice leads to reversible intermediary fibrosis. Hepatology, 37, 1067–1078. Van Eyken, P., Geerts, A., De Bleser, P., Lazou, J. M., Vrijsen, R., Sciot, R., Wisse, E., & Desmet, V. J. (1992). Localization and cellular source of the extracellular matrix protein tenascin in normal and fibrotic rat liver. Hepatology, 15, 909–916. Vassy, J., Rigaut, J. P., Briane, D., & Kraemer, M. (1993). Confocal microscopy immunofluorescence localization of desmin and other intermediate filament proteins in fetal rat livers. Hepatology, 17, 293–300. Vermeulen, K., Berneman, Z. N., & Van Bockstaele, D. R. (2003). Cell cycle and apoptosis. Cell. Prolif., 36, 165–175. Vyas, S. K., Leyland, H., Gentry, J., & Arthur, M. J. (1995). Rat hepatic lipocytes synthesize and secrete transin (stromelysin) in early primary culture. Gastroenterology, 109, 889–898. Watanabe, T., Niioka, M., Ishikawa, A., Hozawa, S., Arai, M., Maruyama, K., Okada, A., & Okazaki, I. (2001). Dynamic change of cells expressing MMP-2 mRNA and MT1-MMP mRNA in the recovery from liver fibrosis in the rat. J. Hepatol., 35, 465–473. Weber, K. T. (1997). Fibrosis, a common pathway to organ failure: Angiotesin II and tissue repair. Semin. Nephrol., 17, 467–491. Wei, H., Lu, H., Li, D., Zhan, Y., Wang, Z., & Huang, X. (2001). The expression of AT1 receptor on hepatic stellate cells in rat fibrosis induced by CCl4. Chin. Med. J. (England), 114, 583–587. Wei, H. S., Lu, H. M., Li, D. G., Zhan, Y. T., Wang, Z. R., Huang, X., Cheng, J. L., & Xu, Q. F. (2000). The regulatory role of AT 1 receptor on activated HSCs in hepatic fibrogenesis: Effects of RAS inhibitors on hepatic fibrosis induced by CCl(4). World J. Gastroenterol., 6, 824–828. Will, H., Atkinson, S. J., Butler, G. S., Smith, B., & Murphy, G. (1996). The soluble catalytic domain of membrane type 1 matrix metalloproteinase cleaves the propeptide of progelatinase A and initiates autoproteolytic activation. Regulation by TIMP-2 and TIMP-3. J. Biol. Chem., 271, 17119–17123.

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20.

ORTHOTOPIC LIVER TRANSPLANTATION

Gagandeep Singh, Pankaj Rajvanshi and Sanjeev Gupta INTRODUCTION Over the past four decades, liver transplantation has been performed widely as a life-saving procedure. However, in several parts of the world, liver transplantation has been prevented by the high costs involved and the lack of proper expertise. Additionally, organ donation has failed to keep pace with the number of patients requiring liver transplantation, a state of affairs that has led to serious consideration of a variety of alternative approaches. However, there are two general approaches to liver transplantation. One is orthotopic liver transplantation (OLT), which is performed most frequently, requires replacement of the liver by a donor organ. In this case, the transplanted liver is placed in the space made available following removal of the native liver. Trimming of the donor organ may be necessary if the recipient individual is of small body size. In contrast, heterotopic or auxiliary liver transplantation does not require removal of the native liver because the liver or a liver segment is transplanted at an ectopic site, such as the splenic bed, the right or left paravertebral gutter or the pelvis.

Some Historical Notes Pioneering work by several investigators in the post-World War II era established the early groundwork for organ transplantation. Animal studies in the 1950s showed that liver transplantation was technically feasible. In 1963, Thomas Starzl The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 525–542 © 2004 Published by Elsevier Ltd. ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15020-4

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Fig. 1. Landmarks in Liver Transplantation: Some of the important advances in the field are depicted, along with a steady rise in the number of OLT performed annually in the U.S. (Some data are from UNOS, Richmond, VA).

