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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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.
10.
11.
12.
13.
14.
15.
16.
17.
18.
DRUG METABOLISM AND HEPATOTOXICITY J. Michael Tredger
207
ASSEMBLY AND SECRETION OF HEPATIC VERY-LOW-DENSITY LIPOPROTEIN Geoffrey Gibbons
229
BILIRUBIN METABOLISM Peter L. M. Jansen and E. Edward Bittar
257
CLINICAL BIOCHEMISTRY OF THE LIVER Neil McIntyre
291
ALCOHOLIC LIVER DISEASE S. F. Stewart and C. P. Day
317
FULMINANT HEPATIC FAILURE Watson Ng, Ian D. Norton and D. Brian Jones
361
PRIMARY BILIARY CIRRHOSIS James Neuberger
383
CHRONIC ACTIVE HEPATITIS Ian G. McFarlane
399
HEPATITIS B VIRUS F. Fred Poordad
427
CURRENT ISSUES IN HEPATITIS B VACCINES Jane N. Zuckerman and Arie J. Zuckerman
439
THE MOLECULAR VIROLOGY OF HEPATITIS C VIRUS Timothy L. Tellinghuisen and Charles M. Rice
455
vii
19.
20.
21.
22.
THE ROLE OF THE HEPATIC STELLATE CELL IN LIVER FIBROSIS Timothy J. Kendall and John P. Iredale
497
ORTHOTOPIC LIVER TRANSPLANTATION Gagandeep Singh, Pankaj Rajvanshi and Sanjeev Gupta
525
BIOLOGICAL PRINCIPLES AND NOVEL THERAPIES IN LIVER CELL TRANSPLANTATION Sanjeev Gupta, Mari Inada, Vinay Kumaran and Brigid Joseph
543
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
The Intrahepatic Biliary Tree
5
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 <15 m in diameter; interlobular bile ducts are 15–100 m in diameter; septal ducts are 100–300 m in diameter; area ducts 300–400 m in diameter; segmental ducts are 400–800m in diameter; and hepatic ducts 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.
2.
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 (<15 m in diameter) have an unknown secretory function, are resistant to drug or toxin induced liver injury and have the capacity to proliferate and differentiate with injury of large intrahepatic bile ducts.
proliferative capacity depend on the nature of bile duct injury. In the classic bile duct injury model, the bile duct ligated (BDL) rat, only large bile ducts proliferate (Alpini et al., 1998). In contrast, when large bile ducts are injured by CCl4 or ANIT administration, a loss of large bile duct secretory capacity is associated with small bile duct proliferation and de novo expression of the secretin receptor, apical CFTR Cl− channels and Cl− /HCO− 3 exchanger in small ducts, to compensate for the loss of large cholangiocyte functions (LeSage, Glaser, LeSage, Glaser, Alvaro et al., 1999; LeSage et al., 2001; Marucci et al., 1999). Proliferation of small cholangiocytes is also induced by the feeding of the bile acid, taurocholate, which also causes proliferation of large cholangiocytes (Alpini et al., 2001). We propose that cholangiocytes lining small ducts have the capacity to differentiate into secreting epithelium, primarily as a response to loss of large bile duct function.
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BILE DUCT STRUCTURE Intrahepatic bile ducts are lined by columnar cells, which posses a basement membrane (Benedetti et al., 1996; Schaffner & Popper, 1961). There is a conspicuous ER and Golgi as well as a vacuolar compartment, but they are less well developed compared to hepatocytes (Benedetti et al., 1996; Sasaki et al., 1967; Schaffner & Popper, 1961). The basolateral membrane has an underlying basement membrane (Benedetti et al., 1996; Schaffner & Popper, 1961). Functional tight junctions have been identified between cholangiocytes (LaRusso et al., 1991). The apical membrane has microvilli, thereby increasing the effective surface area (LaRusso et al., 1991). Intrahepatic bile ducts are classified by diameter: hepatic 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) (Kanno et al., 2000; Ludwig, 1987; Marzioni et al., 2002). Small bile ducts are lined by 4 to 5 cuboidal cholangiocytes and larger bile ducts consist of 10 to100 cholangiocytes (Kanno et al., 2000; Ludwig, 1987; Marzioni et al., 2002). In cholangiocytes lining large bile ducts, the Golgi apparatus is well developed and is located between the apical pole and the nucleus (Benedetti et al., 1996). In contrast, cholangiocytes lining small bile ducts have less cytoplasm and the nucleus/cytoplasm ratio is high (Benedetti et al., 1996). The different nucleus/cytoplasm ratio observed between small and large bile ducts (Benedetti et al., 1996) may represent cholangiocytes in large ducts being more differentiated cells, which express membrane receptor, transporters, and channels that are responsible for ductal secretion. In contrast, the smaller cytoplasm in small cholangiocytes suggests that small cholangiocytes may be undifferentiated primitive cells.
ASPECTS OF THE VASCULAR SUPPLY The intrahepatic bile ducts are nourished by the peribiliary plexus, which originates from the hepatic artery (Gaudio et al., 1996; Ohtani et al., 1983; Terada et al., 1989; Yamamoto & Phillips, 1984). The peribiliary plexus is most notable around large bile ducts, and less discernible around small bile ducts (Gaudio et al., 1996). Blood flowing through the peribiliary plexus enters the periportal sinusoids (Gaudio et al., 1996; Ohtani et al., 1983; Terada et al., 1989; Yamamoto & Phillips, 1984). These anatomic relationships have two potential physiologic implications. First, substances absorbed from intrahepatic bile ducts can be transferred back to the hepatic sinusoids, returned to hepatocytes, and then potentially secreted into bile (Gaudio et al., 1996; Yamamoto & Phillips, 1984). This exchange between cholangiocytes and hepatocytes has been termed “cholehepatic shunting” and
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has been suggested to occur for both bile acids and certain drugs (see below) (Hofmann, 1989). Second, blood flow is countercurrent to the direction of bile flow (Ohtani et al., 1983). Countercurrent bile and blood flow may be physiologically advantageous for bile formation (vid´e infra).
DUCTAL BILE SECRETION Studies using isolated cholangiocytes and isolated intrahepatic bile duct units (IBDU) have revealed the nature of the transport mechanisms underlying secretinstimulated ductal secretion (Alpini et al., 1996, 1998; Alpini, Glaser, Robertson et al., 1997; Alpini, Glaser, Rodgers et al., 1997; Cho & Boyer, 1999; Cho et al., 1995; Mennone et al., 1995; Roberts et al., 1993). Cholangiocytes modify canalicular bile by secretion of Cl− and HCO− 3 (Alpini et al., 1988, 1989, 1996, 1997; Alvaro et al., 1993, 1997; Fitz et al., 1993; LeSage et al., 1996; Mennone et al., 1995; Roberts et al., 1993). On the basolateral membrane, Na+ /H+ exchanger − and the Na+ : HCO− 3 symporter mediate HCO3 uptake (Alvaro et al., 1993), and on the apical membrane, cAMP-activated CFTR Cl− channel (Alpini et al., 1997; Fitz et al., 1993) and Cl− /HCO− 3 exchanger (Alpini et al., 1996, 1997; Alvaro et al., 1993, 1997; LeSage et al., 1996; Mennone et al., 1995; Roberts et al., 1993) secrete bicarbonate into the lumen. Both cAMP and Ca2+− activated Cl− channels are present on the apical membrane (Alpini et al., 1997; Fitz et al., 1993). Cholangiocyte secretory functions are increased by the hormones secretin (Alpini et al., 1988, 1989, 1996, 1997, 1998, 2002; Alvaro et al., 1993; Kato et al., 1992; LeSage et al., 1996), bombesin (Cho et al., 1995) and vasoactive intestinal peptide (VIP) (Cho & Boyer, 1999), whereas the gastrointestinal hormones somatostatin (Tietz et al., 1995), gastrin (Glaser et al., 1997) and insulin (LeSage et al., 2002) decrease secretion due to up and down regulation of cAMP, respectively (Alpini et al., 1996, 1997, 1998; Glaser et al., 1997; LeSage et al., 2002). ATP (Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998) and bile acids (Alpini et al., 1997, 1999, 2001) regulate ductal secretion by signaling events at the apical membrane of the cholangiocytes by the Na+ - dependent bile acid transporter, ASBT (Alpini et al., 1997; Lazaridis et al., 1997). It will be recalled that ATP signals through purinergic receptors located on the apical membrane of cholangiocytes (Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998). Regional Distribution of Bile Ductal Secretion Secretin-regulated secretion by cholangiocytes occurs exclusively in large bile ducts (>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 (<8 m in diameter) derives from small intrahepatic bile ducts (Fig. 2, lower panel). Large (but not small) cholangiocytes, and IBDU isolated from rats expressing sensitivity to secretin, somatostatin (SSTR2 ) receptors, and the Cl− /HCO− 3 exchanger respond to secretin and somatostatin by manifesting changes in cAMP levels (Alpini et al., 1996, 1997). As a direct demonstration of regionalization of secretory events in the biliary tree, large but not small isolated intrahepatic IBDU respond to secretin by showing an increase in duct lumen (Alpini et al., 1997). Similar to rats, human bile duct secretion is also regional in distribution since only large-size bile ducts express the Cl− /HCO− 3 exchanger (Martinez-Anso et al., 1994). Although one cannot draw a direct correlation between IBDU size reported in our previous study and the bile duct diameter in human liver sections, as defined by Ludwig’s classification (Ludwig, 1987), it would seem reasonable to suggest that the small IBDU would best be characterized as ductules in the Ludwig classification and the larger IBDU would represent the interlobular ducts (Kanno et al., 2000). The exclusivity of secretion to only large ducts is possibly due to the presence of the peribiliary plexus, the vascular element for cholangiocytes being limited to ducts greater than 15 m in diameter (Gaudio et al., 1996).
Regulatory Hormones and Neurotransmitters Secretin, bombesin and VIP increase ductal secretion (Alpini et al., 1988, 1989, 1996, 1997, 2002; Cho & Boyer, 1999; Cho et al., 1995; Kato et al., 1992). Insulin, gastrin and somatostatin decrease cholangiocyte secretion (Glaser, 1997; LeSage, 2002; Tiets, 1995). Acetylcholine (ACh) and ␣−1 adrenergic agonists potentiate (Alvaro et al, 1997; LeSage et al., 2001), whereas insulin, dopamine and −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Fig. 2. [Upper Panel] Small [A] and Large [B] IBDU were Isolated as Described in Materials and Methods and Cultured in MEM for 12–24 Hours at 37 ◦ C. Note: A lumen is clearly visible (arrow), with surrounding epithelial cells lining the duct lumen. The photomicrogram was obtained with DIC optics to enhance image contrast and, due to a narrow depth of focus, provides a clear outline of the duct lumen. Original magn., X2100. [lower panel] Frequency distribution of diameters of small and large cholangiocytes purified by counterflow elutriation and immunoaffinity separation from normal rat liver (A–D). Note that cells differ in size and morphological appearance (A–B). Orig. magn., X625.
Functional Heterogeneity of Intrahepatic Cholangiocytes
Fig. 2.
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endothelin agonists inhibit (Caligiuri et al., 1998; Glaser et al., 2003; LeSage et al., 2002) secretin-stimulated ductal secretion. Secretin, gastrin, and somatostatin receptors are present only in large cholangiocytes (Alpini et al., 1996, 1997, 1998), whereas endothelins ETA and ETB receptors are expressed by both small and large cholangiocytes (Caligiuri et al., 1998). Secretin binds to secretin receptor on the basolateral membrane of cholangiocytes (Alpini et al., 1994) resulting in increased cAMP levels (Alpini et al., 1996, 1997; Kano et al., 1992). Bombesin and VIP increase duct secretion independent of cAMP synthesis (Cho et al., 1995; Cho & Boyer, 1999) and the origin of ductal choleresis (small vs. large ducts) is unknown. In vivo, secretin induces a much larger bicarbonate-rich chloresis in rats with enhanced ductal hyperplasia induced by BDL (Alpini et al., 1988; Glaser et al., 1997; Tietz et al., 1995), cirrhosis (Alpini et al., 1997), or chronic ANIT feeding (LeSage et al., 2001) compared to normal rats where secretin-induced choleresis is minimal (Alpini et al., 1988, 1989; LeSage, 1996). Similarly, bombesin and VIP increase bile flow and bicarbonate to a much greater degree in BDL rats compared to controls (Cho et al., 1995; Cho & Boyer, 1999). Increased secretion in response to secretin, bombesin and VIP is consistent with an increased number of cholangiocytes in BDL rats producing more ductal bile flow (Alpini et al., 1988, 1989). Insulin, gastrin and somatostatin inhibit the expression of secretin receptor and secretin-stimulated cAMP synthesis, thus preventing secretin’s choleric effects (Glaser et al., 1997; LeSage et al., 2002; Tietz et al., 1995). Both gastrin and insulin increase intracellular Ca2+ and PKC-␣ activity in cholangiocytes (Glaser et al., 1997; LeSage et al., 2002), which are required for inhibition of secretin-stimulated cAMP synthesis and ductal secretion. ACh enhances secretin-stimulated ductal bile secretion through interaction with M3 ACh receptor subtypes on cholangiocytes (Alvaro et al., 1997). ACh increases secretin-stimulated (but not basal) activity of the Cl− /HCO− 3 exchanger in IBDU and secretin-stimulated cAMP synthesis in isolated cholangiocytes (Alvaro et al., 1997). The potentiation of secretinstimulated ductal bile secretion is dependent on Ca2+ but not PKC (Alvaro et al., 1997). FK-506 and cyclosporine inhibit ACh potentiation of secretin-stimulated Cl− /HCO− 3 exchanger, demonstrating that calcineurin most likely mediates the cross-talk between calcium and adenylyl cyclase pathways (Alvaro et al., 1997).
Paracrine and Autocrine Control of Ductal Secretion Extracellular ATP and adenosine have well defined roles as stimulants of electrolyte and fluid secretion in bile ducts (Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998). In cholangiocytes, ATP binds to apical P2Y2 (P2U)
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receptors (Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998). ATP, released from hepatocytes, is capable of stimulating receptors on both adjacent hepatocytes and after reaching the biliary tree, stimulating receptors on the apical domain of cholangiocytes (Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998). Thus, ATP may play a role in coordinating the hepatocyte and ductal components of bile formation, a process that has been termed “hepatobiliary coupling” (Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998). ATP in bile is a potent stimulus for cholangiocyte Cl− and fluid secretion and activates basolateral NHE (Elsing et al., 1996; Zsembery et al., 1998). ATP also increases Cl− /HCO− 3 exchanger activity in cholangiocytes pretreated with cAMP analogs (Melero et al., 2002; Strazzabosco et al., 1997). Consequently, the release of ATP from cholangiocytes may regulate secretion of cholangiocytes downstream (paracrine signaling). It remains to be determined if cholangiocytes lining small bile ducts can release ATP and signal downstream cholangiocytes in large ducts, thus providing a mechanism for the regulation of ductal secretion by cross-talk between small and large cholangiocytes. The presence of multiple stimulatory (i.e. secretin, bombesin, vasoactive, ACh, and ATP) (Elsing et al., 1996; Roman et al., 1999; Schlenker et al., 1997; Zsembery et al., 1998) and inhibitory (i.e. endothelin, insulin, gastrin and somatostatin) (Alpini et al., 1998; Caligiuri et al., 1998; Glaser et al., 1997; LeSage et al., 2002; Tietz et al., 1995) stimuli to cholangiocyte secretion likely reflects the need for fine regulation of cholangiocyte secretion from both circulatory and nerve inputs. The presence of these hormone receptors primarily in large bile ducts supports the concept that ductal secretion originates primarily from large ducts (Alpini et al., 1996, 1997).
BILE ACID TRANSPORT Until recently it was assumed that bile acids, after hepatocyte secretion, were simply conducted to the intestine by bile ducts (Hofmann, 1989). Studies with bile duct ligated rats suggest that bile acids may move across the biliary epithelium (Lamri et al., 1992). The absorbed bile acids return via the peribiliary plexus to the hepatocytes for secretion into bile (Gurantz & Hofmann, 1984; Palmer et al., 1987). This shunting of bile acids back and forth between hepatocytes and cholangiocytes has been termed “cholehepatic shunting” (Gurantz & Hofmann, 1984; Palmer et al., 1987). More recently, interest in bile acid transport in bile ducts has risen sharply due to the identification of bile acid transport in cholangiocytes (Alpini, Glaser, Rodgers et al., 1997; Lazaridis et al., 1997). Studies by us (Alpini et al., 1997)
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showed genetic and protein expression for the apical Na+ -dependent ASBT and the 14-KD ileal cytosolic binding protein (IBABP) in cholangiocytes. ASBT is structurally identical to the ileal bile acid transporter which is the sole transporter involved in the reclamation of bile acids from the ileum (Aldini et al., 1992). ASBT is expressed in large but not small (less than 15 m) bile ducts (Alpini et al., 1997). We have suggested that the presence of ASBT in small ducts would counterpoise secretion of bile acids at the level of the canalicular membrane, thus reducing overall bile acid-induced bile flow (Alpini et al., 1997). Bile acids interact with cholangiocytes, both in vitro (Alpini, Glaser, Robertson, Phinizy, Rodgers et al., 1997) and in vivo in bile acid fed animals (Alpini, Glaser, Ueno et al., 1999; Alpini, Ueno et al., 2001) resulting in cholangiocyte proliferation and increases in ductal bile secretion of cholangiocytes (Alpini, Glaser, Robertson, Phinizy, Rodgers et al., 1997; Alpini, Glaser, Ueno et al., 1999; Alpini et al., 2001). Corresponding to the presence of ASBT only in large cholangiocytes (Alpini et al., 1997), bile acids stimulate proliferation and secretion only in large cholangiocytes in vitro (Alpini et al., 1997). This observation is probably ascribable to the requirement of bile acid uptake by ASBT, since only intracellular bile acids can signal cholangiocyte proliferation and secretion (Alpini et al., 1997). Chronic feeding of taurocholate and taurolithocholate induces de novo expression of ASBT, and activation of proliferative and secretin-stimulated secretory capacity of small cholangiocytes (Alpini et al., 2001), which normally do not express ASBT (Alpini et al., 1997) and are unresponsive to secretin (Alpini et al., 1996, 1997) and mitotically dormant (Alpini et al., 1998).
Physiologic Advantages of Cholangiocyte Heterogeneity in Ductal Secretion Limiting secretin-stimulated ductal secretion only to large ducts (Alpini et al., 1996, 1997) may provide the liver with a number of theoretical physiological advantages. First, secretion from large ducts might provide a washing effect to prevent retention of precipitable material or mucus proteins in larger intrahepatic bile ducts. Second, limited secretion in large ducts theoretically takes advantage of the counterflow effect of the opposite direction of blood and bile flow within the biliary system. With blood flow directed from large to small ducts, concentration gradients created between large ducts and the circulation due to secretion would not be dissipated in small ducts, which do not express secretory transporters (Alpini et al., 1996, 1997). Third, enhanced cholehepatic shunting of solutes (e.g. bile acids) would be more effective since bile acids absorbed by large bile ducts (Alpini et al., 1997) could enter the peribiliary plexus, where small ducts, with a minimal
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peribiliary plexus could not participate in cholehepatic shunting. These concepts concerning physiologic advantages of cholangiocyte heterogeneity are conjectural in nature and need to be experimentally tested.
Cholangiocyte Proliferative and Repair Responses Whereas cholangiocytes are mitotically dormant (Alpini et al., 1998; LeSage et al., 1996), they proliferate in most human liver diseases (Alpini et al., 2002). They proliferate following experimental bile duct injury caused by BDL (Alpini et al., 1988, 1989) or ANIT feeding (Alpini et al., 1988; Kossor et al., 1995; LeSage et al., 2001). Cholangiocytes also proliferate following partial hepatectomy, and restore bile duct mass to normal within one week (LeSage et al., 1996). Thus far, all models of cholangiocyte proliferation show an associated increase in ductal secretion (Alpini et al., 1988, 1989, 1999; LeSage et al., 1996, 2001).
Regional Distribution of Bile Duct Proliferation In the intrahepatic biliary epithelium, there are specific compartments from which cholangiocytes proliferate (i.e. small and large sized ducts) and that differentially respond to injury, hepatic toxins or diets (Alpini et al., 1998; LeSage, Benedetti et al., 1999; LeSage, Glaser et al., 1999; LeSage et al., 2001). Following BDL, large cholangiocytes existing in large bile ducts proliferate, thus resulting in a 10 to 20-fold increase in the number of intrahepatic bile ducts (Alpini et al., 1998). By contrast, following partial hepatectomy, the regrowth of bile ducts occurs as the result of proliferation of both small and large bile ducts (LeSage et al., 1996). Administration of a single dose of CCl4 to normal or BDL rats and chronic ANIT feeding to normal rats induces a transient reduction of bile ducts (ductopenia) due to cholangiocyte loss (by apoptosis) (LeSage, Glaser et al., 1999; LeSage, Penedetti et al., 1999; LeSage et al., 2001). In large bile ducts, there is a loss of secretory and proliferative activities, whereas small bile ducts are more resistant to CCl− 4 and ANIT-induced damage. They proliferate and secrete to compensate for loss of large cholangiocyte function. The differential response of small and large ducts to CCl4 is likely due to the presence of cytochrome P450 2E1, the enzyme that initiates CCl4 hepatoxicity (Clawson et al., 1989; Handler & Goldstein, 1996) in large but not small cholangiocytes (LeSage, Benedetti et al., 1999; LeSage, Glaser et al., 1999). Variable sensitivities of large and small ducts to other forms of injury or carcinogens may be due to differential expression of other enzymes or proteins in large and small cholangiocytes. Phase I or mixed-function oxygenase
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enzymes e.g. microsomal cytochrome P-450, aminopyrine-N-demethylases, G-6 PO4 , and NADPH cytochrome C reductase, and phase II or glutathione redox cycle enzymes (e.g. GSH-peroxidase, UDP-glucuronosyltransferase, and glutathioneS-transferase), and drug-metabolizing enzymes are heterogeneously expressed by cholangiocytes (Lakehal et al., 1999; LeSage et al., 1999; Mathis et al., 1989). Proliferative stimuli can trigger a focal proliferative response in other epithelia besides cholangiocytes. For example, with prolonged hormonal stimulation, replication of pancreatic cells is restricted to intralobular but not interlobular ducts (Elsasser et al., 1990). EGF-induced proliferation in the kidney is restricted to proximal tubular cells (Han et al., 2002). In the liver, periportal hepatocytes have a greater proliferative capacity than perivenous hepatocytes following partial hepatectomy (Lee et al., 1998). It could well be that the differential blood supply with possibly its circulating growth factors such as vascular endothelial growth factor (VEGF) may be considered as an additional explanation for the differential proliferation of small and large ducts. Keeping in mind that the peribiliary plexus surrounds solely large ducts, proliferation of the plexus occurs only around the large ducts following BDL (Gaudio et al., 1996). It therefore has been postulated that VEGF which is released by only large cholangiocytes supports proliferation of large bile ducts after BDL and also (by a paracrine mechanism) supports the growth of the peribiliary plexus lying adjacent to the large ducts. This implies that small cholangiocytes do not play a role in the proliferation of adjacent blood vessels, which is consistent with the expansion of small bile ducts in the BDL model. When hepatocyte regeneration in the experimental animal is impaired, small bile ducts proliferate and invade adjacent hepatocyte parenchyma. These ductal cells are referred to as oval cells; their association with defective regeneration has led to the view that they are the progeny of facultative stem cells (Alison et al., 1998).
Cholangiocyte Apoptosis Apoptosis or programmed cell death is activated in an organism as a defense mechanism against the accumulation of damaged cells (Guicciardi & Gores, 2002). Apoptosis is considered an important mechanism in cholangiocyte death, leading to ductopenia (Guicciardi & Gores, 2002). In ANIT or CCl4 treated rats, the triggering of small bile duct proliferation may depend on the presence of cholangiocyte apoptosis in these models (LeSage, Benedetti et al., 1999; LeSage, Glaser et al., 1999; LeSage, Glaser, Ueno et al., 2001), whilst the failure of proliferation of small bile ducts to occur in BDL rats may be due to the absence of cholangiocyte apoptosis in this model (LeSage, Glaser, Ueno et al., 1999).
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It is now known that bile acids are cytoprotective, and anti-apoptotic in relation to cholangiocytes. For example, after feeding taurocholate to BDL, CCl4 -treated rats (Marucci et al., 2003), small cholangiocytes [which are de novo activated in this model of CCl4 -induced damage of large ducts (LeSage, Benedetti et al., 1999; LeSage, Glaser et al., 1999)] are found to be mitotically quiescent and unresponsive to secretin. Studies done in vitro using large cholangiocytes obtained from BDL rats show that taurocholate stops the inhibitory effects of CCl4 on apoptotic, proliferative and secretory capacities of large cholangiocytes from occurring (Marucci et al., 2003). Taurocholate protects against the effects of apoptosis and loss of function in large cholangiocyte. These functions depend on the activation of PI3-K and AKT expression (Marucci et al., 2003). As will be recalled, Bcl-2 modulates apoptosis in the liver and is upregulated in BDL rats (Kurosawa et al., 1997), an effect that protects hepatocytes from bile salt induced apoptosis (Kurosawa et al., 1997). Liver Bcl-2 is particularly expressed in cholangiocytes. This observation has led Que et al. (1997) to postulate that Bcl protects cholangiocytes against exposure to bile salt in high concentrations. It is also noteworthy that Bcl-2 expression in small bile ducts exceeds that in larger ducts, suggesting the presence of greater resistance in smaller ducts to apoptosis (Charlotte et al., 1994). A parallel situation exists in respect of annexin-V in murine liver: it is expressed mainly by small intrahepatic bile ducts and plays a role in the regulation of apoptosis (Diakonova et al., 1997). Such findings could partly account for the higher resistance found in small ducts to agents/drugs that induce apoptosis. Cholangiocyte Heterogeneity: Non-transport Related Proteins In normal and cholestatic human liver bile ducts, whether hepatic, segmental, area, or septal, and peribiliary glands express pancreatic enzymes such as lipase, ␣-amylase, and trypsin (Terada et al., 1992, 1994). The human bile duct system has its own pattern of blood group antigen expression with sialylated Lewisa antigens present primarily in large septal bile ducts (Okada et al., 1988). Very recently, the microarray technique has been used to demonstrate the existence of over 80 other proteins that are differentially expressed by large and small cholangiocytes (Ueno et al., 2003). Both the physiological and pathological significance of differential expression of this wide variety of proteins has not yet been explored.
DISEASE STATES Damage to bile ducts causes several chronic cholestatic disorders (cholangiopathies) (Alpini et al., 2002; Roberts et al., 1997). In these cholangiopathies,
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there often coexists cholangiocyte death and proliferation, ductal remodeling, inflammation and fibrosis (Alpini et al., 2002; Roberts et al., 1997). Cholestasis is almost always present and may be the cause or promoter for the progression of the disease (Alpini et al., 2002; Roberts et al., 1997). CF is an example of biliary cirrhosis secondary to a dysfunction of cholangiocyte ion transport (Alpini et al., 2002; Roberts et al., 1997). Thus, dysfunctional biliary electrolyte transport may also promote the cholestasis in other cholangiopathies (Alpini et al., 2002; Roberts et al., 1997). Most primary cholangiopathies appear to be due to an autoimmune induced process (Alpini et al., 2002; Roberts et al., 1997). Cytokines and proinflammatory mediators likely induce apoptotic and proliferative responses in cholangiocytes, activate fibrogenesis, and alter the transport functions of cholangiocytes (Alpini et al., 2002; Roberts et al., 1997). Cholangiopathies differentially affect the biliary epithelium, leading to selective alteration and destruction of specific sized ducts (Alpini et al., 2002; Roberts et al., 1997).
Primary Biliary Cirrhosis PBC is the prototypic disease in humans of bile duct damage (Roberts et al., 1997). PBC is characterized by spotty rather than diffuse proliferation/loss of certain sized ducts (i.e. small interlobular bile ducts) (Alpini et al., 2002; Roberts et al., 1997). The etiology of PBC remains elusive. Current studies suggest that the interlobular bile duct destruction is immune based, and commonly associated with autoimmune diseases (Alpini et al., 2002; Roberts et al., 1997). Patients with PBC have autoantibodies that interreact with components of mitochondrial multi-enzyme complexes (Alpini et al., 2002; Roberts et al., 1997). In addition to binding to mitochondria, autoantibodies in the patients are thought to be against the autoantigen pyruvate dehydrogenase complex (PDC) dihydrolipoamide acetyltransferase (E2) which bind to the plasma membrane of cholangiocytes specifically in PBC (Alpini et al., 2002; Jones et al., 1995; Roberts et al., 1997).
Primary Sclerosing Cholangitis PSC is associated with inflammation and prominent fibrosis of intrahepatic and extrahepatic bile ducts (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). The sclerosis of the bile ducts may be the result of multiple factors, including autoimmune, bacterial, congenital, drug, or viral agents (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al.,
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1997). The etiology of PSC remains poorly understood, despite a large number of studies evaluating differing hypotheses. PSC has a propensity to primarily affect extrahepatic and large bile ducts in the liver, although a small duct variant of PSC has been described where the large ducts are spared (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997).
Cholangiocarcinoma Cholangiocarcinoma occurs somewhat frequently in patients with PSC (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). Cholangiocarcinoma has a strong predilection for involving the major bile duct bifurcation (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). Cholangiocarcinoma arising out of small bile ducts (peripheral cholangiocarcinoma) is rare (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). Cholangiocarcinoma is not seen in patients who have a small duct variant of PSC (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). Most of the other risk factors for cholangiocarcinoma have long-standing inflammation and injury of cholangiocytes (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). p53 overexpression and K-ras mutations occur commonly in patients with PSC and biliary tract cancer and are associated with a shortened life survival (Alpini et al., 2002; Kanno et al., 2000; Marzioni et al., 2002; Roberts et al., 1997). The mechanisms responsible for the control of cholangiocarcinoma growth are poorly understood. However, there are studies showing that gastrin (by increasing PKC-␣) (Kanno et al., 2001) and ␣−2 adrenergic stimulation (through modulation of Raf-1 and B-Raf activities) (Kanno et al., 2002) are able to inhibit the growth of cholangiocarcinoma.
Cystic Fibrosis Although CF is primarily considered as a pulmonary disease, liver disease has been increasingly diagnosed during recent years, probably due to an increased suspicion that there is a connection between the two entities (Alpini et al., 2002; Roberts et al., 1997). Given that data assessing the effects of defective CFTR on cholangiocyte biology have not yet been obtained, it seems likely that impaired secretory function of cholangiocytes maybe responsible for reduced bile flow and alkalinity (Alpini et al., 2002; Roberts et al., 1997). Although a state of functional CFTR is absent or reduced in large bile ducts in the liver (Alpini et al., 2002;
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Roberts et al., 1997), there is also the distinct possibility that other Cl− channels (e.g. Ca2+ - dependent) (Roman et al., 1999; Schlenker et al., 1997) are present in both small and large cholangiocytes, and that these channels act to maintain bile duct function by substituting for CFTR activity. However, to date, no clear association between specific CFTR mutations and the presence of liver disease has been observed. Treatment with ursodeoxycholic acid, aimed at improving biliary secretion in terms of bile viscosity and bile acid composition, is currently the most effective therapeutic modality in CF-associated liver disease (Paumgartner & Beuers, 2002).
Polycystic Kidney Liver Disease (PKLD) In autosomal dominant PKLD the genetic defect results in the slow growth of multiple epithelial cysts within the renal and liver parenchyma (Perrone et al., 1997). Cysts appear in the intrahepatic biliary tree in PKLD (Perrone et al., 1997). The abnormality appears to develop out of large bile ducts since the cystic ductal cell also secretes Cl− and HCO− 3 like normal large cholangiocytes but secretion is diminished, probably as a result of reduced Cl− /HCO− 3 exchanger activity in the apical membrane of cystic ductal cells as compared with the normal cholangiocytes (Perrone et al., 1997).
Biliary Atresia Biliary atresia is a destructive, inflammatory process of the intrahepatic and extrahepatic bile ducts, which leads to obliteration of the biliary tract and to biliary cirrhosis. It is the most common cause of cholestasis in infants and children. The pathogenesis of biliary atresia is unknown. When larger ducts are involved in biliary atresia the prognosis is poor. Most recent studies have focused on dysregulation of ductal morphogenesis and environmental factors (viruses or metabolic insults) in combination with genetic or immunologic susceptibility. Reovirus type 3 and Group C rotavirus have been implicated in biliary atresia. A dysregulation of ductal morphogenesis is supported by the frequent coexistence with other developmental anomalies, particularly visceral organ symmetry. This association suggests abnormalities in developmental genes that cause the failure of the ductal remodeling process at the hilum. Recently, a mouse model with insertional mutation in the proximal region of mouse chromosome 4 has been described with features of biliary atresia and abdominal situs inversus.
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POTENTIAL FUNCTIONS OF SMALL CHOLANGIOCYTES We propose that small ducts act as a reserve cell population in the liver, which is ready to respond to injury. Following bile duct injury, small ducts proliferate to provide compensatory secretory function, whereas large ducts undergo repair (LeSage et al., 1999, 2001). Other investigators have proposed that small ducts function as progenitor cells for biliary cells and hepatocytes (Theise et al., 1999). Upon injury, this cell may be activated to proliferate and produce progeny, which can differentiate into either bile duct cells or hepatocytes (Theise et al., 1999). Although our studies with CCl4 , ANIT and partial hepatectomy failed to show that activation of small cholangiocytes produced hepatocyte precursor cells (e.g. expression of ␣-fetoprotein or albumin), other studies using the allyl alcohol model, Solt-Farber model and furan induced liver injury, show that cells within small ducts or closely adjacent to small ducts have the potential to differentiate into either bile duct cells and hepatocytes or develop into neuroendocrine cells, intestinal-type adenocarcinomas, hepatocellular carcinomas and cholangiocarcinomas (Edakuni et al., 2001; Factor et al., 1994; Novikoff & Yam, 1998; Pack et al., 1993; Petropoulos et al., 1985; Sell, 1990). These studies show that small cholangiocytes (or cells closely adjacent to small ducts) have great plasticity with capacities to differentiate into a variety of cell types (LeSage et al., 1999). Most of the secretory functions of cholangiocytes appear to be limited to cholangiocytes lining large bile ducts (Alpini et al., 1996, 1997, 1998), yet small ducts may also secrete based on mechanisms unrelated to cAMP/CFTR. Small cholangiocytes are rich in cytoplasmic vesicles (Benedetti et al., 1996) and vesicledependent fluid secretion is present in cholangiocytes (Marinelli et al., 1997, 1999). Annexin-V is also present in small cholangiocytes (Katayanagi et al., 1999); it has been shown to play a role in cytoskeletal-dependent vesicular transport. Small cholangiocytes have been shown to express ETA and ETB (Caligiuri et al., 1998). Since endothelin exerts its functions through activation of Ca2+ (Pinzani et al., 1996), we propose that small cholangiocytes participate in ductal bile secretory activity through a Ca2+ -regulated pathway that is cAMP-independent (Alpini et al., 1996; Alpini, Glaser, Robertson, Phinizy et al., 1997; Alpini, Glaser, Robertson, Rodgers et al., 1997; Glaser et al., 1997, 2003; LeSage, Alvaro, Benedetti et al., 1999; LeSage, Benedetti et al., 1999; LeSage et al., 1996, 2001) (e.g. calciumdependent Cl− channel). If this is the case, then cholangiocytes sequentially modify bile in spatially separate processes as it passes through the axis of the biliary tree.
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SUMMARY We have summarized the recent findings that demonstrate that cholangiocytes are heterogeneous with regard to morphology, secretory activity to gastrointestinal hormones/peptides and bile salts and proliferative/apoptotic responses to liver injury/toxins. On the basis of these findings, we propose a scheme for mapping the morphological, secretory, proliferative and apoptotic events in the intrahepatic biliary tree (Fig. 3). This model proposes that bile ducts are morphologically heterogeneous with small ducts lined by small cholangiocytes, and large ducts
Fig. 3. Mapping of the Morphological, Secretory, Proliferative, and Apoptotic Events in the Intrahepatic Biliary Tree. Note: This model proposes that bile ducts are morphologically heterogeneous with small ducts (on the left) lined by small cholangiocytes and large ducts (on the right) lined by large cholangiocytes. Large but not small bile ducts express secretin and somatostatin receptors, CFTR and Cl− /HCO− 3 , and respond to these two hormones with changes in ductal secretion in both normal and BDL rats. Small and large ducts differentially proliferate in response to BDL, partial hepatectomy, and CCl4 administration. In BDL rats, only the large ducts proliferate; in partial hepatectomy, both the small and large ducts proliferate, and in CCl4 -treated rats only the small bile ducts proliferate promptly. Cholangiocyte apoptosis is not observed in BDL rats, while in CCl4 -treated rats, cholangiocyte apoptosis is confined to the large bile ducts.
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lined by large cholangiocytes. Large but not small bile ducts, express secretin and somatostatin receptors, CFTR, and Cl− /HCO− 3 , and respond to these two hormones with changes in ductal secretion. Pathologically, small and large ducts respond differentially to specific injury/toxins. Following BDL, only large cholangiocytes proliferate, whereas following CCl4 administration, damage and loss of large duct function leads to de novo proliferation and secretion of small cholangiocytes (resistant to CCl4 ) in order to compensate for the loss of large duct function. In partial hepatectomy, both small and large cholangiocytes respond with increases in proliferation and secretion. The presence of cholangiocyte heterogeneity may well endow the biliary system with physiological advantages involving bile secretion, cholehepatic shunting, and countercurrent bile and blood flow. Finally, human cholangiopathies differentially target the small and large ducts leading to cholangiocyte proliferation/loss.
FUTURE DIRECTIONS Further studies are needed to determine why cholangiopathies are restricted to specific sized ducts. The observations of cholangiocyte heterogeneity in rat models need to be extended to include animal models that more closely resemble normal human biliary function under normal conditions and in cholangiopathies in disease. Further studies are also necessary for evaluating the role of small ducts in the overall contribution of ductal secretion during normal bile formation and as a compensatory role in diseases in which ductal bile secretion is reduced (e.g. CF). In ductopenia, the repair of bile ducts becomes critical, yet the mechanisms for duct morphogenesis are unknown. The roles of cholinergic, adrenergic, dopaminergic and serotoninergic innervation regulating the heterogeneous responses of bile ducts to agonists, injury/toxins and viruses may also be critical to the maintenance of bile duct structure after injury. Since microvascular proliferation may be important in repair of bile ducts after injury, studies are needed to evaluate the role of the blood supply and circulating factors (e.g. VEGF) in the regulation of cholangiocyte function. Since some cholangiopathies (e.g. PBC) likely develop by an immunological mechanism, it may be relevant to study the heterogeneous expression of specific antigens, which may regulate the pathogenesis of these immune mediated cholangiopathies.
ACKNOWLEDGMENTS Portions of the findings presented here were supported by a grant award to Dr. Alpini from Scott & White Hospital and Texas A&M University, by an NIH
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grant DK58411 and by VA Merit Award to Dr. Alpini, and by an NIH grant DK 54208 to Dr. LeSage.
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Glaser, S. S., Rodgers, R. E., Phinizy, J. L., Robertson, W. E., Lasater, J., Caligiuri, A., Tretjak, Z., LeSage, G. D., & Alpini, G. (1997). Gastrin inhibits secretin-induced ductal secretion by interaction with specific receptors on rat cholangiocytes. American Journal of Physiology, 273, G1061–G1070. Guicciardi, M. E., & Gores, G. J. (2002). Bile acid-mediated hepatocyte apoptosis and cholestatic liver disease. Digestive and Liver Diseases, 34, 387–392. Gurantz, D., & Hofmann, A. F. (1984). Influence of bile acid structure on bile flow and biliary lipid secretion in the hamster. American Journal of Physiology, 247, G736–G748. Han, H. J., Yoon, B. C., Lee, S. H., Park, S. H., Park, J. Y., Oh, Y. J., & Lee, Y. J. (2002). Ginsenosides inhibit EGF-induced proliferation of renal proximal tubule cells via decrease of c-fos and c-jun gene. Planta Medicine, 68, 971–974. Handler, J. A., & Goldstein, R. S. (1996). Xenobiotic metabolism and toxic responses of intrahepatic biliary epithelium. In: A. E. Sirica & D. S. Longnecker (Eds), Biliary and Pancreatic Ductal Epithelia. Pathobiology and Pathophysiology (pp. 181–199). Hofmann, A. F. (1989). Current concepts of biliary secretion. Digestive Disease Science, 34, 16S–20S. Jones, D. E., Palmer, J. M., James, O. F., Yeaman, S. J., Bassendine, M. F., & Diamond, A. G. (1995). T-cell responses to the components of pyruvate dehydrogenase complex in primary biliary cirrhosis. Hepatology, 21, 995–1002. Kanno, N., Glaser, S., Chowdhury, U., Phinizy, J. L., Baiocchi, L., Francis, H., LeSage, G., & Alpini, G. (2001). Gastrin inhibits cholangiocarcinoma growth through increased apoptosis by activation of Ca2+ -dependent protein kinase C-alpha. Journal of Hepatology, 34, 284–291. Kanno, N., LeSage, G., Glaser, S., Alvaro, D., & Alpini, G. (2000). Functional heterogeneity of the intrahepatic biliary epithelium. Hepatology, 31, 555–561. Kanno, N., LeSage, G., Phinizy, J. L., Glaser, S., Francis, H., & Alpini, G. (2002). Stimulation of alpha2-adrenergic receptor inhibits cholangiocarcinoma growth through modulation of Raf-1 and B-Raf activities. Hepatology, 35, 1329–1340. Katayanagi, K., Van de Water, J., Kenny, T., Nakanuma, Y., Ansari, A. A., Coppel, R., & Gershwin, M. E. (1999). Generation of monoclonal antibodies to murine bile duct epithelial cells: Identification of annexin V as a new marker of small intrahepatic bile ducts. Hepatology, 29, 1019–1025. Kato, A., Gores, G. J., & LaRusso, N. F. (1992). Secretin stimulates exocytosis in isolated bile duct epithelial cells by a Cyclic AMP-mediated mechanism. Journal of Biological Chemistry, 267, 15523–15529. Katz, N., & Jungermann, K. (1993). Metabolic heterogeneity of the liver. Hepatic transport and bile secretion: Physiology and pathophysiology. N. Tavoloni & P. D. Berk (Eds) (pp. 55–70). Kossor, D. C., Goldstein, R. S., Ngo, W., DeNicola, D. B., Leonard, T. B., Dulik, D. M., & Meunier, P. C. (1995). Biliary epithelial cell proliferation following alpha-naphthylisothiocyanate (ANIT) treatment: Relationship to bile duct obstruction. Fundamental Applications Toxicology, 26, 51–62. Kurosawa, H., Que, F. G., Roberts, L. R., Fesmier, P. J., & Gores, G. J. (1997). Hepatocytes in the bile duct-ligated rat express Bcl-2. American Journal of Physiology, 272, G1587–G1593. Lakehal, F., Wendum, D., Barbu, V., Becquemont, L., Poupon, R., Balladur, P., Hannoun, L., Ballet, F., Beaune, P. H., & Housset, C. (1999). Phase I and phase II drug-metabolizing enzymes are expressed and heterogeneously distributed in the biliary epithelium. Hepatology, 30, 1498– 1506. Lamri, Y., Erlinger, S., Dumont, M., Roda, A., & Feldmann, G. (1992). Immunoperoxidase localization of ursodeoxycholic acid in rat biliary epithelial cells. Evidence for a cholehepatic circulation. Liver, 12, 351–354.
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LaRusso, N. F., Ishii, M., & Vroman, B. T. (1991). The ins and outs of membrane movement in biliary epithelia. Transactions of the American Clincial and Climatological Association, 102, 245–259. Lazaridis, K. N., Pham, L., Tietz, P., Marinelli, R. A., deGroen, P. C., Levine, S., Dawson, P. A., & LaRusso, N. F. (1997). Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter. Journal of Clinical Investigation, 100, 2714–2721. Lee, V. M., Cameron, R. G., & Archer, M. C. (1998). Zonal location of compensatory hepatocyte proliferation following chemically induced hepatotoxicity in rats and humans. Toxicoligic Pathology, 26, 621–627. LeSage, E. G., Alvaro, D., Benedetti, A., Glaser, S., Marucci, L., Baiocchi, L., Eisel, W., Caligiuri, A., Phinizy, J. L., Rodgers, R., Francis, H., & Alpini, G. (1999). Cholinergic system modulates growth, apoptosis, and secretion of cholangiocytes from bile duct-ligated rats. Gastroenterology, 117, 191–199. LeSage, G. D., Benedetti, A., Glaser, S., Marucci, L., Tretjak, Z., Caligiuri, A., Rodgers, R., Phinizy, J. L., Baiocchi, L., Francis, H., Lasater, J., Ugili, L., & Alpini, G. (1999). Acute carbon tetrachloride feeding selectively damages large, but not small, cholangiocytes from normal rat liver. Hepatology, 29, 307–319. LeSage, G., Glaser, S., Alvaro, D., Marzioni, M., Ueno, Y., Francis, H., & Alpini, G. (2001). Alpha1 (but not beta-1) adrenergic agonists potentiate secretin-stimulated ductal secretion in bile duct ligated (BDL) rats through cross-talk between Ca2+ -dependent PKC and adenylyl cyclase pathways. Hepatology, 34, A1219. LeSage, G., Glaser, S. S., Gubba, S., Robertson, W. E., Phinizy, J. L., Lasater, J., Rodgers, R. E., & Alpini, G. (1996). Regrowth of the rat biliary tree after 70% partial hepatectomy is coupled to increased secretin-induced ductal secretion. Gastroenterology, 111, 1633–1644. LeSage, G. D., Glaser, S. S., Marucci, L., Benedetti, A., Phinizy, J. L., Rodgers, R., Caligiuri, A., Papa, E., Tretjak, Z., Jezequel, A. M., Holcomb, L. A., & Alpini, G. (1999). Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver. American Journal of Physiology, 276, G1289–G1301. LeSage, G., Glaser, S., Robertson, W., Phinizy, J. L., Rodgers, R., & Alpini, G. (1996). Partial hepatectomy induces proliferative and secretory events in small cholangiocytes. Gastroenterology, 110, A1250. LeSage, G., Glaser, S., Ueno, Y., Alvaro, D., Baiocchi, L., Kanno, N., Phinizy, J. L., Francis, H., & Alpini, G. (2001). Regression of cholangiocyte proliferation after cessation of ANIT feeding is coupled with increased apoptosis. American Journal of Physiology. Gastrointestinal Liver Physiology, 281, G182–190. LeSage, G. D., Marucci, L., Alvaro, D., Glaser, S. S., Benedetti, A., Marzioni, M., Patel, T., Francis, H., Phinizy, J. L., & Alpini, G. (2002). Insulin inhibits secretin-induced ductal secretion by activation of PKC alpha and inhibition of PKA activity. Hepatology, 36, 641–651. Ludwig, J. (1987). New concepts in biliary cirrhosis. Seminars in Liver Disease, 7, 293–301. Marinelli, R. A., Pham, L., Agre, P., & LaRusso, N. F. (1997). Secretin promotes osmotic water transport in rat cholangiocytes by increasing aquaporin-1 water channels in plasma membrane. Evidence for a secretin-induced vesicular translocation of aquaporin-1. Journal of Biological Chemistry, 272, 12984–12988. Marinelli, R. A., Tietz, P. S., Pham, L. D., Rueckert, L., Agre, P., & LaRusso, N. F. (1999). Secretin induces the apical insertion of aquaporin-1 water channels in rat cholangiocytes. American Journal of Physiology, 276, G280–G286. Martinez-Anso, E., Castillo, J. E., Diez, J., Medina, J. F., & Prieto, J. (1994). Immunohistochemical detection of chloride/bicarbonate anion exchangers in human liver. Hepatology, 19, 1400–1406.
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Marucci, L., Alpini, G., Glaser, S., Alvaro, D., Benedetti, A., Francis, H., Phinizy, J. L., Marzioni, M., Mauldin, J., Venter, J., Baumann, B., Ugili, L., & LeSage, G. (2003). Taurocholate feeding prevents CCl4 -induced damage of large cholangiocytes through a PI3 kinase dependent mechanism. American Journal of Physiology, 284, G290–G301. Marzioni, M., Glaser, S. S., Francis, H., Phinizy, J. L., LeSage, G., & Alpini, G. (2002). Functional heterogeneity of cholangiocytes. Seminars in Liver Disease, 22, 227–240. Masyuk, T. V., Ritman, E. L., & LaRusso, N. F. (2001). Quantitative assessment of the rat intrahepatic biliary system by three-dimensional reconstruction. American Journal Pathology, 158, 2079– 2088. Mathis, G. A., Walls, S. A., D’Amico, P., Gengo, T. F., & Sirica, A. E. (1989). Enzyme profile of rat bile ductular epithelial cells in reference to the resistance phenotype in hepatocarcinogenesis. Hepatology, 9, 477–485. Melero, S., Spirli, C., Zsembery, A., Medina, J. F., Joplin, R. E., Duner, E., Zuin, M., Neuberger, J. M., Prieto, J., & Strazzabosco, M. (2002). Defective regulation of cholangiocyte Cl− /HCO3 (− ) and Na+ -H+ exchanger activities in primary biliary cirrhosis. Hepatology, 35, 1513–1521. Mennone, A., Alvaro, D., Cho, W., & Boyer, J. L. (1995). Isolation of small polarized bile duct units. PONAS. USA, 92, 6527–6531. Nathanson, M. H., & Boyer, J. L. (1991). Mechanisms and regulation of bile secretion. Hepatology, 14, 551–566. Nielsen, S., Smith, B. L., Christensen, E. I., Knepper, M. A., & Agre, P. (1993). CHIP28 water channels are localized in constitutively water-permeable segments of the nephron. Journal of Cellular Biology, 120, 371–383. Novikoff, P. M., & Yam, A. (1998). Stem cells and rat liver carcinogenesis: Contributions of confocal and electron microscopy. Journal of Histochemistry and Cytochemistry, 46, 613–626. Ohtani, O., Kikuta, A., Ohtsuka, A., Taguchi, T., & T, M. (1983). Microvasculature as studied by the microvascular corrosion casting/scanning electron microscope method. I. Endocrine and digestive system. Archives of Histology and Cytology Japan, 46, 1–42. Okada, Y., Jinno, K., Moriwaki, S., Shimoe, T., Tsuji, T., Murakami, M., Thurin, J., & Koprowski, H. (1988). Blood group antigens in the intrahepatic biliary tree. Journal of Hepatology, 6, 63–70. Pack, R., Heck, R., Dienes, H. P., Oesch, F., & Steinberg, P. (1993). Isolation, biochemical characterization, long-term culture, and phenotype modulation of oval cells from carcinogenfed rats. Experimental Cell Research, 204, 198–209. Palmer, K. R., Gurantz, D., Hofmann, A. F., Clayton, A. F., Hagey, L. R., & Cecchetti, S. (1987). Hypercholeresis induced by norchenodeoxycholate in biliary fistula rodent. American Journal of Physiology, 252, G219–G228. Paumgartner, G., & Beuers, U. (2002). Ursodeoxycholic acid in cholestatic liver disease: Mechanisms of action and therapeutic use revisited. Hepatology, 36, 525–531. Perrone, R. D., Grubman, S. A., Murray, S. L., Lee, D. W., Alper, S. L., & Jefferson, D. M. (1997). Autosomal dominant polycystic kidney disease decreases anion exchanger activity. American Journal of Physiology, 272, C1748–C1756. Petropoulos, C. J., Yaswen, P., Panzica, M., & Fausto, N. (1985). Cell lineages in liver carcinogenesis: Possible clues from studies of the distribution of alpha-fetoprotein RNA sequences in cell populations isolated from normal, regenerating, and preneoplastic rat livers. Cancer Research, 45, 5762–5768. Pinzani, M., Milani, S., De Franco, R., Grappone, C., Caligiuri, A., Tosti-Guerra, C., Maggi, M., Failli, P., Ruocco, C., & Gentilini, P. (1996). Endothelin 1 is overexpressed in human cirrhotic liver and exerts multiple effects on activated hepatic stellate cells. Gastroenterology, 110, 534–548.
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Que, F. G., Gores, G. J., & LaRusso, N. F. (1997). Development and initial application of an in vitro model of apoptosis in rodent cholangiocytes. American Journal of Physiology, 272, G106– G115. Roberts, S., Kuntz, S., Gores, G., & LaRusso, N. (1993). Regulation of bicarbonate-dependent ductular secretion assessed by lumenal micropuncture of isolated rodent intrahepatic bile ducts. PONAS USA, 90, 9080–9084. Roberts, S. K., Ludwig, J., & LaRusso, N. F. (1997). The pathobiology of biliary epithelia. Gastroenterology, 112, 269–279. Roman, R. M., Feranchak, A. P., Salter, K. D., Wang, Y., & Fitz, J. G. (1999). Endogenous ATP release regulates Cl− secretion in cultured human and rat biliary epithelial cells. American Journal of Physiology, 276, G1391–1400. Sasaki, H., Schaffner, F., & Popper, H. (1967). Bile ductules in cholestasis: Morphologic evidence for secretion and absorption in man. Laboratory Investigations, 16, 84–95. Schaffner, F., & Popper, H. (1961). Electron microscopic studies of normal and proliferated bile ductules. American Journal of Pathology, 38, 393–410. Schlenker, T., Romac, J. M., Sharara, A. I., Roman, R. M., Kim, S. J., LaRusso, N., Liddle, R. A., & Fitz, J. G. (1997). Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes. American Journal of Physiology, 273, G1108–1117. Sell, S. (1990). Is there a liver stem cell? Cancer Research, 50, 3811–3815. Strazzabosco, M., Mennone, A., & Boyer, J. L. (1991). Intracellular pH regulation in isolated rat bile duct epithelial cells. Journal of Clinical Investigation, 87, 1503–1512. Terada, T., Ishida, F., & Nakanuma, Y. (1989). Vascular plexus around intrahepatic large bile ducts in normal livers and portal hypertension. Journal of Gastroenterology and Hepatology, 1, 276–278. Terada, T., Kono, N., & Nakanuma, Y. (1992). Immunohistochemical and immunoelectron microscopic analyses of alpha-amylase isozymes in human intrahepatic biliary epithelium and hepatocytes. Journal of Histochemistry and Cytochemistry, 40, 1627–1635. Terada, T., Morita, T., Hoso, M., & Nakanuma, Y. (1994). Pancreatic enzymes in the epithelium of intrahepatic large bile ducts and in hepatic bile in patients with extrahepatic bile duct obstruction. Journal of Clinical Pathology, 47, 924–927. Theise, N. D., Saxena, R., Portmann, B. C., Thung, S. N., Yee, H., Chiriboga, L., Kumar, A., & Crawford, J. M. (1999). The canals of Hering and hepatic stem cells in humans. Hepatology, 30, 1425–1433. Tietz, P. S., Alpini, G., Pham, L. D., & LaRusso, N. F. (1995). Somatostatin inhibits secretin-induced ductal hypercholeresis and exocytosis by cholangiocytes. American Journal of Physiology, 269, G110–118. Ueno, Y., Alpini, G., Yahagi, K., Kanno, N., Moritoki, Y., Fukushima, K., Glaser, S., LeSage, G., & Shimosegawa, T. (2003). Evaluation of differential gene expression by microarray analysis in small and large cholangiocytes isolated from normal mice. Liver International (in press). Yamamoto, K., & Phillips, M. J. (1984). A hitherto unrecognized bile ductular plexus in normal rat liver. Hepatology, 4, 381–385. Zsembery, A., Spirli, C., Granato, A., LaRusso, N. F., Okolicsanyi, L., Crepaldi, G., & Strazzabosco, M. (1998). Purinergic regulation of acid/base transport in human and rat biliary epithelial cell lines. Hepatology, 28, 914–920.
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APPENDIX ANIT = ␣-naphthylisothiocyanate BDL = bile duct ligation CCl4 = carbon tetrachloride cAMP = adenosine 3 , 5 -monophosphate CF = cystic fibrosis CFTR = cystic fibrosis transmembrane regulator IBDU = intrahepatic bile duct units PBC = primary biliary cirrhosis PSC = primary sclerosing cholangitis
3.
THE ACTIN CYTOSKELETON IN LIVER FUNCTION
R. Brian Doctor and Matthew Nichols INTRODUCTION The aim of this chapter is to detail how actin and its associated proteins direct signaling and transport functions at the apical membrane of liver epithelial cells. Within hepatocytes and cholangiocytes, the two epithelial cell types in the liver, filamentous actin is distributed along the plasma membrane and concentrated at the apical membrane domain (see Fig. 1). Fundamental aspects of the actin cytoskeleton have long been appreciated. The last decade, however, has seen an explosion in the discovery of the molecular mechanisms that underlie these actin-dependent functions. This chapter is presented in three sections. The first section highlights poignant molecular features of actin and actin-associated proteins. Actin-associated proteins are responsible for harnessing the potential of the actin cytoskeleton to perform a number of regulated tasks within cells. With this foundation in hand, the chapter’s second section deals with the apical domain of liver epithelial cells where many of the molecular characteristics of the actin cytoskeleton are applied to carry out a multitude of functions that occur within this domain. Whenever possible, these features will be related directly to hepatocyte and cholangiocyte functions. The third section of the chapter describes the initial steps that have been taken to understand the molecular pathophysiology of specific liver diseases that are rooted back to alterations in the actin cytoskeleton.
The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 49–79 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15003-4
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Fig. 1. Filamentous Actin Concentrates at the Plasma Membrane Domains. Note: Staining of liver sections with rhodamine-phalloidin, which binds to filamentous but not monomeric actin, shows filamentous actin is concentrated at the cell periphery. The upper panel shows staining in a cord of hepatocytes. While the sinusoidal (basal) and lateral membranes are stained, staining of the canalicular (apical) membranes is particularly intense (arrowheads). The same is true in cholangiocytes that line the intrahepatic bile duct (lower panel). This large duct shows modest staining along the basolateral domains of the cells and intense staining at the apical domain (arrowheads).
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PROTEINS AND PROPERTIES OF THE ACTIN CYTOSKELETON The Cellular Cytoskeleton The eukaryotic cytoskeleton is comprised of three distinct types of filaments: intermediate filaments, microtubules and microfilaments. While evidence continues to emerge that these three filamentous systems are interactive, each has discernible roles in providing structural integrity, organization and shape to eukaryotic cells. Intermediate filaments (e.g. keratin) are formed from individual extended proteins that form coiled-coil dimers which then associate in a lateral fashion to form stable, elastic cables. In epithelial cells, these keratin cables course through the body of cells while binding at sites of cell-cell (i.e. desmosome) and cell-matrix (i.e. hemi-desmosome) contact. The intermediate filament system confers mechanical stability both to the individual cells and to epithelial layers. This mechanical stability is demonstrated in humans with loss of function mutations in specific keratin genes that are normally expressed in the basal cell layer of the epidermis. Termed epidermolysis bullosa simplex, these subjects develop severe blistering of the skin with even modest abrasion. Histologic observation shows that these modest abrasions completely disrupt the integrity of the basal cells. Unlike intermediate filaments, microtubules and microfilaments are formed from the polymerization of globular dimers and monomers, respectively. Microtubules and microfilaments are highly dynamic structures capable of undergoing rapid polymerization and depolymerization. Emanating from the supra-nuclear microtubule organizing center, microtubules, microtubule associated proteins and microtubular motor proteins allow for the targeted delivery of vesicles, organelles and proteins throughout the cell. Microfilaments, or actin filaments, are formed through the head-to-tail addition of actin monomers. In contrast to intermediate filaments and microtubules, actin filaments are concentrated at the plasma membrane where they perform a number of functions. These functions include structural support for the membrane, conferring cell shape, restriction and retention of proteins in the membrane, regulation of integral membrane protein function, cell motility and vesicle movement. A broad range of actin-associated proteins bind and moderate the activities of the actin cytoskeleton and allow for the execution of actin cytoskeletal functions. Three Distinct Genes Encode for Actin There are several distinct actin genes in the mammalian genomes, each coding for a ∼43 kD protein. ␣-Actin genes are expressed within smooth, cardiac and
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striated muscle cells while - and ␥-actin are more broadly expressed. Comparison of the amino acid sequences shows ␣-actin is 94% identical to both - and ␥actin. -Actin and ␥-actin are 98% identical to each other with only a insertion of three glutamic acid residues after the initiating methionine and a valine to isoleucine conversion of the tenth residue of -actin accounting for the only amino acid differences. While the ␣-, - and ␥-actin proteins have similar biochemical properties, multiple actin genes can be expressed within a single cell type. ␣Actin filaments may be more resistant to ADF/cofilin degradation (Vartiainen et al., 2002) but other functional differences of the distinct actins within cells are largely unknown. The actin gene products can be discretely localized within the same cell. - and ␥-actin are concurrently expressed in gastric parietal cells but -actin is concentrated around the canalicular membranes while ␥-actin is distributed predominantly along the basolateral membranes (Yao et al., 1995). The comparative expression and localization of - and ␥-actin has not been described in liver epithelial cells.
Actin Monomers Polymerize into Filaments The most significant functional feature of actin monomers are their ability to polymerize into extended filaments. Actin monomers are a polarized protein with a nucleotide binding cleft in the center of the protein. Polymerization occurs through “head” to “tail” interactions. Either ATP or ADP binds with high affinity in the nucleotide cleft. Bound ATP is hydrolyzed by the protein’s slow ATPase activity. Placed in an ATP-supplemented physiologic salt solution, actin monomers endure a lag period before undergoing a period of rapid polymerization. The lag period is the rate limiting step in filament formation as nuclei of three bound monomers are initially formed. Once formed, actin monomers rapidly bind to the filament ends until the concentration of actin monomers is reduced to the Km for the binding interaction. This is termed the critical concentration. When the monomer concentration drops to the critical concentration, net polymerization and depolymerization are at equilibrium. When examined microscopically, the addition of actin monomers onto a filament occurs preferentially on one end, termed the “plus” or “barbed” end, of the filament. The opposing end is termed the “minus” or “pointed” end. “Barbed” and “pointed” ends were originally used because of the appearance of filament ends when they were decorated with myosin heads in early studies of actin filaments. While these terms are still used today, this chapter will use “plus” and “minus” for conceptual ease. The preferential addition to the “plus” end and deletion from the “minus” end is due to three properties of actin and actin
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polymers. First, the rates of monomer association/dissociation are much greater on the “plus” end vs. the “minus” end of the filament. Second, the ATPase activity of actin is greater when bound within a filament. Third, the binding affinity of monomers is greater for ATP-bound actin than for ADP-bound actin. Thus, when ATP-actin monomers are added to a solution with pre-existing filaments, there is a rapid association at the “plus” end that exceeds the rate of ATP hydrolysis. Thus, there is a cap of ATP-actin on the “plus” end. The slower association rate at the “minus” end, when coupled with the higher ATPase activity in polymerized actin, allows ATP hydrolysis to occur prior to the addition of another monomer. This addition will be of a lower affinity due to the presence of ADP-actin. As a result, when the monomer concentration is near the critical concentrations for the filament ends, ATP-actin will add preferentially to the “plus” end, ADP-actin will dissociate preferentially from the “minus” end and individual actin monomers can be observed to “treadmill” from the “plus” end to the “minus” end of filaments.
Cells Harness the Potential of Actin Polymerization Actin filaments on their own have little functional utility but cells have evolved specific actin-associated proteins that harness the potential of the actin polymerization. These include proteins to nucleate de novo filaments, moderate the rates of “plus” end polymerization, orchestrate the depolymerization of “minus” end of filaments, stabilize the filaments when appropriate, utilize stabilized filaments to anchor molecular scaffolds and form tracks to permit directed mechanical movement (see Table 1).
Arp2/3 Complex Nucleates Actin Filaments The rate-limiting step in actin polymerization is the nucleation of the filament. The molecular mechanisms permitting and directing actin nucleation within cells was an enduring mystery until a complex of seven proteins, termed the Arp2/3 complex, was discovered. The Arp2/3 complex utilizes actin-related proteins (Arps), to mimic a nucleated actin site and initiate the formation of new actin filaments (Mullins & Pollard, 1999). Not surprisingly, filament nucleation is a tightly regulated process and cells have evolved a number of mechanisms to limit, direct and coordinate the de novo synthesis of filaments. Among the best understood is the Wiskott-Aldrich Syndrome Protein (WASP). When activated to unfold at specific membrane sites, WASP then recruits and activates the Arp2/3 complex to initiate actin polymerization. As described later,
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Table 1. Examples of Actin associated Proteins, their Functions and their Regulation. Activity
Protein
Regulation
Comments
Nucleation
Arp2/3 Complex
WASP
Critical for de novo filament formation
Cdc42 PI(4,5)P2 Monomer binding Capping
Severing
b-Thymosin Profilin CapZ Adducin
none PI(4,5)P2 PI(4,5)P2 Ca2+ /calmodulin phosphorylation
Actin monomer buffer Induces accelerated polymerization Regulates availability of (+) ends Recruits spectrin to capped ends
ADF/Cofilin
Phosphorylation PI(4,5)P2 pH Ca2+ PI(4,5)P2 pH
Pivotal protein in filament turnover
Gelsolin
Lateral Binding Cross-Linking
Tethering
Motor
Tropomyosin
Shelters filaments from severing
Villin
Ca2+
a-Actinin Spectrin Filamin
PI(4,5)P2 Phosphorylation
ERM Proteins
Phosphorylation
Ankyrin
PI(4,5)P2 Phosphorylation
Myosins
Deconstructs actin meshworks Caps severed filaments
Ca2+ /Calmodulin Phosphorylation PI(4,5)P2 (myoX)
Actin bundling and severing activity in microvilli Parallel actin filament bundling Orthogonal actin filament bundling Actin gelation and membrane attachment Links actin, membrane and regulatory proteins Site-specific localization of membrane proteins Family of actin mechanoenzymes
the directed initiation of actin filament formation is important in a number of cellular processes. Beta-Thymosin and Profilin Control “Plus” End Polymerization In epithelial cells, the total actin concentration far exceeds the critical concentration for actin polymerization yet only about half of the cellular actin is incorporated into
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filaments. This is a result of a large portion of the actin monomers being complexed with abundant, low molecular weight monomer binding proteins. -Thymosin is an abundant actin monomer binding protein with a dissociation constant near the critical concentration for the “plus” end of the filament. When bound with an actin monomer, it disallows the addition of the monomer to a filament end. Consequently, -thymosin behaves as an “actin buffer” to sequester excess monomers when polymerization is inhibited and provide a surplus of actin monomers when actin polymerization is induced. Profilin is another abundant low molecular weight actin monomer binding protein. Activated by PI(4,5)P2 , profilin promotes exchange of ATP for ADP on actin monomers and increases the affinity of actin monomer binding to the “plus” end of filaments. The physiologic significance of the thymosin/profilin system within cells is that it provides a system to drive and sustain actin polymerization to a much greater extent than if there was no buffered pool of monomers available.
Disassembly of Filaments is an Essential Part of Actin Dynamics Equally as important as the regulated polymerization of actin filaments is the coordinated disassembly of actin filaments. Filament disassembly can occur via severing filaments along the length of a filament or by accelerating the removal of monomers from the “minus” end of filaments. Disassembly is important in remodeling of filaments that are no longer required or are an impediment to cellular events. Their disassembly also supplies monomers needed for actin polymerization at other cellular sites. There are a number of different filament severing proteins with distinct modes of severing and regulation. The related gelsolin proteins are Ca2+ regulated actin severing proteins. With increased Ca2+ , gelsolin binds the lateral aspect of filaments, cleaves the filament and caps the “plus” end of the resultant product. PI(4,5)P2 binding liberates the gelsolin cap from the actin filaments. Gelsolin and functionally related proteins such as scinderin play a pivotal role in degrading cortical meshworks of actin filaments to permit vesicular access to and from the plasma membrane. Villin is a gelsolin family member expressed within the apical microvilli of epithelial cells and plays a significant role in Ca2+ regulated disassembly of microvilli. Actin depolymerizing factor (ADF) or cofilin is another family of actin filament disassembly proteins with an essential role in cellular physiology (Bamburg, 1999). ADF/cofilin can either bind laterally onto an actin filament or bind to the “minus” end of filaments. In its activated form, laterally bound ADF/cofilin induces an increased “twist” in the actin filament such that an actin filament completes
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an entire rotation in 57 nm instead of 74 nm in a native filament (McGough et al., 1997). This increased molecular strain renders the filament susceptible to severing. Activated ADF/cofilin also binds to the “minus” end of filaments and increases the rate of monomer depolymerization. Monomer dissociation from the “minus” end is the rate limiting step in filament dynamics. By increasing the rate of “minus” end dissociation and the number of “minus” ends following filament severing, ADF/cofilin increases the net availability of monomers and, consequently, increases the rate of “plus” end polymerization. ADF/cofilin is regulated by multiple means including inhibition by phosphoryaltion on serine-3, low intracellular pH and elevated levels of PI(4,5)P2 . Different cell types express different isoforms of ADF/cofilin. The ADF isoform in epithelial cells has a comparatively high rate of filament dissociation, allowing for more rapid monomer dissociation from “minus” ends and, consequently, accelerated polymerization at “plus” ends.
Filaments Can Be Stabilized From Dynamic Turnover To oppose the depolymerizing activities of proteins such as gelsolin and ADF/cofilin, other proteins stabilize filaments and inhibit monomer exchange at filaments ends. CapZ is a well-characterized “plus” end capping protein and was initially described for capping actin filaments along the Z-line in muscle cells. On quiescent filaments, CapZ can hold “plus” ends in a ready state for subsequent polymerization. In motile cells, “plus” end capping by CapZ optimizes the generation of force that results from actin polymerization by capping the longer, less efficient filaments. Further, capping of filaments that are out of the line of desired extension increases the efficiency of vectorial polymerization and directional cell motility. The affinity of CapZ for “plus” ends is negatively regulated by PI(4,5)P2 . The covering of the lateral aspects of filaments can also affect filament dynamics. Tropomyosin is a prominent lateral binding protein that spans across seven adjacent actin subunits along an actin filament. When bound, tropomyosin impedes the access and activities of gelsolin, ADF/cofilin and Arp2/3.
Stabilized Filaments are Cross-Linked Into Structural Lattices An individual actin filament is not, in general, a particularly useful structural unit. Instead, cells express filament cross-linking proteins that bundle individual filaments into parallel arrays or link them into orthogonal meshworks. The “tightest” actin bundles, such as those found in epithelial microvilli, are formed
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by single proteins with two actin binding motifs. A single microvillus may contain multiple distict bundling proteins. The co-expression of these bundling proteins allows for the orderly genesis, stabilization and degradation of a microvillus. Other parallel actin bundles, such as those found in stress fibers, are bundled by actin bundling dimers that contain a single actin binding domain and a rod domain that is capable of self-association. ␣-Actinin, an actin crosslinking dimer from the spectrin family, is the most noted example of an actin bundling dimer. Actin bundling dimers create an interfilament spacing and allow myosins (i.e actin motor proteins) and their cargoes to run along the filament bundle with reduced steric hinderence. Away from apical microvilli, epithelial membranes are supported by a meshwork of actin filaments termed “cortical actin.” Crosslinking this orthogonal array of filaments integrates the individual physical properties of a filament into a cohesive gel. Two broadly expressed but distinct orthogonal crosslinking proteins include spectrin and filamin. ␣- and -spectrin chains form antiparallel dimers and dimer pairs associate in a head-to-head direction to form spectrin tetramers that extend up to 200 nm in length. The tails of the spectrin tetramers associate with adducin, an actin binding protein that mediates the spectrin tetramer crosslinking of actin filaments. Similarly, filamin is an extended protein dimer with an ␣-actinin-related actin binding motif, a rod domain comprised of multiple repeats and a carboxy terminus dimerization motif. Filamin extends to 160 nm in a flexible V-shaped dimer with ends of the V interacting with actin filaments.
Actin Filaments are Tethered to the Plasma Membrane While bundled and orthogonal, arrays of actin filaments are important structural elements, the plasma membrane must be physically linked to these arrays to confer structural properties to the membrane. The predominant means of linking the plasma membrane to the underlying actin cytoskeleton is for actin-associated proteins to bind, either directly or indirectly, to specific integral membrane proteins. Not surprisingly, orthogonal crosslinking proteins tether to integral membrane proteins. Filamin links the actin cytoskeleton to the plasma membrane through a number of integral membrane proteins. Its role in providing structural support for the plasma membrane is best displayed in a spontaneous filamin knock-out melanoma cell line (Cunningham et al., 1992). These cells have lowered membrane tensions and undergo dramatic, continuous blebbing and recovery of their plasma membrane. Spectrin also links the membrane to the underlying actin cytoskeleton. Spectrin’s structural role is exemplified in erythrocytes (RBC) where spectrin confers the elasticity required in RBC deformation. Mutations in the spectrin gene
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account for a number of hereditary hemolytic anemias. In cells as functionally diverse as RBC, neurons and epithelial cells, the actin-anchored spectrin lattice also restricts and retains integral membrane proteins within specific membrane domains. In epithelial cells such as hepatocytes and cholangiocytes, the basolateral distribution of Na+ -K+ -ATPase allows for the efficient vectorial absorption of Na+ . Linkage by ankyrin-spectrin-actin complexes provides for the polarized retention of Na+ -K+ -ATPase in the basolateral membrane. Proteins of the Protein 4.1 superfamily also form important actin-membrane linkages. These are multi-domain proteins that marry an actin binding domain to an additional domain capable of linking directly or indirectly to the plasma membrane. Among the superfamily members, ezrin-radixin-moesin (ERM) family members are of particular importance in epithelial cells. ERM proteins are also A-kinase associated proteins (AKAP). The AKAP site permits the specific localization of protein kinase A (PKA) to cytoskeletal complexes and allows targeted distribution of the activated PKA subunits. In liver epithelial cells, radixin is concentrated at the canalicular membrane in hepatocytes while ezrin is concentrated at the apical membrane in cholangiocytes (Fouassier et al., 2001). Ezrin is the best characterized ERM family member in epithelial cells. Its most intriguing membrane linkage occurs through ERM binding phosphoprotein 50 (EBP50). EBP50 is comprised of two PSD95-Dlg-ZO1 (PDZ) domains and an ezrin binding region. PDZ proteins were concurrently discovered in the postsynaptic density of neurons (PSD95), in Drosophila melanogaster (Discs large) and the tight junctions of epithelial cells (ZO1). Subsequently, hundreds of PDZ proteins have been identified. These proteins form critical scaffolds in the organization of a wide range of membrane micro-domains and many PDZ proteins are linked to the underlying actin cytoskeleton. The most common function of PDZ domains is the recognition and binding of cytoplasmic C-terminal tails of specific integral membrane receptors, transporters and channels. Concentrated along the microvillar membrane of specific epithelial cells, the actin-ezrin-EBP50 complex has been implicated in both the regulated distribution and activity of numerous proteins. EBP50 is expressed at the apical membrane in both hepatocytes and cholangiocytes where its binding partners include Mrp2 and CFTR, respectively (Hall et al., 1998; Hegedus et al., 2003; Short et al., 1998).
The Actin Cytoskeleton is Used to Generate Force and Movement Since its discovery as the thin filaments in muscle sarcomeres, actin filaments have been associated with force generation and movement. Anit-parallel bundles of the myosin II mechanoenzyme make up the thick filaments that ratchet along the actin
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thin filaments. Over a dozen myosin family members have since been identified and found to serve a number of functions in various cell types. Each myosin family member has a highly conserved head domain that utilizes ATP hydrolysis to drive its motor function. In contrast, the tail domain is highly variable and specifies the cargo that the myosin will carry. Along specific epithelial microvilli, myosin I-like protein is a mechanical link between the microvillar actin filaments and the adjacent membrane. It has also been localized to on late endosomes, inferring a functional role in endocytic trafficking (Raposo et al., 1999). Myosins V and VI also play important roles in exocytic and endocytic vesicle trafficking in epithelial cells. Interestingly, myosin V and VI motors are respectively “plus” end and “minus” end directed motors. This would allow these two motor proteins and their vesicular cargoes to travel in opposite directions along a polarized set of actin filaments. In the last decade, the energy released in actin polymerization has been shown to be harnessed by cells and converted into a motive force. The most striking example resides at the leading edge of motile cells where actin polymerization drives membrane protrusion forward. Non-motile cells also utilize actin polymerization. In phagocytic cells such as Kupffer cells in the liver, actin polymerization drives plasma membrane up and around a bound particle to allow its engulfment. Alternatively, epithelial cells utilize the force generated in actin polymerization to drive the internalization of endocytic vesicles from the plasma membrane into the cell interior.
Actin Filments are Regulated at the Plasma Membrane The activities of actin cytoskeletal proteins are regulated in a variety of ways (Table 1). While intracellular Ca2+ levels and protein phosphorylation have been known to modify the activities of several actin associated proteins, two additional elements, phosphatidyl inositol (4,5) bis phosphate (PI(4,5)P2 ) and the Rho family of small GTPases, have more recently emerged as central players in the spatial and temporal regulation of the actin cytoskeleton.
Phosphatidyl inositol (4,5) bis-phosphate Phosphoinositides comprise a small percentage of the membrane composition but play central roles in regulating the cellular events. The phosphoinositide PI(4,5)P2 plays a particularly important role in regulating a wide variety of actinassociated proteins. In general, increased levels of PI(4,5)P2 increase actin polymerization while decreased levels of PI(4,5)P2 decrease actin polymerization.
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Fig. 2. PI(4,5)P2 is an Intermediate in the Phosphoinositide Signaling Pathways. Note: There are a number of means for generating or eliminating (PI(4,5)P2 . The most prominent pathways are shown. PI(4,5)P2 can be generated by phosphorylation of PI(4)P or dephosphorylation of PI(4,5,6)P3 . PI(4,5)P2 can be eliminated by its dephosphorylation to PI(4)P, phosphorylation to PI(4,5,6)P3 or hydrolysis to IP3 and diacylglycerol. The specific pathway of elimination is important since each elimination product itself has some cell signaling activity (denoted by grey arrows).
PI(4,5)P2 is an intermediate lipid product whose levels are controlled by balancing synthesis (e.g. phosphorylation of PI(4)P at the 5-position), additional phosphorylation (e.g. phosphorylation of PI(4,5)P2 to PI(3,4,5)P3 ), dephosphorylation (e.g. dephosphorylation of PI(4,5)P2 to PI(4)P) and degradation (i.e. hydrolysis to IP3 and diacylglycerol) (see Fig. 2). The specific PI(4,5)P2 elmination pathways are important since the by-products of the elimination pathways are themselves potent cell signaling molecules. Localized at membranes, proteins with specific PI(4,5)P2 binding domains are regulated by PI(4,5)P2 at specific sites of the membrane. The most noted PI(4,5)P2 bind motif is the pleckstrin homology domain but other PI(4,5)P2 binding motifs have also been identified.
Rho Family of Small Monomeric GTPases The Ras superfamily of small GTPases serve as “molecular switches” to regulate a wide variety of cellular events. Two branches of the Ras superfamily, the Rho and adenosine ribosylation factor (Arf) families, exert part of their regulatory activities through modulation of the actin cytoskeleton. Within the Rho family, the cellular effects of three members, Rho, Rac and Cdc42, have been studied in fair detail and found to have differential effects even within the same cell. Prenylation of the Rho family members concentrates their effects at membranes and is required for their activity. Active when bound with GTP, Fig. 3 shows the general pathways in which Rho superfamily members regulate their activity. There are currently 20 identified Rho GTPase family members (Cas et al., 1999). A sign of the significance of these proteins, Rho GTPase regulatory proteins,
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Fig. 3. The Activities of the Rho Family of GTPases are Regulated by Binding of Guanine Nucleotides. Note: The twenty-member Rho GTPase family, including Rho, Rac and Cdc42 members, cycle between active and inactive states as dictated by the bound guanine nucleotide. Guanine nucleotide exchange factors (GEF: ∼60 identified) activate Rho GTPases by enhancing GTP-GDP exchange. GTPase activating proteins (GAP: ∼70 identified) increase the GTPase activity of Rho GTPases to inactivate the Rho protein. Finally, guanine nucleotide exchange inhibitors (GEF: 4 identified) pull inactive Rho GTPases from the membrane. Prenylation of Rho proteins is required for activity and likely anchors Rho GTPases at the membrane. (This figure was fashioned from a previously published figure of Rho GTPase regulation; see Nature 420, 629–634, 2002).
including guanine nucleotide exchange factors (GEF), GTPase activating proteins (GAP) and guanine nucleotide exchange inhibitors (GDI) far out number the individuat Rho GTPases. In isolated fibroblasts, activation of Rho increases stress fiber density, activation of Rac increases lamellapodia formation and activation of Cdc42 increases filopodia formation (see Fig. 4). The role and mechanisms of Rho GTPases in regulating actin-dependent events in polarized, differentiated epithelial cells remains in its infancy but they have been clearly implicated in regulating events ranging from basolateral vesicle secretion to site-directed actin nucleation to apical endocytosis.
Rho GTPases and PI(4,5)P2 are Interactive While presented separately, the phosphoinositide and Rho GTPase regulatory pathways are interwoven to have complimentary effects on control of the actin cytoskeleton at membranes. For example, the Arp2/3 activator WASP has
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Fig. 4. Varied Actin Cytoskeletal Responses to Distinct Rho GTPases. Note: Though closely related, the activation of distinct Rho GTPase family members result in different responses by the actin cytoskeleton. When compared to control cells (A), injection of activated Rho results in the formation of stress fibers (B), injection of activated Rac results in lamellapodial formation (C) and injection of activated Cdc42 results in filopodia formation (D). Each of these Rho GTPases have effects on the actin cytoskeleton in differentiated epithelial cells but, as exemplified in this figure, their effects are distinct from each other. (This figure was modified with permission from A. Hall, Science 279, 509–514, 1998).
binding sites for both Cdc42-GTP and PI(4,5)P2 . While Cdc42-GTP activates WASP activity on its own, this activation is further enhanced by the presence of PI(4,5)P2 . Alternatively, Rac-GTP activates PI(4)P5-kinase, the enzyme responsible for catalyzing the formation of PI(4,5)P2 from PI(4)P. Importantly, the coordinated activation/inhibition of the different Rho family members and phosphoinositides provides the cell a broad dynamic range in the temporal activation/ inhibition of actin-dependent events within discrete micro-domains of individual epithelial cells.
FUNCTION OF THE ACTIN CYTOSKELETON AT THE APICAL DOMAIN OF LIVER EPITHELIAL CELLS Over the last decade, multiple roles of the actin cytoskeleton in epithelial cell physiology have come to light. Concentrated at the plasma membrane in every region of the cell, the actin cytoskeleton interacts with the plasma membrane, imparts tension into the membrane and provides the membrane with structural support. Within the varied domains of epithelial cells, the organization of the actin cytoskeleton is markedly distinct. A meshwork of actin, termed “cortical actin,” underlies the basolateral membrane. At sites along the basal membrane where integrin proteins adhere to the underlying matrix, cables of actin filaments are specifically grounded. In cultured epithelial cells, these actin cables appear as prominent actin stress fibers. In addition to its structural role, the actin cytoskeleton associated with the cell-matrix adhesion sites participates in bi-directional
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communication between the extracellular matrix and the cell interior. At the transition between the basolateral and apical membranes of epitheial cells, actin filaments are specifically bound to the tight junction and adherens junction complexes and form a circumferential ring at the level of the junctional complex. Beneath the apical membrane at the level of the junctional complex, a network of spectrin-stabilized actin filalments form the terminal web matrix. The apical membrane of numerous epithelial cell types have microvilli, specialized membrane structures formed by underlying parallel bundles of actin filaments that emanate from the terminal web and terminate at the microvillar tips. Enormous progress has been made in understanding how the actin cytoskeleton in the terminal web and microvilli impacts seemingly every event that transpires within the apical membrane domain. To provide a contextual background, the following section will first review the physiology of apical membrane events within hepatocytes and cholangiocytes. Subsequently, the role of the actin cytoskeleton in moderating these apical membrane activities will be examined. As shown in Fig. 5, the featured actin-dependent activities include: (1) trafficking exocytic vesicles to the apical membrane; (2) accessing the plasma membrane; (3) retaining specific proteins in the membrane; (4) moderating the activities of the protein while in the membrane; (5) endocytosing the protein from the membrane; (6) trafficking endosomes to the cell interior, and (7) recycling vs. degrading the protein.
Physiology of the Apical Membrane Domain in Hepatocytes and Cholangiocytes Hepatocytes and cholangiocytes are the two epithelial cell types in the liver. Hepatocytes comprise ∼80% of the liver parenchyma and perform the majority of the liver’s functions. Key among these functions is the uptake, modification and secretion of bile constituents from the blood, across the cell and into the canalicular space. This vectorial transport of specific compounds requires the polarized distribution and regulation of specific proteins in both the basolateral (i.e. sinusoidal) and apical (i.e. canalicular) membranes. The polarized apical distribution of proteins is established, in part, by the directed delivery of proteins to the apical membrane. In hepatocytes, this includes both the transcytotic and direct delivery of proteins from the Golgi to the apical membrane. The apical membrane of hepatocytes contains at least four ATP binding cassette (ABC) proteins that are responsible for the transport of bile acids (spgp), phosphatidyl choline (mdr2), organic cations (mdr1) and organic anions including conjugated bilirubin (Mrp2). The fates and functions of Mrp2 will be used to exemplify
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Fig. 5. Actin-Associated Events in the Apical Domain of Epithelial Cells. Note: The actin cytoskeleton impacts a number of functional activities at the apical membrane of epithelial cells. These include: (1) trafficking exocytic vesicles to the apical membrane; (2) accessing the plasma membrane; (3) retaining specific proteins in the membrane; (4) moderating the activities of the protein while in the membrane; (5) endocytosing the protein from the membrane; (6) trafficking endosomes to the cell interior; and (7) recycling versus degrading the protein. See text for details.
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the roles of the actin cytoskeleton in regulating transport functions at the apical membrane of hepatocytes (see Fig. 6). Cholangiocytes line the intrahepatic bile ducts and comprise only 2–4% of the liver cell mass but secrete a bicarbonate-rich fluid that contributes up to 40% of the bile volume. This secretion results from the coordinated transepithelial movement of Cl− and HCO− 3 along with the resultant osmotic flux of water into the lumen of the bile duct. Cystic fibrosis transmembrane conductance regulator (CFTR) is an ABC protein with cAMP-mediated Cl− channel activity that plays a pivotal role in driving secretion in cholangiocytes. As with Mrp2 in hepatocytes, CFTR will be used to highlight the functions of the actin cytoskeleton at the apical domain of cholangiocytes. These two ABC transport proteins have specific linkages to the actin cytoskeleton. These linkages occur through a scaffolding complex of ezrin-radixin-moesin (ERM) proteins and the ERM binding phosphoprotien 50 (EBP50) PDZ domain protein (see Fig. 6). While the activity of channel or transporter proteins can be directly regulated by moderating their open probability or turnover rate, respectively, moderating protein density within the membrane continues to emerge as a central means of regulating their total, effective activity. In studies of EBP50 binding with 2-adrenergic receptor, EBP50 played an essential role in receptor recycling to the plasma membrane (Cao et al., 1999). Vesicular trafficking as a means of regulating membrane protein activity occurs in hepatocytes and cholangiocytes and is reflected in their high rates of plasma membrane turnover (Doctor et al., 2002; Kilic, Doctor & Fitz, 2001). In cultured hepatocytes and cholangiocytes, membrane areas equivalent to ∼1.5% of the total plasma membrane per min are delivered and retrieved from the plasma membrane of cultured hepatocytes and cholangiocytes. Activation of a number of signaling pathways can moderate this rate significantly. Furthermore, in cultured cholangiocytes, filamentous actin is associated with a subpopulation of endocytosed membranes, 40% of endocytosed membranes are recycled back to the plasma membrane and disruption of the actin cytoskeleton greatly diminishes exocytic rates. With an emphasis on vesicular trafficking, the following section will focus on how the actin cytoskeleton regulates physiologic events at the plasma membrane.
Trafficking of Exocytic Vesicles to the Apical Membrane The microtubule (MT) system is historically known for its targeted delivery of membrane vesicles to the various regions of cells. Once formed and targeted, apically-bound vesicles are transported to the apical domain along microtubules. Kinesin, an anterograde microtubule motor protein, docks onto and carries vesicles
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Fig. 6. ABC Transporters are Linked to the Actin Cytoskeleton Through an ERM-PDZ Protein Complex. Note: Specific ABC transporters are sequestered into signaling-effector complexes in the apical domain of liver epithelial cells. (A) The paradigm consists of the C-terminal tail of the ABC transporter being bound by a PDZ protein, which is in turn linked to an ERM protein. ERM proteins concurrently bind actin as well as PKA. (B) In cholangiocytes, CFTR is bound to ezrin via EBP50. (C) In hepatocytes, Mrp2 is bound to radixin via EBP50. These actin-associated complexes are important in the regulated distribution and activity of these ABC proteins.
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hundreds of microns to the cell periphery. Not yet to its fusion site, the actin cytoskeleton has been implicated in the faithful targeting of apical vesicles. The notion of cooperative microtubule-actin trafficking of vesicles was bolstered by the recent observation that myosin V and conventional kinesin are capable of direct binding (Huang et al., 1999). Such a multifunctional motor complex would allow kinesin to deliver vesicles along the MT track to the membrane domain and then switch tracks onto actin filaments where myosin V delivers the vesicle to the appropriate site for membrane fusion and exocytosis (see Fig. 5). An additional barrier to the delivery of exocytic vesicles to the apical membrane of epithelial cells is the actin cytoskeleton of the terminal web. In cell types with high secretory rates, the Ca2+ -activated severing protein scinderin is a key regulator of the spatial and temporal release of exocytic vesicles. While hepatocytes and cholangiocytes do not appear to express significant levels of scinderin, gelsolin, a Ca2+ dependent actin severing protein, is expressed in these liver cells. The addition of gelsolin into cultured epithelial cells, increases exocytosis at the apical membrane (Muallem et al., 1995). This regulated severing activity provides a means for site-specific insertion of receptors, channels and transporters into the apical membrane.
Membrane Proteins are Confined and Retained by the Actin Cytoskeleton The cytoplasmic surface of the plasma membrane is a crowded space with protein concentrations approaching 1 gr/ml (Sheetz, 1993). The actin cytoskeleton assists in the organization of this domain by sequestering integral membrane proteins with appreciable cytoplasmic tails into molecular corrals. These actin-based corrals restrict lateral diffusion of proteins into ∼0.6 m micro-domains of the membrane (Edidin, Kuo & Sheetz, 1991). Physical association of proteins to the actin cytoskeleton can further restrict proteins within membrane microdomains and increase their half-life within the membrane. The general contribution of the actin cytoskeleton in retaining proteins in the canalicular membranes of hepatocytes was shown in WIF-B cells, a polarized hepatocyte cell culture model (Tuma, Hyasae & Hubbard, 2002). Normally restricted to the canalicular membrane of polarized cells, cytochalasin D disruption of the actin cytoskeleton greatly diminished the retention of 5 -nucleotidase and aminopeptidase N in the canalicular membrane. Interestingly, the apical concentration of Mrp2 was not affected by the same treatment. This suggests Mrp2 has additional, actin-independent mechanisms that contribute to its retention in the apical membrane or that recovery of Mrp2 from the apical membrane requires the actin cytoskeleton. The consequences of direct protein-protein linkage was first demonstrated by the basolateral retention of
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Na+ -K+ -ATPase in polarizing MDCK cells (Hammerton et al., 1991). Na+ -K+ ATPase is tethered in the basolateral membrane of most epithelial cells through a linear actin-spectrin-ankyrin-Na+ -K+ -ATPase protein complex. During epithelial development, a twelve-fold increase in the dwell time of Na+ -K+ -ATPase in the basolateral membrane coincides with the period in which the spectrin-ankyrin cytoskeletal elements are ordered along that domain. Along the apical membrane of hepatocytes and cholangiocytes, the ABC transporters Mrp2 and CFTR are linked to the actin cytoskeleton by a linear actin-ERM-EBP50-ABC protein complex (see Fig. 6). As described further below, disruption of these complexes results in the loss of apical polarity of the ABC transporters. Actin Cytoskeleton Regulation of Apical Membrane Protein Activities In addition to increasing their residence time in the membrane, submembranous molecular corrals and scaffolding proteins cluster functionally interactive proteins within membrane microdomains and coordinate their cooperative activities. While the actin cytoskeleton can directly regulate some ion channel activities, the formation of signaling-effector complexes onto actin scaffolds provides for a dynamic means of coordinating events within membrane microdomains. With nearly 400 PDZ domain proteins identified in the human genome, PDZ domain proteins have moved to the forefront as organizers of supramolecular complexes (Sheng & Sala, 2001). PDZ proteins are able to generate supramolecular complexes by expressing a variety of different protein-protein binding domains and by expressing multiple PDZ domains within the same protein. Within epithelial cells, PDZ domain proteins contribute to the polarized distribution, molecular organization, trafficking and regulated activity of specific integral membrane proteins. While generally understated, PDZ proteins like EBP50, PSD95, ZO-1 and a multitude of others are all anchored to the actin cytoskeleton. In cholangiocytes, dominant-negative disruption of the CFTR-EBP50 interaction results in the complete ablation of the cAMP-mediated Cl− secretory response in cholangiocytes (Fouassier et al., 2001). Furthermore, CFTR regulates a number of other ion conductance pathways. Though never demonstrated, it is tempting to speculate that PDZ proteins such as EBP50 might mediate the molecular organization and interaction of the functionally linked ion conductance pathways. Endocytosis of Apical Membrane Proteins Just as insertion of a receptor, channel or transporter into the plasma membrane is a potent means of up-regulating their activity, removal of the protein from the
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membrane is a potent means of down regulating their activity. Not surprisingly, this also is a highly regulated process in which the actin cytoskeleton plays a central role. There are multiple means by which different cell types internalize membranes, including phagocytosis, macropinocytosis, clathrinmediated endocytosis (CME), caveolae-mediated endocytosis and endocytosis by mechanisms that are independent of either clathrin or caveolae. Each of these distinct modes of membrane internalization involves different actin-dependent steps for internalization. Within liver epithelial cells, CME is the best characterized means of internalization. CME involves the invagination of the membrane into clathrin-coated pits, dynamin-dependent fission of the vesicle from the plasma membrane and translocation of the vesicle away from the membrane. While not essential in all cells for CME to occur, the actin cytoskeleton enhances the efficacy of the endocytic steps. This is particularly true at the apical domain of polarized, differentiated epithelial cells where a dense, complex actin cytoskeletal network must be negotiated (Gottlieb et al., 1993; Jackman et al., 1994; Shurety et al., 1996). CME is a multi-step process that includes; (1) the organization of the endocytic site; (2) invagination of the plasma membrane; (3) fission and release of the membrane vesicle; (4) dissolution of the cortical actin; and (5) internalization of the endocytic vesicles. Each of these steps involves the actin cytoskeleton.
Organization of the Endocytic Site Endocytic vesicles do not emerge randomly from the membrane but, instead, repeatedly emerge from the same area. This indicates that proteins responsible for developing sites of endocytosis are sequestered or restricted to these microdomains (Gaidarov et al., 1995). The actin cytoskeleton contributes to this “corraling” of clathrin since disruption of actin filaments results in a 7-fold increase in the diffusion area of the coated pit membranes.
Invagination of the Plasma Membrane During invagination of clathrin coated membranes, actin filaments are induced to form around the neck region. This induction is coordinated by intersectin. Intersectin is an scaffolding protein capable of binding a large number of clathrinassociated proteins including clathrin, AP2, dynamin, WASP and Cdc42. Spatial and temporal analysis shows the appearance of filamentous actin coincides with the recruitment of dynamin to the neck region (Merrifield et al., 2002).
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Fission and Release of the Membrane Vesicle The fission and release of clathrin coated vesicles from the plasma membrane is significantly affected by the actin cytoskeleton. Treatment of epithelial cells with cytochalasin D to disrupt the actin cytoskeleton resulted in the accumulation of clathrin coated vesicles along the apical membrane (Shurety, Bright & Luzio, 1996). The molecular mechanisms underlying the actin-dependent fission of membrane vesicles are unclear. Different forms of molecular machinery for mechanical “pinching” of vesicles, including dynamin, actin polymerization (Arp2/3 complex, WASP and Cdc42) and myosin VI all reside in this region and could contribute to the pinching off of vesicles from the plasma membrane. While the dynamin GTPase is clearly involved in the vesicle fission process, it remains unresolved if dynamin functions as a mechanical “pinchase” or coordinates vesicle pinching through the recruitment and organization of the actin cytoskeleton. Dynamin does recruit actin filaments to the neck region. This recruitment, combined with the Arp2/3 nucleation machinery, may be utilized to drive the elongation or apposition of the membranes in the neck region of the budded vesicle. Similarly, myosin VI could play a similar role. In contrast to most other myosins, myosin VI is a “minus” end directed motor. Filaments coursing down from microvilli into the terminal web are polarized with their “plus” end anchored at the plasma membrane. Thus, myosin VI would attach and drive the intracellular movement of CME vesicles.
Dissolution of the Cortical Actin Similar to the need of exocytic vesicles to traverse the cortical actin barrier to gain access to the plasma membrane, endocytic sites are likely to require mechanisms to either remove the underlying cortical actin (e.g. gelsolin recruitment and activation) or inhibit its formation over the site of endocytosis.
Internalization of Endocytic Vesicles Once detached from the membrane, vesicles are actively trafficked away from the plasma membrane by the actin cytoskeleton. Myosin-dependent trafficking and actin polymerization both may play a role in the intracellular movement of vesicles. Myosin VI, a “minus” end directed motor is found on both attached and detached endocytic vesicles and could carry the vesicles towards the cell interior along polarized actin filaments (Buss, Luzio & Kendrick-Jones, 2001). Recent
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observations of actin tails behind endocytic vesicles indicates cells harnass the actin polymerization machinery that is used to drive cell membranes away from the cell (e.g. cell motility, phagocytosis) to move endocytic vesicles into the cell interior. Various microscopic techniques have now identified actin tails emanating from endocytic vesicles and actin nucleating proteins, including Arp2/3 and N-WASP, labeling the endocytic vesicle surface (Tauton et al., 2000).
Rac Directs Vesicle Recycling Once driven into the cell interior, CME vesicles lose their clathrin coat and form into early endosomes. At a crossroad, these endosomes either sort into the lysosomal degradation pathway or return to the membrane via the recycling pathway. Arf6 and Rac are small GTPases that are important mediators of actin cytoskeleton dynamics. Both Arf6 and Rac contribute to faithful vesicular recycling in epithelial cells. Likewise, the faithful recycling of 2 -adrenergic receptor requires its interaction with EBP50 binding partner (Cao et al., 1999). Alternatively, myosin Ia associates with endosomal fractions and overexpression of truncated, non-functional myosin Ia impairs trafficking to lysosomes (Raposo et al., 1999).
ACTIN AND THE ETIOLOGY OF SPECIFIC LIVER DISEASES Recent studies of cellular mechanisms of diseases have discovered direct links to alterations in the actin cytoskeleton. While abnormalities within the actin protein itself would most likely be lethal, alterations in the broader actin cytoskeleton could have non-lethal yet deleterious effects on cellular function and have profound effects at the tissue and organ level. The next section will describe disease processes in which alterations in the actin cytoskeleton promotes specific diseases. These processes have implications in a broad range of liver diseases that include transplantation viability, cholestatic liver disease, genetic diseases of the liver and hepatitis B infection.
Ischemia Results in Profound Structural and Functional Alterations Ischemia, which means insufficient blood flow, has severe effects on organ function and occurs both in disease states (e.g. coronary artery disease) and clinical protocols
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(i.e. surgery and organ transplantation). Metabolic inhibition of isolated or cultured epithelial cells mimics the ATP depletion that occurs in ischemic tissues and is an effective model to study the resultant cellular and molecular alterations. Foremost among them, ischemia is perhaps the clearest example of a disease process in which the actin cytoskeleton is profoundly disrupted and the genesis of the alterations is understood at a molecular level (Doctor & Fouassier, 2002). In ATP depleted cells, there is a change in the amount and distribution of Factin. Paradoxically, despite a profound decrease in the cellular ATP/ADP ratio, there is a relative increase in the amount of F-actin. This is likely attributed to the dysregulation of actin binding proteins and resultant increase in available monomers for polymerization. Normally concentrated at the cell periphery, Factin in ischemic or ATP-depleted cells redistributes from the cell periphery to a cytoplasmic/perinuclear localization. These changes in F-actin coincide with significant structural and functional alterations. Along the apical membrane of ischemic or ATP-depleted epithelial cells, the most striking change occurs within the microvilli. In ATP-depleted cholangiocytes, microvilli initially elongate and then the microvillar density plummets to near zero. Multiple pathways appear to contribute to this disruption. First, the ATP-depleted state results in net dephosphorylation of ezrin. This dephosphorylation results in its detachment from the microvillar actin filaments and redistribution of ezrin into the cell interior. Second, microvillar Ca2+ concentrations are elevated during ischemia and ATP-depletion which, in turn, activate the filament severing capabilities of the villin bundling protein. Third, the actin severing and depolymerizing activities of ADF/cofilin are activated by dephosphorylation and low pH. These conditions prevail in metabolically inhibited epithelial cells. Further, in ischemic proximal tubule cells, ADF/cofilin becomes concentrated at the apical domain. These structural alterations in microvilli are paralleled by functional deficits at the apical membrane. In ATP-depleted cholangiocytes, the concentration of Na+ glucose contransporter 1 and activity of -glutamyl transpeptidase in the apical membrane are significantly decreased. In vivo studies of ischemic cholangiocytes also show significant internalization of the apical membrane protein leucine aminopeptidase. These changes suggest that membrane protein internalization is a result of ischemia-induced alterations in the actin cytoskeleton and its interaction with the plasma membrane.
Relationship Between Biliary Flow and Apical Structure and Function Non-ischemic disruption of the apical actin cytoskeleton also results in significant epithelial disease. Enterocytes from individuals with microvillus inclusion disease have decreased levels of actin, a loss of apical microvilli and an increase in apical
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cytoplasmic vesicles containing apically targeted proteins. Microvillus inclusion disease is an autosomal recessive disorder characterized by refractory diarrhea from birth and is usually fatal. Similarly, experimental models of cholestatic liver disease display marked alterations in the organization of the canalicular domain in hepatocytes. Hepatocytes from obstructive cholestatic livers have significantly decreased numbers of motile cytoplasmic vesicles and large intracellular pseudocanaliculi (Torok, Larusso & McNiven, 2001). In general, these hepatocytes have impaired transcytotic trafficking and a loss of domain specificity of canalicular membrane proteins (Steiger, Meier & Landmann, 1994). The psuedocanaliculi are associated with a cortical actin meshwork, have luminal microvilli and retain the ability to actively transport charged molecules into their lumen. Modification of the pericanalicular actin cytoskeleton can also induce intrahepatic cholestasis. Treatment of rats with phalloidin, a toxin that binds to the lateral aspect of actin filaments and disrupts their dynamics, induces an accumulation of actin microfilaments around bile canaliculi, decreases contractility of bile canaliculi and diminishes intrahepatic bile flow (Stieger & Landmann, 1996). This loss in bile flow is accompanied by a redistribution of Mrp2 and other apically targeted proteins from the canalicular membrane to intracellular microsomal structures (Rost, Kartenbeck & Keppler, 1999). These experimental models highlight the importance of the actin cytoskeleton in the physiologic secretion of bile and demonstrate the potential role of the actin cytoskeleton in the pathogenesis of cholestatic liver diseases.
Genetic Diseases have Direct Links with the Actin Cytoskeleton With actin and its numerous associated proteins being vital to so many cellular functions, it is not surprising to find genetic alterations in actin associated proteins that result in disease. Mutations in proteins of the band 4.1 superfamily provide a glimpse into the genetic diseases of the actin cytoskeleton that range from cancer to cystic fibrosis. Merlin is an ERM-like protein of the band 4.1 superfamily that colocalizes with the actin cytoskeleton and shares ∼45% sequence identity with the ERM proteins. Under normal conditions, merlin is a tumor suppressor protein and negative growth regulator. Mutations in the merlin gene result in the autosomal recessive disorder neurofibromatosis 2 that leads to an increased incidence of nervous system tumors including meningiomas, schwannomas, and astrocytomas (Gutmann, 1997). Cystic fibrosis is a lethal autosomal recessive disorder resulting from mutations in the genes encoding CFTR and is manifested as impaired epithelial chloride secretion. In the normal liver, CFTR is expressed in cholangiocytes where it plays a significant role in hormonally regulated alkaline fluid secretion from
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the intrahepatic bile duct. As was shown in Fig. 6, CFTR is an ABC transport protein that is sequestered into an actin-anchored complex that contributes to its polarized distribution and cAMP-regulated activity. Normally, CFTR is linked to this complex through an interaction of its C-terminal tail with the PDZ domain of EBP50. Interestingly, while generally of a milder phenotype, ∼10% of CFTR mutations result in deletion of this C-terminal region (see Fig. 7). While recently challenged (Gutmann, 1997), experimental truncation of the C-terminal region resulted in a loss of CFTR polarity (Moyer et al., 1999). Further, disruption of the cytoskeletal association of CFTR by dominant-negative transfection of the PDZ1 domain of EBP50 results in an ablation of cAMP-mediated Cl- secretion in cholangiocytes (Fouassier et al., 2001). While the relative contributions of membrane trafficking, protein retention and regulated channel activation require additional investigation, the association of CFTR with the actin cytoskeleton is instrumental in its physiologic function and contributes significantly to the pathology incystic fibrosis. In parallel with cystic fibrosis and mutations in CFTR, mutations in Mrp2 results in the autosomal recessive disorder Dubin-Johnson Syndrome (DJS). Mrp2 is an organic anion transporter that secretes conjugated bilirubin from hepatocytes into the bile. DJS is characterized by chronic conjugated hyperbilirubinemia. In hepatocytes, Mrp2 associates with PDZ proteins, including EBP50, through its C-terminal tail. EBP50 can then tether Mrp2 to the actin cytoskeleton by interacting with the actin-binding AKAP protein radixin (Rdx) that is concentrated at the canalicular membrane in hepatocytes (see Fig. 6). The importance of this complex in regulated Mrp2 function is highlighted in radixin knock-out studies (Kikuchi et al., 2002). In Rdx-/-mice, serum concentrations of conjugated bilirubin increase soon after birth and mild liver injury develops. This pattern of congenital hyperbilirubinemia parallels that seen in humans with DJS. While the cellular mechanisms underlying the loss of Mrp2 function continue to be studied, a decrease in Mrp2 concentration in the bile cannalicular membrane indicates the actin-associated radixin complex contributes to the canalicular localization of Mrp2 (see Fig. 7).