and colleagues at the University of Colorado in Denver performed the first OLT in man (see Fig. 1), although this and several subsequent patients survived only briefly (Starzl et al., 1963). Other investigators, notably, Sir Roy Calne in England, also joined the effort to perform OLT. Although subsequent progress in surgical methods improved outcomes, not more than one third of the patients treated with liver transplantation survived for one year. Rejection of the transplanted liver was a major problem. However, the advent of cyclosporine in 1979 completely changed the prospects of organ transplantation (Kahan, 1989). Immunosuppressive regimes using cyclosporine very significantly improved outcomes of liver transplantation. Thus, the 1980s witnessed a burgeoning interest in liver transplantation programs, and by 1986, over 40 liver transplantation centers had already been established in the United States alone. Research in immunosuppression yielded additional agents, such as tacrolimus (Allison, 2000). The first genetically engineered molecules to make a successful debut for immunosuppression was OKT3, a monoclonal antibody directed against the CD3 receptor on T lymphocytes (Cosimi et al., 1987). Insights into better and longer preservation of donor organs contributed enormously in programmatic development. The number of liver transplantations being performed has increased with greater experience. However, the widening gap between the number of available donor livers and patients on waiting lists has become a cause for concern. A major limitation in recent years has been organ donation in relatively fixed numbers, at a time when improved outcomes have broadened the overall scope of OLT. Interest in transplanting a part of the liver harvested from a related

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donor (living-related liver transplantation) or transplanting a donor organ into more than one recipient (split-liver transplantation) are innovations meant to overcome some of these limitations. Indeed, these procedures are rapidly becoming universal, as more centers acquire expertise in living-related and split liver transplants.

Donor Organ The most common sources of donor livers are brain-dead individuals on life-support. Presence of transmissible infectious disease, e.g. rabies, human immunodeficiency viruses, or other serious infections, would be obvious exclusionary criteria. Other conditions preventing organ donation include systemic infection, disseminated malignancy and irreversible liver damage. Active replication of hepatitis B virus (HBV) adversely affects the transplanted liver because consequent liver disease is markedly accelerated by immunosuppression and these patients have to be carefully chosen. On the other hand, there is ongoing debate concerning transplantation of livers from and into patients with chronic hepatitis C virus (HCV). The debate is sustained by a relatively benign course of HCV in many carriers, relatively slower progression of HCV-induced liver disease following infection of a transplanted liver and the possibility of augmenting the organ donor pool if HCV positive individuals were permitted to donate organs, e.g. for use in HCV carriers. Organ transplantation would no doubt flourish were it possible to identify universal organ donors. However, this is not yet the case. The nature of our immune systems dictates that transplantation antigens must regulate who might be a suitable recipient for a given donor liver. In the absence of “self” recognition, tissues are rejected in normal individuals with the exception of genetic identity, such as autologous tissues, e.g. skin grafts, or tissues obtained from monozygotic twins, also termed isogeneic donors. In other situations, the display of major histocompatibility complex (MHC) antigens directs tissue tolerance. In humans, the MHC is represented by the Human Leukocyte Antigen (HLA) system. The transplantation antigens have been further classified into class I and class II HLA antigens, which are responsible for specific effector limbs of the immune response. Additional antigenic systems have also been identified, e.g. the ABO system in red blood cells. For unknown reasons, liver is less immunogenic than other organs, such as the heart (Bishop & McCaughan, 2001). Whereas transplantation of kidneys or heart without HLA-matching may result in accelerated rejection of allografts, matching is sought for only the main blood group antigens (ABO system) in the case of the liver (Opelz et al., 1999). Analysis of HLA-compatibility

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is not routinely performed. On the other hand, analysis of ABO-compatibility and graft survival does show a significant advantage for tissue matching in liver recipients. In view of varying degrees of allogenicity of donor livers, all liver recipients currently require immunosuppression. Whether some individuals will be able to minimize or to discontinue immunosuppression, following development of a “chimeric” immune system with donor cells (Starzl et al., 2000) has been under continued investigation.