Infectious Pathogens Hijack the Host Actin Cytoskeleton A variety of bacterial and viral pathogens “hijack” the host actin cytoskeleton to promote cellular uptake, intracellular motility and infection of adjacent cells. Their utilization of the host’s actin cytoskeleton is essential to their virulence and pathogenicity. A striking example of “actin hijacking” is found in Listeria monocytogenes. Listeria expresses the protein ActA at an extracellular pole. ActA induces actin filament nucleation and polymerization through the recruitment and
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Fig. 7. Pathogenic Effects Following Disruption of Actin-Associated ABC Transporter Complexes. Note: Cystic fibrosis and DubinJohnson Syndrome are related to mutations in CFTR and Mrp2, respectively. (A) Ten percent of CFTR mutations are traced to C-terminus truncations, the site of EBP50 binding. Experimental truncations or dominant-negative dissociation of CFTR from the actin-associated complex results in a loss of cAMP-mediated CFTR Cl− secretion. (B). Similarly, radixin knock-out mice lose their apical Mrp2 distribution and develop Dubin-Johnson Syndrome-like conjugated hyperbilirubinemia.
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activation of the host’s Arp2/3 complex. Similar to the extension of the plasma membrane during phagocytosis or driving the intracellular movement of endocytic vesicles, this polymerization propels the bacteria through the host’s cytoplasm. When stained for F-actin, infected cells show a “comet tail” of F-actin behind the Listeria. This mechanism is not unique to Listeria. Shigella flexneri bacteria and Vaccinia virus also recruit and utilize actin polymerization to drive their motility within host cells. Helicobacter pylori, an invasive human pathogen that causes inflammation of the gastric epithelial mucosa, induces effacement of microvilli and reorganization of the actin cytoskeleton and adjacent cellular proteins directly below the site of bacterial attachment (Segal, Falkow & Tomkins, 1996). Several H. pylori virulence factors, including vacuolating cytotoxin, VacA, and a cytotoxin-associated antigen, CagA, have been implicated in this reorganization. Enteropathogenic Escherichia coli (EPEC) induces a similar loss of brush border microvilli and reorganization of the host’s actin cytoskeleton directly beneath the extracellular bacterium. The pathogenicity of enterotoxigenic Bacteroides fragilis, a noninvasive bacterium associated with diarrheal disease, is related to toxin-mediated alterations of the apical F-actin structure at the level of the tight junctions of cultured human intestinal epithelial cells. This disruption of the actin cytoskeleton diminishes the paracellular permeability barrier function and induces the diarrheal disease that is associated with B. fragilis. Chronic Hepatitis B virus (HBV) infection and progression to cirrhosis are well-recognized risk factors for the development of hepatocellular carcinoma. HBV infection modifies the actin cytoskeleton and increases the metastasis of infected hepatocytes. Hepatitis B virus X protein (HBx), a 17-kD protein encoded by the HBV genome, induces rearrangement of the actin cytoskeleton (Lara-Pezzi et al., 2001). CD44, the major receptor for hyaluronin, is a plasma membrane glycoprotein involved in the regulation of tumor cell growth and metastasis (Sherman et al., 1994). Through its cytoplasmic tail, CD44 associates with ERM proteins. Moesin then links it to the actin cytoskeleton. F-actin, moesin and CD44 are each clustered at the HBx-induced pseudopodial tips. Though the mechanism remains unclear, ERM proteins are associated with increased migration or metastatic potential in a wide variety of cell types and is likely responsible for enhancing metastasis in hepatocellular carcinoma cells.
CONCLUDING REMARKS As touched on in the first section of the chapter, the last decade has seen explosive growth in our molecular understanding of the proteins and pathways that govern
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the biochemistry of actin filaments. Cell biologists have made enormous strides in applying this molecular understanding to begin unraveling the mysteries underlying the role of the actin cytoskeleton in cellular functions. Some of these advances were highlighted in the second section of the chapter that described the multiplicity of roles the actin cytoskeleton plays in directing events within the apical domain of liver epithelial cells. As covered in the third section of the chapter, understanding the physiology of the actin cytoskeleton has already spilled over into the study of liver disease pathogenesis. The following decade will undoubtedly see enormous continued progress in these three areas and will hopefully lead to the development of specific treatments for actin-related diseases.
REFERENCES Bamburg, J. (1999). Proteins of the ADF/cofilin family: Essential regulators of actin dynamics. Ann. Rev. Cell. Dev. Biol., 15, 185–230. Buss, F., Luzio, J., & Kendrick-Jones, J. (2001). Myosin VI, a new force in clathrin mediated endocytosis. FEBS Letters, 508, 295–299. Cao, T. T., Deacon, H. W., Reczek, D., Bretscher, A., & Von Zastrow, M. (1999). A kinase-regulated PDZ-domain interaction controls endocytic sorting of the b2-adrenergic receptor. Nature, 401, 286–290. Cunningham, C. C., Gorlin, J. B., Kwiatkowski, D. J., Hartwig, J. H., Janmey, P. A. et al. (1992). Actin-binding protein requirement for cortical stability and efficient locomotion. Science, 255, 325–327. Doctor, R., Dahl, R., Fouassier, K., Kilic, G., & Fitz, J. (2002). Cholangiocytes exhibit dynamic, actin-dependent apical membrane turnover. Am. J. Physiol., 282, C1042–1053. Doctor, R., & Fouassier, L. (2002). Emerging roles of the actin cytoskeleton in cholangiocyte function and disease. Sem. Liver Dis., 22, 263–275. Edidin, M., Kuo, S. C., & Sheetz, M. P. (1991). Lateral movements of membrane glycoproteins restricted by dynamic cytoplasmic barriers. Science, 254, 1379–1382. Fouassier, L., Duan, C., Sutherland, E., Simon, F., Yun, C. et al. (2001). ERM binding phosphoprotein 50 is expressed at the apical membrane of rat liver epithelia. Hepatology, 33, 166–176. Gottlieb, T. A., Ivanov, I. E., Adesnik, M., & Sabatini, D. D. (1993). Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells. J. Cell. Biology, 120, 695–710. Gutmann, D. (1997). Molecular insights into neurofibromatosis 2. Neurobiol. Dis., 3, 247–261. Hall, R., Ostegaard, L., Premont, R., Blitzer, J., Rahman, N. et al. (1998). A C-terminal motif found in the b2-adrenergic receptor, P2Y1 receptor and CFTR determines binding to the Na+ H+ exchanger regulatory factor family of PDZ proteins. Proc. Natl. Acad. Sci. USA, 95, 8496–8501. Hammerton, R. W., Krzeminski, K. A., Mays, R. W., Ryan, T. A., Wollner, D. A. et al. (1991). Mechanism for regulating cell surface distribution of Na+ -K+ -ATPase in polarized epithelial cells (see comments). Science, 254, 847–850.
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Hegedus, T., Sessler, T., Scott, R., Thelin, W., Bakos, E. et al. (2003). C-terminal phosphorylation of Mrp2 modulates its interaction with PDZ proteins. Biochem. Biophys. Res. Comm., 320, 454–461. Huang, J., Brady, S., Richards, B., Stenoien, D., Resau, J. et al. (1999). Direct interaction of microtubuleand actin-based transport motors. Nature, 397, 267–270. Jackman, M. R., Shurety, W., Ellis, J. A., & Luzio, J. P. (1994). Inhibition of apical but not basolateral endocytosis of ricin and folate in Caco-2 cells by cytochalasin D. J. Cell. Science, 107, 2547–2556. Kikuchi, S., Hata, M., Fukumoto, K., Yamane, Y., Matsui, T. et al. (2002). Radixin deficiency causes conjugated hyperbilirubinemia with loss of Mrp2 from bile canalicular membranes. Nat. Genetics, 31, 320–325. Kilic, G., Doctor, R., & Fitz, J. (2001). Insulin stimulates membrane conductance in a liver cell line: Evidence for insertion of ion channels through a PI3 kinase-dependent mechanism. J. Biol. Chem., 276, 26762–26768. Lara-Pezzi, E., Serrador, A., Montoya, M., Zamora, D., Yanez-Mo, M. et al. (2001). The hepatitis B virus X protein induces a migratory phenotype in a CD44-dependent manner. Hepatology, 33, 1270–1281. McGough, A., Pope, B., Chiu, W., & Weeds, A. (1997). Cofilin changes the twist of F-actin: Implications for actin filament dynamics and cellular function. J. Cell. Biol., 138, 771–781. Merrifield, C., Feldman, M., Wan, L., & Almer, W. (2002). Imaging actin and dynamin recruitment during invagination of single clathrin coated pits. Nat. Cell. Biol., 4, 691–698. Moyer, B. D., Denton, J., Karlson, K. H., Reynolds, D., & Wang, S. et al. (1999). A PDZ-interacting domain in CFTR is an apical membrane polarization signal. J. Clin. Invest., 1353–1361. Muallem, S., Kwiatkowska, K., Xu, X., & Yin, H. (1995). Actin filament disasssembly is a sufficient trigger for exocytosis in non-excitable cells. J. Cell. Biol., 128, 589–598. Mullins, R., & Pollard, T. (1999). Structure and function of the Arp2/3 complex. Curr. Opin. Struct. Biol., 9, 244–249. Raposo, G., Cordonnier, M., Tenza, D., Menichi, B., Durbach, A. et al. (1999). Association of myosin Ia with endosomes and lysosomes in mammalian cells. Mol. Biol. Cell., 10, 1477–1494. Rost, D., Kartenbeck, J., & Keppler, D. (1999). Changes in the localization of the rat canalicular conjugate export pump Mrp2 in phalloidin-induced cholestasis. Hepatology, 29, 814–821. Segal, E., Falkow, S., & Tomkins, L. (1996). H. pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation. Proc. Natl. Acad. Sci. USA, 93, 1259–1264. Sheng, M., & Sala, C. (2001). PDZ domains and the organization of supramolecular complexes. Ann. Rev. Neurosci., 24, 1–29. Sherman, L., Sleeman, J., Herrlich, P., & Ponta, H. (1994). Hyaluronate receptors: Key players in growth, differentiation, migration and tumor progression. Curr. Opin. Cell. Biol., 6, 726–733. Short, D., Trotter, K., Reczek, D., Kreda, S., Bretscher, A. et al. (1998). An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J. Biol. Chem., 273, 19797–19801. Shurety, W., Bright, N., & Luzio, J. (1996). The effects of cytochalasin D and PMA on the apical endocytosis of ricin in polarized Caco-2 cells. J. Cell. Sci., 109, 2927–2935. Steiger, B., Meier, P., & Landmann, L. (1994). Effects of obstructive cholestasis on membrane traffic and domain specific expression of plasma membrane proteins in rat liver parenchymal cells. Hepatology, 20, 201–213. Stieger, B., & Landmann, L. (1996). Effects of cholestasis on membrane flow and surface polarity in hepatocytes. J. Hepatol., 24, 128–134.
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Tauton, J., Rowning, B., Coughlin, M., Wu, M., Moon, R. et al. (2000). Actin dependent propulsion of endosomes and lysosomes by recruiting N-WASP. J. Cell. Biol., 148, 519–530. Torok, N., Larusso, E., & McNiven, M. (2001). Alterations in vesicle transport and cell polarity in rat hepatocytes subjected to mechanical or chemical cholestasis. Gastroenterology, 121, 1176– 1184. Tuma, P., Nyasae, N., & Hubbard, A. (2002). Nonpolarized cells selectively sort apical proteins from cell surface to a novel compartment but lack apical retention mechanisms. Mol. Biol. Cell., 13, 3400–3415. Vartiainen, M., Mustonen, T., Mattila, P., Ojala, P., Theleff, I. et al. (2002). The three mouse ADF/cofilins evolved to fulfill cell-type specific requirements for actin dynamics. Mol. Biol. Cell., 13, 183– 194. Yao, X., Chaponnier, C., Gabbiani, G., & Forte, J. (1995). Polarized distribution of actin isoforms in gastric parietal cells. Mol. Biol. Cell., 6, 541–557.
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4.
MECHANISMS OF BILE FORMATION AND CHOLESTASIS
M. Sawkat Anwer INTRODUCTION The liver, the largest gland in the body, plays a central role in the metabolism and excretion of endogenous and exogenous solutes. Because of its location between the digestive tract and the general circulation, and its ability to efficiently extract a wide variety of compounds, the liver is ideally suited to remove solutes absorbed from the intestine. Bile, the exocrine secretion of the liver, provides a route of excretion for many of these substances. Bile also assists in digestion and absorption of fat by providing bile acids and phospholipids to the duodenum, and plays an immunological role by delivering IgA to the intestine. Bile being an aqueous solution (97.5% water) is more suitable for the excretion of water soluble compounds. However, the presence of micelle forming bile acids above their critical micellar concentration (CMC) allows solubilization of lipids in bile. Thus, water-soluble as well as lipid-soluble compounds are excreted via bile. Only solutes that are excreted into the bile directly contribute to bile formation and include compounds like bile acids, cholesterol, phospholipid, bilirubin, proteins, inorganic ions, glutathione, drugs and toxins. Since biliary solutes are, in most part, derived from sinusoidal blood, transhepatic transport of solutes is intimately related to bile formation. Hepatocytes, the major cell type in the liver, and bile ductular cells are responsible for the transhepatic transport of solutes and bile formation.
The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 81–118 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15004-6
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The term cholestasis was originally coined by morphologists to describe the presence of bile plugs. Recently this definition has been modified to include any condition that results in decreased bile formation with consequent serum accumulation of solutes destined for bile, i.e. when transhepatic solute transports are compromised. Cholestasis is characterized by morphological, biochemical, physiological and clinical changes that include alterations of the cytoskeletal and intracellular organelles, and elevation of canalicular marker enzymes (like alkaline phosphatase and ␥-glutamyl transferase) and solutes destined for bile (like bilirubin, bile acids) in the serum. If the delivery of bile to the intestine is severely compromised, impaired intestinal lipid digestion leads to the development of steatorrhea. Additionally, accumulated bile constituents may produce membrane damage resulting in further impairment of the liver’s ability to secrete bile. The accumulation of bile constituents in blood may also damage extra-hepatic tissues. This vicious cycle of cell injury makes it difficult to distinguish the primary mechanism from the secondary changes. Nevertheless, studies with various experimental models of cholestasis have allowed us to propose certain mechanisms. This chapter summarizes our present understanding of cellular mechanisms involved in hepatic solute transport, bile formation and cholestasis. More details can be found in recent reviews cited in appropriate sections of this chapter.
HEPATIC SOLUTE TRANSPORT Transport of a solute through hepatocytes involves uptake at the sinusoidal membrane, intracellular transport and metabolism, and excretion across the canalicular membrane. This is accomplished with the help of a number of transport mechanisms located at sinusoidal as well as canalicular membranes (see Fig. 1). Solutes can also bypass hepatocytes and enter bile passively via tight junctions along the paracellular pathway. Solutes that are transported by hepatocytes can be divided into the following groups: (a) inorganic ions; (b) organic anions; (c) lipids; (d) organic cations; (e) organic neutral compounds; and (f) macromolecules.
Inorganic Ions Inorganic ions are excreted in bile and are the major determinant of bile osmolarity and hence, the osmotic flow of water. A number of transport mechanisms have been identified at sinusoidal as well as canalicular membranes. Those located at the sinusoidal membrane include Na+ -K+ exchange, Na+ -H+ exchange, Na+ -HCO3 − cotransport and SO4 2+ -OH− exchange (Anwer, 1993;
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Fig. 1. Transporters participating in bile formation: hepatic uptake of bile acid (BA), organic anions (OA) and organic cations (OC) is mediated primarily by Na+ /taurocholate cotransporting polypeptide (NTCP), the family of organic anion transporting proteins (OATPs) and organic cation transporters (OCTs), respectively. The Na+ -H+ exchanger (NHE) and Na+ -HCO− 3 cotransporter (NBC) at the sinusoidal membrane of hepatocytes and basolateral membrane of cholangiocytes are involved in intracellular pH regulation and HCO− 3 excretion. Multi-drug resistance proteins (MRP3 and MRP1) mediate sinusoidal efflux of organic anions, including toxic bile acids, while MRP2 and BSEP (Bile salt export pump) mediate canalicular excretion of conjugated organic anions and bile acids, respectively. MDR1 and MDR2 (multidrug resistance gene products) are involved in biliary excretion of organic cations and phospholipids, respectively. Chloride/bicarbonate exchange is mediated by an anion exchanger (AE) at canalicular as well as apical membrane of cholangiocytes. Cystic fibrosis transmembrane conductance regulator (CFTR) act as chloride channels, and reabsorption of conjugated bile acid from the biliary tree is mediated via an Na+ -dependent bile acid transporter (IBAT).
Kanno et al., 2001; Trauner et al., 1999). 3Na+ /2K+ exchange, mediated by Na+ -K+ -ATPase, is an electrogenic primary active (direct ATP requirement) transport process, and is responsible for maintaining the transmembrane potential difference and concentration gradients of Na+ (extracellular > 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 (<50%), are transported by a Na+ coupled cotransport process (Na+ /bile acid cotransporter). Unconjugated bile acids and other organic anions are taken by Na+ -independent transport processes. Fatty acids and certain amino acids are also transported by Na+ -coupled transporters, which are different from the Na+ /bile acid cotransporter. The Na+ /bile acid cotransporter has been cloned from rat (ntcp) and human (NTCP). There is a 77% amino acid identity between ntcp and NTCP, and NTCP consists of 349 amino acids, i.e. 13 amino acids less at the carboxy terminal
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compared to Ntcp (Meier & Stieger, 2000). Recent studies suggest that ntcp/NTCP is the major transporter of conjugated bile acids uptake into hepatocytes. The gene encoding for ntcp and NTCP is located on rat chromosome 6q24 and human chromosome 14q241.–24.2, respectively (Meier & Stieger, 2000). Whether there is another transporter mediating Na+ /bile acid cotransport remains unclear (Wolkoff & Cohen, 2003). This uptake process is electrogenic which transports two Na+ with each bile salt. Hepatic uptake of bile acid is affected by the length and charge of the side chain as well as the number of hydroxyl groups. Na+ dependent cholate uptake is mediated by the Na+ /bile acid cotransporter at lower concentrations (<100 m) and may involve tertiary active transport at higher concentrations (Anwer, 1993). A number of organic anions are taken up by hepatocytes via Na+ -independent transport mechanisms (Anwer, 1991; Kuipers & Vonk, Kullak-Ublick et al., 2000). These include unconjugated bile acids, bilirubin, thyroid hormones, cholephilic dyes (BSP, ICG), drugs (rifamycin). The driving force for this transport appears to be the high intracellular GSH concentration (Wolkoff & Cohen, 2003). Thus, this transporter may be an organic anion/GSH exchanger. Studies aimed at cloning the Na+ -independent transporter resulted in the discovery of a family of transporters (Hagenbuch & Meier, 2003) known as organic anion transporting polypeptides both in rodents (oatps) and humans (OATPs). The oatps/OATPs are members of a growing gene family expressed in various tissues and encode for transporters with overlapping as well as distinct substrate specificity. In rat liver, for example, Na+ -independent uptake of organic anions are mediated by oatp1 and oatp 2. Oatp1 has been found to transport a variety of substrates, including bile acids, steroid hormones, and even some organic cations (Meier & Stieger, 2000; Meng et al., 2002). By contrast, oatp2 is a high affinity digoxin transporter which also transports bile acids and estrogen conjugates. The members of the OATP-1A and 1B subfamilies are involved in drug transport while OATP-C is involved in bilirubin transport. Although oatps are able to transport bile acids, their contribution to overall bile acid uptake under physiological conditions remains unclear. Recent studies suggest that there are basolateral transporters involved in efflux of organic anions from hepatocytes to blood. These are multidrug resistance associated proteins, MRP1 and MRP3 (Kullak-Ublick et al., 2000; Trauner et al., 1999). Although their expression under physiological conditions is low, they may play an important role in cholestasis and proliferating cells (M¨uller & Jansen, 1998). Organic anions can traverse biological membranes by passive non-ionic diffusion, and uptake by this mechanism may be significant for certain di- and monohydroxy unconjugated bile acids. Although such a mechanism is not concentrative, intracellular conjugation and binding followed by canalicular excretion can result in substantial biliary excretion of these bile acids. Similarly, other organic
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anions are also concentrated in bile without a concentrative uptake across the sinusoidal membrane. Although special transport mechanisms at the sinusoidal membrane are not necessary, active sinusoidal transport allows for efficient transhepatic transport. Organic anions usually undergo metabolic alterations before excretion across the canalicular membrane. Conjugated bile acids are excreted unchanged while unconjugated forms are usually hydroxylated and/or conjugated before excretion into bile. Conjugation is not necessary for hydrophilic bile acids, but appears to be essential for biliary excretion of lipophilic bile acids (Anwer, 1993). Unconjugated bile acids are primarily conjugated with the amino acids taurine or glycine. Certain bile acids, like lithocholate and ursodeoxycholate, are also conjugated with sulfate and glucuronic acid. Some of these conjugates can reflux back into the circulation (mediated via MRP1/MRP3) to be excreted by the kidneys specially under cholestatic conditions (M¨uller & Jansen, 1998). Bilirubin is primarily conjugated with glucuronic acid and excreted as mono- and diglucuronide. It appears that the conjugation step determines the maximal biliary excretion rate of bilirubin, and inadequate hepatic conjugation may explain the fasting hyperbilirubinemia seen in Bolivian squirrel monkeys. Mechanisms by which organic anions are transported from the sinusoidal to the canalicular pole are unclear (Anwer, 1993). The liver cytosol contains a number of organic anion binding proteins, which include Y protein with glutathione-Stransferase activity, fatty acid binding protein or Z protein and Y1 protein with 3␣-hydroxy dehydrogenase and steroid sulfotransferase activity. However, their role in intracellular transport has not been clearly established. It appears that the 33 kD Y1 bile acid binder may play a role in minimizing the reflux of bile acids into the circulation, and redistribution in other intracellular compartments. Since some of these proteins also possess enzymatic activities, it is likely that they facilitate the metabolism of organic anions. Some studies indicate that microtubule-dependent vesicular pathways may be involved in intracellular transport (Anwer, 1993; Crawford, 1996). This hypothesis is based primarily on studies with colchicine, which depolymerizes microtubules. Colchicine does not affect basal bile flow and bile acid excretion. However, under conditions of increased hepatic bile acid flux, colchicine decreases bile flow and excretion of bile acid, lipid and bilirubin diglucuronide. It is proposed that intracellular transport of a part of bilirubin diglucuronide may involve cotransport with bile acid via a microtubule dependent vesicular pathway. Such a mechanism may also explain taurocholate-induced transcellular transport of horseradish peroxidase and fluid phase markers. The contribution of this pathway to overall biliary excretion under basal conditions is probably small, and may increase with increasing bile acid flux through hepatocytes. However, in view
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of the recent findings that microtubules are involved in the translocation of canalicular transporters, the results obtained with colchicines may also reflect the inability of the transporter to translocate to the canalicular membranes (Anwer, 1998; Crawford, 1996). Canalicular excretion is considered to be the rate limiting step in the overall transcellular transport of most organic anions. Vectorial transport from blood to bile is facilitated by more than one transport mechanism present at the canalicular membrane. It is thought that these transport systems are responsible, at least in part, for 100–1000 fold higher concentrations in bile compared to blood. Our present understanding of transport systems responsible for canalicular excretion of organic anions is greatly facilitated by studies in patients with the Dubin-Johnson syndrome, and with animal models such as mutant corridale sheep with chronic conjugated hyperbilirubinemia and mutant rats with organic anion excretory defects (Jansen & Oude Elferink, 1988). These mutant rats include Wistar GY rats, TR− rats and a mutant Sprague-Dawley strain. With the introduction of expression cloning a number of transporter have already been cloned and we are beginning to understand the molecular basis of solute transport across the canalicular membrane. Most of these transporters belong to the superfamily of ABC (ATP-binding cassette) transporter requiring direct ATP hydrolysis for the transport function (Kullak-Ublick et al., 2000; Meier & Stieger, 2000; Trauner et al., 1999). Two such transporters, namely BSEP (also known as sister of pglycoprotein) and MRP2, are involved in organic anion excretion. Bile acids are transported by the bile salt export pump, BSEP. MRP2 (mrp2 in rodents) is also known as the canalicular multi-specific organic anion transporter (cMOAT). As the name implies, it transports a wide variety of amphipathic organic anions, such as leukotriene C4 and substrates conjugated with glutathione, glucuronic acid and sulfate.
Lipids Biliary lipids consisting mainly of phospholipids and cholesterol are the second most, after bile acids, important organic constituents of bile. While it has long been known that bile acids stimulate biliary excretion of phosphilpids and cholesterols, the canalicular transporter for phospholipids has only recently been identified. Studies with mdr2 knockout mice revealed that the mdr2 gene product is essential for biliary lipid, but not bile acid secretion (Elferink & Groen, 2002). The knockout mice do not secrete either phospholpid or cholesterol. Further studies demonstrated that the murine mdr2, as well as the human orthologue MDR3 are responsible for phosphatidylcholine (PC) transport. Elferink and
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Groen (2002) suggested that the transporter functions as a flippase by translocating its substrate from the inner to the outer leaflet of the canalicular membrane and thereby creating a PC-rich domain. By destabilizing the PC-rich domain, biliary excreted bile acids facilitate vesiculation of the PC-rich membrane bilayers; this is followed by release of lipid-rich vesicles. Morpholigical evidence in support of such a hypothesis is available. Whether cholesterol excretion is also mediated by a canalicular transporter is not yet known. The possibility that ABCA1, an ABC transporter related to Tangier disease, may be involved in cholesterol transport could not be substantiated in Abca1 knockout mice (Groen et al., 2001).
Organic Cations Organic cations are mostly aromatic amines and are comprised of a number of important drug groups like cholinergics, local anesthetics, antibiotics, antineoplastics and antihelmintics, as well as endogenous solutes like choline, thiamine and nicotinamide. Some of these cations are taken up by hepatocytes and excreted in the bile. Based on kinetic and inhibition studies in perfused livers and isolated hepatocytes, it has been suggested that at least two Na+ -independent transport mechanisms (Type I and Type II) are involved in hepatic uptake of exogenous organic cations (Groothuis & Meijer, 1996). Monovalent cations, like procainamide ethobromide, appear to be transported by the Type I system, while multivalent cations, like d-tubocurarine, are transported by the Type II system. In the case of cardiac glycosides and taurocholate, they inhibit uptake via Type II, but not Type I. More recent studies suggest that OCT1 (organic cation transporter I) expressed at the basolateral membrane of hepatocytes (Koepsell, 1998) represents the type I transport system found in rats and humans (van Montfoort et al., 2001). Interestingly, oatp1 and oatp2 have also been shown to transport organic cations (van Montfoort et al., 1999). Whereas oatp2 may represent the type II cation transport system in rats, OATP-A may transport substrates of both type I and II cation transport systems (van Montfoort et al., 2001). The mechanisms underlying the canalicular transport of organic cations are poorly understood with the exception of solutes transported by MDR1 and MDR3. Although MDR1 (mdr1a, b in rodents) is involved in transporting cationic and cytotoxic chemotherapeutic agents (vinblastin and doxorubicin), no endogenous substrate for this transporter has yet been identified. In as much phosphatidylcholine, an organic cation, is transported by MDR3 (Elferink & Groen, 2002), it
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can be considered an organic cation transporter. Whether MDR3 can also transport other types of organic cations is not known. While additional transporters may be involved in biliary organic cation secretion, no such transporters have yet been identified. Since tetraethylammonium (TEA) transport into canalicular membrane vesicles is stimulated by the pH-gradient and inhibited by other organic cations, it has been argued that the canalicular transport may involve organic cation/H+ exchange (Moseley et al., 1992). However, for such a mechanism to be effective, the presence of a local pH-gradient needs to be postulated, since data indicates that intracellular and canalicular lumen pH lies in the region of 7.0 and 7.25, respectively. Organic cations also accumulate within lysosomes and mitochondria (Groothuis & Meijer, 1996). The accumulation within endocytotic vesicles may involve cation/H+ exchange and may be facilitated by the intraluminal acidic environment (Van Dyke et al., 1992). Whether or not vesicular accumulation is an intermediate step in biliary excretion of organic cations remains to be investigated.
Organic Neutral Compounds Of the neutral compounds that undergo hepatobiliary transport ouabain, a cardiac glycoside, is thus far the best characterized. Ouabain is taken up by a Na+ independent, saturable, energy-dependent and concentrative process (Anwer, 1993), but the nature of the driving force is not yet known. Hepatic uptake of ouabain is inhibited by other neutral steroids such as cortisol, aldosterone, estradiol and testosterone. Hepatic uptake of cortisol, estrone, estradiol and testosterone is also carrier-mediated (Anwer, 1993). Cortisol uptake, for example, is inhibited competitively by cortisone and corticosterone and non-competitively by dexamethasone, estrone and testosterone. Thus, hepatic uptake of neutral compounds, like that of organic anions, may also involve more than one transporter with overlapping substrate specificity. Recent studies with rat oatps indicate that both oatp1 and oatp2 can transport ouabain, although oatp2 appears to have extended preference for neutral compounds (Meier & Stieger, 2000). Neutral compounds, like endogenous steroid hormones and exogenous cardiac glycosides, are excreted in bile (Anwer, 1993; Bohan & Boyer, 2002). Ouabain is concentrated in bile and is not metabolized. Since steroid hormones are conjugated with glucuronic acid, sulfate or glucosiduronate, it is likely that these conjugates are excreted by canalicular organic anion transport systems, MRP2 (see above); 17-estradiol glucuronide has been shown to be a substrate for mrp2 (Trauner et al., 1999). Whether ouabain is transported by mrp2 remains unknown. Glucose
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appears in human and rat bile when its plasma concentration exceeds 350 and 280 mg/dl, respectively. It is held that passively excreted glucose is completely reabsorbed by a specific transport mechanism under normal physiological conditions (Anwer, 1993).
Macromolecules A number of proteins, appearing in bile, are derived either from plasma or hepatobiliary cells (hepatocytes and bile duct cells). The major fraction of bile proteins (about 80%) appears to originate in plasma. Plasma proteins may gain access to bile via paracellular or transcellular pathways. The paracellular pathway involves sieving across the tight junctions, and is primarily responsible for the biliary excretion of low molecular weight proteins, like horseradish peroxidase, and complex carbohydrates, like inulin. The transcellular pathway involves endocytosis followed by vesicular transcytosis, and finally, exocytosis of vesicular contents into bile. Three forms of endocytosis (non-specific, absorptive and receptor-mediated endocytosis) have been recognized at the sinusoidal membrane (Crawford, 1996). Non-specific or fluid-phase endocytosis involves uptake of a small volume of extracellular fluid containing solutes, resulting from the constitutive process of membrane invagination and internalization. This process is non-discriminatory and responsible for non-saturable hepatic uptake of fluid-phase markers such as inulin, sucrose and horseradish peroxidase. The process of adsorptive endocytosis is similar to fluid-phase endocytosis except that non-specific binding of the ligand to the plasma membrane takes place before endocytosis. Because of this binding, the process is more efficient and faster than fluid-phase endocytosis. Receptor-mediated endocytosis is a quantitatively more important uptake mechanism for macromolecules (Crawford, 1996) which involves binding of the ligand to specific receptors distributed along the plasma membrane, clustering of receptor-ligand complex in coated pit regions, invagination and pinching off of the coated pit followed by dissociation of clathrin (the major protein of the coat) to form coated vesicles inside the cell. Acidification of vesicles (also called endosomes) by H+ -ATPase results in the dissociation of ligands from receptors. The receptor segregates and recycles to the plasma membrane, while the ligand is delivered to lysosomes for degradation or excreted directly into bile by exocytosis. A small fraction of the internalized ligand is returned to the cell surface still bound to the receptor through a pathway referred to as diacytosis or retroendocytosis. Receptor mediated endocytosis is involved in hepatic removal of macromolecules, for example, insulin, epidermal growth factor, asialoglycoproteins and polymeric
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IgA. The polymeric IgA complexed with its receptor (secretory component) bypasses lysosomal degradation, and is excreted into bile. IgA is one of the major bile proteins in rats, mice, rabbits, cats, dogs, and chickens, whereas it is a minor component in guinea pigs and sheep (Engelking & Anwer, 1992). It is an interesting observation that IgA transport to bile in human partly occurs across biliary epithelial cells expressing the secretory component. This may partly occur via interaction with the asialoglycoprotein receptors. Since asialoglycoprotein receptors are unique to the liver, receptor-mediated endocytosis via this receptor has been exploited to target foreign genes to hepatocytes (Smith & Wu, 1999).
GENERAL MECHANISM OF BILE FORMATION Endogenous bile flow rates vary markedly from animal to animal and range from 1.2 to 160 l/min/kg body weight (Cornelius, 1976). Primary bile produced at canaliculi (canalicular bile) is further modified downstream in bile ductules (ductular bile). Canalicular bile flow is conventionally divided into two fractions: bile acid dependent bile flow (BADF) and bile acid independent bile flow (BAIF). In addition, bile formation is regulated by acinar heterogeneity and paracellular permeability. The following is a brief description of mechanisms involved in bile formation in relation to the anatomical site and general solute transport; the details of which can be found in previous reviews (Anwer, 1993; Nathanson & Boyer, 1991). Regulatory aspects of these transporters will be touched upon later.
Bile Acid-Dependent Bile Flow (BADF) The formation of bile depends on biliary excretion of osmotically active solutes, which provide the osmotic force necessary for water movement during bile formation. Thus, the local osmotic gradient created by the secretion of bile acid anion and its accompanying cation (principally Na+ for electroneutrality) provides the driving force for BADF. However, the osmotic effect of bile acid and its counterion alone can not explain the observed BADF of 7–160 l per mol of bile acid excreted in different species. This is because bile acid secretion is associated with an increase in biliary excretion of osmotically active solutes notably Na+ , K+ , Cl− and HCO3 − in excess of that required for electroneutrality. Anwer (1993) proposed that the osmotic effect of secreted bile acid and its counterion provide the primary driving force for water movement into the canaliculi. This results in the translocation of osmotically active permeant solutes by solvent drag and diffusion, which, in turn, determines the movement of total amount of water
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Fig. 2. Proposed mechanism of bile acid-dependent bile formation: Bile acids are secreted into canaliculi as anions followed by Na+ to maintain electroneutrality. The resulting increase in osmotic activity (mostly due to an increase in [Na+ ]) leads to the movement of water and electrolyte into canaliculi by solvent drag and diffusion. As bile moves along the pericanalicular ductules, protonated bile acids are reabsorbed, resulting in an increase in − + biliary [HCO− 3 ]. H and HCO3 are continually generated from CO2 spontaneously and/or via the action of carbonic anhydrase present on the luminal surface. The increase in [HCO− 3] increases osmolarity thus leading to additional bile formation. The loss of bile acid from the monomeric pool due to reabsorption is rapidly replenished from the micellar pool to maintain CMC, resulting in minimal decreases in osmolarity. Reabsorbed bile acids are eventually resecreted following reuptake by hepatocytes as anions and/or in the protonated form to produce more bile. Arrows represent the direction of net movement during formation of bile acid-induced HCO− 3 -rich bile. Modified after Anwer (1993).
associated with bile acid secretion (Fig. 2). This mechanism may explain species differences in BADF if permeability characteristics of the canaliculus vary among different species. A similar mechanism may also contribute to the choleretic effect of exogenous compounds that are concentrated in bile. Certain unconjugated bile acids, such as cholate, ursodeoxycholate and norbile acids, produce higher BADF than their corresponding conjugated bile acids. Since these bile acids, unlike their conjugates, also increase biliary [HCO3 − ], increased biliary HCO3 − excretion is considered to be responsible for this HCO3 − rich hypercholeresis. A mechanism based on “cholehepatic recycling” has been
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postulated to explain bile acid-induced HCO3 − -rich hypercholeresis. According to this hypothesis (see Fig. 2), the increase in [HCO3 − ] is due to reabsorption from bile of unconjugated bile acids in the protonated form, and the hypercholeresis is due to recycling of reabsorbed bile acids. This hypothesis is consistent with a number of observations (Anwer, 1993). A large number of organic compounds that are concentrated in bile also increase canalicular bile formation, at least in part, by mechanisms similar to BADF. In addition to direct osmotic effects, other mechanisms may also be involved. These agents may increase bile formation by increasing biliary [HCO3 − ] like unconjugated bile acids. It has been suggested that procaine and furosemide may increase bile formation, in part, by inhibiting reabsorption of electrolytes and fluid from canaliculi. The nature of such a reabsorptive mechanism, however, remains to be elucidated. Since furosemide increases biliary excretion of glutathione, it is also possible that some of these agents stimulate biliary secretion of osmotically active organic solutes.
Bile Acid-Independent Bile Flow (BAIF) A number of exogenous and endogenous agents (e.g. glucagon, theophylline, barbiturates and SC2644), that are not concentrated enough to act as osmotic choleretics can increase canalicular bile flow without increasing bile acid excretion. BAIF may be due to biliary excretion of a number of different solutes. These can be divided into two broad categories: inorganic and organic solutes. Sodium is the predominant inorganic cation in bile, and Cl− and HCO3 − are the major inorganic anions. Over the years, all three ions have been implicated in the generation of BAIF. However, unequivocal evidence in support of specific transport mechanisms responsible for BAIF is still lacking. It has been argued that active transport of inorganic ions may not provide an osmotic gradient since tight junctions are readily permeable to these ions. On the other hand, based on current knowledge of electrolyte and water transport in other epithelia, it is reasonable to assume that BAIF, at least in part, is dependent on biliary electrolyte excretion. Like other fluid transporting epithelia, hepatocytes have a polarized distribution of inorganic ion transporters (Fig. 1). It is conceivable that coupled operation of these transporters, as proposed for HCO3 excretion by ductutal cells (see Fig. 3) could lead to the vectorial transport of HCO3 − from the sinusoid into the canaliculus. Such a mechanism could account for the HCO3 − -dependent fraction of BAIF, the partial dependency of BAIF on Na+ and Cl− , and cAMPinduced canalicular bile formation. Alternatively, these ion transport mechanisms, by regulating intracellular events, may affect biliary excretion of other osmotically
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Fig. 3. Proposed mechanism of ductular bicarbonate secretion. Vectorial transport of HCO3 − from blood to bile may be accomplished by coupled operation of the Na+ -H+ exchanger (NHE) and the Na+ -HCO3 − cotransporter (NBC) at the basolateral membrane, and the Cl− -HCO3 − exchanger (AE) at the apical membrane. Secretin, acting via adenylate cyclase (AC)/cAMP/PKA, stimulates Cl− efflux by phosphorylating CFTR. Increases in Cl− levels in the lumen activate the Cl− -HCO3 − exchanger, resulting in increased HCO3 − excretion. The efflux of Cl− also stimulates the electrogenic Na+ -HCO3 − cotransporter at the basolateral membrane by depolarizing cholangiocytes. The effect of secretin is inhibited by gastrin, acting via phospholipase C (PLC) and somatostatin (see text for details). Modified after Anwer (1993) and Kanno et al. (2001).
active solutes and thereby, BAIF. In this case, biliary excretion of inorganic ions may mainly be due to solvent drag and diffusion. Biliary excretion of sulfate may involve sulfate/HCO3 − exchange, a pathway potentially shared by other organic anions. It is thus likely that HCO3 − may affect bile formation by facilitating biliary excretion of osmotically active solutes. A number of organic solutes (bilirubin, glutathione, and amino acids) are concentrated in bile in addition to bile acids, and could provide the osmotic gradient for bile formation. With the exception of glutathione, roles of other organic solutes
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in BAIF are unclear. Bilirubin in physiological concentrations does not increase BAIF, although choleresis occurs when infused at high doses. Certain amino acids (aspartate, glutamate, methionine and cysteine) are concentrated in bile, but their role in BAIF has not yet been established. There is growing evidence that biliary excretion of glutathione and its conjugates may be partially responsible for BAIF, and solutes other than glutathione may also be involved in BAIF (Anwer, 1993; Nathanson & Boyer, 1991). It is likely that the combined osmotic activity of a number of biliary excreted organic solutes contributes to BAIF but the contribution of each solute may only be apparent under experimental conditions designed to enhance biliary excretion of that particular solute.
Acinar Heterogeneity in Hepatic Bile Formation Acini represent the structural and functional unit of the liver. Liver microanatomy shows it as being divided into acini, each of which is a three-dimensional microvascular unit in which all hepatocytes are perfused by the same terminal portal venule and hepatic arteriole. The perfusion of this unit is unidirectional from the acinar axis (formed by the terminal hepatic venule, the hepatic arteriole and the bile ductule) to the acinar periphery where two or more terminal hepatic venules usually empty the acinus. The acinus is arbitrarily divided into three zones. Zone 1 represents hepatocytes surrounding the portal venules (periportal hepatocytes) and zone 3 represents hepatocytes surrounding the hepatic venule (perivenous hepatocytes). Hepatocytes in-between these two zones are represented by zone 2 or intermediate zone. There are morphological and functional differences among hepatocytes of the liver acinus; these are discussed in detail elsewhere (Traber et al., 1988). Functional heterogeneity among hepatocytes of different acinar zones has been assessed by producing selective damage to zone 1 (allyl alcohol) and zone 3 (bromobenzene) cells or by comparing results obtained during antegrade and retrograde perfusions (Traber et al., 1988). The uptake of taurocholate and BSP appears similar in all zones since no difference in uptake during antegrade and retrograde perfusion of the liver could be found. However, biliary excretion of taurocholate is faster during antegrade compared to retrograde perfusion, indicating a slower excretion by zone 3 hepatocytes. Moreover, taurocholate accumulates predominantly in zone 1 hepatocytes when a tracer dose is injected. When zone 3 hepatocytes are damaged by bromobenzene or carbon tetrachloride, bile flow decreases by 30–40%. Such results suggest that under physiological conditions, hepatocytes of zone 1 and probably zone 2 are primarily involved in biliary excretion of bile acids, and hence BADF, and hepatocytes of zone 3
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contribute to BAIF. The contribution of zone 3 hepatocytes to BADF, however, increases with increasing bile acid load.
Ductular Bile Formation Canalicular bile composition and volume may be modified by ductular cells. This modification of analicular bile may involve net secretion or absorption of water, and is highly species-dependent (Anwer, 1993; Kanno et al., 2001). Results based on erythritol clearance studies indicate considerable ductular reabsorption in dogs, minimal involvement in rats and pronounced secretion in humans under basal conditions. Early evidence for ductular secretion is based on the choleretic effect of secretin, and secretion in isolated segments of the bile duct. Secretin choleresis, mediated via cAMP, is generally associated with an increase in biliary [HCO3 − ] and pH, and results from ductular secretion of HCO3 − . Recent studies indicate the presence of an Na+ -H+ exchanger and a Na+ -HCO3 − cotransporter at the basolaterial membrane, and a Cl− -HCO3 − exchanger with CFTR at the apical membrane (Fig. 1). Coupled operations of these transporters have been proposed to explain HCO3 − secretion (Fig. 3). Studies of isolated cholangiocytes have provided further insight into the hormonal regulation of ductular HCO3 − secretion, which is a highly regulated process (Kanno et al., 2001). Secretin increases ductular HCO3 − secretion, an effect inhibited by somatostatin, gastrin, endothelin and insulin. Bombesin and acetylcholine increase ductular bile flow by stimulating Cl− -HCO3 − exchange and secretin-stimulated cAMP production, respectively. Secretin-induced HCO3 − secretion in cholangiocytes involves a series of events (Fig. 3). Secretin stimulates adenylate cyclase leading to increases in cAMP and activation of cAMP-dependent protein kinase (PKA) which in turn activates CFTR by phosphorylation. This leads to the opening of the Cl− channel followed by activation of the apical Cl− -HCO3 − exchanger due to a favorable Cl− gradient. Depolarization induced by Cl− efflux leads to the activation of the basolateral Na+ -HCO3 − cotransporter and influx of HCO3 − . Somatostatin, acting via its receptor, inhibits the effect of secretin by decreasing cAMP formation. Gastrin, on the other hand, inhibits secretin-induced cAMP formation by activating protein kinase C. Although it is generally accepted that water is reabsorbed during ductular passage of bile, the available supporting evidence for this view is limited. Bile ducts have been shown to absorb sugar and bile acids, but whether the reabsorption of organic solutes contributes significantly to water reabsorption remains to be established.
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Role of Paracellular Pathway Biliary excreted solutes can enter canaliculi via the paracellular, as well as transcellular pathway, with osmotic choleretics transported via the transcellular pathway. Subsequent movement of fluid and inorganic ions into canaliculi is thought to take place across the canalicular membrane, as well as via the paracellular pathway. Paracellular movement is passive down the electrochemical gradient and tight junctions are the major permeability barriers of the paracellular pathway (Anwer, 1993). Tight junctions allow passage of small electrolytes and proteins, appear to be selectively permeable to cations, and are mostly impermeable to organic anions. An increase in paracellular permeability is associated with decreased bile acid excretion, bile formation and increased regurgitation of bile acid in different experimental models. Thus, tight junctions are considered to play an important role in bile formation by providing effective barriers against reflux of osmotically active solutes from canaliculi. However, other studies show that drug-induced cholestasis precedes an increase in paracellular permeability, and vasopressininduced increases in tight junction permeability are not associated with a major effect on bile flow. Thus, there are some unsettled questions about the degree to which bile formation is affected by paracellular permeability. Since tight junctions are selectively impermeable to organic anions e.g. bile acids, and bile formation depends on bile acid excretion, a change in tight junction permeability may not affect bile formation unless the change is large enough to allow regurgitation of bile acids.
CELLULAR MECHANISMS OF CHOLESTASIS Cholestasis can be divided into two major categories, extrahepatic and intrahepatic cholestasis. Extrahepatic cholestasis results from overt extrahepatic blockage (e.g. stone, malignancy) or from duct obstruction visible at the microscopic level (e.g. the diverse “vanishing duct” syndrome). After bile duct obstruction, constituents of bile are secreted for a period of time. The back pressure of obstruction may disrupt canalicular tight junctions leading to regurgitation of biliary contents into the bloodstream. While physical obstruction to bile flow underlies extrahepatic cholestasis, the mechanism of intrahepatic cholestasis entails alterations in cellular mechanisms involved in bile formation. Our present understanding of the cellular mechanisms of bile formation and cholestasis is based on studies on experimental models of cholestasis. Intrahepatic cholestasis is produced by a number of agents, like estrogen, anabolic steroids, certain bile acids, endotoxins, etc.
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(Bohan & Boyer, 2002). Other models include bile duct ligation to simulate extrahepatic cholestasis, animals with congenital cholestasis, such as mutant TR− , Groningen Yellow and Eisai hyperbilirubinuria rats (Jansen & Oude Elferink, 1988; Kuipers & Vonk, 1991), and patients with cholestatic disorders (Elferink & Groen, 2002; Jansen et al., 2001; Lee & Boyer, 2000). It is evident that the maintenance of normal bile formation requires transport of appropriate solutes across sinusoidal and/or canalicular membranes. Hepatic solute transport is, in turn, dependent on cellular mechanisms responsible for the synthesis and delivery of transporters to the appropriate membrane domains, maintenance of both ion gradients, and adequate energy supply. Considering the complexity and the degree of coordination required at the cellular level to produce bile, more than one mechanism is likely to be involved in the pathogenesis of cholestasis. Our understanding of the pathogenesis of cholestasis is based on investigations to define physiological regulation of various transporters, and their deregulation in experimental models of cholestasis. In addition, studies on the expression of transporters in cholestatic diseases have provided valuable information on the role of specific transporters in the pathogenesis of some of these diseases. It is becoming clear that there are a number of ways hepatocellular functions, and consequently, solute transport, can be adversely affected. Thus, multiple hypotheses have been proposed to explain the mechanism of cholestasis. Transport proteins, like other proteins, are synthesized in the ER, processed in the Golgi complex, and then translocated to their intended site of action, basolateral membrane for NTCP and canalicular membrane for BSEP, for example. A transporter has to be inserted into the membrane for it to transport its solute across that membrane. This is a complex regulated process, which requires the participation of various signaling molecules along with vesicles, microtubules, and microfilaments. A breakdown in this regulated process can lead to a decreased amount or an absence of a transport protein at its intended site, resulting in decreased or no transport function, and hence, cholestasis. In addition, the transporter activity may be decreased directly, leading to cholestasis. Indeed, cholestasis is associated with down-regulation of ntcp and mrp2 (Lee et al., 2000; Trauner et al., 1997) and up-regulation of mrp3 (Donner & Keppler, 2001; Soroka et al., 2001) with a relatively preserved expression of bsep (Lee et al., 2000). Mutations in a gene encoding for a particular transporter may result in a lack of important transport function leading to cholestasis. For example, mutations of BSEP, MDR3 and MRP2 (cMOAT) are implicated in type 2 progressive familial intrahepatic cholestasis (PFIC2), PFIC3 and Dubin-Johnson syndrome, respectively (De Vree et al., 1998; Paulusma et al., 1997; Strautnieks et al., 1998). The result of a point mutation may lead to loss of transport activity and/or inability to translocate to the plasma membrane, as recently demonstrated for BSEP
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(Wang et al., 2002). Apart from genetic defects, regulation of transporters at the level of transcription, translation, post-translational modifications, translocation to the plasma membrane and transporter activity may be altered by chemicals/disease processes, thereby leading to decreased bile formation, and hence, cholestasis. Progress has been made in our understanding of the transcriptional and posttranslational regulation of various transporters, and how these processes may be altered in cholestasis. It would appear that post-translational changes are early events, while the transcriptional changes are delayed effects of cholestasis. It is becoming evident that nuclear receptors (Chiang, 2003; Karpen, 2002) and STAT (Anwer, 1998), a member of the signal tranducers and activators of transcription, play an important role in the transcriptional regulation of various transporters, while the post-translational regulation is mediated via classical second messengers. It is now well established that the interaction of hormones/growth factors with their receptors at the plasma membrane leads to the generation of second messengers that activate a cascade of key enzymes that produce the biological effects. In hepatocytes, activation of cell surface receptors results in the formation of cAMP or cGMP, increases in cytosolic Ca2+ and activation of kinases, including PKC, PI3K and MAPK (see Fig. 4). These second messengers and kinases are involved in various aspects of bile formation and the signal transduction pathways involve a cascade of factors/enzymes, the details of which are still unclear. What follows is a summary of recent findings as they relate to the role of second messengers in the regulation of bile formation and the pathogenesis of cholestasis.
Role of Nuclear Receptors and STAT Expression of NTCP and OATP-C is down regulated in patients with cholestasis (Oswald et al., 2001; Zollner et al., 2001). The suggestion has been made that nuclear receptors (NRs) play a role in the mechanism of this down regulation. What is known so far is that nuclear receptors are involved in transcriptional regulation of organic anion transporters, bile acid synthesis and other hepatic functions (Chiang, 2003; Karpen, 2002). Nuclear receptors, by responding to intracellular changes in sensitive ligands, alter the expression of target genes. There are over 150 members of the NR family and those shown to affect organic anion transporters are mentioned here. These NRs (Class II NR) form heterodimers with retinoid X receptor (RXR) before binding to response elements in the promoter region of the target gene, and therefore regulating the initiation of transcription. Farnesol X receptor (FXR), one of the RXR partner NRs, binds bile acids with high affinity. Studies based on a cholic acid feeding model have shown that FXR knockout mice, compared to wild type mice, express low levels of bsep, and are unable to down regulate ntcp and
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Fig. 4. Major signal transduction pathways involved in bile formation: Interaction of glucagon/epinephrine and Arginine vasopressin/phenylephrine with their receptors leads to the activation of Adenylyl cyclase (AC) and phospholipase C (PLC), respectively. AC converts ATP into cAMP and PLC hydrolyzes phosphoinositide(4,5)-bisphosphate(PIP2) into inositol(1,4,5)-trisphosphate (IP3) and diacyglycerol (DAG). IP3 and cAMP, acting via PKA increase [Ca2+ ]i by releasing Ca2+ from its cytoplasmic store. DAG activates nPKCs and cPKCs. Insulin and hepatocyte growth factor (HGF), acting via receptor tyrosine kinase (RTK), activate PI3K (phosphoinositide-3-kinase) and MAPK (mitogen activated protein kinase) pathways. TC and cAMP also activate the PI3K pathway and cell swelling and TUDC activates PI3K as well as the MAPK pathway. Some of the effects mediated via these signaling pathways are listed in respective boxes. BA = bile acid, OA =organic anion, OC = organic cation.
cholesterol-7-hydroxylase expression. Thus, FXR acts as a sensor of intracellular bile acid levels and is involved in the coordinate regulation of bile acid uptake, synthesis and expression. FXR is thought to affect ntcp expression by a complex mechanism involving a small heterodimer partner (SHP). The following mechanism (Fig. 5) has been proposed to explain the down regulation of ntcp in cholestasis (Karpen, 2002; Chiang, 2003). The expression
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Fig. 5. Transcriptional regulation of organic anion transporters by nuclear receptors: Bile acids (BA) stimulate expression of BSEP and SHP by activating FXR response elements in the BSEP and SHP promoters. SHP, in turn, interferes with RXR:RAR transactivation of NTCP promoter, resulting in decreased expression of NTCP. HNF1␣ transcriptionally activates NTCP and OATP-C. BA decreases NTCP and OATP-C expression by suppressing HNF1␣ via its inhibitory effect on HNF4␣. SHP also inhibits HNF4␣-mediated transactivation of the HNF1␣ promoter and autoregulates its own promoter by repressing LRH-1 (liver receptor homologue). Modified from Chiang (2003) and Karpen (2002).
of Ntcp is activated by retinoic acid receptor (RAR) as a heterodimer with RXR (RAR:RXR). Increased intracellular bile acid in cholestasis activates the FXR response element in the SHP promoter leading to the expression of SHP, which in turn down regulates RXR:RAR activation of ntcp. More recently Jung and KullakUblick (2003) have suggested that bile acid-induced suppression of hepatocyte nuclear factor (HNF1␣), via activation of HNF4, is implicated in cholestasisinduced down regulation of NTCP and OATP-C, both of which are transcriptionally activated by HNF1␣. Bile acids can also suppress HNF1␣ via SHP. Thus, the down regulation of ntcp may also be mediated via suppression of HNF1␣. The down regulation of ntcp in pregnancy may be due to suppression of HNF1␣ and RXR:RAR (Arrese et al., 2003). Although bsep expression is decreased in FXR knockout mice, and although FXR acts as a potent activator of the BSEP promoter (Ananthanarayanan et al., 2001), bile acid feeding does not increase bsep expression. In addition, an increased bile acid flux, induced by bile acid feeding,
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decreases ntcp expression, and increases bsep expression without affecting the level of FXR (Wolters et al., 2002). Thus, additional factors beyond FXR may be involved in the transcriptional regulation of bsep. The preceding considerations bolster the notion that transcriptional regulation of transporters is a consequence of cholestasis as the effects are initiated by an increase in intracellular bile acid concentration. These effects are thus recognized as cellular responses to decreased intracellular bile acid levels, and thereby limit or minimize bile acid-induced cellular toxicity. Another nuclear receptor, namely pregnane X receptor (PXR) in rodents, and steroid and xenobiotic receptor (SXR) in humans, are activators of OATP2, MDR1 and MRP2 genes. However, it remains to be seen whether PXR/SXR plays a role in steroid-induced cholestasis. Prolactin transcriptionally up-regulates ntcp in rats (Ganguly et al., 1997), an effect mediated via STAT5 binding to ntcp-GLEs (interferon -gamma-activated sequence-like elements). Glucocorticoids, which upregulate intestinal bile acid transporter, do not affect hepatic taurocholate uptake (Nowicki et al., 1997), indicating a lack of regulation of ntcp by glucocorticoid-responsive element. Interestingly, reduced ntcp expression in pregnant rats in late gestation was associated with an increased level of STAT5 (Arrese et al., 2003), an effect thought to be due to an increased level of prolactin.
Role of Cyclic AMP Glucagon and secretin, acting via cAMP, rapidly stimulate bile formation by hepatocytes and cholangiocytes, respectively. Cyclic AMP stimulates sinusoidal Na+ /Taurocholate (TC) cotransport, transcytotic vesicle trafficking and canalicular secretion of bile acids, organic anions and HCO3 − in hepatocytes (Anwer, 1998; Benedetti et al., 1994; Gr¨une et al., 1993; Hayakawa et al., 1990) and HCO3 − secretion and exocytosis in cholangiocytes (Kato et al., 1992; Kanno et al., 2001). Unlike cAMP, cGMP does not stimulate hepatobiliary bile acid transport, but it does stimulate bile formation by increasing biliary HCO3 − excretion (Myers et al., 1996). The ability of cAMP to stimulate microtubule-dependent vesicle trafficking in hepatocytes (Hayakawa et al., 1990) led to the suggestion that cAMP stimulates various solute transport systems by translocating the transporters to the plasma membrane. This is supported by findings that cAMP increases ntcp (Dranoff et al., 1999; Mukhopadhayay et al., 1997) in sinusoidal membranes and mrp2 (Roelofsen et al., 1998), mdr2 and mdr3 (Gatmaitan et al., 1997) and bsep (Kipp et al., 2001) in canalicular membranes. However, while the translocation to canalicular membrane is dependent on microtubules (Boyer & Soroka, 1995; Gatmaitan et al., 1997), cAMP-mediated translocation to the basolateral membrane is dependent
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on microfilaments (Dranoff et al., 1999; Webster & Anwer, 1999). Secretin stimulated biliary HCO3 − secretion is mediated via cAMP, which activates the CFTR Cl− channel prior to activation of the Cl− -HCO3 − exchanger (Kanno et al., 2001). Expression of the hepatocyte and the cholangiocyte Cl− -HCO3 − exchanger is diminished in primary biliary cirrhosis (Medina et al., 1997), thus raising the distinct possibility that abnormalities of HCO3 − transport may play a role in this cholestatic disorder. The effect of cAMP is believed to be mediated via cAMP-dependent kinase, also known as protein kinase A or PKA (Anwer, 1998; Kanno et al., 2001), although the role of PKA has been directly evaluated in cAMP mediated stimulation of Na+ /TC cotransport (Gr¨une et al., 1993) and cholangiocyte Cl− -HCO3 − exchange (Alvaro et al., 1997) and cAMP-mediated cell survival (Webster & Anwer, 1998). Signaling pathway(s) downstream of cAMP/PKA leading to vesicle movement and consequent transporter translocation may involve activation of the PI3K pathway and increases in cytosolic Ca2+ (Figs 6 and 7). In addition, PKA may stimulate vesicle movement by phosphorylating microtubule-associated proteins (Davidson et al., 1992). There is suggestive evidence that the cAMP signaling pathway may be altered in cholestasis. Experimental cholestasis induced by bile duct ligation and endotoxin is associated with decreased expression of ntcp, mrp2 and bsep (Lee et al., 2000; Trauner et al., 1997). These decreases are more pronounced in respect of ntcp and mrp2 than bsep. Bile duct ligation also decreases the ability of glucagon to increase cAMP in hamster hepatocytes (Matsuzaki et al., 1997). This appears to be due to decreased expression of ␣-subunit(s) of stimulatory as well as inhibitory G proteins (Bouscarel et al., 1998) coupling the receptor to adenylyl cyclase. Altogether, these observations suggest that the cAMP signaling pathway may be down-regulated in cholestasis. It is noteworthy that a more pronounced decrease in bile acid uptake by ntcp compared to bile acid secretion by bsep may represent a hepatocellular defense mechanism against the accumulation of intracellular bile acids, and hence, exacerbation of cholestasis. This is further supported by the finding that the down-regulation of canalicular mrp2 is associated with an upregulation of mrp3 (Donner & Keppler, 2001), which exports bile acids across the sinusoidal membrane (Hirohashi et al., 2000).
Role of Calcium Changes in extracellular as well as intracellular Ca2+ ([Ca2+ ]i ) have been shown to affect bile formation. An extracellular Ca2+ below 50 M is associated with a decrease in bile formation. This is due to an increase in tight-junction permeability,
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Fig. 6. Role of cAMP, Ca2+ and PKC in bile formation: Cyclic AMP, acting via PKA, stimulates translocation of ntcp to the basolateral membrane and Bsep to the canalicular membrane. Increases in [Ca2+ ]i , due to influx or release of Ca2+ from intracellular stores by PKA or IP3, activate Ca2+ -calmodulin (Ca2+ /CAM)-dependent kinases, which activates the Na+ -H+ exchanger (NHE), augments PKA-induced Ntcp translocation, and increases tight-junction permeability via phosphorylation of myosin light-chain. PKC inhibits cAMPmediated increases in bile acid uptake by diminishing PKA-mediated increases in [Ca2+ ]i and stimulates bile acid excretion, probably by phosphorylating bsep. nPKC may produce cholestasis by re-targeting mrp2 to the basolateral membrane, while cPKC␣ may stimulate mrp2 translocation to the canalicular membrane. TUDC may reverse TLC-induced cholestasis by reversing the effect of TLC on cPKC␣ and nPKC.
resulting in reflux of secreted solutes, and is not due to a direct effect on either hepatic uptake or biliary excretion of TC (Anwer & Clayton, 1985; Reichen et al., 1985). Changes in [Ca2+ ]i , on the other hand, affect hepatic transport of bile acids. This effect of [Ca2+ ]i has been studied using calcium mobilizing agents, such as arginine vassopressin (AV), calcium ionophores (A23187 or ionomycin), and intracellular calcium chelators (MAPTA or BAPTA). Chelation of [Ca2+ ]i by MAPTA, BAPTA or EDTA decreases basal rate of Na+ /TC cotransport in hepatocytes (Bouscarel et al., 1996; Gr¨une et al., 1993). Thus, resting [Ca2+ ]i plays an important role in maintaining basal Na+ /bile acid cotransport. Interestingly, basal Na+ /TC cotransport is also inhibited in response to increases in [Ca2+ ]i produced by calcium ionophores and AV in hamster hepatocytes (Bouscarel et al., 1996), but not in rat hepatocytes (Gr¨une et al., 1993; Kuhn & Gewirtz, 1988).
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Fig. 7. Role of PI3K and MAPK signaling pathway in bile formation: Activation of PI3K by TC, cAMP, TUDC and cell swelling leads to the formation of PIPs which can activate the Ras/ERK pathway and PKB/PKC. Cyclic AMP mediated activation of PKB/PKC may be involved in microfilament-dependent Ntcp translocation. TUDC and cell swelling induced microtubule-dependent Bsep translocation may involve PI3K/PIPs/Ras/Raf/Erk pathway. The effect of cAMP is not mediated via the Erk pathway, which is inhibited by cAMP. TUDC, cell swelling and cAMP may stimulate Bsep translocation via P38 MAPK. Erk and p38 MAPK pathways may converge on MNK1/MSK1.
This may be attributable to species differences in the regulation of Na+ /bile acid cotransport by [Ca2+ ]i ; other explanations are discussed by Bouscarel et al. (1999). In contrast to its effect on uptake, AV increases bile acid excretion transiently in perfused rat livers (Kuhn et al., 1990) and bile acid efflux in isolated rat hepatocytes by decreasing substrate affinity for the transporter (Kuhn & Gewirtz, 1988). Whether these effects are due to increases in [Ca2+ ]i or activation of PKC (see Fig. 4) was not investigated. AV and phenylephrine stimulate the sinusoidal Na+ -H+ exchanger, an effect mediated via receptor-mediated increases in [Ca2+ ]i as well as activation of PKC (Anwer & Atkinson, 1992; Anwer, 1994). Intracellular Ca2+ also plays a role in cAMP-stimulated bile acid uptake in that the effect of cAMP depends on its ability to increase [Ca2+ ]i from IP3 -sensitive pools (Gr¨une et al., 1993). The calcium signal is transducted via calmodulin to calmodulin-dependent kinases and phosphatases which produce biological effects by phosphorylating and dephosphorylating other proteins, respectively (Lukas et al., 1988). In hepatocytes
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(Anwer, 1994; Gr¨une et al., 1993), effects of calcium on AV-induced stimulation of the Na+ -H+ exchanger and cAMP-induced stimulation of the Na+ /TC cotransporter are mediated via calmodulin (Fig. 6). Cyclic cAMP-induced dephosphorylation of Ntcp (Mukhopadhayay et al., 1998a) is mediated via the action of the calmodulin-dependent phosphatase, protein phosphatase 2B, activated by cAMP-induced increases in [Ca2+ ]i (Webster et al., 2002a). The exact role of the calcium-dependent signaling pathway in hepatic bile formation and cholestasis is not yet clearly understood. Increases in [Ca2+ ]i have been associated with choleresis as well as cholestasis. For example, cholestatic (TLC) as well as choleretic (TUDC) bile acid increase [Ca2+ ]i (Anwer et al., 1988; Beuers et al., 1993a), and TUDC can reverse cholestasis produced by TLC (Scholmerich et al., 1990). Some would suggest that increases in [Ca2+ ]i may lead to increased tight-junctional permeability, and hence, cholestasis (Nathanson et al., 1992a, b) and this effect may be mediated via myosin light chain phosphorylation (Yamaguchi et al., 1991). TUDC may stimulate Ca2+ -dependent stimulation of vesicular exocytosis by enhancing Ca2+ entry into hepatocytes, which may be compromized in cholestasis (Beuers et al., 1993b). However, increases in [Ca2+ ]i do not influence exocytosis in normal hepatocytes (Bruck et al., 1994). Studies to define the role of [Ca2+ ]i have been complicated by the fact that some agents used to increase [Ca2+ ]i also activate PKC (Fig. 4). In addition, increases in [Ca2+ ]i by different agents may lead to activation of different downstream kinases, including PI3K (Benzeroual et al., 2000) and PKB (Yano et al., 1998), resulting in various effects in normal and cholestatic hepatocytes. It is likely that such effects are associated with temporal and spatial changes in [Ca2+ ]i induced by different agents (Nathanson et al., 1994, 1995).
Role of Protein Kinase C Protein kinase C is a family of at least 12 isozymes (Newton, 2003) including conventional (cPKC␣, , I, II and ␥), novel (nPKC␦, , and ), atypical (aPKC and ) isoforms, and PKC. These isoforms differ in their dependency on Ca2+ and phospholipids, such that cPKCs are dependent on Ca2+ and diacylglycerol (DAG), while nPKCs are Ca2+ -independent, and aPKCs are independent of both Ca2+ and DAG. The PKCs present in rat hepatocytes include cPKC␣, nPKC␦, nPKC, and aPKC with the presence of cPKCII being controversial (Beuers et al., 1999; Jones et al., 1997; Stravitz et al., 1996). The role of PKC in bile formation has been studied using known activators (phorbol esters) and inhibitors of PKC. Observations indicate that agents known to activate PKC produce cholestasis (Corasanti et al., 1989), inhibit basal and
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cAMP-stimulated bile acid uptake (Bouscarel et al., 1996; Gr¨une et al., 1993), stimulate biliary excretion of bile acids (Kuhn et al., 1990) and organic cations (Steen et al., 1993). Evidence is also available that PKC is involved in ATPdependent modulation of cation channels (Fitz et al., 1994). PKC inhibits cAMPstimulated bile acid uptake, at least in part, by reducing the ability of cAMP to increase [Ca2+ ]i (Gr¨une et al., 1993). AV increases bile acid efflux by decreasing Km (Kuhn and Gewirtz, 1988), while cPKC␣ phosphorylates Bsep (Noe et al., 2001). Thus, if the effect of AV is mediated via PKC, cPKC␣ may stimulate bile acid excretion by phosphorylating Bsep, and thereby, enhancing substrate affinity (see Fig. 6). Activation of PKCs has been implicated in bile acid-induced cholestasis and apoptosis. GCDC-induced apoptosis requires activation of cPKC␣ and nPKC␦, but not nPKC (Jones et al., 1997) and the non-apoptotic effect of TCDC is due to activation of aPKC (Rust et al., 2000). On the other hand, a cholestatic bile acid, TLC, activates nPKC in isolated hepatocytes (Beuers et al., 1999) and inhibits cPKC␣ in isolated perfused rat livers (Beuers et al., 2001). The effect of TLC on nPKC appears to be mediated via the PI3K signaling pathway (Beuers et al., 2003). TUDC may reverse TLC-induced cholestasis by activating cPKC␣ and/or inhibiting nPKC (Beuers et al., 1996, 2001). TUDC-induced activation of cPKC␣ may be involved in the translocation of Mrp2 to the canalicular membrane, and consequent restoration of organic anion excretion in cholestatic livers (Beuers et al., 2001). This is likely since PKC (isoforms not studied) re-targets canalicular Mrp2 to the basolateral membrane. This could contribute to cholestasis (Kubitz et al., 2001). Re-targeting may also be mediated via nPKC (Fig. 6) in light of evidence suggesting that bile formation and cholestasis can be mediated via PKC isoform-dependent processes. However, the role of PKC isoforms, as well as the downstream targets of PKC isoforms have not yet been clearly established.
Role of PI3K Signaling Pathway Phosphatidylinositol-3-kinase (PI3K) is one of the phosphatidylinositol (PI) kinases that phosphorylate at specific positions of the inositol ring. The resulting phosphorylated PIs (PIPs) have been implicated in a wide variety of biological effects produced by hormones/growth factors. PIPs act as second messengers in signal transduction pathways involved in vesicle trafficking, cell survival, cell proliferation, cell migration, and transport of glucose and bile acids (Anwer, 1998; Rameh & Cantley, 1999; Toker, 2000). Folli et al. (1997) first demonstrated a role of PI3K in bile formation by showing that wortmannin, a specific inhibitor of PI3K, diminished bile formation, bile acid secretion and vesicle trafficking in isolated
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perfused rat liver. Since then various studies have provided evidence supporting a role for PI3K in the regulation of hepatobiliary transporters. Take for example, PI3K, which is involved in: (1) Cell swelling and cAMP-mediated translocation of ntcp (Webster & Anwer, 1999; Webster et al., 2000); (2) ATP-dependent transport of bile acids and other organic anions across the canalicular membrane (Misra et al., 1999b); (3) TC- and cAMP-induced translocation of bsep and mrp2 to the canalicular membrane (Misra et al., 1998, 1999a); (4) TUDC -induced increases in bile acid secretion (Kurz et al., 2000); (5) ATP release and chloride secretion in cholangiocytes (Feranchak et al., 1999); and (6) Insulin-mediated membrane recruitment (Kilic et al., 2001). Cell swelling also stimulates biliary bile acid excretion, translocation of bsep and mrp2 to the canalicular membrane (Kubitz et al., 1991; Schmitt et al., 2001) and activates PI3K (Krause et al., 1996; Webster et al., 2000). Thus, PI3K may also be involved in cell swelling induced translocation of bsep and mrp2. However, other mechanisms have been proposed (see below). Further insight has been gained into the PI3K signaling pathway involved in the regulation of transporters in bile formation. For example, cell swelling and cAMP activate wortmannin-sensitive PKB and p70S6K , but p70S6K is not involved in the stimulation of Na+ /TC cotransport and ntcp translocation (Webster & Anwer, 1999; Webster et al., 2000). The possibility of participation of the PI3K/PKB signaling pathway is supported by results showing that the inhibition of PKB activation decreases cell swelling and cAMP-mediated stimulation of Na+ /TC cotransport and ntcp translocation (Webster et al., 2002b). Translocation of the glucose transporter appears to be mediated via P13K/PKB, as well as the PI3K/aPKC signaling pathway (Hernandez et al., 2001; Standaert et al., 1999). Thus, it is likely that the PI3K/aPKC signaling pathway may also be involved in ntcp translocation (Fig. 7). Whether either of these signaling pathways is also involved in PI3K mediated translocation of canalicular transporters remains unknown but this possibility seems real. Mechanisms by which the translocation of hepatobiliary transporters is mediated via the PI3K signaling pathway are still unclear. It is known that translocation of ntcp to the sinusoidal membrane, and of bsep and mrp2 to the canalicular membrane is dependent on microfilaments (Dranoff et al., 1999; Vlahos et al., 1994) and microtubules (Boyer & Soroka, 1995; Gatmaitan et al., 1997), respectively, and several canalicular transporters, like bsep & mrp2, and the polymeric immunoglubulin A receptor traffic on the same vesicle (Soroka et al., 1999). PI3K products, like PIP, PIP2 and PIP3 , are involved in vesicle trafficking (Rameh & Cantley, 1999; Wurmser et al., 1999). It is thus reasonable to speculate that the PI3K/PKB(aPKC) signaling pathway stimulates exocytosis by increasing
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vesicle trafficking along the cytoskeleton. This results in the fusion of intracellular vesicles to the plasma membrane, leading to plasma membrane translocation of various transporters stored in the intracellular vesicles (Fig. 7). In addition, PI3Kmediated activation of the MAPK (mitogen activated protein kinase) cascade (Toker, 2000) may also be involved (Fig. 7).
Role of MAPK Signaling Pathway The mitogen activated protein kinases (MAPKs) include a group of protein kinases that are activated by a variety of signals (Schaeffer & Weber, 1999; Seger & Krebs, 1995; Wilkinson & Millar, 2000). Mammalian cells have 2 major types of MAPKs: (1) ERK1/ERK2 (Extracellular signal-regulated kinase) activated in response to activation of receptor tyrosine kinase by growth factors; (2) SAPKs (stress activated protein kinases) activated by UV radiation, inflammatory cytokines, DNA damaging agents and inhibitor of protein synthesis. SAPKs include two distinct subfamilies: JNKs (c-Jun amino-terminal kinases) and p38 MAPK. These MAPKs mediate signal transduction from the cell surface to the nucleus. They participate in cell proliferation and differentiation under controlled activation and oncogenesis during uncontrolled activation. Activation of these MAPKs requires sequential phosphorylation of various kinases. The MAP signaling pathways involving ERK1/2 and p38 have also been implicated in biliary excretion of bile acids. Hypo-osmotic cell swelling increases the capacity of biliary TC excretion. Although this effect requires a G-proteinand tyrosine kinase-dependent activation of ERK1/2, it is independent of PKC (Noe et al., 1996). Similarly, TUDC-induced increases in bile acid excretion are dependent on ERK1/2 activation, but not on G-protein, tyrosine kinase or protein kinase C (Schliess et al., 1997). In addition, cAMP, which inhibits ERK1/2 in hepatocytes (Webster & Anwer, 1999), reduced TUDC-induced activation of ERK1/2 and increases in bile acid excretion (Schliess et al., 1997). The effect of TUDC appears to be mediated by a PI3K-dependent activation of a Ras/ERK pathway (Kurz et al., 2000). Cell swelling- and TUDC-induced increases in bile acid secretion and bsep translocation to the canalicular membrane also require activation of PI3K-independent activation of p38 MAPK (Kurz et al., 2001). In contrast to bile acid secretion, cell swelling-induced stimulation of hepatic uptake of bile acid is not dependent on ERK1/2 (Webster et al., 2000). These studies indicate that stimulation of bsep translocation and the consequent increase in bile acid excretion can be mediated via activation of ERK1/2 as well as p38 MAPK pathway (Fig. 7). Since ERK1/2 and p38 MAPK can activate the
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downstream kinases (Schaeffer & Weber, 1999), like MAPK signal-integrating kinase 1 (MNK1) and mitogen- and stress-activated protein kinase 1 (MSK1), it is possible that bsep translocation along the cytoskeleton is mediated via one of these kinases (see Fig. 7). The role of PI3K and MAPK pathways in cholestasis has not been directly evaluated. Considering their role in bile formation, these pathways are likely to be altered in cholestasis. On the other hand, agents that stimulate these pathways would be expected to produce anti-cholestatic effects, as is the case with the anticholestatic effects of ursodeoxycholate (Lazaridis et al., 2001). So far as the cholestatic effect of TLC is concerned, it may be mediated via PI3K-dependent activation of nPKC (Beuers et al., 2003).
Role of Protein Phosphatases Cellular functions are regulated by reversible phosphorylation; that is, by protein kinases and phosphoprotein phosphatases. This is taken to mean that the levels of phosphoprotein in the steady state is governed by the kinase-phosphatase equilibrium. While several studies have been made of the role of various kinases in bile formation, investigations of the role of protein phosphatases are sparse. Inhibitors of protein phosphatase 1 and 2A (PP1/2A) such as okadaic acid and microcystin, have produced evidence suggesting that PP2A regulates microtubuledependent vesicle movement in hepatocytes (Hamm-Alvarez et al., 1996). Okadaic acid, which inhibits PP2A in hepatocytes, blocks the ability of cAMP to stimulate TC uptake, and to translocate and dephosphorylate ntcp, and increase [Ca2+ ]i in hepatocytes (Mukhopadhayay et al., 1998b). Thus, cAMP-induced increases in [Ca2+ ]i are dependent on PP2A activity. The ability of cAMP to dephosphorylate Ntcp is inhibited by a calcium chelator (Mukhopadhayay et al., 1998a). Cyclic AMP activates Ca2+ /calmodulin-dependent PP2B (also known as calcineurin), while an inhibitor of PP2B reduces cAMP-induced dephosphorylation of ntcp (Webster et al., 2002a). Activation by phosphorylation of Oatp1 by extracellular ATP is reduced by okadaic acid (Glavy et al., 2000). However, okadaic acid fails to affect basal fluid secretion, and the basal activity of Cl− -HCO− 3 exchanger. Inactivation of secretin-induced activation of CFTR by okadaic acid remains a possibility (Alvaro et al., 1997). Together, these results suggest that protein phosphatases, notably PP2A and PP2B, may regulate both vesicular transport and organic anion uptake in hepatocytes, and secretin-induced secretion by cholangiocytes by involving dephosphorylating proteins. It would therefore be of interest to determine whether protein phosphatases are key players in the regulation of other transporters and whether they are altered in cholestasis.
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SUMMARY It is clear that our understanding of the mechanism of transhepatic solute transport and bile formation has increased over the years. Bile formation involves vectorial transport of solutes from blood to bile, and depends on the coordinated activities of various solute transporters located at the basolateral and apical membranes of hepatocytes and cholangiocytes. Cholestasis is the result of compromised vectorial transport of solutes destined for bile. Similarly, our understanding of various transporters, their substrates and locations has increased steadily as is the cellular mechanism regulating these transporters. It is now more clear that choleretic and cholestatic agents modify the function of these transporters through various signal transduction pathways. Nuclear receptors are considered to regulate transcription of various transporter genes. Cyclic AMP, acting via PKA and the PI3K signaling pathway, stimulates transhepatic transport of bile acids by translocating ntcp and bsep to the sinusoidal and the canalicular membrane, respectively. The PI3K signaling pathway plays a role in biliary excretion of bile acids and TLC-induced activation of PKC. TUDC stimulates bile acid excretion via the P38 MAPK signaling pathway. TUDC also reverses TLC cholestasis by stimulating PKC-mediated translocation of mrp2 to the canalicular membrane. Calcium, acting via Ca2+ /calmodulin dependent kinases/phosphatases, augments cAMP-mediated translocation of ntcp, increases tight-junctional permeability by phosphorylating myosin light-chain, and stimulates sinusoidal Na+ -H+ exchange. PKC stimulates bile acid secretion, most likely by phosphorylating bsep, and Na+ H+ exchange. However, our understanding of the role of specific PKC isoforms in bile formation is still incomplete. The mechanisms by which PI3K and MAPK signaling pathways stimulate transporter translocation along the cytoskeleton have not yet been elucidated. We still know little about the role of protein phosphatases and nuclear receptors in bile formation and cholestasis.
REFERENCES Alvaro, D., Mennone, A., & Boyer, J. L. (1997). Role of kinases and phosphatases in the regulation of fluid secretion and C1− -HCO3 − exchange in cholangiocytes. Am. J. Physiol., 273, G303–G313. Ananthanarayanan, M., Balasubramanian, N., Makishima, M., Mangelsdorf, D. J., & Suchy, F. J. (2001). Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid receptor. J. Biol. Chem., 276, 28857–28865. Anwer, M. S. (1993). Transheptatic solute transport and bile formation. Adv. Vet. Sci. Comp. Med., 37, 1–29. Anwer, M. S. (1994). Mechanism of activation of the Na+ -H+ exchanger by arginine vasopressin in hepatocytes. Hepatology, 20, 1309–1317.
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Trauner, M., Meier, P. J., & Boyer, J. L. (1999). Molecular regulation of hepatocellular transport systems in cholestasis. J. Hepatol., 31, 165–178. Van Dyke, R. W., Faber, E. D., & Meijer, D. K. (1992). Sequestration of organic cations by acidified hepatic endocytic vesicles and implications for biliary excretion. J. Pharmacol. Exp. Ther., 261, 1–11. van Montfoort, J. E., Hagenbuch, B., Fattinger, K. E., Muller, M., Groothuis, G. M., Meijer, D. K., & Meier, P. J. (1999). Polyspecific organic anion transporting polypeptides mediate hepatic uptake of amphipathic type II organic cations. J. Pharmacol. Exp. Ther., 291, 147–152. van Montfoort, J. E., Muller, M., Groothuis, G. M., Meijer, D. K., Koepsell, H., & Meier, P. J. (2001). Comparison of “type I” and “type II” organic cation transport by organic cation transporters and organic anion-transporting polypeptides. J. Pharmacol. Exp. Ther., 298, 110–115. Vlahos, C. J., Matter, W. F., Hui, K. Y., & Brown, R. F. (1994). A specific inhibitor of phosphatidylinositiol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem., 269, 5241–5248. Wang, L., Soroka, C. J., & Boyer, J. L. (2002). The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II. J. Clin. Invest., 110, 965–972. Webster, C. R. L., & Anwer, M. S. (1998). Cyclic AMP mediated protection against bile acid induced apoptosis in cultured rat hepatocytes. Hepatology, 27, 1324–1331. Webster, C. R. L., & Anwer, M. S. (1999). Role of the PI3K/PKB signaling pathway in cAMP-mediated translocation of rat liver Ntcp. Am. J. Physiol., 277, G1165–G1172. Webster, C. R. L., Blanch, C., & Anwer, M. S. (2002a). Role of PP2B in cAMP-induced dephosphorylation and translocation of NTCP. Am. J. Physiol. Gastrointest. Liver Physiol, 283, G44–G50. Webster, C. R. L., Blanch, C. J., Philips, J., & Anwer, M. S. (2000). Cell swelling-induced translocation of rat liver Na+ /taurocholate cotransport polypeptide is mediated via the phosphoinositide 3-kinase signaling pathway. J. Biol. Chem., 275, 29754–29760. Webster, C. R. L, Srinivasulu, U., Ananthanarayanan, M., Suchy, F. J., & Anwer, M. S. (2002b). Protein kinase B/Akt mediates cAMP- and cell swelling-stimulated Na+/taurocholate cotransport and Ntcp translocation. J. Biol. Chem., 277, 28578–28583. Wilkinson, M. G., & Millar, J. B. (2000). Control of the eukaryotic cell cycle by MAP kinase signaling pathways. FASEB J., 14, 2147–2157. Wolkoff, A. W., & Cohen, D. E. (2003). Bile acid regulation of hepatic physiology: I. Hepatocyte transport of bile acids. Am. J. Physiol. Gastrointest. Liver Physiol., 284, G175–G179. Wolters, H., Elzinga, B. M., Baller, J. F., Boverhof, R., Schwarz, M., Stieger, B., Verkade, H. J., & Kuipers, F. (2002). Effects of bile salt flux variations on the expression of hepatic bile salt transporters in vivo in mice. J. Hepatol., 37, 556–563. Wurmser, A. E., Gary, J. D., & Emr, S. D. (1999). Phosphoinositide 3-kinases and their FYVE domain-containing effectors as regulators of vacuolar/lysosomal membrane trafficking pathways. J. Biol. Chem., 274, 9129–9132. Yamaguchi, Y., Dalle-Molle, E., & Hardison, W. G. (1991). Vasopressin and A23187 stimulate phosphorylation of myosin light chain- 1 in isolated rat hepatocytes. Am. J. Physiol., 261, G312–G319. Yano, S., Tokumitsu, H., & Soderling, T. R. (1998). Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Nature, 396, 584–587. Zollner, G., Fickert, P., Zenz, R., Fuchsbichler, A., Stumptner, C., Kenner, L., Ferenci, P., Stauber, R. E., Krejs, G. J., Denk, H., Zatloukal, K., & Trauner, M. (2001). Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases. Hepatology, 33, 633–646.
5.
THE ROLE OF BILE ACIDS IN THE MODULATION OF APOPTOSIS
Cec´ılia M. P. Rodrigues, Rui E. Castro and Clifford J. Steer INTRODUCTION In health, bile acids are essential for solubilizing lipids in the intestinal lumen; both their synthesis and transport drive bile formation and provide a degradation pathway for cholesterol. However, bile acids are inherently toxic compounds. The proposed mechanisms of bile acid-induced cell damage range from binding to cell membranes to the induction of apoptotic cell death. In contrast to the toxic effects of several hydrophobic bile acids, ursodeoxycholic acid is an endogenous molecule used over the last several decades for the treatment of certain liver diseases. More recently, it has been 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. They interrupt apoptosis by blocking classic pathways and can also significantly activate survival pathways. Thus, by acting as a general modulator of cell survival, ursodeoxycholic acid may act as a primary therapeutic agent in the treatment of neurodegenerative disorders in which increased levels of apoptosis contribute to the pathogenesis of the disease. Indeed, tauroursodeoxycholic acid is neuroprotective in pharmacologic and transgenic animal models of Huntington’s disease, improves graft survival in
The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 119–145 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15005-8
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Parkinsonian rats, and protects against neurological injury after acute ischemic and hemorrhagic stroke.
BILE ACID BIOSYNTHESIS AND PHYSIOLOGICAL ROLE Bile acids are a class of acidic steroids having a cyclopentanoperhydrophenanthrene nucleus (ABCD-ring) containing 19 carbons and most commonly a C5 side chain with a terminal carboxylic acid, as shown in Fig. 1. They are synthesized in the liver from neutral sterols by a complex series of chemical reactions, which are catalyzed by a variety of enzymes located in various subcellular compartments of the liver cell (Russell, 2003). Most of the enzymes involved in bile acid synthesis have been isolated to various degrees of purity. cDNAs have been reported for several enzymes including the rate-limiting enzyme for the bile acid biosynthetic pathway, cholesterol 7␣-hydroxylase (CYP7A1). As indicated by Anwer in the preceding chapter, bile acid biosynthesis in humans and most animal species arises primarily from the cholesterol metabolic pathway. Although cholesterol can be converted into steroid hormones, 90–95% of the daily turnover is the result of its conversion into primary bile acids, predominantly cholic and chenodeoxycholic acids. These bile acids are formed in the liver by a sequence of reactions leading to modifications of the ABCD-ring nucleus as well as sidechain oxidation. The initial and rate-limiting step in bile acid biosynthesis is the 7␣-hydroxylation of cholesterol. CYP7A1 is a cytochrome P450 enzyme located
Fig. 1. The 5-Cholanoic Acid Nucleus is the Basic Structure of C24 -Bile Acids in Mammalian Species. Note: Positions of the principal functional groups in the ABCDring, and possible conjugated derivatives are indicated by arrows. Ursodeoxycholic acid is a 3␣,7-dihydroxy molecule often conjugated at C-24 through an amide bond to the amino acids glycine and taurine.
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in the endoplasmic reticulum and its transcription is repressed by primary bile acids but activated in a feed-forward manner by dietary cholesterol. Subsequently, the conversion of 7␣-hydroxycholesterol to bile acids involves oxidoreduction and hydroxylation, preparing the sterol intermediates for either the cholic acid or the chenodeoxycholic acid pathways. Although this pathway is considered the most important for bile acid synthesis in humans, several alternative pathways have been identified, which under pathologic conditions and/or immature liver function may become quantitatively important. The primary bile acids are conjugated with the amino acids glycine or taurine (see Fig. 1), and then secreted via the bile ducts and gallbladder into the lumen of the small intestine. Here, they act as detergents to emulsify dietary lipids and fat-soluble vitamins. While these emulsified nutrients are taken up by enterocytes in the proximal segments of the gut, the bile acids continue to move distally until absorbed in the ileum by the ileal bile acid transporter. They are then transported to the liver via the portal circulation, taken up by the hepatocyte and resecreted into bile; uptake by the hepatocytes is efficient, intracellular storage is small, and canalicular secretion is rapid and complete. This applies to about 95% of the bile acids delivered into the small intestine, because the remaining 5% are lost to the colon and eventually excreted from the body. New hepatic synthesis must replace these unrecovered bile acids. The total bile acid pool (2–3 g) is constantly maintained under normal conditions primarily by hepatic synthesis (0.2–0.6 g/day). Fecal bile acid excretion (0.2–0.6 g/day) represents the major avenue of bile acid loss and gives an accurate measure of daily synthesis. In health, urinary excretion (2–6 mg/day) is of little quantitative significance. The biliary bile acid pool also includes conjugates of deoxycholic and lithocholic acids. These secondary bile acids are not formed in the liver, but rather result from the metabolism of primary bile acids in the intestine. Microbial biotransformations include deconjugation and 7␣-dehydroxylations. Although these are quantitatively the most important reactions, oxidoreduction and epimerization of hydroxy groups at various positions of the bile acid nucleus also take place in the colon. An important example of this metabolism is the formation of ursodeoxycholic acid, which occurs by oxidation of chenodeoxycholic acid to 7-oxolithocholic acid, followed by bacterial reduction yielding the 7-isomer. Bile acid synthesis plays a key physiological role, especially in lipid homeostasis, as it represents a major metabolic pathway to counterbalance the cholesterol supply. In addition, the expression of genes that synthesize cholesterol, fatty acids and bile acids are regulated by intermediates and/or end-products of the bile acid pathway itself. Bile acids are also essential to provide the driving force for the formation and secretion of bile and, therefore, are crucial elements in the development and maintenance of an efficient enterohepatic circulation. Because of
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their detergent properties, bile acids are crucial in the solubilization and absorption of fat and fat-soluble vitamins in the small bowel. In the colon, they have cathartic effects, and have been suggested to promote colonic carcinogenesis. However, bile acids have an amphipathic structure, which allows these water-soluble compounds to interact with proteins and insert into lipid bilayers. These effects will have severe influences on cell function and structure, particularly when intracellular concentrations of bile acids exceed certain limits.
BILE ACID EFFECTS IN CHOLESTATIC LIVER DISEASES Cholestasis is a common pathophysiological feature of many chronic human liver diseases leading to impaired bile formation, and potentially injuring target liver cells such as hepatocytes and cholangiocytes. Although the exposed bile ducts receive the initial insult in many of these disorders, progression of the disease appears to ultimately result from the accumulation of toxic bile acids within the hepatocyte. Indeed, earlier studies have demonstrated that interruption in bile flow leads to high concentrations of toxic hydrophobic bile acids in the liver. This can then directly induce cell injury and exacerbate immunologic, toxic, or genetic insults to the tissue. Bile acids were thought to be cytotoxic by acting as detergents on cell membranes. Although this might be true at the site of the canalicular membrane, where bile acid concentrations allow mixed micelle formation, it is less plausible in the liver. In fact, serum bile acid levels are below their critical micellar concentration, bile acid cytotoxicity does not always correlate with hydrophobicity and, further, the liver usually adapts to cholestasis and reduces intracellular bile acid concentrations. To this end, it has been suggested that cholestasis results in decreased expression of the basolateral uptake bile acid transporter, increased expression of export transporters, and increased bile acid sulfation to enhance excretion in urine. Based upon morphologic features, bile acid-induced toxicity and subsequent cell death may be described as necrotic and/or apoptotic, thus leaving unveiled the cellular mechanism of liver injury. Necrosis is characterized by swelling and disruption of plasma membrane integrity with release of intracellular constituents. In contrast, apoptosis is typically associated with cell shrinkage, cytoplasmic and nuclear condensation and fragmentation, and formation of apoptotic bodies surrounded by plasma membranes. The prominence of classic Councilman bodies and cell dropout, rather than extensive cell death, in the liver of cholestatic patients suggests that apoptosis may play an important role in cholestasis. However, the predominant type of liver injury may depend upon several factors such as the cell type, the level of exposure and the metabolic status of the cells.
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Role of Apoptosis in Cholestasis Numerous studies have demonstrated that bile acid concentrations which typically occur during cholestasis trigger apoptosis in liver tissue and both primary rat hepatocyte cultures and HuH-7 human hepatoma cells (Rodrigues & Steer, 2000). Although the cytotoxicity of hydrophobic bile acids to hepatocytes and a variety of other cell types has been attributed to the membrane-disruptive effects from their detergent properties, it is now apparent that more basic cellular mechanisms of injury may also be involved. It is well established that the activation and function of a group of cysteine proteinases, known as caspases, with specificity for aspartic acid residues are crucial in mediating apoptosis (Thornberry & Lazebnik, 2000). These cytosolic proteases are synthesized as zymogens that are proteolytically activated by specific pro-apoptotic pathways. The downstream effects include protein cleavage of key substrates in the cell, disruption of the cytoskeletal framework and nucleus, as well as crippling of a variety of mechanisms for DNA repair. The shorter prodomain caspases (e.g. caspases-3, -6, and -7) are effector or executioner proteases that are cleaved and activated by large prodomain caspases (e.g. caspases-2, -8, -9, and -10) thought to function as initiators. Activation of the latter, in turn, requires the involvement of certain adaptor proteins that bind protein-interaction motifs in the prodomain of initiator caspases. The major mechanisms for caspase activation have been identified, defining two basic pathways of apoptosis involving either death receptors or mitochondria (Green, 2000). Upon activation, the death receptors transduce signals by recruiting death domain-containing proteins, which then initiate caspase activation. The activated caspases, such as caspase-8, efficiently cleaves procaspase-3, and other executioner caspases, and apoptosis proceeds. The second pathway is activated by a variety of stress-induced signals, which are transduced to the mitochondria by members of the BH3-only subfamily of Bcl-2 proteins, acting on the Bax subfamily, and causing the latter to oligomerize and insert into the mitochondrial membrane. Cytochrome c is then released and along with Apaf-1 and caspase-9 activates effector caspases, such as caspase-3, which appear to play critical roles in apoptosis. At least one link between death receptor signaling and the mitochondrial pathway exists in the BH3-only subfamily protein Bid, another Bcl-2 family member. Caspase-8 cleaves Bid, which then targets mitochondria to trigger Bax oligomerization and cytochrome c release. A protein called Smac/DIABLO is released from mitochondria along with cytochrome c during apoptosis. This protein functions to promote caspase activation by associating with the Apaf-1 apoptosome and counteracting the inhibitor of apoptosis proteins (IAPs).
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Several studies have shown that protease activation, mitochondrial dysfunction, and certain patterns of cellular distribution of Bcl-2-related proteins play a role in determining the fate of hepatocytes in various models of cholestasis (Fig. 2). The involvement of the death receptor pathway during bile acid-induced apoptosis has also been investigated in isolated hepatocytes. In fact, it was demonstrated that pathophysiological concentrations of bile acids can induce a ligand-independent death receptor pathway (Faubion et al., 1999). Activation of this apoptotic pathway appears to involve subsequent recruitment of the Fas-associated death domain, activation of caspase-8 as well as downstream effector caspases, including cathepsin B. The function of the Bcl-2 family of proteins in the regulation of apoptosis has been investigated in patients with primary biliary cirrhosis as well as in bile ductligated rats and bile acid-fed animals. Certain members of this family inhibit, while others promote, apoptosis. For example, Bcl-2 and Bcl-xL (Bcl-x long; protein product of long splice variant of bcl-x) are repressors of apoptosis, while Bax and Bcl-xS (Bcl-x short) promote apoptosis. Although it has been shown that Bcl-2, Bcl-xL , and Bax are expressed in the liver, only cholangiocytes and not hepatocytes normally express the anti-apoptotic Bcl-2. However, induction of cholestasis by bile duct ligation leads to Bcl-2 protein expression in hepatocytes, rendering positive cells less vulnerable to apoptosis induced by toxic bile acids (Kurosawa et al., 1997). The ability to limit apoptosis during cholestasis by increasing Bcl-2 expression may represent an adaptive phenomenon to protect hepatocytes as patients with primary biliary cirrhosis show increased nuclear DNA fragmentation and also de novo Bcl-2 expression (Koga et al., 1997). In addition, feeding deoxycholic acid to rats increased the abundance of the anti-apoptotic gene product Bcl-2, while simultaneously increasing translocation of Bax to the mitochondrial membrane (Rodrigues et al., 1998). Bile acids are recognized to play key regulatory functions in gene expression by binding transcription factors involved in a number of cell processes and via post-transcriptional regulation of mRNA levels. Indeed, two cognate nuclear receptors for bile acids have already been described explaining how bile acids can directly modulate gene transcription (Chawla et al., 2001). Several lines of evidence from cell culture studies and bile acid-fed animal models suggest that the cytotoxicity of bile acids may be attributed to mitochondrial dysfunction (Rodrigues & Steer, 2000). The central role of mitochondria in apoptosis was initially suggested in cell-free systems using Xenopus egg extracts in which nuclear condensation and DNA fragmentation were found to be dependent on the presence of mitochondria, and more specifically of cytochrome c. In humans with cholestasis, swollen liver mitochondria are common, and in bile ductligated rats isolated mitochondria show impaired state III respiration. In addition,
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Fig. 2. Schematic Representation of Modulation of Apoptosis by Bile Acids. Note: Toxic bile acids induce apoptosis by activating both death receptor- and mitochondrial-mediated pathways. Activation of the death receptor recruits adaptor molecules and procaspases. Active initiator caspases, such as caspase-8, then act on procaspase-3 that when activated cleaves key substrates in the cell to orchestrate apoptotic death. In addition, stress-induced signals are transduced to the mitochondria by members of the BH3-only subfamily, acting on the Bax subfamily of pro-apoptotic Bcl-2 proteins. The mitochondria release cytochrome c, Smac/DIABLO, the apoptosis inducing factor (AIF), and possibly other apoptogenic proteins. If, however, the cells express Bcl-2 (or Bcl-xL ), then inhibition of the Bid effect will prevent the release of mitochondrial factors. Cytochrome c induces the oligomerization of Apaf-1, which recruits and activates procaspase-9 that then activates procaspase-3. Caspase-8 also cleaves and activates Bid, which translocates to the mitochondria and triggers Bax-subfamily proteins to induce cytochrome c release. Conversely, UDCA prevents apoptosis by stabilizing the mitochondrial membrane, thus inhibiting the PT opening as well as Bax translocation. Finally, toxic bile acids also partially activate an EGFR/MAPK survival pathway, while UDCA strongly activates survival signals that prevent mitochondrial dysfunction and apoptosis. See text for more complete discussion.
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bile acids disrupt state III respiration when added to isolated mitochondria or permeabilized hepatocytes, and deplete ATP in hepatocytes undergoing cell death. Finally, the mitochondrial permeability transition (PT), which is characterized by rapid permeability of mitochondria to low molecular weight solutes, mitochondrial swelling, and collapse of the transmembrane potential, has been implicated as a mechanism of cell death induced by bile acids. Indeed, incubation of isolated rat liver mitochondria with hydrophobic bile acids cause morphologic changes associated with PT as well as mitochondrial depolarization, and cytochrome c release (Rodrigues et al., 1999). The onset of PT clearly precedes and is required for caspase activation, poly(ADP-ribose) polymerase cleavage, and nuclear fragmentation. Finally, toxic bile acids may cause translocation of the pro-apoptotic protein Bax from the cytosol to mitochondria, where it also triggers the release of cytochrome c. A possible explanation of this observation is that Bax displays several distinct detergent-induced conformations that expose defined regions of the protein allowing several degrees of hetero- and homodimerization. Interestingly, liver mitochondria from bile duct-ligated rats demonstrated resistance to bile acid-induced PT, suggesting an adaptive mechanism to minimize toxicity during cholestasis (Lieser et al., 1998). Simultaneous with the opening of the PT pore, the mitochondrial transmembrane potential collapses, thereby uncoupling the respiratory chain and inhibiting ATP biosynthesis during cell injury. This results in production of reactive oxygen species, including superoxide anion and peroxides, and subsequent release of mitochondrial proteins. Several studies have indeed suggested that oxidant stress may play a role in hepatic injury during cholestasis. Accumulation of hydrophobic bile acids can generate free radicals in purified liver mitochondria, isolated rat hepatocytes, and in the bile duct-ligated rat. In addition, it was recently demonstrated that antioxidants reduce bile acid-induced hepatocyte apoptosis by preventing oxidant stress and subsequent stimulation of the PT (Yerushalmi et al., 2001).
Anti-Apoptotic Properties of Ursodeoxycholic Acid In contrast to the toxic effects of several hydrophobic bile acids, ursodeoxycholic acid (UDCA), improves liver function tests in patients with hepatobiliary disorders (Lazaridis et al., 2001). Both the unconjugated form and its amidated conjugates tauroursodeoxycholic acid (TUDCA) and glycoursodeoxycholic acid (GUDCA) are also effective at protecting against the toxicity of hydrophobic bile salts (Rodrigues & Steer, 2000). In fact, the therapeutic effects of UDCA in cholestasis may result, in part, from its ability to inhibit apoptosis (Fig. 2). It has been reported that toxic bile acids fed to rats induce apoptosis in the liver and UDCA inhibits this
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effect in vivo, in part, by preventing translocation of the pro-apoptotic protein Bax from the cytosol to the mitochondria (Rodrigues et al., 1998). These studies were subsequently extended to show that UDCA plays a unique role in modulating the apoptotic threshold in both hepatic and non-hepatic cells, in response to a variety of agents acting through different apoptotic pathways (Rodrigues et al., 1998). In contrast to the effects of hydrophobic bile acids, cells exposed to UDCA alone exhibited no significant apoptotic changes. However, when combined with deoxycholic acid, UDCA almost completely inhibited the morphologic alterations in primary rat hepatocytes and HuH-7 cells associated with the hydrophobic bile acid. In addition, UDCA inhibited the apoptosis induced by ethanol, transforming growth factor 1 (TGF-1), anti-Fas antibody, and okadaic acid, suggesting a basic mechanism that is common to each of the different apoptotic pathways. Interestingly, UDCA was equally protective against these apoptotic agents in nonhepatic cells, such as Saos-2, HeLa and Cos-7 cells. At least one explanation for this ubiquitous anti-apoptotic effect appears to involve the mitochondrial membrane. Indeed, it was demonstrated that UDCA, as well as its taurine and glycine conjugates, prevent the release of cytochrome c and subsequent caspase activation, as well as the change in transmembrane potential induced by several apoptotic stimuli (Rodrigues et al., 1999). Additional mechanisms of action for UDCA may also be engaged, where the bile acid interferes with alternate and upstream molecular targets. In fact, UDCA through direct or indirect mechanisms was a potent inhibitor of TGF-1-induced transcriptional activation of E2F-1-mediated apoptosis (Sol´a et al., 2003). Both transgene overexpression and caspase inhibition suggest that UDCA can specifically modulate the E2F1/p53/Bax pathway, abrogating E2F-1-induced p53 and p53-associated Bax expression, independently of its effect on mitochondria and/or caspases. However, it did not significantly reduce the loss of pRb induced by TGF-1. UDCA also inhibited the down regulation of Bcl-2 by TGF-1, which is consistent with decreased p53 stabilization and/or reduced degradation of nuclear factor B (NFB). Thus, the ability of UDCA to inhibit TGF-1-induced apoptosis appears to involve both stabilization of the mitochondrial membrane and inhibition of Bax translocation, as well as modulation of the E2F-1 apoptotic pathway. As detailed above, Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. Bax can directly destabilize the lipid bilayer structure of the outer mitochondrial membrane, promoting the formation of an apoptotic pore and allowing the subsequent efflux of proteins. It was recently determined that the stabilizing effects of UDCA on membranes could potentially counteract such an action. Mitochondria were isolated from rat liver, exposed to recombinant Bax protein and electron paramagnetic resonance spectroscopy and spin label analysis used to determine
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membrane perturbation (Rodrigues et al., 2003). The results indicated that Bax induced increased polarity of the membrane lipid core in both the superficial and deeper membrane regions, consistent with membrane insertion. In fact, the domain perturbation was associated with the observed increase in membrane permeability. In addition, Bax insertion into the mitochondrial membrane(s) markedly disturbed protein order structure. Pretreatment with TUDCA almost completely abolished Bax-induced lipid and protein structure perturbation, thus inhibiting membrane permeabilization. UDCA was also shown to partially prevent apoptosis via the death receptor pathway, in primary mouse hepatocytes co-cultured with fibroblasts that express the Fas ligand (Azzaroli et al., 2002). However, a nitric oxide derivative of UDCA, but not UDCA, was recently shown to protect against liver damage in murine models of autoimmune hepatitis and to prevent caspase activation in HepG2 cells treated with a Fas agonistic antibody (Fiorucci et al., 2001). In this case, cysteine Snitrosylation by intracellular nitroxide formation, rather than a specific influence of UDCA, was suggested as the main mechanism responsible for caspase inhibition. Questioning the pivotal position of mitochondria in apoptosis, recent studies have suggested an additional endoplasmic reticulum stress-mediated pathway that bypasses mitochondria to directly activate caspases. A variety of conditions can induce oxidative stress of the endoplasmic reticulum and lead to apoptosis, which may have an important role in several liver diseases including nonalcoholic steatohepatitis (NASH), cholestasis, and alcohol-induced liver disease. Although not fully understood, this pathway seems to involve disruption of calcium homeostasis and caspase-12 activation. It appears that UDCA can also prevent endoplasmic reticulum-stress mediated apoptosis (Xie et al., 2002).