Organ Preservation When an organ is removed from a donor, it is necessary to preserve it in the best possible condition until transplantation. Previously, the time constraints imposed by preservation methods required organ transplantation within a few hours, often under emergency surgery conditions. However, development of conditions for preserving organs for extended time periods has permitted organ transplantation under better conditions. The establishment of centralized agencies to identify donor organs, maintain a priority list of recipients based on medical need, and coordinated regional procurement for national distribution are major elements of effective transplantation programs. The United Network of Organ Sharing (UNOS) based in Richmond, Virginia is the coordinating and tracking organization for cadaveric solid organs in the United States. A guiding principle in organ preservation is avoidance of warm ischemiareperfusion to prevent tissue injury. Another principle is to decrease the metabolic activity and oxidative damage in the organ. Blood is flushed out of the organ to prevent vascular occlusion by thrombus formation, followed by infusion of cold preservation solution. Until late 1987, the safe outer limit for preserving the human liver was 6–8 hours. Initially, the donor livers were simply perfused with cold solutions containing plasma. Subsequently, solutions were devised to more generally represent constituents found in cells, which significantly prolonged the viability of donor livers. With the introduction of the University of Wisconsin (UW) solution in 1988, quite a significant advance in organ preservation was made (Kalayoglu et al., 1988). The UW solution contains lactobionate and raffinose, which are high molecular weight sugars that prevent intracellular entry of free water. In addition, glutathione, adenosine and other components serve as scavengers of free oxygen radicals, which are released during organ reperfusion and contribute to endothelial injury and graft failure. The UW solution has extended the period of safe liver preservation for up to 24 hours. Although seemingly little, this gain in organ preservation has greatly helped in harvesting organs from wide geographic areas for distribution at short notice to virtually all parts of the United States.

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Surgical Procedures Surgery for OLT proceeds in three distinct stages: the dissection phase, anhepatic phase and the reperfusion phase. Briefly, it is during the dissection phase, that the host liver is mobilized and vascular structures are prepared for resection and reanastomosis with the donor liver. The vena cava above the liver is divided to maximize the available caval length, the infrahepatic cava divided at its junction with the caudate lobe of the liver, and the native organ is then removed. The anhepatic phase lasts from the period between removal of the liver and revascularization of the donor liver. The native liver is replaced with the donor organ followed by vascular anastomosis, first the vena caval ends, and then the portal vein. The liver graft is allowed to reperfuse via the portal vein and to drain into the inferior vena cava. Arterial reconstruction is then undertaken and the patch of donor aorta around the celiac axis is anastomosed to a segment of the recipient hepatic artery. The reperfusion phase encompasses the period during which the liver graft is fully vascularized and biliary reconstruction is completed. During removal of the host liver, it is necessary to temporarily cross-clamp the portal vein and often the inferior vena cava. Patients with liver disease generally tolerate such venous stasis, possibly due to potential collateral channels returning blood, although bleeding during the anhepatic phase or postoperative renal failure may be encountered as complications. In contrast, use of venovenous bypass with canulae placed into the femoral vein and the transected portal vein results in splanchnic decompression with blood returning through the left axillary vein. Use of veno-venous bypass has greatly improved the safety of liver transplantation.

Split and Living-Related Liver Transplantation To increase utilization of existing donor liver supply, which seems to have peaked in the United States at under 5000 livers per year, split-liver transplantation has become more popular. In split-liver transplantation, the liver is divided into anatomically and physiologically adequate portions before transplantation into two recipients (Otte et al., 1990). The liver is subjected to either an in situ split during donor harvest surgery or subsequently on the operating room back table. In living-related transplantation, which was first reported in Brazil and Australia, a portion of the liver is removed from the donor (usually a parent) and transplanted into the recipient, mostly a child. The technical portion of this surgery is essentially similar to split-liver transplantation. Cumulative experience obtained with patients who underwent living-related liver transplantation have been reported from Japan (where cadaveric organs are not available for transplantation), as well as from

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other places (Akabayashi et al., 2003; Jabbour et al., 2001; Kim-Schluger et al., 2002). The procedure has now been extended to include adults and is being done with increasing frequency. Liver resection surgery does carry risks for the donor, including hemorrhage, sepsis and death, although the overall frequency of such complications has not been documented. Use of auxiliary liver transplantation without having to remove the native liver has advantages. First, auxiliary liver transplantation avoids the surgical hazards of liver removal. Second, the recipient continues to benefit from the native organ, particularly in settings where either significant metabolic function is present or the host liver is acutely injured, and could potentially recover with sufficient time. And finally, where the host liver has the capacity to recover, cessation of immunosuppression leads to rejection of the auxiliary liver without requiring any further action. The initial interest in auxiliary liver transplantation was tempered by the onset of atrophy in the transplanted organ due to the lack of portal blood. Whether trophic signals in the portal blood, e.g. hormones, growth factors, etc., were required for maintaining the liver was a possibility (Terpstra et al., 1988). Subsequently, however, technical advances produced ways of providing portal blood to the auxiliary liver. This led to the development of auxiliary partial OLT (APOLT), where the portal blood supply is allocated to both transplanted and native livers. APOLT is associated with much more efficient survival of the liver graft. Although interest in auxiliary liver transplantation has waxed and waned over the years, patients with acute liver failure, as well as metabolic deficiency states have recently been treated by APOLT with encouraging results (Azoulay et al., 2001; Durand et al., 2002).

Indications for Liver Transplantation The indications for OLT have changed over time and may be classified in several ways with the most frequent indications listed in Table 1. In view of its “irreversible” nature, OLT was reserved initially for the most advanced cases, where no cures were available. However, as survival of patients after OLT began to routinely approach up to 90% at one year, patients were often treated at relatively earlier stages of diseases, often in efforts to simply improve quality of life by eliminating persistent symptoms, e.g. intense pruritus. Acquired liver disease resulting from multiple etiologies constitutes the single largest patient group receiving OLT. The other end of the spectrum represents patients with acute liver failure who will die without OLT. Dramatic results are obtained following OLT in carefully selected patients with acute liver failure, commonly due to viral hepatitis, drugs, poisons, hepatic

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Table 1. Common Indications for Liver Transplantation. Acute liver failure Viral hepatitis Autoimmune hepatitis Acetaminophen and other drugs Toxins, e.g. poisonous mushrooms Hepatic venous outflow obstruction Other causes Neoplastic Nonresectable hepatocellular carcinoma Epithelioid hemangioendothelioma Hepatoblastoma Neuroendocrine tumors, e.g. carcinoid, etc.

Chronic liver disease Primary biliary cirrhosis Primary sclerosing cholangitis Alcoholic liver disease Chronic viral hepatitis Miscellaneous causes Congenital Biliary atresia Polycystic disease of the liver

vein thrombosis, etc. Diagnostic challenges may be posed by initial presentations of autoimmune hepatitis and Wilson’s disease with acute liver failure as these disorders generally present more insidiously. Fatal subacute liver failure may also develop in patients who discontinue penicillamine therapy in the setting of previously well-controlled Wilson’s disease (Schilsky et al., 1994). The very nature of acute or subacute liver failure, which manifests suddenly and advances rapidly, requires urgent attention, although multidisciplinary approaches toward supportive care have decreased the high mortality (90–100%) in acute liver failure (Schafer & Shaw, 1989). Prompt OLT can be life saving in patients with acute liver failure, although delays in organ availability or presence of limiting conditions, e.g. sepsis, intracranial hypertension, irreversible brain damage, etc., may preclude OLT. Of course, it is critical to determine whether a patient with acute liver failure would recover without OLT. The most useful predictors of mortality in acute liver failure utilize weighted indexing of multiple parameters (O’Grady et al., 1992). In acetaminophen-induced acute liver failure, blood pH, prothrombin activity, serum creatinine and severity of encephalopathy serve as positive predictors of mortality (Table 2). In patients with nonacetaminophen-induced acute liver failure, age, duration of jaundice, serum factor V level and degree of hyperbilirubinemia help in predicting mortality and guiding therapies. Patients with chronic cholestasis (impaired bile flow) are often excellent candidates for OLT (Weisner et al., 1992). Advanced cholestasis may manifest with extrahepatic manifestations, such as serious bone disease. In patients with primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC) that produce cholestatic manifestations due to immunological biliary injury, the natural history is characterized by an indolent course, and identifying the best time for OLT could be difficult. However, models have been produced to predict

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Table 2. Selected Predictors of Mortality in Acute Liver Failure.a Parameter

Predictive Accuracy

Acetaminophen-induced liver failure pH 100 sec Serum creatinine >300 umol/l Encephalopathy grade 3–4

0.81 0.71 0.69 0.83

Nonacetaminophen-induced liver failure Age 40 years Jaundice for >7 days before encephalopathy Prothrombin time >50 sec >100 sec Serum bilirubin >300 umol/l

0.57 0.83 0.78 0.46 0.81

a Adapted

from O’Grady, J. G., Smith, H. M., Davies, S. E., et al. J. Hepatology (1992), 14, 104–111.