Bile Acid Activation of Survival Signaling Pathways Bile acids may prevent and counteract their inherent cytotoxicity by activating cell survival pathways (Fig. 2). These pathways include the NF-B, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, calcium-dependent signal transduction cascades, and even cyclic adenosine monophosphate (cAMP). Indeed, bile acids have been shown to regulate several genes, such as cholesterol 7␣-hydroxylase, major histocompatibility complex class I, and cyclooxygenase-2, through activation of PKC. Its activation is observed in a matter of minutes and at bile acid concentrations that are within the physiological range. In addition, hydrophobic bile acids seem to be better activators of PKC than more hydrophilic species (Stravitz et al., 1995). With respect to apoptosis, the PKC family includes anti-apoptotic members, such as PKC-␣, and pro-apoptotic elements, such as PKC-␦. Thus, the ultimate net effect
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of bile acids may depend on the signal balance between pro- and anti-apoptotic PKC isoenzymes or even activation of MAPKs. Receptor protein tyrosine kinases and activated PKC have been shown to contribute to the activation of several downstream kinase cascades, such as the MAPKs. MAPKs are a superfamily of homologous enzymes that integrate and transduce intracellular and extracellular signals into the nucleus, serving in three parallel cascades, including the extracellular signal-regulated kinase pathway (ERK), the p38 pathway, and the JNK pathway. Although specific biological functions of these pathways are difficult to determine, because of their extensive cross talk, the ERK pathway generally transduces growth factor signals involved in differentiation, proliferation and secretion, while the others function as stress signal transducers. Each MAPK cascade is characterized by a cassette of three enzymes, which sequentially phosphorylate and activate a downstream kinase and finally transcription factors. That cytotoxic stresses can signal the MAPK pathway via activation of the epidermal growth factor receptor (EGFR) supports the notion that certain stresses may have a self-limiting effect upon their toxicity due to activation of the MAPK pathway. Recent studies show that deoxycholic acid causes ligand-independent activation of EGFR and Fas receptors in primary hepatocytes (Qiao et al., 2001). In this case, EGFR signaling, via the MAPK pathway, counteracted deoxycholic acid-induced apoptosis. Further, UDCA can also stimulate the activation of the intracellular MAPK signaling (Qiao et al., 2002). This UDCA/EGFR/MAPK pathway represents a survival cascade that inhibits bile acid-mediated cytotoxicty. In fact, when UDCA-induced MAPK signaling was abolished with inhibitors, bile-acid induced apoptosis was potentiated. The UDCA/EGFR/MAPK pathway probably inhibits apoptosis by blocking mitochondrial dysfunction. Finally, the PI3K signaling pathway appears to represent an important component of the net effect of survival pathways induced by bile acids. In fact, PKC isoforms are downstream effectors of PI3K-dependent survival signals, as is Akt, the cellular homolog of the viral oncoprotein -Akt, known to suppress apoptosis in several cell types. PKC- was found to be a critical component of the taurochenodeoxycholate-induced PI3K signaling cascade, and its activation resulted in NF-B-dependent transcriptional activity (Rust et al., 2000). Activation of NF-B represents an important survival pathway, which results in the induction of genes that regulate the Bcl-2 family and caspase function.
Alternative Mechanisms of Ursodeoxycholic Acid Effects There is increasing experimental and clinical evidence to suggest that other mechanisms are involved in the protective effect of UDCA in cholestatic
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disorders (Paumgartner & Beuers, 2002). These include its ability to prevent bile duct injury by toxic bile acid species, and stimulate impaired hepatocellular secretion of hydrophobic bile acids. Conjugates of UDCA counteract the effects of high concentrations of more hydrophobic bile acids in vitro, perhaps through modulation of structure and composition of mixed phospholipid-rich micelles in bile. However, because millimolar concentrations of bile acids are present only within the biliary lumen, this protective effect will be restricted to the biliary tree. The effects of UDCA on cholangiocytes are apparently mediated by calcium and PKC-␣-dependent mechanisms, which is similar to its anticholestatic effect in hepatocytes as discussed below. Further, UDCA has been shown to stimulate secretion of bile acids and other organic compounds in the bile fistula rat, in isolated hepatocytes and perfused rat liver and, more recently, in patients with primary biliary cirrhosis and primary sclerosing cholangitis. A possible explanation for UDCA-induced choleresis involves its ability to regulate the expression of transporter proteins for biliary secretion in the hepatocyte. In addition, UDCA may stimulate hepatobiliary vesicular exocytosis and insertion of carrier proteins into the apical membrane of the hepatocyte. Carriermediated transport across the canalicular membrane is the rate-limiting step in hepatocellular secretion of numerous cholephilic substrates including bile acids, and bilirubin and glutathione conjugates. Canalicular membrane transport activity is modulated by the insertion and probably retrieval of transport proteincarrying vesicles into this domain. This mechanism is defective in experimental cholestasis and, therefore, it has been hypothesized that TUDCA stimulates hepatobiliary exocytosis and insertion of the apical conjugate export pump, Mrp2, into the canalicular domain of hepatocytes (Beuers et al., 2001). In fact, TUDCA induces a sustained increase of cytosolic free calcium in isolated hepatocytes by depleting inositol 1,4,5-triphosphate-sensitive microsomal calcium stores, and by increasing calcium influx across the plasma membrane (Beuers et al., 1993). It also selectively translocates the calcium-sensitive ␣-isoform of PKC, a mediator of calcium-stimulated exocytosis, to hepatocellular membranes (Beuers et al., 1996). This results in a higher canalicular transport and, consequently, in the reestablishment of the biliary efflux. It has also been suggested that TUDCA increases the canalicular bile acid transport via activation of MAPKs (Schliess et al., 1997). The therapeutic efficacy of UDCA might be related to its ability to modulate cell-mediated immunity, such as the reversal of aberrant expression of the major histocompatibility complex class I molecules in hepatocytes (Calmus et al., 1990). However, the conclusions taken from these results must be carefully considered, as this immunomodulatory effect may be due to the replacement of
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immunosuppressor hydrophobic bile acids, rather than a direct effect of UDCA on the immune system.
BILE ACIDS FOR THE TREATMENT OF NEURODEGENERATIVE DISORDERS UDCA modulates cell death at several levels suggesting an essential function of this bile acid in regulating apoptosis. Its therapeutic role has been established in the treatment of certain liver diseases and, perhaps, should also be considered for non-liver diseases in which increased levels of apoptosis contribute to their pathogenesis. Mitochondrial dysfunction and subsequent oxidative damage have been implicated in several neurobiological disorders, such as acute stroke and chronic neurodegenerative diseases. It was initially described that neuronal cell death associated with these disorders was necrotic in nature, but recent data suggest that apoptotic cell death also plays a crucial role. As a general modulator of cell survival, TUDCA may be a potential therapeutic agent. Several recent studies have examined the mitoprotective and anti-apoptotic properties of TUDCA in a variety of cell types and several models of neurological disorders, including Huntington’s disease (HD), Parkinson’s disease, and acute ischemic and hemorrhagic stroke.
TUDCA is Neuroprotective in Pharmacologic and Transgenic Animal Models of Huntington’s Disease HD is a genetically dominant neurological disorder caused by abnormal expansion of the trinucleotide (CAG) repeat sequence in exon 1 of the gene Htt encoding the huntingtin protein. Transgenic mice overexpressing the protein with a normal polyglutamine tract do not develop the disease phenotype. In contrast, mice expressing either an intact or truncated protein with the polyglutamine expansion develop neuropathology reminiscent of the human disease. This expansion results in specific cell loss in the neostriatum and mitochondrial insufficiency as prominent features of HD neuropathology. In fact, mitochondrial compromise in HD is demonstrated by abnormal energy metabolite concentrations and utilizations, impaired striatal mitochondrial respiratory chain complex II/III activity and increased stress-induced mitochondrial depolarization, free radical production, and associated oxidative damage. A consequence of mitochondrial dysfunction in neuronal cells may be the activation of certain apoptotic pathways. DNA fragmentation is elevated in HD neostriatum and the frequency of apoptotic cells
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is positively correlated with the length of CAG repeats. In addition, lymphoblasts isolated from HD patients are more susceptible to cell death mediated through the mitochondrial pathway of apoptosis. Finally, caspases, crucial for the initiation and execution of apoptosis are elevated and activated in HD brain. Expression of a dominant negative caspase-1 mutant resulted in extended lifespan and delayed onset of neuropathology and behavioral deficits in transgenic HD mice (Ona et al., 1999). Chronic administration of 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, provides a relatively accurate animal model of HD, closely resembling the pathology and symptoms of this neurodegenerative disease. Metabolic compromise, followed by activation of excitatory amino acid receptors, and free radical production appears to play a major role in the pathogenesis of the lesion. Several drugs have been shown to have a neuroprotective effect in 3-NPtreated rats as a result of decreased excitotoxicity. More recently, various studies have reported positive results with caspase inhibition. For example, transgenic mice expressing a caspase-1 dominant negative mutant were resistant to 3-NP (Andreassen et al., 2000). The systemic administration of TUDCA in the 3-NP rat model of HD significantly improved the morphologic striatal lesions (Keene et al., 2001). Indeed, TUNEL labeling of striatal cells was markedly reduced by TUDCA treatment, whereas lesion sizes were smaller and striatal volumes noticeably larger (Fig. 3). Moreover, behavioral studies correlated well with histological findings, since the significant neuroproptection resulted in almost complete prevention of hyperactive behavior associated with 3-NP administration and maintained neophobia characteristic of cognitively intact animals. In addition to this in vivo evidence, other studies have also characterized the role of apoptosis in 3-NP-induced death in cultured neuronal cells and shown that TUDCA markedly reduced the mitochondrial perturbations associated with apoptosis (Rodrigues et al., 2000). Coincubation with TUDCA was able to rescue 3-NP-induced changes of nuclear morphology (Fig. 3), mitochondrial swelling and membrane disruption. Similarly, cytochrome c efflux was partially prevented by TUDCA in both isolated mitochondria and intact cells. In contrast, none of these processes was inhibited by coincubation with the hydrophilic bile acids, hyodeoxycholic acid or taurocholic acid, or the PT inhibitor cyclosporine A. Thus, TUDCA appears to act by blocking 3-NP-induced mitochondrial membrane perturbation via a megapore opening-independent effect. In contrast, as discussed above, the inhibition of deoxycholic acid-induced mitochondrial disruption and cytochrome c release occurred primarily through inhibition of the PT, suggesting two potentially independent mechanisms of TUDCA cytoprotection. TUDCA effects may, in fact, involve prevention of both non-selective and selective permeabilization of the mitochondrial membrane. Membrane stability
The Role of Bile Acids in the Modulation of Apoptosis Fig. 3. TUDCA Inhibits 3-NP-Induced Cell Death in vitro and in vivo. Note: A, Fluorescence microscopy of Hoechst staining after incubation of neuronal RN33B cells with either no addition (a), 3-NP (b), TUDCA (c), or 3-NP plus TUDCA (d). Apoptotic cells were identified by morphological changes characterized by condensed chromatin, fragmentation, and apoptotic bodies (from Rodrigues et al., 2000). B, 3-NP-induced lesions in a rat model of Huntington’s disease detected by reduced neuronal immunoreactivity. Lesion volume is significantly greater in 3-NP (a) compared with 3-NP plus TUDCA (b) rats sacrificed 6 weeks following 3-NP administration. This effect is amplified in tissue from animals sacrificed 8 months after the final 3-NP injection (c and d). Additionally, long-term 3-NP rats (c) have smaller striatal volumes and larger ventricles than their 3-NP plus TUDCA counterparts (d). From Keene et al. (2001). 133
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would, of course, inhibit cytochrome c release and, thereby, modulate cytochrome c-mediated downstream events, such as caspase activation and endogenous substrate cleavage. 3-NP-induced disruption of the outer mitochondrial membrane may be responsible for the initial cytochrome c release from mitochondria through a non-specific, cyclosporine A-independent permeabilization. This, in turn, could stimulate Bax translocation to the mitochondria and a subsequent amplification of cytochrome c efflux via a Bax-mediated process. In fact, during 3-NP induced apoptosis, Bax protein levels decreased in the cytosol and increased proportionally in mitochondria. Such observations support the alternate and apparently PT-independent mechanism of cytochrome c release. Both selective and non-selective permeabilization of the mitochondrial membrane appear to be significantly inhibited by TUDCA. These data suggested that TUDCA could possibly act as a therapeutic agent for regulating cell survival in HD. Thus, the initial studies were extended to a transgenic mouse model of HD that provides certain advantages over the pharmacologic 3-NP model and, therefore, may more accurately reflect the true pathophysiology of HD. Specifically, it results from genetic rather than chemical alterations, and involves chronic versus acute pathophysiology. We examined the effects of TUDCA in the well-characterized R6/2 transgenic mouse model of HD, which contains a trinucleotide CAG expansion (∼150 repeats) of Htt exon 1. Mutant, N-terminal huntingtin fragments are strong inducers of caspase activation and apoptosis. The mice are found to exhibit severe neuropathophysiology and associated neurodegeneration with concomitant sensorimotor deficits, and typically die at ∼14 weeks of age. In addition, they exhibit prominent striatal atrophy and formation of neuronal intranuclear inclusions. Administration of TUDCA beginning at age 6 weeks and continuing until 12 weeks led to a marked reduction in striatal cell apoptosis and degeneration of R6/2 transgenics as shown in Fig. 4 (Keene et al., 2002). In fact, the TUDCAtreated transgenic animals were not significantly different from vehicle and TUDCA-treated wild-type mice. We then examined if TUDCA, by preventing neuronal degeneration and death, would also reduce striatal atrophy. Control R6/2 mice exhibited prominent atrophy compared with control and TUDCAtreated wild-type mice, whereas measurements from TUDCA-treated R6/2 mice showed significantly less cerebral atrophy compared with untreated transgenic animals. Striatal volumes were subsequently determined using Nissl stained sections from control and TUDCA-treated animals. Both untreated and TUDCAtreated R6/2 mice showed reduced striatal volumes when compared with wildtype controls; however, the reduction with TUDCA was almost 50% less than that seen in the untreated transgenic animals. In addition, we investigated whether TUDCA treatment could inhibit mutant huntingtin aggregation, as formation
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Fig. 4. TUDCA is Neuroprotective in a Transgenic Animal Model of Huntington’s Disease. Note: A, TUNEL-stained sections show that control vehicle (a) and TUDCA-treated (b) wild type control mice exhibit less apoptosis compared with control transgenic mice (c). TUDCA-treated R6/2 mouse striata (d) contained significantly fewer apoptotic cells compared with untreated transgenic mice. B, Nissl-stained sections show that wild type vehicle (a) and TUDCA (b) controls had larger striatal volumes compared with R6/2 controls (c). TUDCA-treated mouse striatal volume (d) was significantly larger than untreated R6/2 mice. The striatum of untreated wild type animals (a) is outlined (broken line) for reference and superimposed (solid line) on striata from each of the other groups (b–d). C, Immunohistochemistry using an antibody specific for ubiquitin shows no ubiquitinated neuronal inclusions in vehicle (a) and TUDCA-treated (b) wild type mice. In contrast, vehicle (c) and TUDCA-treated (d) R6/2 mouse striatum contained extensive aggregate formation of the huntingtin protein. Quantitative analysis revealed significantly fewer aggregates in TUDCA-treated transgenic animals compared with R6/2 control mice. Further, inclusions were significantly smaller in the TUDCA group (inset). From Keene et al. (2002).
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of intracellular inclusions composed of huntingtin and ubiquitin is a common pathological hallmark of HD in the transgenic mouse models. TUDCA decreased both the average size and number of individual intracellular inclusions (Fig. 4), significantly improving HD pathology. Finally, and not surprisingly, locomotor and sensorimotor abilities improved substantially in HD mice treated with TUDCA. Age and task difficulty influenced the observed degree of behavioural recovery.
Improved Survival and Function of Nigral Transplants in a Rat Model of Parkinson’s Disease Transplantation of human embryonic dopamine neurons in patients with Parkinson’s disease is currently being evaluated in clinical trials. Although transplantation of nigral tissue ameliorates functional deficiencies characteristic of Parkinson’s disease, a major hurdle for successful neural grafting is the poor survival of dopaminergic neurons grafted in recipient patients. Further, an improvement in survival of dopamine neurons could circumvent the limited availability of human embryonic dopamine tissue that also remains a major barrier in the clinical application of neural transplantation. Although a variety of factors may contribute to the high frequency of incomplete symptomatic recovery, the loss of transplanted dopamine neurons appears to be paramount. A number of animal experiments and clinical studies have shown that the survival rate of grafted dopamine neurons is only about 5–10% when dopaminergic neurons are implanted using a cell suspension technique. The enhancement of dopamine neuronal survival is, therefore, a pertinent issue in improving the clinical response to cell implantation. There is growing evidence showing that the majority of cell death in neuronal grafts results from apoptosis when dopamine neurons are implanted into the brain. In fact, the number of implanted cells increased several-fold with disruption of the apoptotic pathways by caspase inhibitors or other anti-apoptotic agents. In addition, apoptosis normally takes place within the first several days after transplantation. Thus, the majority of cell death in nigral grafts occurs either immediately or shortly after transplantation. These studies suggest that anti-apoptotic agents should be included in the preparation of the cell suspensions prior to transplantation and during the first few days after transplantation to prevent apoptosis and improve graft survival. Several factors such as mechanical damage, anoxia, and nutritional insufficiency during the implantation procedure may lead to the production of reactive oxygen species and immediate cell death in the graft area. In addition, the new environment in the striatum does not favor nigral survival as the neural grafts may lack sufficient
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neurotrophic factor support. Based on this notion, a variety of strategies have been designed to prevent cell death of neural transplants. In recent studies, treatment of embryonic nigral cells immediately after donor tissue preparation with caspase inhibitors has been shown to increase survival rate of transplanted dopamine neurons and improve graft function (Schierle et al., 1999). Prompted by these results, an in vivo transplantation model has been used to determine whether TUDCA could block apoptotic pathways, enhance dopamine neuron survival and improve nigral graft function (Duan et al., 2002). First, cell culture experiments examined the effects of TUDCA on the survival of dopamine neurons in serum-free conditions. The results showed that the number of tyrosinehydroxylase-positive neurons in the TUDCA-treated cultures was significantly greater than control cultures at 7 days (Fig. 5). In addition, by TUNEL assay the number of apoptotic cells was dramatically reduced with TUDCA. In the transplantation study, TUDCA was added to the media when nigral tissue was trypsined and dissociated from day 14 rat embryos. Cell suspensions containing TUDCA were then stereotaxically injected into the striatum of adult rats subjected to an extensive unilateral 6-hydroxydopamine lesion of the nigrastriatal dopamine pathway. At two weeks post-transplantation, four of six rats that received a cell suspension incubated with TUDCA exhibited at least 50% reduction in amphetamine-induced rotation scores compared to pre-transplantation values. In contrast, none of the control animals showed 50% reduction in motor asymmetry. Further, at 4 days after transplantation, the number of apoptotic cells was much smaller in the graft areas of TUDCA-treated groups, whereas at 6 weeks postgrafting tyrosine-hydroxylase positive cells were more than 3-fold increased. The areas of the host striatum that were reinnervated by the grafts were larger in the TUDCA-treated group than in controls (Fig. 5). Thus, TUDCA may prove to have therapeutic benefit over other drugs because it is devoid of any toxicity, allowing it to be used not only during graft preparation to reduce the number of donor embryos required, but also after transplantation to improve cell survival and behavioral recovery.
Neuroprotection by TUDCA in Animal Models of Acute Stroke Neuronal cell death from acute stroke is a complex process and appears to involve a variety of different pathways and potential mechanisms. The ensuing cell death was originally thought to be primarily necrotic. However, a number of recent studies suggest that apoptosis plays a key role in neuronal cell death from ischemic and hemorrhagic stroke. In the ischemia-reperfusion injury, neurons exhibit several features of apoptosis, including chromatin condensation, TUNEL
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Fig. 5. TUDCA Improves Survival of Dopaminergic Neurons in vitro and After Transplantation in a Rat Model of Parkinson’s Disease. Note: A, Dopamine producing tyrosine hydroxylase-positive cells were less abundant in untreated (a) than in TUDCAtreated (b) cultures at 7 days in vitro. B, Photomicrographs of grafted striatum processed for tyrosine hydroxylase immunhistochemistry illustrate typical grafts from controls (a) and TUDCA-treated neuronal grafts (b) at 6 weeks post-transplantation. A cell suspension technique was used to perform the neural transplants, after obtaining the ventral mesencephalic tissue from embryos with a gestational age of day 14. From Duan et al. (2002).
labeling, and caspase activation. Bax, a pro-apoptotic member of the Bcl-2 family, is increased in the core and in cells at the periphery of the ischemic infarct showing DNA fragmentation. In contrast, the anti-apoptotic proteins Bcl-2 and Bcl-xL are increased in neurons adjacent to an infarct that survive ischemic brain injury. Caspase-3, a well-characterized executioner caspase in apoptosis, is also
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activated following transient cerebral ischemia. In addition, there is evidence that caspase-1 activation could play a pivotal role in ischemic neurodegeneration. Not surprisingly, caspase inhibitors afford significant ischemic and excitotoxic neuronal damage (Hara et al., 1997). The mechanism of neuronal loss after hemorrhagic stroke is less well described, in part, because of a lack of adequate experimental models. However, caspase inhibitors have recently been shown to inhibit DNA fragmentation after intracerebral hemorrhage from collagenase injection into the rat striatum (Matsushita et al., 2000). Also, antisense inhibition of tumor necrosis factor-␣ reduced the number of apoptotic cells after intracerebral hemorrhage in rats (Mayne et al., 2001). Prompted by these studies, a well-characterized rat model of transient focal cerebral ischemia was studied to assess the protective effect of TUDCA (Rodrigues et al., 2002). Marked cell death with prominent TUNEL-labeling was observed within the ischemic penumbra, as well as mitochondrial swelling and caspase activation. A single intravenous dose of TUDCA given 1 or 2 h after middle cerebral artery occlusion, resulted in improved neurologic function and reduced infarct volumes by ∼50% 2 and 7 days after reperfusion (Fig. 6). In addition, TUDCA significantly reduced the number of TUNEL-positive cells, prevented mitochondrial swelling and membrane disruption, and partially inhibited downstream caspase activation and endogenous substrate cleavage associated with apoptosis. Finally, the neuroprotective effect of TUDCA was long lasting, suggesting that the benefit was not simply due to a delay in cell death. These initial studies have more recently been extended to test the potential benefit of TUDCA in a collagenase-induced hemorrhagic model of stroke (Rodrigues et al., 2003). Intracerebral hemorrhage remains a devastating cerebrovascular event with a mortality rate approaching 50%. Therapies have been largely ineffective in reducing morbidity and mortality, and clinical trials have lagged far behind those for patients with ischemic stroke. Injury is thought to arise from tissue reaction secondary to the hematoma resulting in ischemia, edema, intense inflammation, and ultimately cell death. Characteristic changes of apoptosis were detected after hemorrhagic stroke, including increased numbers of TUNEL-positive cells, and significant caspase activation in the region immediately surrounding the hematoma. This narrow zone of selective neuronal injury may significantly add to the area of stroke injury unless rescued from apoptosis. In fact, animals treated with caspase inhibitors and genetically engineered caspasedeficient mice showed reduced cell death from ischemic stroke. TUDCA, in turn, significantly reduced the appearance of TUNEL-positive cells, activation of caspase-3-like proteases, and histological damage of the peri-hematoma region (Fig. 7). In addition, the tissue and biochemical changes were also associated with improved neurological function. This data suggested that many neurons can be
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Fig. 6. TUDCA Reduces Infarct Volume and Prevents Mitochondrial Disruption After Middle Cerebral Artery Occlusion in The Rat. Note: A, At two days after reperfusion, 2,3,5-triphenyltetrazolium chloride stained coronal sections of the brain in vehicle-injected rats (a) exhibit greater lesion volumes compared with animals receiving TUDCA (b) given one hour after ischemia. B, Seven days after reperfusion, electron micrographs of brain tissue in vehicle-injected animals (a) show swollen mitochondria, with outer membrane rupture and loss of cristae, as compared with normal appearing mitochondria in TUDCA-treated rats (b). From Rodrigues et al. (2002).
rescued by TUDCA within 3 h, and some up to 6 h after intracerebral hemorrhage. In fact, TUDCA administered up to 3 h after intracerebral hemorrhage significantly reduced the striatal lesions by almost 50%. It is now well established that NF-B activation may play a prominent role in acute stroke injury. NF-B activation increases transcription of a number of different genes, including those involved in inflammation as well as apoptosis. The
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Fig. 7. TUDCA Reduces Neurlogical Injury After Acute Hemorrhagic Stroke in the Rat. Note: A, coronal section of the brain two days after intracerebral hemorrhage (ICH). The ipsilateral (Ipsi) ICH core and its periphery (Peri-ICH) are outlined. B, Nissl-stained striatal sections show that total lesion volume was markedly greater in vehicle-injected controls (a) compared with animals receiving TUDCA (b) given 1 hr before collagenase injection. The vehicle lesion is outlined for reference and superimposed on the striatum of TUDCA animals. From Rodrigues et al. (2003).
precise role of NF-B in stroke, however, remains unclear. In fact, some studies suggest that NF-B activation mediates injury, while others suggest a protective role. NF-B DNA binding activity is increased after experimental intracerebral hemorrhage and activation is frequently colocalized to cells containing fragmented DNA, suggesting a pro-apoptotic role. Also, reduced expression of the proinflammatory cytokine tumor necrosis factor-␣, a key mediator of NF-B activation, has been shown to be neuroprotective after intracerebral hemorrhage. NF-B activation was significantly decreased by TUDCA administration, Bcl-2 levels remained
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elevated, and rats showed improved neurological function. In addition, TUDCA resulted in Bad phosphorylation through activation of the Akt survival pathway. These results suggest that TUDCA can significantly reduce the injury associated with intracerebral hemorrhage. It appears to inhibit apoptosis by preserving mitochondrial membrane stability, as well as through activation of certain survival pathways. The marked anti-apoptotic properties of TUDCA together with its lack of toxicity and potential use up to 6 h post-injury make it an attractive candidate in the treatment of hemorrhagic stroke, as well as perhaps other acute neurological disorders, such as head and spinal cord injuries, where metabolic compromise renders neurons more susceptible to cell death.
SUMMARY Bile acids are inherently toxic molecules, which can have a profound influence on cellular function and structure. The proposed mechanisms of bile acidinduced hepatocyte damage range from a simple detergent effect on binding to plasma membranes to the induction of cell death. Bile acids induce apoptosis via ligand-independent, death receptor pathways, involving the Fas receptor and also through classic mitochondrial pathways. In contrast, the oral administration of UDCA and TUDCA has been used clinically for years to treat cholestasis and a number of other liver diseases. There is now strong evidence that the cytoprotective mechanism of UDCA and its conjugates results from their ability to inhibit apoptosis in hepatic cells. Interestingly, the anti-apoptotic properties of these bile acids are independent of the cell type and death signal, suggesting a common anti-apoptotic mechanism(s). Specifically, UDCA prevents apoptosis by modulating mitochondrial membrane perturbation, opening of the permeability transition pore, Bax translocation, cytochrome c release, and subsequent caspase activation and substrate cleavage. In addition, apoptosis can not only be inhibited by blocking pro-apoptotic pathways but also by activating survival signals for example through the cAMP, Akt, NF-B, MAPK and PI3K-mediated pathways. Bile acids, including UDCA, are potent intracellular signaling molecules with crucial regulatory properties. Thus, by acting as general modulators of cell function, UDCA and TUDCA may play an essential role as therapeutic agents in the treatment of non-liver diseases in which cell survival is compromised, such as in neurodegenerative disorders. Indeed, animal experiments have provided evidence that TUDCA may be effective in ameliorating the HD phenotype in a pharmacological rat model as well as in a transgenic mouse model. The ability of TUDCA to prevent neurologic insult, however, may not be confined only to HD. In fact, treatment of embryonic nigral cells improves graft survival and function
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in a rat model of Parkinson’s disease, while acute neuronal injury associated with ischemic or hemorrhagic stroke may be reduced by administration of TUDCA. As a hydrophilic bile acid, TUDCA is readily water-soluble, can be administered orally or intravenously, and is associated with minimal toxicity, making it a promising drug for potential further clinical application.
ACKNOWLEDGMENTS The authors thank the members of the laboratories and, in particular, Dr. Walter C. Low of the Department of Neurosurgery, University of Minnesota for their help during the course of these studies.
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Rodrigues, C. M. P., Ma, X., Linehan-Stieers, C., Fan, G., Kren, B. T., & Steer, C. J. (1999). Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation. Cell Death Differ, 6, 842–854. Rodrigues, C. M. P., Sol´a, S., Nan, Z., Castro, R. E., Ribeiro, P. S., Low, W. C., & Steer, C. J. (2003). Tauroursodeoxycholic acid reduces apoptosis and protects against neurological injury after acute hemorrhagic stroke in rats. Proc. Natl. Acad. Sci. USA, 100, 6087–6092. Rodrigues, C. M. P., Sol´a, S., Sharpe, J. C., Moura, J. J., & Steer, C. J. (2003). Tauroursodeoxycholic acid prevents Bax-induced membrane perturbation and cytochrome c release in isolated mitochondria. Biochemistry, 42, 3070–3080. Rodrigues, C. M. P., Spellman, S. R., Sol´a, S., Grande, A. W., Linehan-Stieers, C., Low, W. C., & Steer, C. J. (2002). Neuroprotection by a bile acid in an acute stroke model in the rat. J. Cereb. Blood Flow Metab, 22, 463–471. Rodrigues, C. M. P., & Steer, C. J. (2000). Mitochondrial membrane perturbations in cholestasis. J. Hepatol, 32, 135–141. Rodrigues, C. M. P., Stieers, C. L., Keene, C. D., Ma, X., Kren, B. T., Low, W. C., & Steer, C. J. (2000). Tauroursodeoxycholic acid partially prevents apoptosis induced by 3-nitropropionic acid: Evidence for a mitochondrial pathway independent of the permeability transition. J. Neurochem, 75, 2368–2379. Russell, D. W. (2003). The enzymes, regulation, and genetics of bile acid synthesis. Annu. Rev. Biochem., 72, 137–174. Rust, C., Karnitz, L. M., Paya, C. V., Moscat, J., Simari, R. D., & Gores, G. J. (2000). The bile acid taurochenodeoxycholate activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade. J. Biol. Chem, 275, 20210–20216. Schierle, G. S., Hansson, O., Leist, M., Nicotera, P., Widner, H., & Brundin, P. (1999). Caspase inhibition reduces apoptosis and increases survival of nigral transplants. Nat. Med, 5, 97–100. Schliess, F., Kurz, A. K., vom Dahl, S., & Haussinger, D. (1997). Mitogen-activated protein kinases mediate the stimulation of bile acid secretion by tauroursodeoxycholate in rat liver. Gastroenterology, 113, 1306–1314. Sol´a, S., Ma, X., Castro, R. E., Kren, B. T., Steer, C. J., & Rodrigues, C. M. P. (2003). Ursodeoxycholic acid modulates E2F-1 and p53 expression through a caspase-independent mechanism in TGF-1-induced apoptosis of rat hepatocytes. J. Biol. Chem., 278, 48831–48838. Stravitz, R. T., Vlahcevic, Z. R., Gurley, E. C., & Hylemon, P. B. (1995). Repression of cholesterol 7 ␣-hydroxylase transcription by bile acids is mediated through protein kinase C in primary cultures of rat hepatocytes. J. Lipid Res, 36, 1359–1369. Thornberry, N. A., & Lazebnik, Y. (2000). Caspases: Enemies within. Science, 281, 1312–1316. Xie, Q., Khaoustov, V. I., Chung, C. C., Sohn, J., Krishnan, B., Lewis, D. E., & Yoffe, B. (2002). Effect of tauroursodeoxycholic acid on endoplasmic reticulum stress-induced caspase-12 activation. Hepatology, 36, 592–601. Yerushalmi, B., Dahl, R., Devereaux, M. W., Gumpricht, E., & Sokol, R. J. (2001). Bile acid-induced rat hepatocyte apoptosis is inhibited by antioxidants and blockers of the mitochondrial permeability transition. Hepatology, 33, 616–626.
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6.
GROWTH FACTORS AND THE LIVER
Clare Selden INTRODUCTION Unless the liver is subjected to trauma, most hepatocytes are in the resting phase of the cell cycle, G0, the differentiated state; less than one cell in a thousand is undergoing DNA synthesis in the normal liver at any time. However, that the liver has a remarkable capacity to regenerate after traumatic injury has been known for centuries. Classical Greek mythology illustrates the point beautifully with the eagle who plucked out Prometheus’s liver by day only to find it had regrown by morning! Somewhat more recently, Cruveilhier in 1833 (Cruveilhier, 1833) and Budd in 1945 (Budd, 1945) described the event of regeneration of the liver in man. Today, with the advent of CT scanning one can illustrate the phenomenon in patients undergoing hepatic resections for removal of tumors in the liver (Fig. 1). The ability of hepatocytes to undergo mitosis is retained throughout the life of the animal, although in aged animals the response is slower and smaller (Bucher et al., 1964).
THE PROLIFERATIVE STIMULUS? The exact signal for the liver to begin regenerating is unknown. There is evidence that the increased metabolic load on the remaining liver after experimental partial hepatectomy produces some stimulus (Bucher & McGowan, 1985; McGowan et al., 1984; Mead et al., 1990; Ngala Kenda et al., 1984). This could also be reflected in man after hepatic resection. Whatever the initial signal it is apparent The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 147–166 © 2004 Published by Elsevier Ltd. ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15006-X
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Fig. 1. CT Scan of Patient with Fibrolammelar Hepatoma: (a) Before Operation Showing Large Mass in Right Lobe; and (b) 26 Days Post Right Lobe Hepatectomy, Showing Extensive Left Lateral Lobe Regeneration of Remaining Liver.
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that it is blood borne, since in parabiotic rats where one rat undergoes a partial hepatectomy this leads to a proliferative response in the second animal with an intact liver (Moolten & Bucher, 1967). If this experiment was performed with totally hepatectomized rats the effect was still observed suggesting that at least one initial stimulus does not come from within the liver (Greisler et al., 1979) In addition, performing a partial hepatectomy in rats with hepatocytes ectopically transplanted into the spleen 10 months before, leads to a doubling of the mitotic rate of the ectopic hepatocytes (Gupta et al., 1987) (Fig. 2) suggesting the factors are in the systemic circulation, and not simply in the portal blood. Portal hepatotrophic factors, e.g. insulin and glucagon, were initially thought to account wholly for
Fig. 2. Labeling Index of Hepatocytes Transplanted Ectopically into Spleens of Syngeneic Rats 10 Months Prior to a 70% Hepatic Resection in Recipient (Closed Circles). Note: Labeling index is defined as the number of cells actively synthesising DNA as a proportion of the total, expressed as labeled cells per thousand cells. Control rats (open circles) underwent sham hepatectomy. Note that hepatocytes in sham operated animals were proliferating at almost 10-fold more than resting hepatocytes in normal liver (resting labeling index <1 in 1000 hepatocytes). Hepatectomy led to a doubling of the labeling index for ectopically transplanted hepatocytes.
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liver regeneration (Starzl et al., 1973), but this was an oversimplification. Factors produced immediately after removal of or damage to part of the liver, are thought to lead to growth factor production, which induce DNA synthesis in the liver. Although the growth factors are the subject of this chapter; they do not arise independently of other events associated with the cell cycle, so I shall also discuss these briefly.
EARLY EVENTS AFTER A PROLIFERATIVE STIMULUS Within minutes of surgical removal of part of the liver there are changes in the expression of a group of genes associated with cell cycle events, which do not require de novo protein synthesis for their increased activity (Lau & Nathans, 1985). In addition there are alterations in membrane polarization and fluidity (Mahler et al., 1988a, b; Wondergem & Harder, 1980), ionic fluxes (Na+ and Ca++ ) and intracellular pH. There are modifications in the sympathetic innervation occurring during regeneration: norepinephrine, a neurotransmitter for the sympathetic nervous system, is increased after experimental hepatectomy: in vitro, in hepatocyte cultures, this substance acts to enhance the effect of hepatomitogens.
Fig. 3. Diagram Illustrating Various Phases of Cell the Cell Cycle.
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THE CELL CYCLE Each stage of the cell cycle has a prescribed length; during liver regeneration the overall cell cycle time is shortened compared with cells undergoing cell division in the normal liver (Fabrikant, 1968). Figure 3 illustrates the different phases of the cycle and the essential functions of each phase. Growth factors and inhibitors can act at each of the transition points, from G0 to G1, G1 to S or G2 to mitotic phase.
PARADIGMS OF LIVER GROWTH Since the knowledge of growth factors in the liver has been gained mostly from events occurring after a major regenerative stimulus, in this chapter I will discuss the role growth factors and cytokines play during liver regeneration. Much of the experimental evidence has been gleaned in a model of liver growth initiated by a 70% hepatectomy, first described by Higgins and Anderson (1931), although there is also the regenerative response stimulated by disease such as cirrhosis and fulminant liver failure. Since the liver is made up of both parenchymal cells, (hepatocytes) and non-parenchymal cells (including for example Kupffer cells, macrophages, biliary epithelial cells, hepatic stellate fat storing cells (lipocytes) and sinusoidal endothelial cells) (Fig. 4), a regenerative response of all of these cells occurs to restore liver mass and function. It is noteworthy that the time courses of DNA synthesis in each of the major sub-populations are not the same. DNA synthesis in hepatocytes peaks at 24 h, in biliary epithelial cells (cholangiocytes) at 48 h, in Kupffer cells at 72 h, and in sinusoidal endothelial cells at 96 h. A variety of growth factors are involved in initiating proliferation in each of these cell types (Alison, 1986). There are two paradigms of liver growth, that of compensatory hyperplasia in which there is loss of function followed by a restorative burst of cell proliferation achieving the original cell mass, as exemplified by partial hepatectomy, and that of direct hyperplasia in which factors lead to an initial increase in cell number and mass above the original (Hermann et al., 1971) which is later dampened down by apoptosis so that the original liver mass is once again restored (Columbano et al., 1985). In this form of liver growth direct mitogens such as tri-iodothyronine play a part. There is a final common pathway after cyclin D1 but the initial cell signalling pathways differ during these two different mechanisms of liver growth, and the time taken to reach the G1 restriction point when cells are committed to proliferation differs in each form of proliferation. In compensatory hyperplasia, cyclin D activation occurs within 16 h, whereas in direct hyperplasia this stage is reached in less than eight hours.
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Fig. 4. Schematic Showing Relationship of Parenchymal and No-Parenchymal Cells in the Liver.
THE PHASES OF CELL TURNOVER Liver growth can be divided into three phases. Initiation is required which primes cells to make replication competent; this is followed by a proliferation phase during which cell expansion occurs and finally the termination phase when growth arrest occurs to end the regeneration. Each of these is extremely tightly controlled. The initiation step affects quiescent cells in the G0 stage of the cell cycle, so that they are primed to enter G1. During the priming phase a number of factors are activated which by themselves are not sufficient for the completion of cells through the S phase, but which are a necessary step for progression of the cells through the cycle. Briefly, the priming signals are growth factors and transcription factors, and include NFB, STAT3, AP-1 and members of the CCAAT enhancer binding protein family, as well as a myriad of “immediate early genes.” At this stage, cells can be prevented from continuing through the cycle; however, as they cross the G1 restriction checkpoint the process is no longer reversible. Once the
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cells have been primed they require growth factors, such as HGF and TGF-␣ to initiate replication but once cyclin D1 is activated the presence of growth factors is no longer required and the process is self perpetuating (Fausto, 2000). The increased expression of genes, although not only those specifying growth factors, are all involved in the proliferative process and have a temporal relationship to the onset of cell proliferation. The immediate early genes, including c-fos, c-jun and c-myc show increased expression within the first four hours; thereafter between four and eight hours, the delayed genes are expressed, e.g. bclx. After eight hours the cell cycle genes increase such as p53, some cyclins, and between 20 and 48 h genes associated directly with DNA replication and mitosis such as cyclin E and A are increased during which time the cell traverses the S phase, G2 phase and mitotic phase of the cell cycle. One of the major early stimulators of the initiation phase of cell proliferation is the cytokine TNF-␣. Circulating and liver TNF-␣ increases after a partial hepatectomy, probably via Kupffer cells (Iwai et al., 2001; Selzner et al., 2003). TNF-␣ primes cells in the quiescent G0 state which become activated to enter the G1 state. There are two TNF-␣ receptors (TNFR1 and TNFR2), but only TNFR1 appears to play a role in its proliferative effects on the liver. Experiments with knock-out mice showed that in TNFR2 knockout mice liver regeneration after a partial hepatectomy was entirely unaffected. However, in TNFR1 knock-outs, DNA synthesis was dramatically reduced; moreover these animals exhibited an increased mortality 24–40 hours after the hepatectomy (Fausto, 1999; Kirillova et al., 1999). TNF-␣ receptor 1 activation leads to an increase of the transcription factor NFB in the liver within 1–4 hours, in both hepatocytes and Kupffer cells (Cressman et al., 1994). The mechanism of NFB activation occurs via removal of its inhibitor I-B from the NFB heterodimer of P65 (relA) and P50 subunits. This stage of regeneration is known as priming or initiation. Subsequent to NKB activation, Interleukin 6 (IL6) increases between 2 and 12 hours (Fulop et al., 2001; Shiratori et al., 1996). IL6 is produced by nonparenchymal cells, and is probably released by Kupffer cells in response to TNFR1 signaling (Kirillova et al., 1999). In TNFR1 knockouts which have diminished liver regeneration, a single injection of IL6 can restore both DNA synthesis and liver regeneration to normal, implying a pivotal role for IL6 in the regenerative process (Fausto, 1999; Yamada et al., 1997). IL6 is itself a key inducer of several transcription factors, via its binding to its specific receptor IL6R. This ligand-receptor complex binds to a signal transducer, the gp130 protein (Taga & Kishimoto, 1997). A homodimer of two gp130 molecules allows physical interaction with the Janus family of intracellular kinases, in which several distinct tyrosine residues become phosphorylated. The resultant phosphotyrosines, in turn, interact with protein SH2 domains leading to
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activation of STAT signaling molecules (Hemmann et al., 1996). Moreover, IL6 leads, via HNF1, to promoter activation of liver IGF1 binding protein and requires two other classes of transcription factors, STAT3 and AP1, suggesting that the cooperative effect of these transcription factors amplifies hepatic gene expression as an adaptive response to liver injury (Leu et al., 2001). As well as the membrane bound form of IL6 receptor a soluble form may also play a role in regeneration (Peters et al., 2000). To summarize this priming phase, TNF-␣ is released which activates NFB and stimulates secretion of IL6 (Yamada et al., 1997). Interleukin 6 induces activation of several transcription factors, e.g. STAT 3 and CEBP-. Binding of these factors to target genes results in the switch from G0 to G1. The second phase of liver regeneration is that of proliferation. During this phase the classical growth factors Hepatocyte growth factor (HGF) and Transforming growth factor alpha (TGF␣), play their part, via their tyrosine kinase receptors c-met and EGF receptor, respectively. HGF is the most potent of these both in vivo and in vitro (Michalopoulos & Defrances, 1997). After a partial hepatectomy HGF is raised in serum and liver prior to TGF-␣ and indeed stimulates its synthesis. HGF is able to induce histone acetylation in regenerating hepatocytes and may therefore act at different levels of the cell cycle concurrently (Latasa et al., 2001). HGF can also be released from the extracellular matrix; latent HGF is activated by a serine protease (urokinase-type plasminogen activator UPa) which cleaves the single chain inactive HGF to a two chain active form. Hepatic UPa increases during the early stages of liver regeneration, principally via increased UPa receptor expression on hepatocytes. TNFa, as well as inducing IL6, is capable of upregulating synthesis of TGFa both in regenerating liver an isolated hepatocytes, which may provide a further amplification of the proliferative response (Gallucci et al., 2000) (Figs 5 and 6). Insulin like growth factors (IGF I and II) and their binding proteins are synthesised by the liver variably in hepatocytes and non-parenchymal cells, and regulated in a growth hormone-dependent (IGF I) and independent (IGF II) mechanism. Their major role seems to be in fetal and post-natal development, however, IGFBP 1 and 4 mRNAs are increased after a partial hepatectomy and may serve as a fine tuning mechanism for a role of IGFs in liver regeneration (Demori et al., 2000). Over the years a number of less well characterized stimulators of hepatocyte proliferation have been reported. It is difficult to place these mitogens in an obvious sequence of events, nonetheless, they each have been shown experimentally to induce hepatocyte proliferation. A short description of and references to those factors are listed in Table 1.
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Fig. 5. Schematic Structure of Hepatocyte Growth Factor (HGF) Showing Four Kringle Domains in the Alpha Chain. Note: Alpha and beta chains are linked by a disulfide bridge between cyteines 487 and 604. Reproduced with permission from Nakamura et. al. (1989) Nature 342, 440–443.
Fig. 6. HGF Gene Expression in Rat Liver at Various Times After Partial Hepatectomy (15, 45 min, 2, 4, 10 and 24 hours) Illustrated by Northern Blot Analysis. Note: Marked increase in expression at 10 h post-heptatectomy, reduced to near normal levels by 24 h. Reproduced from Selden et al. (1990).
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Table 1. Additional Growth Factors Stimulating Liver Growth. ALR
FAD-linked sulfhydryl oxidsase
Hepatopoietin
HPO
Human derived
Liver regenerating factor 1
LRF1
Induced by peroxisome proliferators
Hepatic stimulator substance Fibroblast growth factor inducible gene 14 ACE inhibitors and Bradykinin
HSS
Organ specific
Fn-14 in mice
Type 1a transmembrane protein Angiotensin converting enzyme inhibitors
Stimulates hepatocyte DNA synthesis; reduces interferon gamma in natural killer cells; controls mitochondrial transcription factor A expression; enhances cytochrome content and oxidative phosphorylation in liver mitochondria Stimulates DNA synthesis in vitro and in vivo via HPO receptor; causes phosphorylation of MAPKK and MAPK; stimulates EGF receptor tyrosine phosphorylation. Activates transcription factor 3 which affects TNF-alpha induced E-selectin gene expression Participates in liver regeneration after liver injury Rapidly induced in murine liver regeneration
Augments liver regeneration after partial hepatectomy
Francavilla et al., 1994b) Lisowsky et al., 2001; Polimeno et al., 2000a; Polimenoet al., 2000b) Li et al., 2000)
(Nawa et al., 2000)
(Margeli et al., 1999) (Feng et al., 2000)
(Ramalho et al., 2001)
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Augmenter of liver regeneration
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Epidermal growth factor (EGF), is also a potent mitogen for hepatocytes in vitro, but is not increased early in liver regeneration, at a time appropriate for a role in hepatocyte replication. However, it also acts as a mitogen for biliary epithelial cells, suggesting it may play a role later in bile duct proliferation (Bissig et al., 2000). Indeed HGF, IL6, somatostatin and bile acids are all factors which promote cholangiocyte proliferation. Recent reports indicate an upregulation of telomerase activity in regenerating liver prior to S phase, paralleled by a prior increase in TERT mRNA. Both EGF in normal hepatocytes in vitro and HGF in cultures of regenerating hepatocytes upregulate telomerase activity, probably via p44/42 MAPK signaling. These growth factors are increased during regeneration and probably play a similar role in vivo (Inui et al., 2001, 2002). As the liver restores its mass by hepatocyte proliferation, new blood vessels are also formed. A variety of growth factors are involved including Vascular endothelial Growth factor (VEGF), which is expressed in the periportal zone of hepatocytes, levels after a partial hepatectomy increase at 48–72 hours, temporal with an increase in sinusoidal cell proliferation (Taniguchi et al., 2001). The hepatic stellate cells (fat storing cells; lipocytes) are synthesizers of HGF as well as responders to that cytokine. Endothelin 1 and Platelet derived growth factor (PDGF) are also associated with HSC proliferation, although their source during liver regeneration is not clear. As well as growth factors per se acting on HSC proliferation, their turnover is modulated by apoptosis regulators such as members of the Bcl family (Saile et al., 1997; Tzung et al., 1997). Much of the evidence for positive stimulators of hepatocyte DNA synthesis has been obtained in vitro with the advent in the seventies of tissue culture systems of primary hepatocytes (McGowan et al., 1981). Not all of the factors which are positive in vitro can be shown to be effective in vivo, probably illustrating the complex nature of the controlling mechanisms. Some hepatotrophic factors can act on the hepatocytes in the absence of other co-factors, while others require a milieu of appropriate factors to elicit the response. The concept of complete (requiring no cofactors) and incomplete mitogens was suggested by Michalopoulos (1990). Besides causing a wave of DNA synthesis and mitosis to restore liver mass, there are several growth factors involved in the equally important phase, that of cessation or termination of proliferation. It is a remarkable fact that the rapid wave of proliferation is controlled very tightly such that the liver does not exceed its original mass by anymore than 10%. This is all the more remarkable in the model of partial hepatectomy where the remnant lobe increases in mass to compensate for the removed segments. How does it know when to stop? It was initially thought that hepatocytes were only capable of undergoing one or two rounds of cell division, which could have provided the mechanism for termination. However, this paradigm
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holds no longer since it is clear that during serial transplantation for repopulation experiments an hepatocyte can divide many, many times (Overturf et al., 1997; Rhim et al., 1994). There are several candidate growth factors and cytokines which play a part in termination, but it is fair to say that the mechanism to date is by no means fully elucidated. Transforming growth factor beta (TGF-) is one such growth factor which acts as an inhibitor of hepatocyte proliferation both in vivo and in vitro (Nagy et al., 1989; Russell et al., 1988). Sinusoidal endothelial cells, HSCs and hepatocytes themselves express TGF beta, although it is interesting that hepatocytes become refractory to TGF- during their proliferative phase of replication (Bissell et al., 1995; Jakowlew et al., 1991). Norepinephrine decreases the mitoinhibitory potency of TGF- and hepatocytes 12–16 hours after hepatectomy are exquisitely sensitive to the effect of norepinephrine on TGF- (Houck et al., 1988; Houck & Michalopoulos, 1989). It is also thought that TGF- protein is released by the extracellular matrix to effect an inhibitory response such that increased gene expression may not be required. TGF- binds to its receptor and signals via the MAD family of transcription factor proteins (Massague, 1996). Further understanding of the termination mechanism involving TGF- comes from transgenic mice over-expressing the cytokine in which the cdc25A protein is down regulated, concomitant with increased histone deacetylation of the p130 repressor complex in the liver (Zahler et al., 2000); thus TGF- 1 regulation of HDAC1 may form the link between chromatin remodeling proteins and inhibition of cyclins via induction of cdcA25. A second line of indirect evidence using adenoviral vector administered type II TGF- receptors indicates enhanced regenerative response when the TGF- protein is unavailable to hepatocytes (Nakamura et al., 2000). Interleukin 1-␣ and  are cytokines produced in Kupffer cells which are increased in expression during the termination phase and have been shown to inhibit hepatocyte proliferation in vitro. In vivo, administration of the IL1 receptor antagonist promoted liver regeneration (Boulton et al., 1997); indirect evidence using an interleukin 1  antagonist, FR167653, which increased DNA synthesis after partial hepatectomy compared with control rats, supports this finding (Hou et al., 2003). One member of the Activin family, Activin A, which is a homodimer of inhibin  A chains, inhibits liver regeneration (Kogure et al., 2000), whereas Activin  C and E appear not to have any effect, at least in knockout mice (Lau et al., 2000). The role played by the activins is likely to be via the extracellular matrix (ECM) (Date et al., 2000). During liver regeneration much of the ECM is lost and in contrast to the normal single cell thick plates, there are clusters of 8–10 hepatocytes, with corresponding loss of sinusoidal spaces. Activin A receptors
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are down regulated during the initial phases of hepatocyte proliferation, possibly rendering them responsive to mitogenic stimuli. In contrast, increased Activin A levels in HSC stimulate those cells to synthesise and secrete fibronectin, an important protein of the ECM involved in appropriate positioning of the “new” hepatocytes (Ichikawa et al., 2001). Other growth and/or transcription factors which are likely to play a part in the termination phase are p53, Sp1, cyclin and cyclin-dependent kinase inhibitors e.g. p21, p19 and p27. However, their precise role has not yet been determined (Albrecht et al., 1997).