survival in these disorders, e.g. the Mayo Model for end stage liver disease that utilizes several independent clinical variables (Kamath et al., 2001). Such insights are extremely helpful in appropriate timing of OLT for achieving optimal results. An ability to perform OLT in an elective manner in relatively well preserved individuals with PBC or PSC yields some of the best outcomes. In the pediatric age group, biliary atresia is a major indication for OLT, which also presents with cholestasis (Carceller et al., 2000). Suitability for OLT generally requires previous biliary decompression with portoenterostomy (Kasai operation). This is possible in only up to 30% of the infants with biliary atresia, because surgery must be performed soon after birth and the correct diagnosis may not be apparent in a timely fashion. Unrelieved biliary obstruction often culminates rapidly in hepatic fibrosis and liver failure. Despite the success of the Kasai operation, biliary cirrhosis is virtually certain to develop as the child gets older. OLT represents the most effective treatment in biliary atresia. In many liver disorders, e.g. chronic viral hepatitis or alcoholic liver disease, it is often impossible to accurately predict the duration of survival. Worldwide, hepatitis B and C viruses and alcohol are the most frequent cause of chronic liver disease. Although healthy HBV and HCV carriers exist, serious liver disease eventually develops in the majority of patients with prolonged viral replication. Often, when cirrhosis supervenes, HBV is integrated into the host genome. Also, several extrahepatic reservoirs of HBV, and some for HCV, have been identified that may contribute to viral persistence, despite removal of the infected liver. OLT in the presence of HBV replication carries great risks, because of graft reinfection and the onset of a peculiarly aggressive form of fibrosing cholestasis that leads to an extremely rapid downhill course (Lau et al., 1992). Therefore, patients with

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active HBV infection were avoided by most liver transplantation centers, although strategies have recently been developed to decrease graft reinfection. On the other hand, in the setting of quiescent or inactive HBV infection, OLT may be well tolerated and outcomes are significantly better (Van Thiel et al., 1994). In contrast with HBV, significant liver disease due to HCV requires a longer period, up to decades. Viremia may be intermittent, continuous or periodic in patients with chronic HCV infection. Despite active HCV replication, however, patients appear to tolerate OLT reasonably well (Lyra et al., 2002). Although graft reinfection is common in patients with chronic HCV carriers, the transplanted liver enjoys greater longevity along with good metabolic function. As a consequence, chronic HCV carriers constitute an acceptable group of patients for OLT. Hepatocellular carcinoma (HCC) may arise in chronic HBV or HCV carriers, as well as in patients with other forms of chronic liver disease. The prognosis of HCC is particularly dismal with median survival from diagnosis of only some 2 months. Liver transplantation may be considered for patients with nonresectable HCC but only after extrahepatic disease has been excluded, although despite best efforts, tumor recurrence could occur after OLT (Yao et al., 2001). Some patients with HCC are cured after OLT, particularly when the tumor is found incidentally. Similarly, some cases with “low grade” malignancies metastasizing to the liver, such as neuroendocrine tumors may be candidates for OLT. The most favorable outcomes of OLT are achieved in relatively uncommon malignancies, such as fibrolamellar HCC, epitheloid hemangioendothelioma, hepatoblastoma, etc., which can potentially be cured (Mc Peake & Williams, 1995). Metabolic disorders represent a most important group of disorders amenable to cure following OLT (Table 3). This topic is dealt with in the next chapter. In several monogenic disorders, the liver is morphologically and functionally intact, and extrahepatic organs, such as the brain, heart or kidneys, are targets. Familial hypercholesterolemia and hemophilia represent such disorders, where OLT can be curative (Lopez-Santamaria et al., 2000; Wilde et al., 2002). In other conditions, the liver suffers damage, as in Wilson’s disease, and here again OLT is curative. Table 3. Genetic Disorders Treated with OLT. Alpha-1 antitrypsin deficiency Allagille’s syndrome Criggler-Najjar syndrome Inborn errors of bile acid metabolism Erythropoeitic protoporphyria Familial hypercholesterolemia Glycogen storage diseases types I and IV Hemophilia

Hyperoxaluria Neville’s syndrome Primary hemochromatosis Protein C deficiency Tyrosinemia Urea cycle deficiency disorders Wilson’s disease

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Obviously, patients with congenital metabolic deficiencies should undergo liver transplantation before irreversible organ damage occurs. Finally, the vexing question of OLT in patients with alcoholic liver disease posed a dilemma until it was demonstrated that outcomes compared favorably with nonalcoholic individuals (Lucey et al., 1992). The concerns were related to the potential of patients resuming an alcoholic or self-destructive life-style, as well as complying poorly with immunosuppressive drug regimes. However, in patients abstinent for at least 6 months, recidivism or return to alcohol is relatively low (10–15%). Many former alcoholic patients have returned to gainful or productive careers after OLT.

Timing of OLT Except for the setting of acute liver failure, OLT needs consideration of an optimal time that provides the patient with the best chance for withstanding surgery and yet not succumbing to the underlying disease. The reasons for adopting a less than aggressive stance include the irrevocable nature of the procedure, and the requirement for lifelong immunosuppression. As indicated above, in some situations, i.e. PBC or PSC, predictive models may be relied upon. Serial monitoring of liver function is helpful, e.g. serum bilirubin >5 mg/dl, serum albumin 60 years used to be considered a barrier to OLT. However, biological rather than chronological age, and presence of associated problems offer better guides. In advanced cardiac disease, OLT becomes difficult as significant blood volume changes occur during and after surgery. Similarly, advanced pulmonary disease, which occasionally might be related to underlying cirrhosis itself, may prevent adequate ventilatory capacity. Chronic renal failure may limit OLT as well. Although simultaneous transplantation of multiple organs is technically feasible, this is an enormous undertaking, and multiple organ transplants are currently limited to only a few centers. Presence of portal vein occlusion or previous biliary surgery necessitates detailed anatomical evaluation before OLT. In acute liver failure, severe intracranial hypertension may cause irreversible brain damage and preclude OLT. Although contraindications might differ from center to center, OLT should be avoided in the presence of disseminated malignancy, active substance abuse, inability to comply with immunosuppression, and advanced systemic sepsis. In general, patients with cholangiocarcinoma tend to do poorly with rapid recurrence of disease, although several patients have successfully undergone OLT (Hassoun et al., 2002). Table 4. Contraindications to OLT. Absolute Advanced cardiopulmonary disease Active substance abuse Advanced systemic sepsis Congenital anomalies precluding transplantation Extrahepatic malignancy

Relative Advanced age Chronic renal failure Extensive prior biliary surgery Portal vein thrombosis

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COMPLICATIONS When all goes well, OLT dramatically alters the situation of a dying patient with encephalopathy and coagulopathy to a fully conscious and ambulatory state followed by an early discharge. However, complications could arise from technical problems during transplantation, infection, drug toxicity, organ rejection, delayed bone disease, as well as impaired growth and nutrition in the young. Technical complications include bleeding from anastomotic sites, primary nonfunction of the liver graft, thrombosis of hepatic artery or portal vein, as well as bile duct stenosis or leakage. In general, the incidence of these complications is low. Among particularly troubling complications are hepatic artery thrombosis, which may cause abscess formation, and biliary tract disease manifesting with anastomotic leak, stricture, obstruction, bleeding or infection. Although thrombosis of the portal vein or the vena cava is uncommon, it is a devastating complication, and usually requires retransplantation of the liver. Increasing experience in dealing with infectious complications in the immunosuppressed patient has facilitated diagnostic distinctions between infection, inflammation and rejection, yet liver biopsies are often required by the experts.

Management of Rejection During alloantigen-related immune activation, a complex cascade of cell activation process has been defined. Simply stated, recognition of specific alloantigens by T lymphocytes results in activation of CD4+ cells, which in turn recruit cytolytic CD8+ cells. A variety of cytokines, particularly interleukins, tumor necrosis factor, interferons, etc., play trophic, and cytopathic roles during the host immune response. Additional cell types, including polymorphonuclear cells or activated macrophages may also play important roles. Preformed antibodies, as well as complement-mediated processes may cause cell injury but “hyperacute” rejection of liver through these processes is almost never encountered. Cytokines are released during endothelial injury, which is responsible for either primary graft nonfunction or dysfunction. The cumulative effects of these processes lead to early or late rejection. Overall, although some element of rejection is apparent in virtually all allografted individuals and although more significant rejection occurs in approximately 60%, only a small proportion (