Maintaining Liver Mass by Apoptosis Another mechanism of termination may be via apoptosis. There is evidence that apoptosis acts physiologically to remove senescent hepatocytes via the Fas/Fas ligand system. After partial hepatectomy there is a significant decrease in Fas mRNA by two hours after surgery. The diminished levels are maintained for 18 hours and fully replenished by six days, indicating that the physiological levels are re-established once the original liver mass is restored; this may help to prevent an overshoot of proliferation (Kiba et al., 2000). Liver caspase-3-protease is increased 18 hours after a partial hepatectomy until 48 hours when the bulk of hepatocyte proliferation has ceased. Interestingly, in a model of direct hyperplasia in which the thyroid hormone T3 induces liver regeneration in normal rats such that the mass increases (Francavilla et al., 1994a; Malik et al., 2003) this increase is modulated by apoptosis to return to original size after 10 days (Ledda-Columbano et al., 1993; Malik & Hodgson, 2002). The cytokeratin status of the liver also impacts on apoptosis. Both cytokeratin 8 and 18 bind the TNF-␣ II receptor to modulate TNF-␣-induced NFB activation. This transcription factor is known to protect against apoptosis induced by TNF-␣ (Caulin et al., 2000; Plumpe et al., 2000). Other metabolic factors such as levels of reactive oxygen species and glutathione content can influence the effect of TNF-␣ on hepatocytes driving either proliferation or apoptosis (Fausto, 2000). Such a balance between apoptosis and regeneration is crucial to maintaining liver mass homeostasis.
GROWTH FACTORS AND LIVER DISEASE I believe the studies described above clearly indicate that growth factors play an important role in liver regeneration after traumatic injury. Evidence for their role in regeneration in man after chemical or virally induced liver failure is less clear.
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The same factors have been implicated, and in particular, HGF and IL-6, appear important, which suggest they may be promising therapeutic candidates. However, almost all studies have looked at the effect of these growth factors given before experimental chemical injury has been induced, which is a far cry from the clinical situation in which fulminant hepatic failure patients may present in the clinic some days after the initial toxic insult. One recent study administered, not IL-6 but hyper IL6, 7 hours after D-galactosamine induced injury in rats and showed an abrogation of liver damage progression (Galun et al., 2000) suggesting that this may have therapeutic potential. However, small animal models of fulminant hepatic failure are not a true correlate of the disease state observed in man.
GROWTH FACTORS AND LIVER DEVELOPMENT Although it is out of the scope of this chapter to discuss in detail the role growth factors play in development, it is worth noting that HGF was recognized as a protein involved in development well before its role as a growth factor in adult liver was recognized. In the development literature the molecule was known as “scatter factor” due to its ability to cause cell movement. Before the observation that these two factors were the same protein, we had demonstrated a significant increase in HGF mRNA in human fetal liver in samples taken from 7–19 weeks of gestation (Selden et al., 1990). Hepatocyte growth factor is now known to be a pleiotrophic factor affecting cell proliferation, motility and morphogenesis (Fig. 7).
Fig. 7. Increased Gene Expression in Human Fetal Liver from 7 to 19 Weeks Gestational Age, Compared with Normal Adult Human Liver. Reproduced from Selden et al. (1990).
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CONCLUDING REMARKS In conclusion, growth factors and cytokines are an important part of the mechanism of liver regeneration and contribute to the proliferation of all the sub-populations of the liver to maintain liver homeostasis after injury (see Fig. 8). They do not, however, appear to be the initiating event of the process, which has still not been fully elucidated.
Fig. 8. Schematic Showing the Involvement of Growth Factors and the Different Cell Populations of the Liver on Hepatocyte Proliferation. Reproduced from Malik et al. (2002). Sem. Cell Develop. Biol. 13(6), 425–432.
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REFERENCES Albrecht, J. H., Meyer, A. H., & Hu, M. Y. (1997). Regulation of cyclin-dependent kinase inhibitor p21(WAF1/Cip1/Sdi1) gene expression in hepatic regeneration. Hepatology, 25, 557–563. Alison, M. R. (1986). Regulation of hepatic growth. Physiol. Rev., 66, 499–541. Bissig, K. D., Marti, U., Solioz, M., Forestier, M., Zimmermann, H., Luthi, M., & Reichen, J. (2000). Epidermal growth factor is decreased in liver of rats with biliary cirrhosis but does not act as paracrine growth factor immediately after hepatectomy [In Process Citation]. J. Hepatol., 33, 275–281. Bissell, D. M., Wang, S. S., Jarnagin, W. R., & Roll, F. J. (1995). Cell-specific expression of transforming growth factor-beta in rat liver. Evidence for autocrine regulation of hepatocyte proliferation. J. Clin. Invest., 96, 447–455. Boulton, R., Woodman, A., Calnan, D., Selden, C., Tam, F., & Hodgson, H. (1997). Nonparenchymal cells from regenerating rat liver generate interleukin-1alpha and −1beta: A mechanism of negative regulation of hepatocyte proliferation. Hepatology, 26, 49–58. Bucher, N. L. R., & McGowan, J. A. (1985). Regulatory mechanism in hepatic regeneration. In: R. Wright, K. M. M. G. Alberti, S. J. Karran & H. Milward-Sadler (Eds), Liver and Biliary Disease: A Pathological Approach (pp. 251–265). London: Saunders. Bucher, N. L. R., Swaffield, M. N., & Ditroia, J. F. (1964). Influence of age upon incorporation of thymidine-2-C14 into DNA of regenerating rat liver. Cancer Res., 24, 509–512. Budd, G. (1945). On the diseases of the liver (1st ed.). London: J. Churchill. Caulin, C., Ware, C. F., Magin, T. M., & Oshima, R. G. (2000). Keratin-dependent, epithelial resistance to tumor necrosis factor-induced apoptosis. J. Cell. Biol., 149, 17–22. Columbano, A., Ledda-Columbano, G. M., Coni, P. P., Faa, G., Liguori, C., Santa, C. G., & Pani, P. (1985). Occurrence of cell death (apoptosis) during the involution of liver hyperplasia. Lab. Invest., 52, 670–675. Cressman, D. E., Greenbaum, L. E., Haber, B. A., & Taub, R. (1994). Rapid activation of posthepatectomy factor/nuclear factor kappa B in hepatocytes, a primary response in the regenerating liver. J. Biol. Chem., 269, 30429–30435. Cruveilhier, J. (1833). Anatomie Pathologique du Corps Humain (Vol. 1, 12 Livraison, Plate 1, 1st ed.). Paris. Date, M., Matsuzaki, K., Matsushita, M., Tahashi, Y., Sakitani, K., & Inoue, K. (2000). Differential regulation of activin A for hepatocyte growth and fibronectin synthesis in rat liver injury. J. Hepatol., 32, 251–260. Demori, I., Balocco, S., Voci, A., & Fugassa, E. (2000). Increased insulin-like growth factor binding protein-4 expression after partial hepatectomy in the rat. Am. J. Physiol. Gastrointest. Liver Physiol., 278, G384–G389. Fabrikant, J. I. (1968). The kinetics of cellular proliferation in regenerating liver. J. Cell. Biol., 36, 551–565. Fausto, N. (1999). Lessons from genetically engineered animal models. V. Knocking out genes to study liver regeneration: Present and future. Am. J. Physiol., 277, G917–G921. Fausto, N. (2000). Liver regeneration. J. Hepatol., 32, 19–31. Feng, S. L., Guo, Y., Factor, V. M., Thorgeirsson, S. S., Bell, D. W., Testa, J. R., Peifley, K. A., & Winkles, J. A. (2000). The Fn14 immediate-early response gene is induced during liver regeneration and highly expressed in both human and murine hepatocellular carcinomas. Am. J. Pathol., 156, 1253–1261.
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Francavilla, A., Carr, B. I., Azzarone, A., Polimeno, L., Wang, Z., Van Thiel, D. H., Subbotin, V., Prelich, J. G., & Starzl, T. E. (1994a). Hepatocyte proliferation and gene expression induced by triiodothyronine in vivo and in vitro. Hepatology, 20, 1237–1241. Francavilla, A., Hagiya, M., Porter, K. A., Polimeno, L., Ihara, I., & Starzl, T. E. (1994b). Augmenter of liver regeneration: Its place in the universe of hepatic growth factors. Hepatology, 20, 747–757. Fulop, A. K., Pocsik, E., Brozik, M., Karabelyos, C., Kiss, A., Novak, I., Szalai, C., Dobozy, O., & Falus, A. (2001). Hepatic regeneration induces transient acute phase reaction: Systemic elevation of acute phase reactants and soluble cytokine receptors. Cell. Biol. Int., 25, 585–592. Gallucci, R. M., Simeonova, P. P., Toriumi, W., & Luster, M. I. (2000). TNF-alpha regulates transforming growth factor-alpha expression in regenerating murine liver and isolated hepatocytes. J. Immunol., 164, 872–878. Galun, E., Zeira, E., Pappo, O., Peters, M., & Rose-John, S. (2000). Liver regeneration induced by a designer human IL-6/sIL-6R fusion protein reverses severe hepatocellular injury. FASEB J., 14, 1979–1987. Greisler, H. P., Voorhees, A. B. J., & Price, J. B. J. (1979). The nonportal origin of the factors initiating hepatic regeneration. Surgery, 86, 210–217. Gupta, S., Johnstone, R., Darby, H., Selden, C., Price, Y., & Hodgson, H. J. (1987). Transplanted isolated hepatocytes: Effect of partial hepatectomy on proliferation of long-term syngeneic implants in rat spleen. Pathology, 19, 28–30. Hemmann, U., Gerhartz, C., Heesel, B., Sasse, J., Kurapkat, G., Grotzinger, J., Wollmer, A., Zhong, Z., Darnell, J. E., Jr., Graeve, L., Heinrich, P. C., & Horn, F. (1996). Differential activation of acute phase response factor/Stat3 and Stat1 via the cytoplasmic domain of the interleukin 6 signal transducer gp130. II. Src homology SH2 domains define the specificity of stat factor activation. J. Biol. Chem., 271, 12999–13007. Hermann, R., Koransky, W., Leberl, C., & Noack, G. (1971). Hyperplasia and hypertrophy of rat liver induced by hexachlorcyclohexane and butylhyroxytoluene. Retention of the hyperplasia during involution of the enlarged organ. Virchows Arch. B. Cell. Pathol., 9(2), 125–134. Higgins, G. M., & Anderson, R. M. (1931). Experimental pathology of liver restoration of liver of white rat following partial surgical removal. Arch. Pathol., 12, 186–202. Hou, Z., Yanaga, K., Kamohara, Y., Eguchi, S., Tsutsumi, R., Furui, J., & Kanematsu, T. (2003). A new suppressive agent against interleukin-1beta and tumor necrosis factor-alpha enhances liver regeneration after partial hepatectomy in rats. Hepatol. Res., 26, 40–46. Houck, K. A., Cruise, J. L., & Michalopoulos, G. (1988). Norepinephrine modulates the growthinhibitory effect of transforming growth factor-beta in primary rat hepatocyte cultures. J. Cell. Physiol., 135, 551–555. Houck, K. A., & Michalopoulos, G. K. (1989). Altered responses of regenerating hepatocytes to norepinephrine and transforming growth factor type beta. J. Cell. Physiol., 141, 503–509. Ichikawa, T., Zhang, Y. Q., Kogure, K., Hasegawa, Y., Takagi, H., Mori, M., & Kojima, I. (2001). Transforming growth factor beta and activin tonically inhibit DNA synthesis in the rat liver. Hepatology, 34, 918–925. Inui, T., Shinomiya, N., Fukasawa, M., Kobayashi, M., Kuranaga, N., Ohkura, S., & Seki, S. (2002). Growth-related signaling regulates activation of telomerase in regenerating hepatocytes. Exp. Cell. Res., 273, 147–156. Inui, T., Shinomiya, N., Fukasawa, M., Kuranaga, N., Ohkura, S., & Seki, S. (2001). Telomerase activation and MAPK pathways in regenerating hepatocytes. Hum. Cell., 14, 275–282.
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Iwai, M., Cui, T. X., Kitamura, H., Saito, M., & Shimazu, T. (2001). Increased secretion of tumour necrosis factor and interleukin 6 from isolated, perfused liver of rats after partial hepatectomy. Cytokine, 13, 60–64. Jakowlew, S. B., Mead, J. E., Danielpour, D., Wu, J., Roberts, A. B., & Fausto, N. (1991). Transforming growth factor-beta (TGF-beta) isoforms in rat liver regeneration: Messenger RNA expression and activation of latent TGF-beta. Cell. Regul., 2, 535–548. Kiba, T., Saito, S., Numata, K., & Sekihara, H. (2000). Fas (APO-1/CD95) mRNA is down-regulated in liver regeneration after hepatectomy in rats. J. Gastroenterol., 35, 34–38. Kirillova, I., Chaisson, M., & Fausto, N. (1999). Tumor necrosis factor induces DNA replication in hepatic cells through nuclear factor kappaB activation. Cell. Growth Differ., 10, 819–828. Kogure, K., Zhang, Y. Q., Maeshima, A., Suzuki, K., Kuwano, H., & Kojima, I. (2000). The role of activin and transforming growth factor-beta in the regulation of organ mass in the rat liver. Hepatology, 31, 916–921. Latasa, M. U., Boukaba, A., Garcia-Trevijano, E. R., Torres, L., Rodriguez, J. L., Caballeria, J., Lu, S. C., Lopez-Rodas, G., Franco, L., Mato, J. M., & Avila, M. A. (2001). Hepatocyte growth factor induces MAT2A expression and histone acetylation in rat hepatocytes: Role in liver regeneration. FASEB J., 15, 1248–1250. Lau, A. L., Kumar, T. R., Nishimori, K., Bonadio, J., & Matzuk, M. M. (2000). Activin betaC and betaE genes are not essential for mouse liver growth, differentiation, and regeneration. Mol. Cell. Biol., 20, 6127–6137. Lau, L. F., & Nathans, D. (1985). Identification of a set of genes expressed during the G0/G1 transition of cultured mouse cells. EMBO J., 4, 3145–3151. Ledda-Columbano, G. M., Coni, P., Simbula, G., Zedda, I., & Columbano, A. (1993). Compensatory regeneration, mitogen-induced liver growth, and multistage chemical carcinogenesis. Environ. Health Perspect., 101(Suppl. 5), 163–168. Leu, J. I., Crissey, M. A., Leu, J. P., Ciliberto, G., & Taub, R. (2001). Interleukin-6-induced STAT3 and AP-1 amplify hepatocyte nuclear factor 1-mediated transactivation of hepatic genes, an adaptive response to liver injury. Mol. Cell. Biol., 21, 414–424. Li, Y., Li, M., Xing, G., Hu, Z., Wang, Q., Dong, C., Wei, H., Fan, G., Chen, J., Yang, X., Zhao, S., Chen, H., Guan, K., Wu, C., Zhang, C., & He, F. (2000). Stimulation of the mitogen-activated protein kinase cascade and tyrosine phosphorylation of the epidermal growth factor receptor by hepatopoietin. J. Biol. Chem., 275, 37443–37447. Lisowsky, T., Lee, J. E., Polimeno, L., Francavilla, A., & Hofhaus, G. (2001). Mammalian augmenter of liver regeneration protein is a sulfhydryl oxidase. Dig. Liver Dis., 33, 173–180. Mahler, S. M., Wilce, P. A., & Shanley, B. C. (1988a). Studies on regenerating liver and hepatoma plasma membranes-I. Lipid and protein composition. Int. J. Biochem., 20, 605–611. Mahler, S. M., Wilce, P. A., & Shanley, B. C. (1988b). Studies on regenerating liver and hepatoma plasma membranes-II. Membrane fluidity and enzyme activity. Int. J. Biochem., 20, 613–619. Malik, R., & Hodgson, H. J. (2002). Thyroid hormone – it’s proliferative and apoptotic effects on the liver. J. Hepatol., 36(Suppl. 1), 270. Malik, R., Mellor, N., Selden, C., & Hodgson, H. (2003). Triiodothyronine enhances the regenerative capacity of the liver following partial hepatectomy. Hepatology, 37, 79–86. Margeli, A. P., Skaltsas, S. D., Spiliopoulou, C. A., Mykoniatis, M. G., & Theocharis, S. E. (1999). Hepatic stimulator substance activity in the liver of thioacetamide-intoxicated rats. Liver, 19, 519–525. Massague, J. (1996). TGFbeta signaling: Receptors, transducers, and Mad proteins. Cell., 85, 947–950.
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McGowan, J. A., Russell, W. E., & Bucher, N. L. (1984). Hepatocyte DNA replication: Effect of nutrients and intermediary metabolites. Fed. Proc., 43, 131–133. McGowan, J. A., Strain, A. J., & Bucher, N. L. (1981). DNA synthesis in primary cultures of adult rat hepatocytes in a defined medium: Effects of epidermal growth factor, insulin, glucagon, and cyclic-AMP. J. Cell. Physiol., 108, 353–363. Mead, J. E., Braun, L., Martin, D. A., & Fausto, N. (1990). Induction of replicative competence (“priming”) in normal liver. Cancer Res., 50, 7023–7030. Michalopoulos, G. K. (1990). Liver regeneration: Molecular mechanisms of growth control. FASEB J., 4, 176–187. Michalopoulos, G. K., & Defrances, M. C. (1997). Liver regeneration. Science, 276, 60–66. Moolten, F. L., & Bucher, N. L. (1967). Regeneration of rat liver: Transfer of humoral agent by cross circulation. Science, 158, 272–274. Nagy, P., Evarts, R. P., McMahon, J. B., & Thorgeirsson, S. S. (1989). Role of TGF-beta in normal differentiation and oncogenesis in rat liver. Mol. Carcinog., 2, 345–354. Nakamura, T., Sakata, R., Ueno, T., Sata, M., & Ueno, H. (2000). Inhibition of transforming growth factor beta prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats. Hepatology, 32, 247–255. Nawa, T., Nawa, M. T., Cai, Y., Zhang, C., Uchimura, I., Narumi, S., Numano, F., & Kitajima, S. (2000). Repression of TNF-alpha-induced E-selectin expression by PPAR activators: Involvement of transcriptional repressor LRF-1/ATF3. Biochem. Biophys. Res. Commun., 275, 406–411. Ngala Kenda, J. F., de Hemptinne, B., & Lambotte, L. (1984). Role of metabolic overload in the initiation of DNA synthesis following partial hepatectomy in the rat. Eur. Surg. Res., 16, 294–302. Overturf, K., Al Dhalimy, M., Ou, C. N., Finegold, M., & Grompe, M. (1997). Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am. J. Pathol., 151, 1273–1280. Peters, M., Blinn, G., Jostock, T., Schirmacher, P., Meyer zum Buschenfelde, K. H., Galle, P. R., & Rose-John, S. (2000). Combined interleukin 6 and soluble interleukin 6 receptor accelerates murine liver regeneration. Gastroenterology, 119, 1663–1671. Plumpe, J., Malek, N. P., Bock, C. T., Rakemann, T., Manns, M. P., & Trautwein, C. (2000). NF-kappaB determines between apoptosis and proliferation in hepatocytes during liver regeneration. Am. J. Physiol. Gastrointest. Liver Physiol., 278, G173–G183. Polimeno, L., Capuano, F., Marangi, L. C., Margiotta, M., Lisowsky, T., Ierardi, E., Francavilla, R., & Francavilla, A. (2000a). The augmenter of liver regeneration induces mitochondrial gene expression in rat liver and enhances oxidative phosphorylation capacity of liver mitochondria. Dig. Liver Dis., 32, 510–517. Polimeno, L., Margiotta, M., Marangi, L., Lisowsky, T., Azzarone, A., Ierardi, E., Frassanito, M. A., Francavilla, R., & Francavilla, A. (2000b). Molecular mechanisms of augmenter of liver regeneration as immunoregulator: Its effect on interferon-gamma expression in rat liver. Dig. Liver Dis., 32, 217–225. Ramalho, F. S., Ramalho, L. N., Zucoloto, S., & Correa, F. M. (2001). Angiotensin-converting enzyme inhibition by lisinopril enhances liver regeneration in rats. Braz. J. Med. Biol. Res., 34, 125–127. Rhim, J. A., Sandgren, E. P., Degen, J. L., Palmiter, R. D., & Brinster, R. L. (1994). Replacement of diseased mouse liver by hepatic cell transplantation. Science, 263, 1149–1152. Russell, W. E., Coffey, R. J. J., Ouellette, A. J., & Moses, H. L. (1988). Type beta transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat. Proc. Natl. Acad. Sci. USA, 85, 5126–5130.
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Saile, B., Knittel, T., Matthes, N., Schott, P., & Ramadori, G. (1997). CD95/CD95L-mediated apoptosis of the hepatic stellate cell. A mechanism terminating uncontrolled hepatic stellate cell proliferation during hepatic tissue repair. Am. J. Pathol., 151, 1265–1272. Selden, C., Jones, M., Wade, D., & Hodgson, H. (1990). Hepatotropin mRNA expression in human foetal liver development and in liver regeneration. FEBS Lett., 270, 81–84. Selzner, N., Selzner, M., Odermatt, B., Tian, Y., van Rooijen, N., & Clavien, P. A. (2003). ICAM-1 triggers liver regeneration through leukocyte recruitment and Kupffer cell-dependent release of TNF-alpha/IL-6 in mice. Gastroenterology, 124, 692–700. Shiratori, Y., Hongo, S., Hikiba, Y., Ohmura, K., Nagura, T., Okano, K., Kamii, K., Tanaka, T., Komatsu, Y., Ochiai, T., Tsubouchi, H., & Omata, M. (1996). Role of macrophages in regeneration of liver. Dig. Dis. Sci., 41, 1939–1946. Starzl, T. E., Francavilla, A., Halgrimson, C. G., Francavilla, F. R., Porter, K. A., Brown, T. H., & Putnam, C. W. (1973). The origin, hormonal nature, and action of hepatotrophic substances in portal venous blood. Surg. Gynecol. Obstet., 137, 179–199. Taga, T., & Kishimoto, T. (1997). Gp130 and the interleukin-6 family of cytokines. Ann. Rev. Immunol., 15, 797–819. Taniguchi, E., Sakisaka, S., Matsuo, K., Tanikawa, K., & Sata, M. (2001). Expression and role of vascular endothelial growth factor in liver regeneration after partial hepatectomy in rats. J. Histochem. Cytochem., 49, 121–130. Tzung, S. P., Fausto, N., & Hockenbery, D. M. (1997). Expression of Bcl-2 family during liver regeneration and identification of Bcl-x as a delayed early response gene. Am. J. Pathol., 150, 1985–1995. Wondergem, R., & Harder, D. R. (1980). Membrane potential measurements during rat liver regeneration. J. Cell. Physiol., 102, 193–197. Yamada, Y., Kirillova, I., Peschon, J. J., & Fausto, N. (1997). Initiation of liver growth by tumor necrosis factor: Deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. Proc. Natl. Acad. Sci. USA, 94, 1441–1446. Zahler, M. H., Irani, A., Malhi, H., Reutens, A. T., Albanese, C., Bouzahzah, B., Joyce, D., Gupta, S., & Pestell, R. G. (2000). The application of a lentiviral vector for gene transfer in fetal human hepatocytes. J. Gene. Med., 2, 186–193.
7.
CHEMOKINE AND CYTOKINE REGULATION OF LIVER INJURY
Kenneth J. Simpson and Neil C. Henderson INTRODUCTION Clinically acute and chronic forms of liver injury are common causes of morbidity and mortality throughout the world. The relative frequency of the various etiological factors varies in different geographical regions. Alcohol and other drug induced liver diseases are more common in developed countries compared with viral hepatitis, which is more common in developing nations. Although complete recovery of liver structure and function is expected after acute liver injury, chronic damage results in hepatic fibrosis, cirrhosis and a predisposition to liver cell cancer. The common forms of liver disease, namely viral hepatitis and alcoholic liver disease are associated with an inflammatory infiltrate in the liver of greater or lesser extent. Both hepatitis B and hepatitis C infections are associated with lymphocytic infiltration of the liver, which is implicated in the pathogenesis of the liver injury. Alcoholic liver disease is often associated with focal spotty necrosis of hepatocytes surrounded by infiltrating neutrophils. The neutrophil infiltration of the liver is most prominent in alcoholic hepatitis. This form of alcohol induced liver disease is more common in young women and is associated with high mortality and lack of effective treatments. Primary biliary cirrhosis is a less common cause of chronic liver disease, which may be increasing in frequency. This condition is associated with a lymphocytic infiltration surrounding and destroying the intrahepatic bile ducts. Primary sclerosing cholangitis is another chronic cholestatic liver disorder associated with lymphocytic destruction of both the intra and extrahepatic bile
The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 167–205 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15007-1
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Fig. 1. Cytokine Networks Involved in the Pathogenesis of Sepsis Related Liver Injury.
ducts. Autoimmune hepatitis can lead to florid inflammation with infiltration of the portal tracts with lymphocytes, which surround the hepatocytes at the limiting plate and lead to hepatocyte death. Even in toxic liver injuries, such as occurs in acetaminophen poisoning, macrophages and neutrophils are found in the liver. Data from animal studies have shown that deletion of hepatic macrophages protects the animal from acetaminophen induced hepatic damage and thus implicates these cells in the pathogenesis of this disorder. Therefore, a wide variety of agents that lead to liver injury are associated with an inflammatory infiltrate that is implicated in the disease pathogenesis. Our understanding of the immunological responses to liver damage is becoming clearer and this new found clarity might lead to the development of novel therapies. It is a discussion of the cytokine and chemokine responses which control these immunological responses which forms the basis of this review (Figs 1–4).
LIVER STRUCTURE AND FUNCTION RELATIONSHIPS The unique anatomical positioning of the liver and rich dual blood supply expose the liver to high concentrations of ingested environmental and pharmaceutical toxins in addition to both intact bacteria or viruses and bacterial components passing through the intestinal wall. The cellular constituents of the liver allow rapidly developing innate immune responses to be seamlessly integrated with the adaptive immune system. The liver contains the largest resident macrophage population in the body. These Kupffer cells line the hepatic sinusoids allowing
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Fig. 2. Intercellular and Interorgan Cytokine Networks Involved in Liver and Lung Injury Following Hepatic Ischemia and Reperfusion.
free interaction with invading bacteria and circulating inflammatory cells. The hepatic sinusoids are also incompletely lined with endothelial cells, the resulting fenestrae allow circulating blood components and cells easy access to hepatocytes, which highly express drug metabolizing enzymes. Unique fat storing stellate cells (Ito cells) also line the hepatic sinusoid and function to regulate sinusoidal
Fig. 3. Contrasting Role of Different Classes of Chemokines on the Pathogenesis of Acetaminophen Poisoning.
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Fig. 4. Alcohol and Intercellular Networks Leading to the Recruitment of Circulating Inflammatory Cells.
blood flow. Liver injury leads to stellate cell activation into a myofibroblast like phenotype and it is these cells that play a central role in matrix formation, scarring and eventual development of cirrhosis. CD4 positive, CD8 positive and double negative lymphocytes are found, predominantly in the portal tracts of the liver. Significant numbers of both NK1.1+ and NK1.1− natural killer cells also populate the liver. Recent data have also shown that the epithelial cells of the biliary tract may also contribute to inflammatory responses via cell-cell interaction, cytokine production and antigen presentation. These resident inflammatory and stromal cells are supplemented by circulating lymphocytes and dendritic cells that constantly traverse the hepatic parenchyma as part of the physiological trafficking of these cells through various tissues. Unfortunately the close approximation of the large number of diverse inflammatory cells can result in an over whelming inflammatory response with resultant liver injury and organ failure. In addition to the direct interaction between different inflammatory cells and stromal cells within the liver, each of the different cellular constituents of the liver are able to produce soluble protein mediators. These mediators such as cytokines and chemokines are able, via binding to specific cell surface receptors, to activate surrounding cells and attract circulating inflammatory cells to the sites of injury. Occasionally, cells synthesizing a particular cytokine or chemokine also express the specific cell surface receptor for this cytokine or chemokine, thus setting up an autocrine loop of cellular activation. Cytokines can broadly be grouped according to their structure or function into interleukins (IL), growth factors, interferons (IFN), tumor necrosis factors
Interleukins
Growth Factors
Interferons
Tumour Necrosis Factors
Chemokines
Interleukin 1 – Interleukin 31
Hepatocyte growth factor Insulin like growth factor I & II Platelet derived growth factor Fibroblast growth factor 1–9 Transforming growth factor ␣ &  1–5 Colony stimulating factors, M-CSF, G-CSF, GM-CSF, erythropoietin, thrombospondin Epidermal growth factors, EGF, TGF-␣, heparin binding EGF like growth factor, amphiregulin Nerve growth factor
Interferon ␣, &␥
Tumour necrosis factor ␣ (cachectin) Tumour necrosis factor  (lymphotoxin)
CXC (alpha) ELR positive: Interleukin 8, Gro ␣,  & ␥, ENA-78, NAP-2 ELR negative: IP10, MIG, SDF CC (beta) MIP1␣ & , MCP 1–4, RANTES, eotaxin
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Table 1. Cytokine Families.
C (gamma) Lymphotactin CX3C Fractalkine
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(TNF) and chemokines (Table 1). However, inclusion in one group does not exclude a cytokine from membership of another, for example IL8 (CXCL8) is both an interleukin and a chemokine. In addition, many of the interleukins are also growth and differentiation factors for both immune and hematopoietic cells. The interleukins and TNF family members are ever expanding groups of cytokines, many of which play important roles in the pathogenesis of liver injury and repair. Presently the chemokines are separated into four distinct structurally and functionally related families according to the relative positioning of the 2 cysteine residues at their amino terminus. CC chemokines have two juxtapositioned cysteines and include monocyte chemoattractant proteins (MCP1–5 {CCL2,8,7,13,12}), eotaxin (CCL11), Regulated on T cell activation, normal T cell Expressed and secreted (RANTES {CCL5}) and macrophage inflammatory proteins (MIP) 1 ␣ (CCL3) and  (CCL4). In general, CC chemokines are chemotactic for mononuclear cells. The CXC chemokine family is characterized by a nonconserved single amino-acid (X) between the two N-terminal cysteines. The CXC family is further subdivided into ELR positive and ELR negative CXC-chemokines. The ELR chemokines have a three amino-acid motif, Glutamine (E), Leucine (L) and Arginine (R) immediately adjacent to the CXC motif. The ELR CXC-chemokines include IL8 (CXCL8), cytokine induced neutrophil chemoattractant (CINC), MIP2, KC, epithelial neutrophil-activating protein 78 (ENA 78 {CXCL5}) and growth related oncogenes (GRO) alpha, beta and gamma (CXCL1–3). The non-ELR CXCchemokine family includes IFN gamma inducible protein 10 (IP10 {CXCL10}) and monokine induced by IFN gamma (MIG {CXCL9}). The ELR motif is important in chemokine ligand binding. The ELR positive chemokines can bind either CXCR1 and/or CXCR2, while the non-ELR chemokines bind CXCR3. The ELR-positive CXC-chemokines are important neutrophil chemoattractants and induce angiogenesis. In contrast, non-ELR CXC-chemokines are chemotactic for mononuclear cells and inhibit angiogenesis. The C and CXC3C family of chemokines contain only a single member, lymphotactin (XCL1) and fractalkine (or neurotactin) respectively (Kennedy et al., 1995). Lymphotactin is a T cell chemotactic factor. Fractalkine (CX3CL1) is a unique chemokine in both its receptor and its structure that contains a transmembrane glycoprotein and a chemokine domain at the end of an extended mucin-like stalk (Bazan et al., 1997). Fractalkine occurs in both membrane-bound and soluble forms and represents a novel regulator of leukocyte chemotaxis and adhesion. Originally chemokines were isolated from stimulated or unstimulated cultured cells and characterized by their biological functions, mainly in relation to their chemoattractant activity. More recently, screening of cDNA libraries has been replaced by searching expressed sequence tag (EST) databases for chemokine like sequences. These strategies to identify new chemokines along with the intense interest of the pharmaceutical
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industry in light of the potential anti-HIV properties of chemokines has lead to an explosion in the numbers of chemokines in both the CXC and CC families. This led to a Consensus Conference to produce a more rational nomenclature for all chemokines and this nomenclature is used throughout this review along with the chemokine’s most common name (Zlotnik & Yoshie, 2000).
CYTOKINE RECEPTORS The functions of cytokines are mediated by binding to specific receptors expressed on the surface of target cells. Cytokine receptors can be classified structurally into 5 different groups (Table 2). The immunoglobulin superfamily contains several extracellular immunoglobulin like domains and include other cell surface molecules such as major histocompatibility complex (MHC), the T cell receptor and intercellular adhesion molecule (ICAM) 1. The hematopoietic growth factor family have a conserved amino acid sequence (tryptophan-serine-X-tryptophanserine or WSXWS) usually located on the extracellular domain just proximal to the transmembrane region of the receptor. The TNF receptor family also includes the CD40 antigen and Fas (Van den Abeele et al., 1995). There are 2 TNF receptors, p55 (TNFRI) and p75 (TNFRII). The interferon receptor family comprises of the receptors for IFN␣/ and IFN␥. Lastly is the chemokine receptor family, characterized by 7 transmembrane alpha-helical structures (Horuk, 1994). Some cytokine receptors are hybrids, for example, the IL6R contains both immunoglobulin and WSXWS domains and most cytokine receptors are not single polypeptide chains, but complexes of two or more. One polypeptide chain binds the cytokine ligand which then dimerizes with a signal transducing polypeptide Table 2. Cytokine Receptors. Immunoglobulin
Haematopoietic
Interleukin 1
Interleukin 2 Type I interferon (␣ ( & ␥) & ) Interleukin 3 Type II interferon (␥) Interleukin 4 Interleukin 5 Interleukin 7 Interleukin 9 Leukaemia inhibitory factor gp 130
Interleukin 6
Interferon
Tumour Necrosis Factor
Chemokine
Tumour necrosis factor Type I/␣/55 kDa Type II//75 kDa Nerve growth factor Fas CD 40
CXCR1 – CXCR5 CCR1 – CCR11 XCR1 CX3CR1 Duffy antigen
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generating a receptor with greater ligand affinity. Signal transducing polypeptides may bind to a number of different cytokine binding chains; for example, gp130 the signal transducing portion of the IL6 receptor can also interact with the receptors binding LIF, oncostatin M and CNTF.
Intracellular Signaling Binding of cytokines to their receptors stimulates a complex system of intracellular signals ultimately leading to the biological effect of the cytokine on its target cell (Darnay & Aggarwal, 1997; Diehl & Rai, 1996b; Foxwell et al., 1992; Mufson, 1997; Schindeler & Darnell, 1995). Some of the growth factor receptors, e.g. platelet derived growth factor (PDGF) and epidermal growth factor (EGF), have intrinsic tyrosine kinase activity. Ligand binding induces receptor dimerization, autophosphorylation and attracts cytosolic proteins with Src homology which subsequently activate the mitogen associated protein kinase (MAPK) pathway. Other intracellular signaling pathways may also be activated by receptors with intrinsic tyrosine kinase activity, including phosphoinositol, protein kinase C and Ras-GTPase. Most cytokine receptors do not posses intrinsic tyrosine kinase activity but associate with soluble cytoplasmic protein tyrosine kinases such as those of the Janus (Jak) or Tyk tyrosine kinase families. Jak phosphorylates members of the STAT (signal transduction and activator of transcription) protein family, which associate with other cytoplasmic phosphorylated STAT proteins, translocate to the nucleus and activate gene transcription by binding to specific sequences in target genes. STAT proteins can also be phosphorylated directly by the membrane bound receptor tyrosine kinases. The specificity of the intracellular signals activated by different cytokines is dependent on the activation of specific STAT proteins and the different target gene sequences recognized by the different STAT protein dimmers. Phosphorylation dependent activation of cytosolic protein phosphatases by tyrosine kinases also affects the degree of protein tyrosine phosphorylation and hence activation of these enzyme systems. Signal transduction of the interferon receptors is also mediated via the Jak/Tyk STAT pathway. TNF␣ induces a wide variety of intracellular second messengers including NF-kB, serine/threonine (stress activated protein kinase, SAPK) and tyrosine kinases, ceramide/sphingomyelin, reactive oxygen species and phosphoinositols (Darnay & Aggarwal, 1997). Members of the chemokine receptor family resemble G-protein coupled receptors and as such are sensitive to inhibition by pertussis toxin. Coupling of the receptor and ligand induces phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate into two potentially active products, inositol 1,4,5-triphosphate (IP3 ) and diacylglycerol (DAG). IP3 mobilizes intracellular calcium and activates calmodulin dependent protein kinases. Both DAG and the elevated intercellular
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calcium can also activate the serine/threonine protein kinase C. Protein kinase C comprises a family of at least 7 different but structurally related enzymes which have different substrate specificities. G proteins can also associate with adenylate cyclase, cGMP phosphodiesterase and Ras, with the activation of other intracellular signaling pathways, including MAPK (mitogen activated protein kinase).
Intracellular Signaling in Hepatocytes Most studies regarding cytokine intracellular signaling have been conducted in cells of the immune system rather than hepatocytes or the nonparenchymal cells of the liver. However, as might be expected hepatocytes and other cells within the liver express many of the intracellular signaling pathways described in other cells. Increased intracellular cGMP is involved in the production of TNF␣ by Kupffer cells during sepsis (Harbrecht et al., 1995). Phosphorylation of Jak1 and to a lesser extent Jak2 with STAT phosphorylation, Ras and MAPK activation has been recently reported following IL4 stimulation of hepatocytes (Chuang et al., 1996). IFN and IL6, but not IL1, TNF␣ and EGF, activate STAT3 in rat hepatocytes and human hepatoma cells (Kordula et al., 1995). In contrast with IL6, which produces rapid and transient activation of STAT1 and STAT3 in HepG2 cells, HGF induces delayed and sustained activation of STAT3 only (Schaper et al., 1997). Insulin inhibits hepatocyte IL6 induced acute phase response by transcriptional inhibition of STAT3 expression (Campos et al., 1996). IL1 induces the SAPK pathway in HepG2 cells, but does not activate Ras or MAPK (Bird et al., 1994). Oxidative stress can also induce the MAPK, which via phosphorylation of protein phosphatases can dephosphorylate and inactivate hepatic p38, a phosphoprotein constitutively active in the liver (Mendelson et al., 1996). In addition, TNF␣ can induce specific hepatocyte phosphatase expression and, hence, modulate signaling via cytoplasmic or membrane bound protein kinases (Ahmad & Goldstein, 1997).
CYTOKINE PRODUCTION The Importance of Intercellular Networking As discussed above, the liver structure is such that many cells able to synthesize cytokines are in close approximation to one another. This allows a rapid and potentially damaging amplification of the immune responses within the liver. Examples of this complex intercellular networking between the innate and adaptive immune responses within the liver, resulting in tissue injury, occur following sepsis (Fig. 1) or ischemia/reperfusion injury (Fig. 2).
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Hepatic Injury During Sepsis The production of cytokines within the liver often depends upon the initial induction of early response cytokines released from the resident macrophage, Kupffer cell, population. Following injection of lipopolysaccaride/endotoxin in experimental animals or infection with Gram-negative bacteria, Kupffer cells are activated via interaction with specific cell surface receptors. Lipopolysaccaride (LPS) binds to a circulating protein (LPS binding protein) which then interacts with CD14 expressed on the Kupffer cell surface. This membrane anchored complex is unable to transduce intracellular signals but after binding with constitutively expressed mammalian Toll-like receptors (TLR) leads to activation of the NFkB transcription factors. These mammalian TLR are important in the initial activation of the innate immune response and are similar both in structure and function to the Drosophila Toll proteins. The first human TLR to be characterized, TLR4, interacts with the CD14/LPS complex. Recent investigation has shown that TLR4 constitutively interacts with MD-2, which is essential for enhanced LPS responsiveness. Other TLR interact with different bacterial components, for example TLR2 binds with peptidoglycan. NF-kB activation leads to expression of a variety of “early response cytokines” of which TNF␣ and IL1 are the best characterized examples. These early response proinflammatory cytokines activate Kupffer cells via an autocrine amplification loop and recruit the stromal cells of the liver (endothelial cells, stellate cells and hepatocytes) to participate in the inflammatory response (paracrine effect) by inducing expression of cytokines and chemokines by these cells (Bukara & Bautista, 2000; Thornton et al., 1990, 1992). TNF␣ and IL1 can also induce the expression of adhesion molecules, such as ICAM-1, on the endothelial surface and the corresponding ligand Mac-1 on circulating inflammatory cells. Adhesion between circulating mononuclear cells and activated endothelium via ICAM-1 can induce both CXC and CC chemokine expression (Lukacs et al., 1994, 1995), that attracts and activates circulating inflammatory cells and further amplifies an inflammatory response. In addition to inducing and amplifying a local inflammatory response, TNF␣ may induce both necrosis and apoptosis of hepatocytes via a direct mechanism and through the induction of nitric oxide expression by the hepatocytes themselves (Kurose et al., 1996). Cell damage may also be mediated via free radical and elastase production induced by TNF␣. The histological changes in the liver and elevated serum transaminases occurring following LPS administration are prevented by prior administration of anti-TNF antibodies, soluble TNF receptors, exogenous recombinant IL10, cyclosporin or dexamethasone (Louis et al., 1997; Santucci et al., 1996). Phosphodiesterase inhibitors can also inhibit hepatic injury in this model by altering the cytokine
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balance in favor of anti-inflammatory cytokines, such as IL10 (Ganter et al., 1997). Pretreatment with IL1, by inducing tolerance, can also inhibit hepatocyte apoptosis induced by TNF␣ in experimental models of sepsis (Bohlinger et al., 1995). Experimental models of hepatic steatosis have shown increased sensitivity to LPS and other injurious agents (including ischemia/reperfusion injury). Kupffer cells from mice with hepatic steatosis have a number of phenotypic alterations, including increased IL6, IL12 and IL18 production either spontaneously or following LPS administration. Recent work had implicated hepatic expression of TNF␣ in the progression of hepatic steatosis into steato-hepatitis. This effect may be mediated by upregulation of mitochondrial uncoupling proteins in hepatocytes. Studies of an alternative model of sepsis induced hepatic injury using cecal ligation and puncture (CLP) have shown early induction of hepatic TNF␣ and IL1. Neutralization of these cytokines limits liver injury and improvesd murine mortality. Subsequently, hepatic expression of the CC chemokines MIP1alpha (CCL3), MIP1beta (CCL4) and MCP1 (CCL2) is dramatically up-regulated secondary to Kupffer cell activation (Matsukawa et al., 1999, 2000). Hepatic expression of the CXC chemokines KC, MIP2 and IP10 (CXCL10) are also induced in this model. Immunoneutralization of MIP2 decreased neutrophil influx into the liver, reduced hepatic injury and improved survival following CLP (Walley et al., 1997). However, neutralization of KC limited liver injury without reducing hepatic neutrophil influx. Following CLP in rats, hepatic expression of the CXC chemokine CINC is up-regulated. However in contrast with KC, immunoneutralization of CINC decreased neutrophil influx into the liver and liver injury. Mice primed with heat killed Propionibacterium acnes develop severe hepatocellular necrosis following lipopolysaccaride injection. This liver injury is associated with hepatic lymphocyte infiltration, Kupffer cell production of IFN␥, IL12 and 18, and both TNF alpha and Fas/Fas ligand induced hepatocellular necrosis. Injection of P. acnes alone leads to a granulomatous hepatitis. Preliminary data suggests that there is up-regulation of hepatic MIP1alpha (CCL3), MIG (CXCL9), and IP10 (CXCL10), and recruitment of Th1 CD4+ T cells to the liver. In contrast, in mice primed with P. acnes, following lipopolysaccaride injection, hepatic expression of these chemokines is reduced and TARC (CCL17) is upregulated in macrophage like cells with recruitment of Th2 type, CCR4 expressing lymphocytes into the liver (Yoneyama et al., 1998). Immunoneutralization of TARC (CCL17) significantly reduced liver injury and improved survival in this murine model. Furthermore, reduced expression of IL4 by recruited CD4+ lymphocytes suggested that neutralization of this chemokine selectively reduced the recruitment of T cells with a Th2 phenotype. There was also a significant reduction in the recruitment of other non-CCR4 expressing effector cells, such as CD8+ T cells and NKT cells, indicating that inhibition of TARC (CCL17)
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has immunomodulatory effects beyond the recruitment of cells expressing CCR4. Interestingly, although development is normal and allergen induced bronchial hyper-reactivity occurs in CCR4 knockout mice, these genetically modified mice are resistant to the toxic effects of LPS injection, including the associated liver injury (Chvatchko et al., 2000). Macrophage derived chemokine (MDC {CCL22}) is also able to bind to CCR4. Recent studies have shown that this CC chemokine plays a central role in the systemic response to sepsis in the murine model of caecal ligation and puncture (Matsukawa et al., 2001). In contrast with the data regarding TARC (CCL17), immunoneutralization of MDC (CCL22) increased murine mortality, increased hepatic expression of TNF-␣, MIP2, KC and MIP1alpha (CCL3) and increased hepatic neutrophil infiltration and liver injury. Recombinant MDC (CCL22) had the opposite effects, suggesting that this chemokine also has an important immunoregulatory role and may be an adjunct to therapy in sepsis. However, these data suggest that therapy may have to be directed against specific chemokines rather than their specific receptor.
Ischemia Reperfusion Injury Another example of the importance of cytokine networks and intracellular communication in the development of hepatic injury occurs consequent to ischemia/reperfusion (I/R). This important clinical problem can occur following hepatic resection or surgery and after hypotension and resuscitation. There are two phases of hepatic injury following I/R; an initial phase (1−3 hours post reperfusion), which is associated with free radical generation and Kupffer cell activation and a later phase (6−24 hours post reperfusion) associated with neutrophil influx into the liver. The accumulation of neutrophils following I/R occurs in both experimental animal models and patients with cirrhosis and are important effectors of hepatic damage. The cascade of events leading to neutrophil accumulation is now better understood following the publication of several recent studies. Free radicals released following reperfusion stimulate the production of PAF and TNF␣ (Serizawa et al., 1996; Suzuki & Toledopereyra, 1994) from Kupffer and endothelial cells. Hepatic expression of IL1␣, another early response cytokine, is also elevated following I/R (Shito et al., 1997). Both PAF and TNF␣ can stimulate the production of each other and therefore further amplify the inflammatory response. Neither TNF␣, nor PAF nor IL1␣ are chemoattractant for neutrophils, but these cytokines can stimulate the release of neutrophilic chemoattractants, such as ENA-78 (CXCL5), MIP2, KC and CINC (Colletti et al., 1996a; Deutschman et al., 1996), from both inflammatory and stromal cells of the liver. Inhibition of early response or chemoattractant cytokines and increasing circulating IL1 receptor
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antagonist attenuates hepatic neutrophil accumulation and damage in animal models of I/R. Furthermore, treatment with anti-inflammatory cytokines, such as IL10 or IL13, result in decreased TNF␣ and MIP2 concentrations which correlate with decreased neutrophil accumulation, edema and hepatic injury. The mode of action of IL10 and IL13 in this situation depends on reduced NF-kB and signal transducer and activator of transcription (STAT) 6 DNA binding, respectively. Recent studies have revealed a central role for ENA-78 (CXCL5) in the hepatic and systemic responses to I/R. TNF␣ induces hepatic ENA-78 (CXCL5) expression in both the ischemic and non-ischemic liver lobes, following I/R in rats (Colletti et al., 1996b). IFN␥ reduces this increase in ENA-78 (CXCL5) concentration (Colletti et al., 2000). This effect of IFN␥ on hepatic ENA78 (CXCL5) concentrations was not associated with any significant reduction in neutrophil infiltration in either liver lobe following I/R, but did reduce the severity of liver injury as assessed by serum aminotransferases. However, immunoneutralization of ENA-78 (CXCL5) reduces both hepatic neutrophil influx and liver injury. Other studies have revealed that systemic release of TNF␣ from the liver following hepatic I/R results in neutrophil mediated lung injury that is dependent on TNF␣ induced ENA-78 (CXCL5) expression (Colletti et al., 1995). Expression of other CXC chemokines also occurs following I/R (Lentsch et al., 1998). Hepatic neutrophil infiltration and damage also occurs following 70% hepatectomy and similar cytokine/chemokine networks have been reported with increased TNF␣ expression inducing local production of the neutrophil chemoattractant, ENA-78 in the liver (Colletti et al., 1996a). LPS stimulated TNF␣ release from monocytes is also increased in patients after partial hepatectomy and therefore similar mechanisms of hepatic damage may occur in humans (Sato et al., 1996). MCP1 (CCL2) is the major CC chemokine released by Kupffer cells during I/R in response to either neutrophil elastase or free radicals. Hepatic MCP1 (CCL2) levels are reduced when either free radical formation or neutrophil elastase are inhibited and these changes are correlated with reduced liver injury. In addition to its mononuclear chemoattractant properties, MCP1 (CCL2) may also amplify the polymorphonuclear leukocyte infiltration and increase tissue injury via its effects on ICAM-1 expression. Not all effects of chemokines noted during I/R are related to leukocyte recruitment, since rats treated with IFN␥, a cytokine known to upregulate IP10 (CXCL10), have increased non-ELR CXC-chemokines, and reduced ELR-positive CXC-chemokines. This shift in the balance of ELR-positive relative to non-ELR CXC-chemokines resulted in reduced liver and lung injury following I/R, but surprisingly no decrease in hepatic neutrophil influx was observed. Thus manipulation of chemokine levels may provide important prophylactic treatment in liver surgery that may lead to I/R injury.
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Other Stimuli to Cytokine and Chemokine Production During Liver Injury Reactive oxygen intermediates are produced by hepatocytes during the metabolism of ethanol, acetaminophen and a variety of other drugs. Free radicals can activate the transcription factor, NF-B, which can induce the transcription of a variety of cytokines and chemokines (Sen & Packer, 1996). Hepatitis B viral infection stimulates the production of TNF␣ (Larapezzi et al., 1996) and human herpes virus 6 may induce IL8 (CXCL8) expression (Inagi et al., 1996) in HepG2 hepatoma cells. Ethanol induces IL8 (CXCL8) (Shiratori et al., 1993) and Gro (CXCL1–3) (Shiratori et al., 1994) production in rodent primary hepatocyte cultures and supernatants from ethanol treated hepatocytes can induce the production of CINC from rat Kupffer cells (Mawet et al., 1996). This latter effect is dependent on hepatocyte ethanol metabolism and is inhibited by 4-methylpyrazole. Cytokine production in the liver is also affected by hormones (van Gool et al., 1990), adrenaline increases LPS induced IL6 production, but reduces TNF␣ synthesis. Low concentrations of corticosterone, similar to those found in vivo, enhance the production of IL6 and TNF␣ by LPS, but higher concentrations of corticosterone inhibit cytokine production. Unopposed amplification of the immune response could lead to the development of autoimmune disease or massive release of proinflammatory mediators with multiorgan failure and death, as is seen during sepsis. Several negative immune modulators exist including anti-inflammatory cytokines (e.g. IL10 and IL4), soluble cytokine receptors (soluble TNF receptors) and cytokine antagonists (IL1 receptor antagonist). IL10, for example, down regulates endotoxin mediated IL6 release from Kupffer and sinusoidal endothelial cells (Knolle et al., 1997). However, not all soluble cytokine receptors are inhibitory; soluble IL6R (p80) can induce IL6 effects on cells not normally expressing this component of the IL6 receptor by binding to the ubiquitously expressed gp130 signal transduction polypeptide (Fernandezbotran et al., 1996).
HEPATOCYTE APOPTOSIS Silent Deletion or Inflammatory Stimulus? Hepatocyte apoptosis appears to be one of the main pathways of liver damage both in animal models and human conditions (Schuchmann & Galle, 2001). Histopathological evidence of apoptosis has been reported in liver biopsies from patients with viral hepatitis, alcoholic liver disease, cholestatic liver disease, acetaminophen induced liver failure and other forms of acute liver damage (Strand
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et al., 1998). Massive hepatocyte apoptosis occurs in Fas antibody treated mice and leads to death secondary to acute liver failure (Ogasawara et al., 1993). Apoptosis in the liver also occurs in acute and chronic alcohol fed animals, bile duct ligated rats (a model of cholestatic liver diseases) and following acetaminophen poisoning. In vitro studies have also demonstrated apoptosis of primary and transformed hepatocytes treated with anti-Fas antibodies, chemotheraputic agents and other toxins such as bile salts, alcohol and acetaminophen. Both in vitro and animal studies have shown that hepatocyte apoptosis can occur via receptor mediated pathways, such as Fas and TNF receptor, or non-receptor mediated pathways. Either of these pathways can proceed via caspase activation or other less characterized non-caspase dependent pathways. Furthermore, agents such as cytotoxic drugs or bile salts can induce hepatocyte apoptosis via both receptor and non-receptor mediated pathways. In some conditions, such as oxidant or bile salt mediated hepatocyte apoptosis, there is p53 dependent upregulation of hepatocyte Fas expression. Induced hepatocyte Fas expression has also been reported in hepatitis C infection, hepatic allograft rejection, Wilson’s disease, adenoviral hepatitis and alcoholic liver disease. In hepatic allograft rejection in transplant recipients, and with in vitro oxidant mediated apoptosis, upregulation of hepatocyte Fas ligand expression has been reported, suggesting that hepatocytes can induce their own suicide or paracrine induction of cell death in surrounding cells. In other conditions Fas ligand expression is induced in the resident liver or the infiltrating inflammatory cells. Animal and in vitro studies have also shown that signaling via the TNF receptor (TNFR) can induce hepatocyte apoptosis. Both Fas and TNFR are members of a super-family of TNF receptors e.g. CD40, TRAMP, TWEAK and TRAIL. Stimulation of these receptors also induces hepatocyte apoptosis or in the case of CD40 can augment Fas mediated apoptosis. Therefore, there is a large body of data to implicate hepatocyte apoptosis in the pathogenesis of a wide variety of liver disorders, which may occur via receptor dependent or independent pathways. Apoptosis is a form of cell death characterized by nuclear chromatin condensation, membrane blebbing and condensation of the cytosol. Plasma membrane integrity is maintained, and apoptosis is classically not associated with an inflammatory response. However, recent data have been reported challenging the view that apoptosis does not induce inflammation. Renal ischemia/reperfusion in a murine model is associated with a monocytic inflammatory infiltrate and apoptosis (Daemen et al., 1999). Caspase inhibition or the inhibition of apoptosis by insulin like growth factor 1 (IGF-1) prevented the development of renal inflammation. Conditional overexpression of the Fas associated death domain protein (FADD) led to the apoptosis of vascular smooth muscle cells (SMC) both in vivo and in vitro (Schaub et al., 2000). Induction of apoptosis led to macrophage recruitment in vivo and monocyte chemotaxis in vitro. In this study, vascular SMC released the
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chemokines IL8 (CXCL8) and MCP1 (CCL2) in a caspase and IL1␣ dependent fashion, and inhibition of MCP1 (CCL2) inhibited monocyte chemotaxis in vitro. Ingestion of apoptotic cells by macrophages leads to down-regulation of inflammatory cytokine and chemokine production, consistent with the thesis that apoptosis does not induce inflammation. However, in addition to the data presented above, several others have reported chemokine production on stimulation of Fas and other members of this family of cell surface receptors. TNF may induce apoptosis of hepatocytes under certain circumstances, but clearly can induce chemokine production from most cell types, including hepatocytes and other cellular components of the liver. Fas ligation induces the production of IL8 (CXCL8) in bronchiolar epithelial cells, rheumatoid synovial cells and cultured colonic epithelial cells. Cross-linking CD40, CD30 and TWEAK can also induce IL8 (CXCL8) production. Non-receptor mediated apoptosis induces the production of IL8 (CXCL8) in neutrophils and human umbilical epithelial cells. In addition to IL8 (CXCL8), death receptor stimulation or apoptosis can induce the expression of CC chemokines such as MCP1 (CCL2), MIP1alpha (CCL3) and RANTES (CCL5). A recent paper reported increased hepatic expression of the CXC chemokines KC and MIP2 in vivo following Fas mediated apoptosis, which was accompanied by hepatic neutrophil infiltration (Faouzi et al., 2001). Although inhibition of KC reduced hepatic neutrophil influx, liver injury assessed by histology and circulating transaminases was similar compared with controls. The massive hepatocyte apoptosis associated with Fas ligation results in massive elevation of serum transaminases, suggesting liver cell lysis. This may stimulate Kupffer cells to produce a variety of proinflammatory cytokines and chemokines. However, our preliminary data have shown induction of low levels of hepatocyte apoptosis in the mouse, with no increases in serum transaminases, are still associated with surrounding inflammatory infiltrate. Furthermore, in contrast with other in vitro cell model systems, peripheral blood derived dendritic cells released MIP3alpha (CCL20) following phagocytosis of apoptotic hepatoma cells (anti-Fas treated Huh-1 cells), suggesting that phagocytosis of apoptotic hepatocytes may also induce release of inflammatory mediators (Shimizu et al., 2001). These data provide compelling evidence that, at least within the liver, apoptosis may induce an inflammatory response.
CIRCULATING CHEMOKINES IN LIVER DISEASE Possible Role in Susceptibility to Infection? Many papers have measured circulating chemokine concentrations in patients with liver disease. IL8 (CXCL8) is perhaps the best studied. The highest concentrations
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are reported in patients with alcoholic hepatitis, with lower concentrations in other histological forms of alcoholic liver disease (Hill et al., 1993; Sheron et al., 1993). Circulating IL8 (CXCL8) is also increased in patients with viral hepatitis, acute GVHD and following liver transplantation (Huang et al., 1996; Shimoda et al., 1998; Tilg et al., 1992). In general, increased levels of IL8 (CXCL8) correlate with reduced survival and impaired hepatic function and arise from increased production and/or reduced hepatic clearance. We have found levels of IL8 (CXCL8) to be significantly higher in hepatic venous compared with portal venous blood, implying increased hepatic production in patients with chronic alcoholic liver disease. The pathophysiological significance of these increased circulating IL8 (CXCL8) concentrations is not clear. In murine studies, increased hepatic neutrophil accumulation occurs in IL8 (CXCL8) transgenic mice and following systemic administration of high doses of MIP2 (Bajt et al., 2001; Simonet et al., 1994). However, the latter study did not find any infiltration of neutrophils into the hepatic parenchyma, and concluded that increased circulating chemokine concentration may direct neutrophils towards, but not into liver tissue. Previous studies using nonspecific chemoattractants, have shown reduced neutrophil chemotaxis in patients with liver cirrhosis, especially alcoholic cirrhosis (Kakimi et al., 2001; Koniaris et al., 2001). This defect was partly due to a circulating inhibitor of neutrophil chemotaxis (DeMeo & Anderson, 1972; Maderazo et al., 1975). Using a modified Boyden chamber technique, we have found reduced neutrophil chemotaxis to IL8 (CXCL8) in patients with liver disorders, including alcoholic cirrhosis and acute liver failure (Mohammed et al., 1998). This defective chemotaxis may partly explain the high risk of infection in these patients. The defective IL8 (CXCL8) induced chemotaxis is worse following simulated gastrointestinal hemorrhage and improves after liver transplantation (Mohammed et al., 1999). Despite the increased circulating CXC chemokine concentrations associated with these conditions, neutrophil CXCR1 and CXCR2 expression is normal (Mohammed et al., 2000). These data suggest that the chemotactic defect of the neutrophils to CXC chemokines in patients with liver failure is due to abnormal chemokine/chemokine receptor interaction and/or post-receptor signaling abnormalities.
ACUTE LIVER INJURY Acute liver damage can produce a variety of clinical syndromes depending on the degree of hepatocellular dysfunction. A spectrum of disorders occurs from asymptomatic elevation of serum liver enzymes to the syndrome of fulminant (acute) hepatic failure (FHF), which is characterized by jaundice, hypoglycemia, encephalopathy and coagulopathy. Unlike progressive liver failure associated with
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cirrhosis, FHF occurs in previously healthy individuals and carries a high mortality without liver transplantation. Histologically, there is massive pan-lobular necrosis of hepatocytes associated with a diffuse mixed inflammatory cell infiltrate of variable severity. The most common etiologies of FHF include acetaminophen poisoning, viral hepatitis or idiosyncratic drug reactions. Recent experimental observations suggest that modification of cytokine expression may provide novel therapeutic potential for treating severe liver injury.
Acetaminophen Poisoning and Liver Regeneration Acetaminophen is a widely available analgesic, frequently used for pain relief and to treat fever. When used in therapeutic doses acetaminophen is safe and effective. Deliberate or unintentional overdose of acetaminophen results in severe and potentially fatal hepatic necrosis. Occasionally, patients taking enzyme inducing drugs such as carbamazepine or consuming excess alcohol develop severe acetaminophen induced liver necrosis even with therapeutic doses of acetaminophen, so-called therapeutic misadventure. Acetaminophen poisoning is the most common cause of FHF in both the United States and United Kingdom. Although N-acetylcysteine (NAC) is an effective antidote, many patients present to hospital outwith the 15 hour period after the overdose during which NAC is maximally effective. Subsequently, liver transplantation is the only effective therapy for severe FHF induced by acetaminophen. Several studies have shown the importance of inflammatory cells within the liver in mediating acetaminophen induced liver injury and the modulating effect of cytokine and chemokine networks. Acetaminophen, like carbon tetrachloride (CCl4 ), induces hepatic necrosis following activation by the cytochrome P450 system. The highly reactive intermediate, NAPQI, accumulates once hepatic glutathione stores are depleted and induces necrosis by binding to intracellular organelles and the plasma membrane. However, Kupffer cells have been implicated in the development of hepatic necrosis following acetaminophen poisoning. Cultured hepatocytes exposed to acetaminophen release unidentified mediators that activate Kupffer cells in vitro. Activated Kupffer cells have been identified in liver tissue following acetaminophen poisoning in experimental animals. Gadolinium chloride pretreatment blocks Kupffer cell function and inhibits hepatic necrosis in a rat model of acetaminophen poisoning, without affecting the metabolism of acetaminophen. Interestingly, pretreatment with lipopolysaccaride, a macrophage activator, also prevented hepatic necrosis in this model (Laskin et al., 1995). Goldin and colleagues, using dichloromethylene diphosphonate containing liposomes, found Kupffer cell blockade only delayed, but did not prevent, hepatic necrosis following acetaminophen poisoning in a murine model. Further investigation of
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the potentially important proinflammatory cytokines produced by Kupffer cells has produced conflicting results. Inhibition of TNF␣ with neutralizing antibodies have shown reduced liver injury in some studies but not others. Administration of exogenous IL10 or soluble TNF receptors also fail to prevent acetaminophen induced liver necrosis in vivo. TNF receptor knockout mice are not resistant to acetaminophen poisoning (Chin et al., 2003). Three of the four chemokine families have also been implicated in acetaminophen liver injury. Our recent studies have implicated fractalkine (CX3CL1) in the hepatic reparative response following acetaminophen poisoning because of its unique function in leukocyte trafficking (Bone-Larson et al., 2000). Constitutive expression of fractalkine (CX3CL1) mRNA was identified in Kupffer cells and hepatocytes, especially the hepatocytes around the central veins. These observations were further explored by studying fractalkine (CX3CL1) knockout and transgenic mice. Six hours following acetaminophen injection, serum transaminases (as a circulating measure of hepatic injury) were significantly increased in the transgenic mice compared with wild type controls. Conversely, serum transaminases were significantly lower in fractalkine (CX3CL1) knock-out mice, at the same time point. To further investigate the potential for fractalkine (CX3CL1) to augment hepatic injury following acetaminophen poisoning via increasing neutrophil influx, hepatic myeloperoxidase, (MPO) was quantified. At 6 and 24 hours post-acetaminophen, significantly higher hepatic MPO levels were found in the transgenic mice compared with the wild type controls, and significantly lower levels of MPO were detected in the liver samples from the knock-out mice compared with wild type controls. In contrast with these data, others have suggested acetaminophen injury may be mediated via extensive Fas mediated apoptosis of hepatocytes (Zhang et al., 2000). Fractalkine (CX3CL1) is protective against Fas mediated apoptosis in certain brain cells. However, we have not found altered mortality and liver injury following Fas injection in fractalkine (CX3CL1) transgenic mice. Other preliminary data have shown up-regulation of fractalkine (CX3CL1) mRNA in the murine concanavallin A hepatitis model. Inhibition of chemokine expression is associated with reduced proinflammatory cytokine production and improved survival. Others have shown that both fractalkine (CX3CL1) and its receptor are expressed in human liver tissue following injury; furthermore, the human hepatoma cell line, HepG2 expresses both fractalkine (CX3CL1) and CX3CL1R and migrate when exposed to fractalkine (CX3CL1) (Efsen et al., 2002). These data suggest that over expression of fractalkine (CX3CL1) in the liver exacerbates liver injury associated with acetaminophen poisoning by increasing neutrophil recruitment and oxidative injuries. In contrast with fractalkine (CX3CL1), MCP1 (CCL2) has effects on experimental acetaminophen poisoning via alteration in the expression of other
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mediators (Dambach et al., 2002; Hogaboam et al., 2000). Hepatic concentrations of MCP1 (CCL2) significantly increase after acetaminophen overdose in mice. However, in CCR2 knock-out mice there was dramatically more injury compared with wild type mice. This increase in acetaminophen hepatotoxicity was related to an increase in hepatic expression of the proinflammatory cytokines, IFN␥ and TNF␣, suggesting that MCP1 (CCL2) can down-regulate proinflammatory cytokine expression. Similar data has been observed in experimental sepsis. Acetaminophen poisoning induces the production of reactive oxygen species in the liver. Free radicals can also stimulate MCP1 (CCL2) production in transformed stellate cells and Kupffer cells in vitro and in vivo in other oxidative liver injury models, e.g. CCL4 (Morra et al., 1999a, b). In contrast with acetaminophen poisoning, anti-oxidant pretreatment with vitamin E, dramatically reduced MCP1 (CCL2) concentrations following carbon tetrachloride. Thus, MCP1 (CCL2) appears to play a conflicting role during oxidative injury in these two murine models, and ongoing studies are needed to elucidate the underlying mechanism. Acetaminophen also increases the hepatic expression of CXC chemokines such as KC and MIP2. MIP2 has effects on the hepatic response to acetaminophen that does not only depend on the recruitment of neutrophils. Initial observations revealed that mice receiving 108 p.f.u of an adenovirus, containing rat MIP2 cDNA followed by acetaminophen had significantly reduced hepatic injury compared with mice that received an empty cassette (Hogaboam et al., 1999a, b). This was associated with a decrease in hepatic neutrophil infiltration and an increase in hepatocyte proliferation. In addition mice receiving the adenovirus expressing MIP2 had reduced liver injury compared with controls, suggesting that increasing hepatic MIP2 may limit adenoviral hepatitis. These effects of adenoviral administration were reproduced following recombinant protein administration; CXC chemokines such as MIP2 and IL8 (CXCL8) limited hepatic injury following acetaminophen poisoning, but non-ELR CXC chemokines had no effect. Perhaps more importantly, the beneficial effects of MIP2 still occurred at delayed time points, when NAC (the antidote for acetaminophen poisoning) was no longer effective. Inhibiting the effects of endogenous MIP2 with neutralizing antibody increased liver injury. Hepatic injury was also increased in CXCR2 knockout mice and following administration of an anti-CXCR2 antibody, demonstrating that these effects of MIP2 are mediated by binding with CXCR2. Increased hepatic expression of other chemokines has been reported. Acetaminophen induces hepatic IP10 (CXCL10) and CXCR3 expression (Bone-Larson et al., 2001). Recombinant IP10 (CXCL10) administered 10 hours after acetaminophen poisoning, when liver injury is established, improves murine survival and hepatic histology. This effect is mediated via induction of CXCR2 expression on hepatocytes and is limited by immunoneutralization of CXCR2
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(Bone-Larson et al., 2001). Others have suggested the hepatoprotective effects of IP10 (CXCL10) are mediated by stimulating hepatocyte growth factor production from hepatocytes (Koniaris et al., 2001). Alternatively, hepatic stellate cells are important in hepatic regeneration following acute injury and these cells express CXCR3 (Pinzani & Marra, 2001). Exposure of stellate cells to IP10 (CXCL10), activates a number of intracellular signaling pathways and induces cellular chemotaxis but not proliferation, providing another potential effect of IP10 (CXCL10) in the response of the liver to injury. Following toxic liver injury the clinical outcome is determined by the balance between ongoing hepatic injury, which may be aggravated by infiltrating inflammatory cells or their cytokine products, and the regenerative processes of the liver. This is a complex balance and is difficult to study in vivo. However, 70% partial hepatectomy (PH) provides a useful model to investigate hepatocyte proliferation in the absence of significant liver injury. ENA78 (CXCL5) and MIP2 are upregulated following PH in rats and immunoneutralization of these chemokines slows the rate of hepatic regrowth, without affecting the recruitment of neutrophils to the liver remnant (Colletti et al., 1998). These chemokines also induced hepatocyte proliferation in vitro, an effect that was dependent on the ELR amino acid motif (Colletti et al., 1998). The complex role of cytokines in hepatic regeneration has been the subject of several recent reviews (Diehl & Rai, 1996a; Hoffman et al., 1994). Many cytokines are hepatocyte mitogens in vitro, but their relative importance in vivo remains unclear. EGF, transforming growth factor alpha (TGF␣) and hepatocyte growth factor (HGF) are complete hepatocyte mitogens, but recent studies have suggested a central role for TNF␣, in hepatic regeneration following both toxic and 70% hepatectomy (PH). Exogenous TNF␣ stimulates hepatic DNA synthesis in rodents and accelerates recovery of liver weight following PH. Although serum TNF␣ concentrations are not elevated following PH, anti-TNF antibodies inhibit regeneration in this model (Bruccoleri et al., 1997). Hepatic regeneration following PH is also impaired in TNFR1 knock-out mice (Yamada et al., 1997). In addition, Kupffer cell blockade augments hepatic regeneration following PH secondary to impaired IL10 release from these cells allowing sustained unopposed TNF␣ production from endothelial cells (Rai et al., 1997). AntiTNF antibodies have an even greater inhibitory effect on hepatic regeneration following PH in chronic ethanol fed rats. TNF␣ released following PH has also been implicated in the activation of several intracellular signaling pathways (e.g. STAT 3) and transcription regulators (e.g. AP1, C/ERB, NF-kB) which may control the expression of protooncogenes and the progression of hepatocytes through the cell cycle during regeneration (Cressman et al., 1995; Diehl et al., 1995; Taub, 1996). Lead nitrate induces liver cell hyperplasia and increases hepatic TNF␣
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concentrations but two other inducing agents, cyproterone acetate or nafenopin, are not associated with increased hepatic TNF␣ expression (Menegazzi et al., 1997). Dexamethasone, a relatively non-specific anti-inflammatory agent, reduces TNF␣ concentrations, without affecting HGF expression, and inhibits hepatocyte proliferation induced by lead nitrate. The TNF␣ responsive cytokine IL6 also plays an important role in hepatocyte proliferation. IL6 knockout mice have delayed liver regeneration following PH.
Viral Hepatitis and Virus Based “Gene” Therapy The advent of transgenic technology has led to its use in the study of hepatic inflammation. IFN␥ transgenic mice develop a chronic hepatitis, which appears mediated by TNF␣ (Okamoto et al., 1996). The HBV transgenic mouse has improved greatly the understanding of the pathogenesis of hepatic damage in HBV infection (Chisari & Ferrari, 1995). The observed reduction in HBV transcription following infection with other hepatotropic viruses or bacteria in HBV transgenic mice appears secondary to the local production of IFN␥ and TNF␣ by macrophages and cytotoxic lymphocytes (Guidotti et al., 1996). An inhibitory effect on HBV replication in these mice can also be induced by exogenous recombinant IL12 administration via the induction of IFN␥ (Cavanaugh et al., 1997). IFN␥ can induce apoptotic cell death in primary hepatocyte culture (Morita et al., 1995) and this mechanism may be important in clearance of viral infected cells in vivo. Adenovirus transfection or liposomal encapsulation, the latter with or without retrovirus infection (Wu & Zern, 1996), have been used to study the transient expression of cytokines in the liver. For example, adenovirus mediated expression of CINC, a CXC chemokine, in the liver is associated with the development of a neutrophilic hepatitis (Maher et al., 1997). IFN␥ is found in the serum of patients with viral hepatitis and acute cellular rejection (Caccionelli et al., 1996). Increased circulating TNF␣ and M-CSF have been reported in patients with viral hepatitis (Spengler et al., 1996). Increased circulating IL6 occurs in patients with HCV infection and is correlated with hepatic inflammatory activity and serum HCV-RNA levels (Malaguarnera et al., 1997). Monocyte production of proinflammatory cytokines is increased in patients with hepatitis C, although others have shown impaired PMA stimulated TNF␣ and IL1 release (Mendoza et al., 1996). Mitogen and HCV peptide stimulated T cells, derived from peripheral blood, produce TNF␣ and IFN␥ but little IL4. In contrast, spontaneous IL10 production and HCV peptide stimulated IL12 production was similar in patients with HCV infection compared with patients with liver cirrhosis and controls (Kakumu et al., 1997). Many studies using RT-PCR, in-situ hybridization or
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immunohistochemistry have demonstrated hepatic expression of a variety of different cytokines in patients with liver disease. Hepatic expression of TNF␣ has been reported in hepatitis B, hepatitis C, autoimmune liver disease and alcoholic hepatitis (Clorentte et al., 1996; Fang et al., 1996). Recent interest has focused on differentiating the T helper cell responses in various inflammatory conditions into Th1, characterized by IFN␥ and IL2 expression and a cell-mediated response, or Th2, characterized by IL4 and IL5 expression and an antibody mediated immune response. A Th2 response may be associated with fibrosis and viral persistence; in contrast, a vigorous Th1 response may damage surrounding hepatocytes (a bystander effect) but result in viral clearance. Hepatic expression of Th1 cytokines has been reported in patients with hepatitis C infection, in addition T cell clones prepared from liver tissue produce Th1 cytokines following stimulation with recombinant HCV proteins (Lohr et al., 1996; Napoli et al., 1996). In contrast with HCV infection, the situation in patients with hepatitis B infection is more complicated. Patients infected with HBV express both IL4 and IFN␥ when the liver is severely inflamed (Bertoletti et al., 1997). Others have shown hepatic expression of Th1 cytokines in patients with acute HBV and active inflammation and Th2 cytokines in patients with chronic infection and low levels of hepatic inflammation (Fukuda et al., 1995). However, it is not clear if this latter finding is related to switching of the immune response from Th1 to Th2, as has been reported in the natural evolution of immune responses in mice. The T helper response in hepatitis B may be dependent on the antigen expressed at a particular time. Hepatitis B core antigen can induce a Th1 response and hepatitis B e antigen induces Th2 or Th0 helper T cells in vivo, this effect can also be modulated by exogenous IFN␣ (Milich et al., 1997). IFN␥ stimulation or viral infection induces expression of MIG (CXCL9), IP10 (CXCL10) and I-TAC (CXCL11) in a variety of cells including monocyte/macrophages, neutrophils and stromal cells such as hepatocytes (Farber, 1997). In vivo treatment with IFN␥ or IL12/IL2 up-regulates both hepatocyte and non-parenchymal cell expression of MIG (CXCL9) (Rowell et al., 1997). Hepatic RANTES (CCL5) is upregulated in patients with hepatitis C infection (Kusano et al., 2000). MIG (CXCL9) expression occurs on sinusoidal epithelial cells in both normal and hepatitis C infected liver tissue, and expression is up-regulated in the latter tissue (Sheilds et al., 1999). This expression of MIG (CXCL9) is associated with hepatic infiltration of CXCR3 positive T cells, suggesting the interaction between CXCR3 and its ligands are important in recruiting lymphocytes to sites of inflammation within liver tissue. Whether this particular chemokine/chemokine receptor interaction is harmful or beneficial to the liver remains to be defined. IP10 (CXCL10) is also expressed in liver tissue from patients with hepatitis C infection and relates to hepatic infiltration with CXCR3 positive T cells within the hepatic
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lobules (Sheilds et al., 1999). Expression of IP10 (CXCL10) is more specific for hepatic inflammation than MIG (CXCL9) and is undetectable in normal liver tissue. In vitro, sinusoidal lining endothelial cells release higher concentrations of IP10 (CXCL10) compared with MIG (CXCL9), following cytokine stimulation. As with MIG (CXCL9), whether this interaction aggravates or attenuates hepatic injury is not clear. Hepatic expression of IP10 (CXCL10) is also induced in a murine model of hepatitis B infection (Kakimi et al., 2001). Interestingly inhibition of IP10 (CXCL10) reduced hepatic inflammation and liver injury, but did not affect the antiviral potential of transferred virus specific cytotoxic lymphocytes, which is dependent on IFN␥ production. Hepatitis C infection is also associated with increased hepatic expression of MCP1 (CCL2), a concomitant increase in hepatic MCP1 (CCL2), and decrease in IP10 (CXCL10) correlated with a better response to interferon treatment in this disease (Narumi et al., 1997). Adenovirus vectors have been used clinically to deliver genes for therapeutic benefit; however, these vectors may induce a potentially fatal hepatitis. Injection with empty control vector leads to hepatitic infiltration with neutrophils and mononuclear cells. This is preceded by increased hepatic expression of MIP2, MCP1 (CCL2), and IP10 (CXCL10). Interestingly, increasing hepatic expression of the CXC chemokine, MIP2, using an adenoviral expression vector, was associated with reduced liver injury. This contrasts with other studies, which have shown acute hepatitis following administration of a CINC expressing adenovirus. These differing reports may relate to the dose of adenovirus administered or the chemokine concentration induced in the liver. Increased dose of adenovirus or hepatic concentration of chemokine appears to be associated with increased liver damage.
Hepatic Allograft Rejection Increased hepatic expression of a variety of chemokines and cytokines have been reported in hepatic allografts associated with the mixed inflammatory cell infiltrate occurring during allograft rejection. IL8 (CXCL8) concentrations are increased in both the graft and serum during rejection. Hepatic expression of IL5, a potent eosinophil chemoattractant, has been reported in patients with acute cellular rejection (Martinez et al., 1993), which can be associated with hepatic eosinophilia. Acute cellular rejection is also characterized by lymphocytic inflammation and hepatic expression of the CC chemokines, MIP1alpha (CCL3) and MIP1beta (CCL4) (Adams et al., 1996). A recent study has shown stromal cell-derived factor (SDF {CXCL12}) expression is induced in biliary epithelium in rejecting hepatic allografts and this is correlated with CXCR4 expressing lymphocyte infiltration. Observational studies have also implicated MIP1alpha (CCL3) and MIP1beta
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(CCL4) in the development of hepatic allograft rejection. Expression of MIP1alpha (CCL3) is up-regulated in sinusoidal endothelium, central veins and infiltrating inflammatory cells in patients with rejection. Endothelial cell MIP1alpha (CCL3) expression is up-regulated early after the liver transplant operation and may thus be important in the initial recruitment of T cells into the graft, with subsequent amplification of the inflammatory response by the expression of MCP1 (CCL2) by the recruited cells (Goddard et al., 2001). Successful treatment of allograft rejection with pulse steroid therapy is associated with down-regulation of endothelial MIP1alpha (CCL3) expression.
CHRONIC LIVER DISEASE Continuing liver cell injury, varying severity of hepatic inflammation and fibrosis are the hallmarks of chronic liver disease. Experimental studies reveal that these features can be reproduced with repeated toxic insults to the liver using CCl4 or concanavallin A (ConA). Clinically, the result of the ongoing injury and scarring, results in cirrhosis and a predisposition to liver cell cancer. The most common causes of chronic liver disease are alcohol and chronic viral hepatitis.
Alcoholic Liver Disease Elevated circulating TNF␣ or IL1 have been observed in patients with alcoholic liver disease, especially those who are malnourished, and have been correlated with survival (Means et al., 1996). In addition to increased TNF␣, elevated IL8 has been reported in patients with alcoholic liver disease (Sheron et al., 1993). Increased circulating IL8 (CXCL8) may have a suppressive effect on neutrophil function as has been reported in IL8 transgenic mice, these mice are unable to mount a neutrophil response to intraperitoneal injection of bacteria (Simonet et al., 1994) as discussed above. LPS induced TNF␣ expression is increased in blood monocytes from patients with alcoholic liver disease due to a decreased responsiveness to the anti-inflammatory effects of IL10 (Lemoine et al., 1995). IL8 (CXCL8) is expressed in the hepatic parenchyma at sites of inflammation during alcoholic hepatitis and correlates with the neutrophilic infiltrate characteristic of this form of liver injury (Maltby et al., 1996). In alcoholic cirrhosis, the expression of IL8 (CXCL8) was more restricted to inflammatory cells and endothelium in fibrous septa and portal tracts (Afford et al., 1998). Experimental models of alcoholic liver disease also reveal increased plasma and hepatic MIP2 concentrations, which also correlate with the degree of necroinflammatory changes in the liver (Nanji et al., 1999). Isolated Kupffer cells from
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alcohol fed rats spontaneously produce 4-fold more MIP2 than Kupffer cells from pair-fed controls, suggesting that Kupffer cells are a major source of MIP2 in this model (Bautista, 1997). In addition to the potential for recruiting neutrophils, MIP2 was also directly toxic to hepatocytes prepared from chronic alcohol fed animals. This effect could be blocked by cyclohexamide, implicating protein synthesis in the hepatotoxic effects of MIP2 (Bautista, 1997). Hepatic IP10 (CXCL10) expression is also increased in experimental models of alcoholic liver disease. The highest expression was reported in the animals with the most liver injury (Nanji et al., 1999). In vitro studies have also shown that IL8 (CXCL8) or CINC can be produced directly by ethanol-challenged hepatocytes or as a result of cell to cell interactions with Kupffer cells. CC chemokine expression has also been implicated in the pathogenesis of alcoholic liver disease. In patients with alcoholic hepatitis, circulating concentrations of MCP1 (CCL2) were increased and spontaneous production of MCP1 (CCL2) was increased in both intrahepatic and peripheral blood mononuclear cells (Fisher et al., 1999). This increased production was correlated with high serum transaminase levels, suggesting that MCP1 (CCL2) may be linked with hepatocyte death in this disease. Furthermore, MCP1 (CCL2), along with other chemokines such as IL8 (CXCL8), MIP1alpha (CCL3) and MIP1beta (CCL4) are all detected in the parenchyma at sites of inflammation in patients with alcoholic hepatitis. Experimental alcoholic liver disease is also associated with increased MCP1 (CCL2) expression. MIP1alpha (CCL3) is also expressed at sites of inflammation during alcoholic hepatitis, and released spontaneously by cultured peripheral blood mononuclear cells in this disease (Fisher et al., 1999).
Concanavallin A Hepatitis and Other Chronic Inflammatory Liver Diseases Concanavallin A (Con-A) injection induces massive T cell activation, infiltration of the liver with neutrophils, CD4+ and CD8+ lymphocytes, and focal hepatic necrosis. Repeated Con-A injection leads to liver fibrosis and scarring. Elevation of serum IL-2, TNF␣ and IFN␥ have been reported. In vitro studies have implicated perforin mediated hepatocyte injury, observing that TNF␣ is not directly cytotoxic to Con-A treated hepatocytes (Watanabe et al., 1996). In contrast, others have shown Con-A is directly cytotoxic to cultured hepatocytes (Leist & Wendel, 1996). However, in vivo administration of anti-TNF antibody, anti IFN␥ antibody or recombinant IL6 prevents hepatic damage without affecting IL2 release or hepatic infiltration of inflammatory cells (Mizuhara et al., 1996). Soluble TNF receptor is also effective in preventing liver damage in this model (Bruck et al., 1997). A recent study found increased plasma MIP2 (but failed to measure hepatic
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concentrations) following Con-A injection, neutrophil influx into the liver and a partial protective effect of immunoneutralization of MIP2, with approximately 50% reduction in plasma transaminases (Nakamura et al., 2001). This protective effect of MIP2 was also associated with a partial reduction in hepatic neutrophil influx. Presumably, other CXC chemokines and CC chemokines are induced in this model and are important in mediating both neutrophil and lymphocyte influx into the liver. Differentiation of the immune response into Th1 or Th2 like responses in patients with primary biliary cirrhosis (PBC) is more confused. Mitogenic stimulation of liver infiltrating T cells from patients with PBC and some with autoimmune hepatitis produce Th2 cytokines (Lohr et al., 1994). However others, studying the intrahepatic expression of IFN␥ and IL4, have suggested a predominance of a Th1 phenotype in PBC (Harada et al., 1997). Defective PPD stimulated Th1 and Th2 cytokine production by peripheral blood cells has also been reported in patients with PBC (Jones et al., 1997). Hepatic expression of IL5, a potent eosinophil chemoattractant, has been reported in conditions such as PBC (Martinez et al., 1995) which can both be associated with hepatic eosinophilia. SLC (CCL21) was cloned from a mouse thymic cDNA library and is expressed in other tissues including spleen, heart and kidney (Yoshie et al., 1997). SLC (CCL21) is chemotactic for mature dendritic cells, na¨ıve and memory T cells, B cells and cultured renal mesangial cells by binding to CCR7 (Yoshida et al., 1998). Murine SLC (CCL21) is also able to bind to and signal through CXCR3, in contrast with human SLC (CCL21) (Soto et al., 1998). Physiologically SLC (CCL21) may function in the homeostatic recirculation of lymphocytes in vivo. Recent data has also implicated SLC (CCL21) in homeostatic proliferation of CD4 positive T cells and progression toward autoimmune disease (Ploix et al., 2001). Others have highlighted the complexity of the chemokine system and the danger of implicating the role of chemokines in vivo from in vitro data, using SLC (CCL21) as an example (Chen et al., 2002a, b). Organ specific transgenic expression of SLC (CCL21) is associated with varying inflammatory infiltrates, depending on the site of expression. Increased expression of SLC (CCL21) in the pancreas was associated with lymphocyte recruitment and the development of lymphoid node-like structures. In contrast, expression in the brain leads to recruitment of eosinophils and neutrophils, possibly due to up-regulation of KC, MIP2 and CCL11, and expression in the skin was not associated with any inflammatory infiltration (Chen et al., 2002a, b). In vivo studies have shown expression of SLC (CCL21) in the small vascular channels associated with portal inflammatory infiltrates in murine granulomatous hepatitis and human cholestatic liver diseases (Grant et al., 2002; Yoneyama et al., 2001). In human studies, the expression of SLC (CCL21) was associated with infiltration with CCR7 expressing lymphocytes. Interestingly, neutralization of SLC (CCL21)
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in the murine model, although reducing portal tract associated lymphoid tissue, interfered with dendritic cell migration limiting local and draining lymph node immune responses. This resulted in failure to eliminate the infective agent inducing the granulomata with persistence of hepatic granulomas and liver damage.
HEPATIC FIBROSIS The stellate cell plays a central role in the development of hepatic fibrosis and cirrhosis. The effect of cytokines on stellate cell biology have been reviewed recently (Friedman, 2003). In a nutshell, stellate cell proliferation and collagen synthesis can be influenced by Kupffer cell (e.g. TGF and TNF␣), endothelial cell (e.g. PDGF) and hepatocyte (e.g. insulin like growth factor and IGF-binding protein) derived factors (Gressner et al., 1995). Stellate cell derived PDGF also has an autocrine effect on the producing cells. IL1, IL4 and IL6 can also modulate stellate cell collagen and cytokine synthesis. Another example of cytokine networking involving hepatic stellate cells (Benyon et al., 1997) is their reported ability to secrete stem cell factor (SCF). SCF activates mast cells and prolongs their survival by inhibiting apoptosis. Studies have correlated the degree of mast cell infiltration with the severity of hepatic fibrosis (Farnell et al., 1995). Hepatic SCF mRNA and protein concentration is increased in fibrotic liver diseases in patients and following bile duct ligation in experimental animals. A salient finding is that human hepatic stellate cells proliferate when exposed to human mast cell tryptase. Therefore, both mast cells and stellate cells may interact via SCF and tryptase to promote fibrogenesis within the liver. MCP1 (CCL2) may play an important role in the perpetuation of hepatic injury and fibrosis. In cirrhosis, MCP1 (CCL2) expression is up-regulated in portal tracts, epithelial cells of regenerating bile ducts and active septa, activated stellate cells and Kupffer cells (Czaja et al., 1994; Marra et al., 1998). Infiltration of monocytes and macrophages into the portal tracts correlated with increased MCP1 (CCL2) expression. In response to injury, hepatic stellate cells undergo a phenotypic change from a lipid storing relatively quiescent cell into myofibroblast like cells capable of increased matrix synthesis, contraction and cytokine and chemokine synthesis. Early studies showed MCP1 (CCL2) expression and release by activated stellate cells both in vivo and in vitro (Marra et al., 1999a). MCP1 (CCL2) is also chemotactic for hepatic stellate cells and stimulates various intracellular signaling pathways including tyrosine kinases and phosphatidyl-inositol-3-kinases, but they do not express CCR2 (Marra et al., 1999b). These data suggest that there is another MCP1 (CCL2) receptor yet to be characterized. Stimulation of intracellular signaling pathways associated with proliferation and survival suggest that MCP1
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(CCL2) may perpetuate the transformed and profibrotic myofibroblast phenotype and also recruit more stellate cells to the site of fibrosis. Thus, therapies aimed at neutralizing this chemokine may decrease fibrogenesis.
PRIMARY AND SECONDARY LIVER CANCER Certain cytokines have been implicated in the development of hepatic cancer. TGF over expression in transgenic mice is associated with a 60% incidence of spontaneous hepatoma. However, the incidence in HGF/TGF double transgenics is only 30%, suggesting coexpression of HGF may protect against hepatocarcinogenesis. Hepatoblastomas are often associated with extramedullary hematopoiesis within the liver and can express the hematopoietic cytokines, erythropoietin, stem cell factor, G-CSF and GM-CSF. Carcinoembryonic antigen can induce Kupffer cell production of TNF␣ and IL1 (Gangopadhyay et al., 1996), these proinflammatory cytokines may induce endothelial cell adhesion molecule expression and thus enhance the potential for metastasis. Elevated circulating concentrations of IL8 (CXCL8) have been reported in patients with hepatoma (Sakamoto et al., 1992) and may contribute to the immunodeficiency of such patients. Transcatheter arterial embolization is a commonly used therapy for hepatoma. Fever and an inflammatory response often follow tumor embolization. This has been associated with increased circulating IL6 concentrations; but systemic TNF␣ or IL1 are unaffected (Matsuda et al., 1994). TNF␣, IL1, IL6 and IFN␥ production have been implicated in the weight loss and cachexia induced by malignant tumors (Argiles & Lopez Soriano, 1997). Implantation of Morris 7777 hepatoma cells in SCID mice induces TNF␣, IL-1 and IL6 production from spleen cells and is associated with profound weight loss (Murray et al., 1997). In contrast, transplantation of MCA sarcoma was not associated with loss or increased proinflammatory cytokine expression (Murray et al., 1997). Rats bearing the Yoshida AH-130 ascites hepatoma have a hypercatabolic state with increased circulating TNF␣ concentrations. Anti-TNF antibodies normalize the muscle protein synthesis abnormalities and disturbances in hormone balance, but are unable to prevent weight loss in this model (Costelli et al., 1993, 1995). Hepatocellular cancer can be an outcome of liver fibrosis and cirrhosis in up to 5% of patients. Lymphocytes infiltrating hepatocellular cancer have strong chemotactic responses to both CC and CXC chemokines in vitro and express high levels of CXCR3 and CCR5. The corresponding ligands for these receptors are detected on vascular endothelium and sinusoidal endothelium. There was decreased expression of CXCR4 on leukocytes and the expression of this receptor
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correlates with hepatic inflammation and fibrosis. Thus, in the case of cancer, the augmentation of certain chemokines to perpetuate the proinflammatory response may provide a beneficial therapeutic option that remains to be investigated. In contrast with hepatitis C infection, in which IP10 (CXCL10) appears to be more important, MIG (CXCL9) expression is strongly expressed on vascular and sinusoidal endothelium in primary hepatic tumors and induces chemotaxis of tumor infiltrating lymphocytes (Yoong et al., 1999). IP10 (CXCL10) was not expressed on endothelium in either primary or secondary hepatic tumors. Thus, in the case of cancer, the augmentation of MIG (CXCL9) expression to perpetuate the proinflammatory response may provide a beneficial therapeutic option that remains to be investigated. SDF1 alpha, the ligand for CXCR4, is reduced in hepatocellular carcinoma, but not in other chronic liver diseases such as hepatitis C infection.
CONCLUSION While our understanding of the cytokines and chemokines involved in liver injury and repair has greatly expanded recently, our therapeutic options for treatment of acute and chronic liver disorders remains limited. Potentially some of the cytokine or chemokine targets discussed above may act as targets for novel therapies such as gene therapy or siRNA knockdown.
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Schaub, F. J., Han, D. K., Liles, W. C., Adams, L. D., Coats, S. A., Ramachandran, R. K., Seifert, R. A., Schwartz, S. M., & Bowen-Pope, D. F. (2000). Fas/FADD-mediated activation of a specific program of inflammatory gene expression in vascular smooth muscle cells. Nat. Med., 6, 790–796. Schindeler, C., & Darnell, J. E. (1995). Transcriptional responses to polypeptide ligands, the JAK/STAT pathway. Ann. Rev. Biochem., 64, 621–651. Schuchmann, M., & Galle, P. R. (2001). Apoptosis in liver disease. Eur. J. Gastroenter. Hepatol., 13, 785–790. Sen, C. K., & Packer, L. (1996). Antioxidant and redox regulation of gene transcription. FASEB J., 10, 709–720. Serizawa, A., Nakamura, S., Suzuki, S., Baba, S., & Nakano, M. (1996). Involvement of PAF in cytokine production and neutrophil activation after hepatic ischaemia-reperfusion. Hepatology, 23, 1656–1663. Sheilds, P. L., Morland, C. M., Salmon, M., Qin, S., Hubscher, S. G., & Adams, D. H. (1999). Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver. J. Immunol., 163, 6236–6243. Sheron, N., Bird, G., Koskinas, J., Portmann, B., Ceska, M., Lindley, I., & Williams, R. (1993). Circulating and tissue levels of the neutrophil chemotaxin interleukin-8 are elevated in severe acute alcoholic hepatitis, and tissue levels correlate with neutrophil infiltration. Hepatology, 18, 41–46. Shimizu, Y., Murata, H., Kashii, Y., Hirano, K., Kunitani, H., Higuchi, K., & Watanabe, A. (2001). CCR6 and its ligand MIP3 alpha might be involved in the amplification of local necroinflammatory response in the liver. Hepatology, 34, 311–319. Shimoda, K., Begum, N. A., Shibuta, K., Mori, M., Bonkovsky, H. L., Banner, B. F., & Barnard, G. F. (1998). Interleukin-8 and hIRH (SDF1-alpha/PBSF) mRNA expression and histological activity index in patients with chronic hepatitis C. Hepatology, 28, 108–115. Shiratori, Y., Takada, H., Hai, K., Kiriyama, H., Mawet, E., Komatsu, Y., Niwa, Y., Matsumura, M., & Shiina, S. (1994). Effect of antiallergic agents on chemotaxis of neutrophils by stimulation of chemotactic factor released from hepatocytes exposed to ethanol. Dig. Dis. Sci., 39, 1569–1575. Shiratori, Y., Takada, H., Hikiba, Y., Nakata, R., Okano, K., Komatsu, Y., Niwa, Y., Matsumura, M., Shiina, S., Omata, M., & Kamii, K. (1993). Production of chemotactic factor, interleukin 8 from hepatocytes exposed to ethanol. Hepatology, 18, 1477–1482. Shito, M., Wakabayashi, G., Ueda, M., Shimazu, M., Shirasugi, N., Endo, M., Mukai, M., & Kitajima, M. (1997). IL1 receptor blockade reduces TNF production, tissue injury and mortality after hepatic ischaemia/reperfusion in the rat. Transplantation, 63, 143–148. Simonet, W. S., Hughes, T. M., Nguyen, H. Q., Trebasky, L. D., Danilenko, D. M., & Medlock, E. S. (1994). Long-term impaired neutrophil migration in mice over-expressing human interleukin-8. J. Clin. Invest., 94, 1310–1319. Soto, H., Wang, W., Strieter, R. M., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Hedrick, J., & Zlotnik, A. (1998). The CC chemokine 6Ckine binds the CXC chemokine receptor CXCR3. Proc. Natl. Acad. Sci. USA, 95, 8205–8210. Spengler, U., Zachoval, R., Gallati, H., Jung, M. C., Hoffman, R., Reithmuller, G., & Pape, G. (1996). Serum levels and in situ expression of TNF alpha and TNF alpha binding proteins in inflammatory liver diseases. Cytokine, 8, 864–872. Strand, S., Hofmann, W. J., Grambihler, A., Hug, H., Volkmann, M., Otto, G., Wesch, H., Mariani, S. M., Hack, V., Stremmel, W., Krammer, P. H., & Galle, P. R. (1998). Hepatic failure and liver
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cell damage in acute Wilson’s disease involve CD95 (APO-1/Fas) mediated apoptosis. Nat. Med., 4, 588–593. Suzuki, S., & Toledopereyra, L. H. (1994). IL1 and TNF production as the initial stimulants of liver ischemia and reperfusion injury. J. Surg. Res., 57, 253–258. Taub, R. (1996). Transcriptional control of liver regeneration. FASEB J., 10, 413–427. Thornton, A. J., Ham, J., & Kunkel, S. L. (1992). Kupffer cell derived cytokines induced the synthesis of a leukocyte chemotactic peptide, interlukin 8, in human hepatoma and primary hepatocyte cultures. Hepatology, 15, 1112–1122. Thornton, A. J., Strieter, R. M., Lindley, I., Baggiolini, M., & Kunkel, S. L. (1990). Cytokine induced gene expression of a neutrophil chemotactic factor/IL8 in human hepatocytes. J. Immunology, 144, 2609–2613. Tilg, H., Ceska, M., Vogel, W., Herold, M., Margreiter, R., & Huber, C. (1992). Interleukin-8 serum concentrations after liver transplantation. Transplantation, 53, 800–803. van den Abeele, P., DeClercq, W., Beyaert, R., & Fiers, W. (1995). 2 TNF receptors, structure and function. Trends Cell. Biol., 5, 392–399. van Gool, J., VanVugt, H., Helle, M., & Aarden, L. A. (1990). The relation among stress, adrenaline, interleukins and acute phase proteins in the rat. Clin. Immunol. Immunopath., 57, 200–210. Walley, K. R., Lukacs, N. W., Standiford, T. J., Strieter, R. M., & Kunkel, S. L. (1997). Elevated levels of macrophage inflammatory protein 2 in severe murine peritonitis increase neutrophil recruitment and mortality. Infect. Immun., 65, 3847–3851. Watanabe, Y., Morita, M., & Akaike, T. (1996). Con-A induces perforin but not Fas mediated hepatic injury. Hepatology, 24, 702–710. Wu, J., & Zern, M. A. (1996). Modification of liposomes for liver targeting. J. Hepatol., 24, 757–763. Yamada, Y., Kirillova, I., Peschon, J. J., & Fausto, N. (1997). Initiation of liver growth by TNF: Deficient liver regeneration in mice lacking type 1 TNF receptor. Proc. Natl. Acad. Sci. USA, 94, 1441–1446. Yoneyama, H., Harada, A., Imai, T., Baba, M., Yoshie, O., Zhang, Y., Higashi, H., Murai, M., Asakura, H., & Matsushima, K. (1998). Pivotal role of TARC, a CC chemokine, in bacteria induced fulminant hepatic failure in mice. J. Clin. Invest., 102, 1933–1941. Yoneyama, H., Matsuno, K., Zhang, Y., Murai, M., Itakura, M., Ishikawa, S., Hasegawa, G., Naito, M., Asakura, H., & Matsushima, K. (2001). Regulation by chemokines of circulating dendritic cell precursors and the formation of portal tract associated lymphoid tissue in granulomatous liver disease. J. Exp. Med., 193, 35–49. Yoong, K. F., Afford, S. C., Jones, R., Aujla, P., Qin, S., Price, K., Hubscher, S. G., & Adams, D. H. (1999). Expression and function of CXC and CC chemokines in human malignant liver tumors: A role for human monokine induced by gamma-interferon in lymphocyte recruitment to hepatocellular carcinoma. Hepatology, 30, 100–111. Yoshida, R., Nagira, M., Kitaura, N., Imagawa, N., Imai, T., & Yoshie, O. (1998). Secondary lymphoid tissue chemokine is a functional ligand for the CC chemokine receptor CCR7. J. Biol. Chem., 273, 7118–7122. Yoshie, O., Imai, T., & Nomiyama, H. (1997). Novel lymphocyte specific CC chemokines and their receptors. J. Leukoc. Biol., 62, 634–644. Zhang, H., Cook, J., Nickel, J., Yu, R., Stecker, K., Myers, K., & Dean, N. M. (2000). Reduction of liver Fas expression by an antisense oligonucleotide protects mice from fulminant hepatitis. Nature Biotechnol., 18, 862–867. Zlotnik, A., & Yoshie, O. (2000). Chemokines: A new classification system and their role in immunity. Immunity, 12, 121–127.
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8.
DRUG METABOLISM AND HEPATOTOXICITY
J. Michael Tredger INTRODUCTION Drug metabolism has become the comfortable generic term describing metabolism of all foreign compounds, xenobiotics or chemicals with no nutritive value (anutrients). It is a process that not only makes therapeutics possible by limiting the persistence of administered drugs but also prevents the accumulation of environmental chemicals and their associated potential for toxicity. Perhaps incongruously, the same pathways and enzymes may also be responsible for the conversion of seemingly innocuous parent drugs and chemicals into products with an enhanced profile of toxicity. The liver makes a substantial contribution to drug metabolism in vivo. This stems from its size, its anatomical position, its blood supply (25–30% of cardiac output via both hepatic artery and hepatic portal vein), its rich endowment of enzymes including the substrates, cofactors and products of intermediary metabolism, and its role in the production and excretion of bile. The same factors predispose the liver to drug-induced toxicity (hepatotoxicity). However, individual susceptibility to hepatotoxins is characteristically variable and a major objective of this chapter is to promote an understanding of how variability may originate within the processes and control of drug metabolism, and is compounded by heterogeneous responses to toxic drug metabolites. More extensive coverage of drug-induced liver injury appears elsewhere (Kaplowitz & DeLeve, 2003; Zimmerman, 1999) and both texts present detailed mechanisms of the hepatotoxicity of a large number of agents not possible within this brief account.
The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 207–228 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15008-3
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DRUG METABOLISM Therapeutic drugs typify many xenobiotics that are well absorbed in that they are frequently hydrophobic and will accumulate in lipoprotein-rich organs. Often, the parent drugs are both poorly extracted by the kidney (due to plasma protein binding) and extensively reabsorbed after renal filtration or biliary excretion. Conversion to more water soluble metabolites therefore promotes their excretion, so reducing the pharmacological activity of drugs and the accumulation (and potential toxicity) of anutrients and environmental contaminants. Drugs may also be removed from liver cells by specific transporter molecules such as p-glycoprotein or organic anion transporters, but these simply transport (and not transform) drugs, so will not be considered further here (but see Bohan & Boyer, 2002). Drugs are of diverse chemical structures, size and shape, ranging from the small aliphatic anti-epileptic valproic acid (mol. wt. 144) to the cyclic peptide immunosuppressant cyclosporine (mol. wt. 1202). The enzymes that catalyze the metabolism of these drugs are equally diverse, although many perform comparable chemical transformations and are therefore classified into families of functionally-related forms (isoenzymes or isoforms). Although the greatest accumulation of drug metabolizing enzymes is within the liver, all tissues show some capacity for transforming drugs, with the intestine, lungs and kidney making especially significant contributions. Since many drugs are administered orally, extensive metabolism in the intestine and particularly the liver may reduce the proportion of unchanged drug reaching the systemic circulation. Such drugs are said to show low bioavailability. If extensive, this first-pass effect may reduce efficacy at the intended site of action and may preclude oral therapy for some drugs (e.g. morphine). There is evidence that drug metabolizing enzymes have evolved from isoforms catalyzing similar reactions on endogenous substrates such as hormones, bile acids and bilirubin. Each isoform may show a unique responsiveness to controlling factors such as substrate, co-substrate and product as well as to a number of regulating factors. The latter not only include several of the endogenous modulators of intermediary metabolism (e.g. age, gender, circadian rhythms, hormones and nutrition) but also a range of exogenous factors, particularly other drugs. Drug metabolizing enzymes can therefore respond rapidly to changes in their prevailing environment, including the quantity of the substrates they metabolize.
Phase I and Phase II Metabolism Transformation to polar drug metabolites may involve Phase I reactions for introducing polar substituents into the parent molecule and/or Phase II reactions
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in which larger polar groups are added (by synthetic or conjugation reactions), increasing both polarity and size. Phase I metabolism of a drug may not be required before phase II reactions occur because suitable sites for conjugation may already exist. Simple increases in polarity promote urinary excretion, while increases in size additionally enhance biliary excretion. Phase I reactions typically involve oxidations, reductions and hydrolyses. Oxidations dominate and the majority of these are catalyzed by mono-oxygenase enzymes. In man, the dominant Phase II reactions involve conjugation with glucuronic acid, inorganic sulphate and the amino acids glycine and taurine. Acetylations and methylations are also common and conjugation with reduced glutathione (GSH) is a pathway involved frequently in detoxifying reactive metabolites. Further details can be found elsewhere (Timbrell, 1999). All phase I and phase II transformations are energy-requiring processes, with the former frequently consuming cofactors such as reduced NADP, and the latter involving activated substrates or co-substrates such as acetyl coenzyme A in the conjugation process (Table 1). Drug metabolizing enzymes catalyzing both phase I and phase II reactions are distributed throughout intracellular organelles with high concentrations in the hepatic smooth endoplasmic reticulum (e.g. mono-oxygenases and glucuronyltransferases), cytosol (e.g. dehydrogenases and sulphotransferases) and mitochondria (some amine oxidases and amino acid transferases). Distinct cell types within an organ (e.g. parenchymal, Kupffer and endothelial cells in liver) may contain different complements of enzymes as may cells of the same type located at different sites (e.g. in hepatocytes across the liver lobule).
Cytochromes P450 Oxidations dominate phase I reactions, and cytochrome P450 catalyzes the largest proportion of these. Cytochrome P450 is the generic term for a superfamily of hemoproteins (mol wt ca. 50 kD) that are membrane bound and catalyze the introduction of molecular oxygen into a substrate with the simultaneous utilization of reduced nicotinamide adenine dinucleotide phosphate (NADPH) – hence, mixedfunction oxidases. All share a common provider of electrons for reducing the cytochromes: NADPH-cytochrome P450 reductase. The DNA sequences of more than 200 cytochromes P450 are now described, including 57 human cytochromes P450 (CYPs) which are the product of 50 genes and additional pseudogenes. More than 20 CYPs are involved in drug metabolism and these are grouped in four families (CYP1–4) each of which shares at least 20% sequence homology (Nelson, 2003) (see Table 2). Individual isoenzymes within a family will metabolize a distinct and limited number of substrates, but overlap will exist with other members
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Table 1. Details of the Common Drug Metabolic Pathways. Enzyme
Reactions
Location
Cosubstrate/Cofactor
Typical Substrates
Phase I pathways CYP-dependent mono-oxygenase
Oxidation, dealkylation
SER
NADPH; molecular O2
Flavine mono-oxygenase
Deamination
SER
NADPH; molecular O2
Aldehyde and xanthine oxidases Alcohol/aldehyde dehydrogenases Nitro- and azo-reductase Peroxidases/prostaglandin synthase
Oxidation Oxidation by H removal Reduction of nitrogen Incorporation of: 0–0
Sol, SER Sol SER, Sol SER
Water NAD+ Various, NAD(P)-based Molecular O2
Many (Tredger & Stoll, 2002) Amines, hydrazines, sulfides etc Caffeine, purines Ethanol/acetaldehyde Azo dyes, nitrobenzene Arachidonic acid, prostaglandins Epoxides, esters, amides etc
Hydrolases Phase II pathways Glucuronyltransferases
Phase III pathways Glutathione conjugate hydrolase
Sol, SER
Water
SER
UDP-glucuronic acid
Inorganic sulfate Adds (or oxidases) GSH Acetate Glycine, taurine Methyl substituent
Sol Sol, SER Sol Mito, Sol Sol, SER
PAPS GSH Acetyl coenzyme A Acetyl coenzyme A S-adenosylmethionine
Degrades GSH conjugates
Sol
Water
Alcohols, phenols, amines, organic acids Alcohols, phenols, amines Acetaminophen Isoniazid, sulfonamides Benzoic acid Thiopurines e.g. azathioprine Glutathionyl acetaminophen
Abbreviations: CYP, cytochrome P450; GSH, ␥-glutamyl-cysteinyl-glycine; mito, mitochondria; NAD(PH): Nicotinamide adenine dinucleotide (phosphate), reduced; PAPS, phosphoadenosyl-phosphosulfate; SER, smooth endoplasmic reticulum; Sol, cytoplasmic.
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Sulfotransferases Glutathione S-transferases Acetyltransferases including Amino-acid transferases Methyltransferases
Cleavage with water Conjugations with: Glucuronic acid
CYPs Induced
Receptor
Inducers
Other Enzymes Induced
CYP1A1, CYP1A2, CYP1B1
Aryl hydrocarbon receptor (Ah/XRE) Constitutive androstane receptor (CAR)
Cigarette smoking, barbecued food, omeprazole Phenobarbital and amobarbital, butobarbitone, glutethimide, heptobarbitone, promethazine, secobarbital Rifampicin, carbamazepine, dexamethasone, phenylbutazone, phenytoin, St John’s Wort, sulfadimidine, sulfinpyrazone; Phenobarbital Fibrate anti-hyperlipidemics
GST, UGT
CYP2B6, CYP2A6, CYP3A
CYP1A1, CYPs2A, CYP2B6, CYP2C8, CYP2C9, CYP3A4, CYP3A5, CYP3A43 CYP2E1
Pregnane X receptor (PXR)
CYPs4A
Peroxisome proliferator-activated receptor (PPAR␣) TR Unknown
CYP Reductase 2E1
EH, GST, UGT Cytochrome P450 reductase ABCB1, SULT, UGT, Cytochrome P450 reductase
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Table 2. Enzyme Induction of Drug Metabolic Pathways.
Cytochrome P450 reductase
Thyroid hormone (T3) Ethanol and chloral hydrate, Isoniazid
Notes: CYPs in bold are primary targets; Items in italics await absolute confirmation; The XRE – is the nuclear binding site for the Ah receptor. The PXR is synonymous with PAR (the pregnane-activated receptor) and SXR (the steroid and xenobiotic receptor). Abbreviations: CYP, cytochrome P450; EH, epoxide hydrase; GST, glutathione transferases; MDR1, multiple drug resistance 1 gene product (P-glycoprotein); SULT, sulfotransferase; TR, thyroid hormone receptor; UGT, glucuronlytransferases.
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of the same family. There are also examples where distinct CYP isoenzymes metabolize the same substrate to different products (e.g. for the anticoagulant warfarin). As well as exhibiting unique characteristics in metabolic profile, each CYP family responds in a unique fashion to chemicals and drugs that induce (increase) or inhibit (decrease) enzyme activity. Consequently, it is possible to predict possible drug interactions if the metabolism of a particular drug can be assigned to a specific CYP which has known responses to enzyme inducers/inhibitors (Tredger & Stoll, 2002). More information on such environmental influences on drug metabolism appears below.
Physiological Variability in Drug Metabolism Normal healthy individuals will exhibit variability in drug metabolic activity which is related to their age and gender, to circulating hormones and to the time of day (circadian rhythms). These factors are briefly considered below and are termed physiological to contrast them with additional metabolic differences arising from genetic variability, pathology (i.e. disease) and external influences such as diet and environmental chemicals. This separation is however largely one of convenience and multiple interactions exist between these modulators of activity.
Age The major effects of age on drug metabolism are evident at the extremes of the lifespan i.e. in neonates and in the elderly. Variability within the heterogeneous human population has made quantitative comparisons difficult, whereas in animals it is clear that drug metabolic enzyme activities are usually significantly lower in both the very young and very old. In man, however, the longer gestation period allows greater maturation of enzymes than in the young of many animals, while age-related falls in drug clearance seem to reflect decreases in cardiac output, liver blood flow, oxygen delivery and liver size irrespective of comparable declines in enzymic activity in the elderly (Le Couteur & McLean, 1998). Neonatal jaundice is probably a good manifestation of the slowly developing function of metabolic enzymes in the very young and relates to impaired bilirubin conjugation with glucuronic acid. The enzymes involved (UDP-glucuronyltransferases) play a major role in drug conjugation but like oxidative pathways, the impairment usually disappears by 2–6 months of age (Ginsberg et al., 2002). However, the extent of the early impairment and rate of development varies from one enzyme to another.
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Subsequently, in childhood, a converse faster rate of metabolism seems to prevail until adulthood. This may relate to both the amount and type of (iso) enzymes present in children and has implications to therapy and prescribing in children (Ginsberg et al., 2002). Similar implications to therapeutics in the elderly derive from the declining clearance of drugs by both the liver and kidneys, particularly for agents with high first pass metabolism (Anantharaju et al., 2002; Zeeh, 2001).
Circadian Rhythms Variations in drug metabolic activity over a 24 h cycle are known to influence the disposition of natural and synthetic steroids as well as drugs including antihistamines, antihypertensive agents and acetaminophen. In animals, the cyclic variations in metabolic activity have been demonstrated to exhibit rhythmic extremes of activity usually differing only one or two-fold. The timing of the extremes may vary from one tissue to another and light cycle is a synchronizing factor, but control of the entire process is incompletely understood. In man, metabolism is simply one aspect of the cyclic variability in drug absorption, distribution, metabolism and excretion which determine circadian rhythms in drug handling. Other contributing factors include gastric acid secretion and pH, gastric emptying, intestinal motility, plasma protein binding, hepatic blood flow, glomerular filtration and perfusion, urinary pH and tubular resorption (Bruguerolle, 1998).
Gender Sex differences in drug handling relate both to absorption and distribution as well as to metabolism (Schwartz, 2003). Drug absorption may be faster in men, but is no more extensive overall. Body size (greater in men) and body fat (greater in premenopausal women) are determinants both of hepatic and renal clearance rates and volumes of distribution. Men generally clear drugs more rapidly than women and this applies to both phase I and phase II metabolic pathways. Exceptions include several CYP3A activities, which are faster in women (e.g. erythromycin demethylation), and rates of acetylation and CYP2C9 and CYP2C19 oxidations, which appear independent of gender. In animals, distinct enzymic isoforms are preferentially transcribed in males vs. females (e.g. cytochrome 2C11 in rats) and their activities are modulated by circulating sex steroid hormones, their presence during neonatal life and their influence on the secretion of growth hormone, thyroxine and other peptide hormones. Gender differences exist particularly in the drug handling and side-effects of general and local anesthetics, salicylates, diazepam and some
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hypoglycemic agents. Variations in hormone levels during the menstrual cycle and pregnancy have been suggested to affect the disposition of ethanol, caffeine, methaqualone and acetaminophen (Schwartz, 2003).
Hormones There is a complex interplay of hormones, cytokines and growth factors with drug metabolizing enzymes which is not fully understood. Modulation of metabolic activity has been associated with glucocorticoids, thyroid hormones, insulin, glucagon, the pituitary and hypothalamus (Zimmerman, 1999). One likely method by which hormones influence enzyme levels is by interacting with specific regulatory sites on the promoter regions of the genes affected. The review by Morgan and colleagues (Morgan et al., 1998) considers the mechanism of such interactions with the cytochromes P450, mainly using animal models where the clearest differences prevail.
Diet and Drug Metabolism Both the intake of food and its composition influence drug metabolizing enzyme activity. Food intake principally influences drug absorption by prolonging gastric emptying times, so enhancing uptake of drugs absorbed in the stomach and reducing that of drugs absorbed in the small intestine. The heavier the meal, the greater is gastric transit time, so fasting generally speeds drug absorption. Starvation (i.e. prolonged fasting) may have additional effects, reducing the availability of cosubstrates for conjugation reactions (especially, GSH and glucuronic acid) and causing a change in activity of some enzymes. For example, starvation enhances CYP2E1 activity, probably via reducing the breakdown of its mRNA, but such effects are not the rule for all enzymes and in all species (Ioannides, 1999). Where even more prolonged, i.e. in malnutrition, the consequences are often deleterious but frequently complex, especially where adverse dietary alterations occur in the amounts and proportions of the major nutrients (carbohydrate, lipid and protein), vitamins and minerals. An even greater complexity results where dietary alterations also alter exposure to a host of dietary components known to modulate intestinal and hepatic enzymes activities and including natural anutrients, food additives and dietary contaminants (Walter-Sack & Klotz, 1996). A well known example is the impairment by grapefruit juice of the metabolism of several CYP3A substrates including the immunosuppressant cyclosporin and calcium channel blockers such as nifedipine.
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Disease and Drug Metabolism Intestinal disease may impair drug absorption, heart disease may reduce blood flow, and drug delivery to metabolic sites, and renal disease will impair drug excretion. Systemic infections and inflammation frequently impair metabolism, often via the action of cytokines (Renton, 2001) and liver disease can impair drug clearance by hemodynamic, excretory or metabolic mechanisms. In cirrhosis and other causes of portal hypertension, liver blood flow is reduced and a collateral circulation may develop, routing blood flow around the liver. This will impair drug delivery and reduce metabolism, particularly for those high first-pass drugs which are cleared extensively when initially presented to the liver. Disorders interrupting bile flow (such as gallstones, biliary sclerosis or cirrhosis, and obstructive tumors) not only impair excretion but may also reduce absorption and metabolism: the absorption of hydrophobic drugs requiring bile for solubilization is especially affected. The same bile salts may be hepatotoxic if accumulated within the parenchyma. Severe hepatocellular damage profoundly impairs metabolism, typically when associated with acute liver damage (e.g. viral hepatitis or drug intoxication) or chronic endstage disease (e.g. cirrhosis). In less severe hepatic disease, drug metabolism is relatively well preserved, although oxidative metabolism frequently deteriorates before the conjugation pathways are affected substantially (Wilkinson, 1997).
Genetics and Drug Metabolism Genotype has both qualitative and quantitative influences on drug metabolism, determining the types of metabolizing enzymes an individual produces and the basal level at which these enzymes are expressed. Such dissimilarities underlie species differences in mammalian drug metabolism, with, for example, pigs being good at glucuronidation and poor at sulfation, while the opposite applies to cats. In man, the differences are more likely related to subtypes of such enzymes. The extent of structural similarity between different enzymes is now used commonly to group enzymes with related functions, such as the cytochromes P450. Those with amino acid sequences greater than 40% identical (homologous) belong to the same family while those with 55% homology are in the same sub-family. Individual forms (isoforms or isoenzymes) share even greater sequence homology and such isoenzymes may show discrete preferences for metabolism of a limited range of substrates, while sharing common features with members of the same sub-family, such as responsiveness to modulators. This categorization has produced logical classification systems and nomenclatures for enzymes within the same family – or a collection of families: a superfamily. For the CYP superfamily, family members
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share the same initial number, sub-families the same letter and individual isoforms are allocated a unique number, e.g. CYP2D6 is isoform 6 in the D sub-family of the CYP2 family. The corresponding gene is CYP2D6. Individual isoforms occur throughout the entire human population but individuals may express distinct variants of the same isoenzyme that differ only minutely e.g. by a single amino acid. Where occurring stably and at a frequency >1%, these variants give rise to multiple forms or polymorphisms of the same isoenzyme. Where related to variants at a single DNA base, these are single nucleotide polymorphisms (SNPs). SNPs may occur in the coding region (exon) of a gene, leading to a variant amino acid sequence (or a silent mutation if the alternative nucleotide triplet codes for the same amino acid) or in non-coding regions such as introns or the promoter. Variants sometimes code for a stop sequence in which case a truncated mRNA product results and an inactive protein. Where a SNP results in a base insertion or deletion, the integrity of triplet coding is lost and alternative products result which are frequently inactive. Promoter region polymorphisms may affect the amount rather than the quality of the gene product, and in some cases will enhance DNA transcription and increase protein product. Comparable overproduction of protein may result when gene replication occurs and the converse absence of product when entire genes are deleted. Therefore, genetic variants can lead to a heterogeneity in expression of polymorphic forms of a particular enzyme. Their activities will range from nothing to many times the population normal and the type or frequency of the underlying polymorphism may differ within ethnic groups. This phenomenon underlies a significant proportion of inter-individual variability in drug metabolic activity, although its extent will differ, depending additionally on the responsiveness of the same genes to exogenous factors (see below). The first recognized polymorphism in drug metabolism was probably that in acetylation status, although the underlying mechanism was not defined for more than forty years. Approximately 90% of Asians but 50% of Caucasians and Afro-Caribbeans are poor acetylators because they lack an active gene product of N-acetyltransferase. Debrisoquine hydroxylation was the first CYP polymorphism to be identified (in 1977) and was linked to polymorphisms in CYP2D6 in man in 1988. There are now known to be more than 70 variants of CYP2D6 (designated CYP2D6*1 to CYP2D6*46 (Nelson, 2003) and at least 30 drugs are transformed by the corresponding isoforms and express polymorphic metabolism, stratified into slow or fast (extensive) metabolizers. About 5–10% of Western Europeans and Americans are poor metabolizers, but only about 1% of Asian and Middle Eastern populations. Superfast metabolizers have been shown to express up to 13 gene copies of the parent (wild type) gene (CYP2D6*1) and their frequency varies some 50-fold among different ethnic populations. To date, at least 21 CYPs have been shown to be polymorphic (Oscarson et al., 2004), although
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the phenotypic effects of the different variants are not all known. Other CYP isoforms contributing significantly to variability in the metabolism of therapeutic agents include CYP2A6, CYP2C9, CYP2C19 and CYP3A5. Polymorphisms are also recognized among the majority of phase I and phase II metabolizing enzymes, including alcohol and aldehyde dehydrogenases, amine oxidases, UDP-glucuronyltransferases, glutathione S-transferases, sulphotransferases and methyltransferases. Aberrant variants of these enzymes may occasionally be linked to disease e.g. Crigler-Najjar patients have a defect in UDP-glucuronyltransferase 1A1 (UGT1A1) that causes a sometimes fatal unconjugated hyperbilirubinemia.
Environmental Influences on Drug Metabolism Drug metabolizing enzymes show a pronounced capability to respond to demand, as well as to physiological regulators (see above) and environmental components. The observation that drugs can affect their own metabolism was first recognized with barbiturates, where progressively increasing doses were required to maintain efficacy as rates of metabolism increased. This process was shown to involve increased synthesis of the drug metabolizing enzymes, a process known as enzyme induction, and was soon recognized to apply not only to the agent in question (autoinduction) but to other drugs. Also frequent is the converse reduction in the rate of a drug’s metabolism, mediated either by the drug itself or more frequently by other drugs or enzyme inhibitors. These processes apply not only to therapeutic drugs but also to many environmental components that share these characteristics.
Enzyme Induction and Enhanced Metabolism Enzyme induction, the increased synthesis of enzyme protein, is the most common mechanism by which drug metabolic activity is increased. Usually associated with de novo synthesis of mRNA and protein, increased activity can also result from synthesis using existing mRNA (by extending its lifetime, as with CYP2E1) or by enhancing the activity of existing protein. The last of these, enzyme activation, is unusual in vivo but may result from configurational changes in enzymes or from increased cosubstrate availability (e.g. where fructose enhances ethanol metabolism with alcohol dehydrogenase by increasing the availability of NAD+ ). Current knowledge suggests that de novo enzyme synthesis is triggered by interaction of an enzyme inducer with sites on gene promoters that modulate gene transcription. The majority of investigations have been with the CYP genes where four major sites, or receptors, have been identified. These bind either the enzyme
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inducer, or its complex with a carrier protein, and are the xenobiotic response element (XRE), the constitutive androgen receptor (CAR), the pregnane X receptor (PXR) and peroxisome proliferator activated receptor (PPAR) (see Table 2). The Ah (aryl hydrocarbon) receptor transports ligands to the XRE. All four nuclear receptors exist as heterodimers in a complex with a common partner, the retinoid X receptor (RXR). The farnesol X receptor (FXR) or bile acid receptor (BAR), and the liver X receptor (LXR) are two additional receptors that heterodimerize with RXR and mediate CYP induction by endogenous compounds (Waxman, 1999). Because discrete CYP families express receptor(s) of different types, only ligands of those receptors will be effective inducers. Table 2 illustrates the association between receptor types and the CYPs and also lists other drug metabolizing enzymes which express the same receptors e.g. UDP-glucuronyltransferases. NADPH-cytochrome P450 reductase (which provides electrons for reducing CYPs) is separately regulated by the thyroid hormone receptor (TR) (Table 2). Although this appears complex, there are two simple paradigms that apply: first, each receptor will bind only a limited range of potential inducers (permitting induction by them), and secondly, each drug metabolizing enzyme gene will express only certain receptors. Therefore, knowing the receptors associated with an enzyme, its response to inducers can be predicted. Similarly, if a new drug is shown to interact with one (or more) of the receptors, its inducing effect can be predicted for the enzymes known to express that receptor. The most clinically significant enzyme inducers are rifampicin, rifabutin, carbamazepine and phenytoin (which interact with PXR), barbiturates, glutethimide and promethazine (with CAR), omeprazole (with XRE) and certain antihyperlipidemic fibrates (with PPAR). Natural anutrients and environmental contaminants seemingly target the same set of receptors e.g. polycyclic aromatic hydrocarbons in cigarette smoke, polychlorinated hydrocarbons and indoles in vegetables such as broccoli (XRE), DDT and other pesticides (CAR). The complementary medicine, St John’s Wort, also appears to interact with PXR. More comprehensive associations of enzymes inducing drugs with CYP isoforms appear elsewhere (Tredger & Stoll, 2002).
Enzyme Inhibition Enzyme inhibition describes the result of competition between two drugs for the same enzyme and leads to a reduced rate of metabolism of one (or both). The competition may be simply between two parent drugs, between one drug and the metabolite of a second or as a result of inactivation of enzyme by the metabolite of a drug. Simple interactions between two parent drugs are the most common,
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rapid in onset, concentration dependent and reversible. Examples include the inhibition of CYP3A by antifungals, anti-virals and calcium channel blockers (e.g. ketoconazole, indinavir and nifedipine, respectively). Where metabolites of one drug bind to the enzyme’s active site and prevent metabolism of the second drug, inhibition may be longer lasting and intensify with repeated dosing. Examples are with lidocaine, nortriptyline and several macrolides such as erythromycin. The final category, known as suicide inhibition, destroys the enzyme completely and is therefore irreversible (except by de novo synthesis). It is a rare event exemplified by inhibition with methoxsalen, spironolactone and some synthetic estrogens such as ethinylestradiol and may be associated with tissue damage or loss of function.
HEPATOTOXICITY Toxic injury to the liver (hepatotoxicity) originates from the adverse effects of the same range of natural anutrients, chemical contaminants and therapeutic agents that are the substrates of drug metabolism. It results in a range of drug-induced toxic lesions that mirror hepatic disease in both appearance and time course and with a correspondingly variable etiology. Both are probably related to the overcoming of endogenous protective mechanisms by the natural or toxic insult. Consequently, their origins may be multifactorial, influenced by both genetics and the environment and representing combinations of events towards the extremes of normality in a heterogeneous human population.
Incidence and Screening Adverse drug reactions are the fourth most common cause of death in the USA and approximately 10% of these affect the liver. An estimated 1000 drugs and chemicals can cause liver injury. Ten to 15% of cases of acute hepatitis are drug related and an even greater proportion of cases of acute liver failure (Kaplowitz, 2003; Zimmerman, 1999). All new medications undergo extensive and costly toxicity screening and many promising drug candidates are lost at this stage. An additional proportion proves hepatotoxic during clinical trials but significant numbers emerge during post-marketing surveilance as the number of patients who are treated increases. This relates to the rare incidence of individual reactions and the rule of three: reactions at a frequency of 1 in 10 need a cohort of 30 patients to identify damage with 95% certainty, while 30,000 patients are required to establish toxicity at a more typical incidence of 1 in 10,000 cases (Kaplowitz, 2003).
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Expression and Diagnosis Hepatotoxicity may arise within hours of ingestion of an acetaminophen dose, or not for decades after exposure to the contrast agent thoratrast (Zimmerman, 1999). Damage may affect all types of liver cells (parenchymal, Kupffer, endothelial, stellate and biliary epithelial) and be expressed as a single or combination of lesions including simple cholestasis, steatosis, granulomata, hepatitis, necrosis, peliosis, hepatic venous occlusion, fibrosis, cirrhosis and neoplasia. Noteworthy is the lack of specificity of these lesions to a drug-related etiology and the corresponding absence of specific diagnostic tests for the majority of lesions (except in some forms of drug-induced autoimmune hepatitis where specific autoantibodies exist). Generally, elevations in liver function tests are the most useful indicator (Larrey, 2002) and liver biopsy may provide invaluable clues when viewed by an experienced histopathologist. Collectively, these factors often make the suspicion of drug-induced hepatotoxicity one of association after exclusion of alternative causes, such as viral infections, and in the absence of specific disease markers e.g. anti-M2 antibodies in primary biliary cirrhosis. Knowledge of the potential for toxicity is valuable and this may be absent with new drugs. Similarly, a temporal relationship to drug ingestion is particularly important, although this becomes increasingly difficult when hepatotoxicity develops over months or even years. Improvement in symptoms after drug withdrawal is diagnostically helpful, but intentional re-challenge is neither safe nor definitive. Scoring systems have been developed which consider these parameters of causality assessment collectively and include the RUCAM and CDS methods (Kaplowitz, 2003; Larrey, 2002).
Pathogenesis The diversity both of the drugs causing injury and the lesions they induce excludes an exhaustive discussion here of the individual underlying mechanisms. These are precisely understood in surprisingly few cases, but a simple classification can be made into predictable and idiosyncratic mechanisms (Zimmerman, 1999). The former applies where toxicity is prevalent among most individuals given the drug and is reproducible in animal models (e.g. with acetaminophen overdose), while idiosyncratic toxins affect only a susceptible minority (e.g. with halothane hepatitis) (Fig. 1). Acetaminophen overdose is the most common cause of drug-induced liver damage, although the drug is very safe if taken as recommended (up to 1 g four times a day). Hepatotoxicity arises when the proportion of drug metabolized via a CYP-dependent intoxication pathway increases after overdose. This produces the
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Fig. 1. Predictable and Idiosyncratic Hepatotoxicity. Note: Panel A shows the predictable origins of acetaminophen hepatotoxicity and panel B the idiosyncratic nature of halothane hepatitis.
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reactive metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), in amounts and at a rate which exceeds endogenous detoxicaton with GSH (see Fig. 1). Consequently, GSH is depleted, and large amounts of NAPQI bind irreversibly (covalently) to protein sulphydryl groups, devastating their structure and function. Damage to mitochondria and the endoplamic reticulum occurs rapidly, leading to energetic crisis, oxidative stress, hepatocyte death, Kupffer cell activation and local vasoconstriction. Hepatic necrosis results, which may be fatal, and liver damage is predictable at high doses of paracetamol (see Fig. 1). General anesthesia with halothane is easy to control and cheap, so the agent is still widely used in the third world despite the rare occurrence of a severe hepatitis-like drug reaction in approximately 1:30,000 individuals. Although reactive metabolites arise from at least two pathways of halothane metabolism, only one of these, the oxidative pathway, has been associated with production of an adduct with the amino acid lysine which is antigenic (see Fig. 1). Only in individuals who develop halothane hepatitis does this lead to production of a circulating antibody, which initiates an immune reaction directed at the major concentration of the neoantigen in the liver. Antibody production therefore underlies the idiosyncratic nature of halothane hepatitis. A unifying theme throughout hepatotoxicity is its origin in an imbalance of intoxication over detoxication pathways. While intoxication pathways produce reactive and potentially toxic metabolites, detoxication removes these likely toxins by further metabolism or reaction with endogenous cytoprotectants that are both chemical (e.g. antioxidants and GSH) and enzymic (e.g. superoxide dismutase and glutathione peroxidase). Detoxication may also involve suppression of the immune response to a metabolite-derived neoantigen. Where intoxication prevails, its products initiate one or more hepatotoxic cascades with discrete adverse outcomes. The severity of liver damage and its rates of progression are dependent on the toxin in question and the combination of initiating events. Among these events (depicted in Fig. 2) are: the impairment of drug and bile acid transport; the formation of complexes (adducts) between reactive metabolites and cell macromolecules such as protein or DNA; the depletion of cytoprotectants such as GSH and free-radical scavengers e.g. vitamins C and A within cells and their organelles; the impairment of mitochondrial function, oxygen availability (for intermediary metabolism) and energy (ATP) production; the initiation of oxidative stress and lipid peroxidation; the activation of stellate cells, initiating fibrosis; the activation of cytokine cascades e.g. with tumor necrosis factor (TNF);
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Fig. 2. Mechanisms of Hepatotoxicity. Abbreviations: ER, endoplasmic reticulum; L, lysosome; M, mitochondria; N, nucleus; P, peroxisome; T, toxic drug metabolite. Note: fragmentation of mitochondrial cristae and ER, denoting damage. Open arrows show drug distribution; chevron arrows show drug metabolite targets; solid arrows show consequences of damage.
the activation of Kupffer cells and recruitment of circulating inflammatory cells. the activation, intracellularly or via Fas ligand at the cell membrane, and release of transcription factors in the Bcl-2 family.
Lethal Hepatocellular Injury Recently, Kaplowitz concisely reviewed how the ultimate summation of these diverse processes is cell death via apoptosis or necrosis (Kaplowitz, 2002). Such fatal cell injury is the outcome only in severe cases of hepatotoxicity, but a common feature is that apoptosis and necrosis occur in parallel, depending upon the local balance of intoxicants and detoxicants. A major distinction between the two processes may be the cell’s energetic status, with controlled cell death characteristic of apoptosis possible when ATP is available, but the chaotic and pro-inflammatory
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events of necrosis prevailing when ATP supplies are exhausted. It is important to stress that first, a delicate balance between these two outcomes probably prevails and secondly, that each liver cell will exist in an almost unique micro-environment of drug concentration, enzyme complement, oxygen tension and susceptibility to the mediators listed above. Consequently, differences in environment at the cellular or even sub-cellular level may be sufficient to mediate the distinct apoptotic or necrotic death of two adjacent liver cells.
Sub-Lethal Hepatocellular Toxicity Where sub-lethal, the manifestations of toxicity reflect both the pharmacological activity of parent drugs and the subcellular targets of their metabolic intermediates and products. A relatively limited number of outcomes result, linked in the majority to the specialist functions of liver cells. The most discrete are perhaps cholestasis and neoplasia. Cholestasis frequently arises when drugs affect the activity of transporter proteins involved in the uptake, intracellular carriage and export of bile acids, although other biliary components (e.g. cholesterol, phospholipids and GSH) may be affected (Bohan & Boyer, 2002; Jansen & M¨uller, 2003). Neoplasia is usually the result of changes to DNA arising from reactive drug metabolites or nucleotide adducts. Steatosis will result from mitochondrial, endoplasmic reticulum or lysosomal impairment (Pessayre et al., 2003; Zimmerman, 1999) and mitochondrial dysfunction appears central to both alcoholic and non-alcoholic fatty liver disease (Farrell, 2002). Cirrhosis may progress from fibrosis originating from the activation of stellate cells or bile acid accumulation. Vascular lesions arise from damage to sinusoidal or terminal venous endothelial cells (Laskin & Gardner, 2003). Kupffer cell activation arises directly or as a secondary consequence of some of these insults, but further triggers recruitment of circulating leucocytes and an immune inflammatory response (hepatitis), as may adhesion molecule up-regulation on sinusoidal endothelial cells (Laskin & Gardner, 2003). Oxidative stress is variably induced by damage to mitochondria, peroxisomes and the endoplasmic reticulum by lipid peroxidation, free radical generation and depletion of GSH (Jaeschke, 2003). Protein adducts of drug metabolites may also prompt immune responses (Obermayer-Straub & Manns, 2003) – or devastatingly disrupt enzyme function and membrane integrity, as with acetaminophen (Nelson & Bruschi, 2003). The rate of onset and severity of hepatotoxicity is linked not only to the consequences of the events just listed, but also to the number and extent that these are involved. Hepatitis linked to cholestasis, for example, is more frequently severe than hepatitis alone and far more so than mild cholestasis, which may
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be relatively benign (e.g. with oestrogenic steroids and cyclosporine). Similarly, alcoholic steatosis per se may be benign but is frequently fatal when associated with alcoholic hepatitis. Acetaminophen is hepatotoxic when grams of the drug produce protein adducts and deplete GSH, so devastating mitochondrial function, membrane integrity and enzymic function.
Susceptibility Commensurate with the central role of drug metabolism in mediating toxic drug reactions is an ability of environmental and genetic modulators to influence an individual’s susceptibility to and outcome from toxicity. All those modifiers of drug metabolism discussed earlier may be influential including gender, age, diet, disease, enzyme inducers and inhibitors and genetic polymorphisms (Zimmerman, 1999). Genetics and enzyme induction are probably most important quantitatively. These may be collectively incorporated into the intoxication/detoxication concept suggested earlier, illustrating how balance may be shifted by a combination of variables almost unique to the individuals affected (Fig. 3). Individual examples include: the greater susceptibility of females than males to immune-mediated hepatotoxicity from drugs such as halothane; the elderly being more likely to
Fig. 3. Components of the Balance Between Intoxication and Detoxication.
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experience drug toxicity associated with drug accumulation because of their slower metabolism (e.g. with ticrynafen (tienilic acid)); children being more likely to experience valproate or aspirin hepatotoxicity; overdose with acetaminophen being more severe in those who drink alcohol chronically, particularly if they are malnourished, but correspondingly less severe where alcohol is ingested together with the overdose; diabetics, particularly those who are obese, being more likely to develop methotrexate hepatotoxicity and patients with osteoarthritis showing an increased propensity to diclofenac-induced hepatitis; the potent enzyme inducing anti-tubercular drug, rifampicin, increasing the risk of hepatitis when used in conjunction with isoniazid; enzyme inhibitors being likely to increase pharmacological toxicity by augmenting accumulation of unmetabolized drug, but increased toxicity resulting from inhibition of a normally detoxifying pathway as for acetaminophen where ranitidine inhibits glucuronidation. Finally, genetic variability can influence both the immune response and toxic hepatitis, with individuals with the HLADRB × 1501 tissue type more susceptible to amoxicillin/clavulanate induced-cholestasis and CYP2D6 poor metabolizers more susceptible to perhexiline maleate-induced fibrosis and cirrhosis. Further details of many of these examples can be found elsewhere (Kaplowitz & DeLeve, 2003; Zimmerman, 1999).
Treatment The first intervention in suspected drug toxicity should be to minimize further exposure and the likelihood of progression to more severe disease. Complete and early withdrawal of the offending drug is optimal, with essential therapy sustained using agents with no hepatotoxic potential. If there is truly no alternative and only minor derangements in liver function occur (<3 times upper normal limits), then therapy should be continued cautiously if frequent monitoring of liver function tests and symptoms shows no deterioration or even improvement. Avoidance of exposure is mandated where environmental compounds or therapy with complementary medicines are suspected as the cause of liver damage. In all cases, resolution of symptoms may be slow (e.g. up to a year with chlorpromazine-induced cholestasis) and incomplete in some cases where progression to fibrosis/cirrhosis has occurred. Although the prevention of drug absorption (by lavage or activated charcoal) may limit absorption after overdose, removal of accumulated drugs is rarely feasible because of their hydrophobicity. Treatments using practical interventions based on the known mechanisms of evolving intoxication are particularly valuable, especially if commenced early. N-Acetylcysteine infusion in the first 12–16 h after acetaminophen overdose is a good example, but later
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treatment is also beneficial. Many other interventions may have theoretical benefit (e.g. inhibition of Kupffer cell activation by pentoxyfylline) but most lack clinical proof of safety and efficacy, and may have unpredicted disadvantages (e.g. TNF-␣ antibodies inhibit liver regeneration as well as cytokine release). Longer-term care should involve symptomatic relief (e.g. with cholestyramine or ursodeoxycholate for pruritus in chronic cholestasis), nutritional support (where severe hepatic impairment is involved) and specialist management of fluid and electrolyte balance, encephalopathy and tissue perfusion (e.g. in a liver intensive care unit) where liver failure develops. Liver transplantation may offer a final therapeutic option.
SUMMARY Interindividual variability in drug metabolic activity arises from genetic variability, physiological modulation and the influence of diet, disease and environmental agents such as other drugs. The same variability can be expected to apply to the generation of reactive and potentially hepatotoxic metabolites from therapeutic agents. However, the ability of these potential toxins to trigger sequences of key events mediating cell damage and dysfunction adds a further layer of variable complexity which determines outcome. The summation of these multiple variables probably underlies the idiosyncratic nature of the majority of hepatotoxins. Recognition of a drug-related etiology continues to be a challenging diagnostic step given the variable pathology induced, but by enabling early drug withdrawal this frequently limits the severity of any evolving damage. Mechanistically-based interventions show the greatest promise in management of emerging toxicity, but clinically safe and effective regimens are surprisingly rare. Given the lengthy and costly development programs for new drugs and the wastefulness of subsequent drug withdrawal, the use of mechanistically-based antidotes incorporated within medications may be a way forward for reducing the potential toxicity of invaluable additions to the pharmacopeia.
REFERENCES Anantharaju, A., Feller, A., & Chedid, A. (2002). Aging liver: A review. Gerontology, 48, 343–353. Bohan, A., & Boyer, J. L. (2002). Mechanisms of hepatic transport of drugs: Implications for cholestatic drug reactions. Semin. Liver Dis, 22, 123–136. Bruguerolle, B. (1998). Chronopharmacokinetics. Clin. Pharmaco, 35, 83–94. Farrell, G. C. (2002). Drugs and steatohepatitis. Semin. Liv. Dis, 22, 185–194. Ginsberg, G., Hattis, D., Sonawane, B., Russ, A., Banati, P., Kozlak, M., Smolenski, S., & Goble, R. (2002). Evaluation of child/adult pharamcokinetic differences from a database derived from the therapeutic drug literature. Toxicol. Sci, 66, 185–200.
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Ioannides, C. (1999). Effect of diet and nutrition on the expression of cytochromes P450. Xenobiotica, 29, 109–154. Jaeschke, H. (2003). In: N. Kaplowitz & L. D. DeLeve (Eds), Drug-induced Liver Disease. New York: Marcel Dekker. Jansen, P. L. M., & M¨uller, M. M. (2003). In: N. Kaplowitz & L. D. DeLeve (Eds), Drug-induced Liver Disease (pp. 97–124). New York: Marcel Dekker. Kaplowitz, N. (2002). Biochemical and cellular mechanisms of toxic liver injury. Semin. Liver Dis, 22, 137–144. Kaplowitz, N. (2003). In: N. Kaplowitz & L. D. DeLeve (Eds), Drug-induced Liver Disease (pp. 1–13). New York: Marcel Dekker. Kaplowitz, N., & DeLeve, L. D. (2003). Drug-induced liver disease. New York: Marcel Dekker. Larrey, D. (2002). Epidemiology and individual susceptibility to adverse drug reactions affecting the liver. Semin. Liver Dis, 22, 145–155. Laskin, D. L., & Gardner, C. R. (2003). In: N. Kaplowitz & L. D. DeLeve (Eds), Drug-induced Liver Disease (pp. 183–211). New York: Marcel Dekker. Le Couteur, D. G., & McLean, A. J. (1998). The aging liver: Drug clearance and an oxygen diffusion barrier hypothesis. Clin. Pharmac, 35, 49–64. Morgan, E. T., Sewer, M. B., Iber, H., Gonzalez, F. J., Lee, Y.-H., Tukey, R. H., Okino, S., Vu, T., Chen, Y.-H., Sidhu, J. S., & Omiecinski, C. J. (1998). Physiological and pathophysiological regulation of cytochrome P450. Drug Metab. Disp, 26, 1232–1240. Nelson, D. R. (2003). Cytochrome P450 Homepage. In: http://drnelson.utmem.edu/hum.html. Nelson, S. D., & Bruschi, S. A. (2003). In: Kaplowitz, N. & DeLeve, L. D. (Eds), Drug-induced Liver Disease. pp. 287–325. New York: Marcel Dekker. Obermayer-Straub, P., & Manns, M. P. (2003). In: N. Kaplowitz & L. D. DeLeve (Eds), Drug-induced Liver Disease (pp. 125–149). New York: Marcel Dekker. Oscarson, M., Ingelman-Sudberg, M., Daly, A. K., Nebert, D. W., & editors (2004). Web site of the human cytochrome P450 allele nomenclature committee. In: http://www.imm.ki.se/CYPalleles. Pessayre, D., Fromenty, B., Mansouri, A., & Berson, A. (2003). In: N. Kaplowitz & L. D. DeLeve (Eds), Drug-induced Liver Disease (pp. 55–83). New York: Marcel Dekker. Renton, K. W. (2001). Alteration of drug biotransformation and elimination during infection and inflammation. Pharmac. Therap, 92, 147–163. Schwartz, J. B. (2003). The influence of sex on pharmacokinetics. Clin. Pharmaco, 42, 107–121. Timbrell, J. (1999). Principles of biochemical toxicology. London: Taylor and Francis. Tredger, J. M., & Stoll, S. (2002). Cytochromes P450 – their impact on drug treatment. Hospital Pharm, 9, 167–173. Walter-Sack, I., & Klotz, U. (1996). Influence of diet and nutritional status on drug metabolism. Clin. Pharmaco, 31, 47–64. Waxman, D. J. (1999). P450 gene induction by structurally diverse xenochemicals: Central role of nuclear receptors CAR, PXR and PPAR. Arch. Biochem. Biophys, 369, 11–23. Wilkinson, G. R. (1997). The effects of diet, aging and disease states on presystemic elimination and oral drug bioavailability in humans. Adv. Drug Del. Rev., 27, 129–159. Zeeh, J. (2001). The aging liver: Consequences for treatment in old age. Arch. Geront. Geriatrics, 32, 255–263. Zimmerman, H. J. (1999). Hepatotoxicity: The adverse effects of drugs and other chemicals on the liver (2nd ed.). Philadelphia: Lippincott, Williams and Wilkins.
9.
ASSEMBLY AND SECRETION OF HEPATIC VERY-LOW-DENSITY LIPOPROTEIN
Geoffrey Gibbons INTRODUCTION In terms of energy per gram, triacylglycerol (TAG) is the most concentrated form of biological energy available and, in mammals, it makes a major contribution to the body’s supply of fuel. For instance, in a typical person weighing 65 kg, the fat stores comprise 15 kg and are capable of providing 550 MJ of energy. Energy stored as glycogen, on the other hand, comprises 0.45 kg, providing a maximum of only 7.65 MJ (Frayn, 2002). TAG is either synthesized endogenously, mainly by the liver and adipose tissue or is obtained from dietary sources via absorption from the gut. The major immediate problems associated with the use of TAG as a fuel are: (i) the controlled transport of a hydrophobic substance in the polar medium of the aqueous plasma; and (ii) presentation of TAG to tissues in a form in which it can be readily utilized (e.g. by muscle) or stored by adipose tissue. To achieve these ends, mammals have developed the amphipathic lipoproteins as stable TAG transport vehicles in the aqueous medium of the blood plasma. The assembly of these TAG-rich lipoproteins by the intestine (as chylomicrons) and by the liver (as very-low-density lipoprotein of VLDL) is an essential part of the process by which dietary and endogenously synthesized TAG is made available for utilization or storage by extra-hepatic tissues.
The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 229–256 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15009-5
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The balance between storage and mobilization of TAG is integrated with that of carbohydrate metabolism to ensure an adequate supply of fuel for energyrequiring tissues in the face of large fluctuations in food intake. In this respect, the liver is an important homeostat for detecting, and responding to, changes in the body’s need for carbohydrate and lipid fuel, particularly during major physiological changes. The liver makes a relatively small contribution to the body’s stores of TAG (Gibbons et al., 2000) and most liver TAG is synthesized from fatty acids resulting from adipose tissue metabolism. The precise function, however, of hepatic TAG secreted as VLDL, in terms of whole body energy expenditure, is controversial (see below). Nevertheless, it is quite well established that changes in the secretion of VLDL are associated with physiological and nutritional transitions. Regulation of VLDL assembly is therefore best understood in terms of fluctuations in whole body energy intake. It thus follows that metabolic abnormalities which disturb the delicate balance between energy intake, storage and expenditure will, in consequence, interfere with VLDL production. To pinpoint this defect(s) at the molecular level, it is essential to understand how VLDL is normally assembled. The importance of this information is becoming more widely recognized in view of the increasing awareness of the linkage between hypertriglyceridemia and the other abnormalities which constitute the Metabolic Syndrome (Zammit, 2002). VLDL is produced by the hepatocytes of the liver and enters the space of Disse ultimately via the basolateral membrane (Hamilton et al., 1991). This is the final act of its passage down the assembly line of the cell’s secretory apparatus. The nascent VLDL appear in the space of Disse as spherical particles ranging from ˚ in diameter. TAG is by far the most abundant lipid and between 250 and 750 A is concentrated in the hydrophobic core of the particle together with a smaller quantity of cholesteryl ester. Thermodynamic stability is conferred upon these particles in two ways. First, by the presence of a hydrophilic “shell” around the core consisting of a monolayer of the more polar lipids, phospholipids and nonesterified cholesterol. And, second, by interaction of the particle lipids with the huge polypeptide apoprotein B100 (apoB100 ). This has a molecular mass of 550 Kd and girdles the exterior of the particle (Fig. 1). The numerous, short, hydrophobic sequences of apoB provide ideal anchoring points by interacting with the fatty acyl chains of the surface phospholipids and, possibly, with the more accessible TAG-molecules of the outer part of the core. Two other, smaller peptides, apoC and apoE, are also associated with VLDL. These are required for targeting the particles sequentially, first to TAG-requiring tissues (apoC), and then to the liver (apoE). This last process removes the TAG-depleted particles from the plasma. ApoB is, however, the structural polypeptide of VLDL, without which the secretion of hepatic lipid is not possible. To understand the process of VLDL assembly, it is essential to recognize that, irrespective of the particle size of VLDL (in human
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Fig. 1. Structure of Very-Low-Density Lipoprotein.
plasma these range from Mr 3–128 × 106 ), each particle contains only 1 molecule of apoB. ApoB remains associated with the particle throughout its metabolism in, and removal from, the plasma compartment. The lipid and apoprotein compositions of nascent and plasma VLDL are shown in Table 1. Table 1. Lipid and Apoprotein Content of VLDL. Particle Nascent VLDL (rat)a Plasma VLDL (rat)b Plasma VLDL (man)c
TAG
PL
C
CE
Apoprotein (apoB + apoC + apoE)
64 73.6 44–60
17.1 12.6 20–23
5.7 2.7 5–8
2.1 1.9 11–14
10.7 9.4 4–11
Notes: Values are expressed as percentages of the total (by weight). TAG = triacylglycerol; PL = phospholipids; C = unesterified cholesterol; CE = cholesteryl ester. a Cole et al. (1982). b Chapman (1986). c Sparks and Sparks (1985).
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So the assembly of VLDL is the process by which the liver packages TAG into the vehicles required for its transport either to adipose tissue for storage, or for immediate consumption by muscle tissue. At these sites, the TAG is offloaded by a process of lipolysis involving lipoprotein lipase (for a review, see Eisenberg, 1988), leaving behind the cholesterol- and apoB-rich carriers. These particles, which include LDL, are potentially atherogenic and one could argue that the complexity of plasma lipoprotein metabolism has evolved out of a need to provide a safe means of disposal of these potentially toxic by-products of TAGtransport. As in the nuclear power industry, the problems involved in utilization of a concentrated energy source are relatively minor compared to the difficulty involved in the safe disposal of the waste products. The main aims of this Chapter are as follows: (i) To document what is currently known of the molecular and cellular mechanisms involved in the construction and secretion of VLDL; (ii) To discuss how nutritional and physiological factors which determine the body’s need to secrete hepatic TAG control the assembly of VLDL and how this process is integrated with lipid metabolism in other body tissues; (iii) To describe how some metabolic abnormalities interfere with VLDL production; and (iv) To discuss the physiological function of VLDL.
THE ASSEMBLY LINE FOR VLDL Synthesis and Translocation of apoB The major problem faced by all secretory proteins is that of translocation from sites of synthesis on the ribosome, across the membrane of the endoplasmic reticulum (ER), into the lumen of the secretory apparatus. The synthesis of these proteins, including that of apoB, begins with the translation of a signal sequence from the appropriate mRNA on a cytosolic ribosome. Binding of the signal peptide to a signal recognition particle (SRP) then temporarily halts translation until the latter binds to a so-called “docking” protein (the SRP-receptor) on the cytosolic surface of the endoplasmic reticulum. This is localized in the vicinity of an incipient protein channel, the assembly, or opening of which is probably dependent upon subsequent dissociation of the SRP, a process which is GTP-dependent (Corsi & Schekman, 1996; Johnson & van Waes, 1999). Opening of the protein channel may occur simultaneously with the resumption of translation. For most secretory proteins, this arrangement ensures that: (i) the protein is directed toward the translocation machinery; and (ii) that translation and translocation occur simultaneously, thus avoiding any conformational mis-folding which may occur in the cytoplasm and which could hinder subsequent translocation. ApoB is not, however, a typical
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secretory protein. Its intracellular behavior is modified by its need to associate with, and transport lipid through the secretory apparatus. One of its many enigmatic features is that it occurs intracellularly as both an ER membrane-bound form and in an aqueous-lumenal form associated with “nascent” lipoprotein. Although this arrangement forms part of the mechanism for the efficient regulation of VLDL secretion (see below), the need to explain this distribution in terms of the machinery of apoB translocation has given rise to major difficulties. Currently, this is a contentious issue (Pease & Leiper, 1996) and there appear to be two plausible explanations. One (Chuck & Lingappa, 1992) requires the presence of “pausetransfer” sequences in apoB which, during translocation, interact with a specific receptor associated with the protein channel. This causes a pausing in the translocation process whilst translation continues. Closing or disassembly of the channel at this stage would trap the partly translocated apoB, like Pharaoh’s chariots, in a viscous sea of membrane lipids. If, on the other hand, the channel remained open and translocation re-started, apoB would be completely translocated into the lumen of the ER. The alternative explanation is that the tight association of apoB with the ER membrane results from the strong binding of the polypeptide emerging from the translocational apparatus, with phospholipids of the inner leaflet of the ER (Pease & Leiper, 1996). In this case, translocation occurs co-translationally without pausing. The relative distribution of membrane-bound and lumenal-apoB would then depend upon the availability of neutral lipid (e.g. triacylglycerol) which would detach apoB, together with its phospholipid framework, via a “budding off” process into the aqueous lumen of the ER (Spring et al., 1992). This product is nascent lipoprotein. In this case, the inner leaflet phospholipids provide a convenient scaffolding which fixes and maintains a secondary structure for the emerging apoB consistent with subsequent additions of TAG (Fig. 2). Approximately 10 min is required for the complete translation and translocation of a molecule of apoB into the ER lumen (Janero & Lane, 1983).
Addition of Lipid to ApoB: The Construction of a Lipoprotein There is general agreement that lipid becomes associated with apoB in at least two distinguishable steps during VLDL assembly (Alexander et al., 1976; Olofsson et al., 1999; Spring et al., 1992). The first step (the initiation step) produces a small, dense, apoB-containing particle (apoB-HDL) and the second involves the association of the precursor particle with an apoB-free droplet containing mainly TAG (the maturation, or bulk lipid addition step). These normally consecutive steps can be uncoupled by Brefeldin A, which inhibits GTP exchange on the small G-protein ARF-1 (Olofsson et al., 1999).
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Fig. 2. Addition of Lipids to Apoprotein B. Source: See text for details.
The exact intracellular site of the secretory apparatus at which apoB becomes associated with lipids has been a controversial issue for many years and is still incompletely resolved. The major question is whether the bulk of TAG transfer to apoB occurs in the ER or in the Golgi complex and a show of hands amongst the various workers in the field would give the verdict to the former, although by a small majority. The main obstacles to a consensus view are, first, the use of several different model systems. These include the human hepatoma cell line HEP-G2, primary cultures of rat and chicken hepatocytes, and, finally, whole rat
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liver. Each of these in vitro model systems has its own advantages and drawbacks, and none may be entirely representative of the assembly process as it actually occurs in the intact human liver (Gibbons, 1994). The second difficulty arises because of the changing structure of the maturing VLDL in successive organelles of the secretory apparatus isolated by cell fractionation. Correct interpretation of these cell-fractionation experiments is often compromised by the difficulty in isolating pure membrane fractions originating exclusively from a single organelle. This difficulty is highlighted by the discovery that conventional methods of preparation of Golgi vesicles results in substantial contamination with endocytotic vesicles containing lipoproteins (e.g. LDL derivatives) of exogenous origin (Hamilton et al., 1991). The consensus opinion is, however, that a certain amount of TAG is added to apoB simultaneously with apoB translocation (i.e. in the rough endoplasmic reticulum (RER); Fig. 2). This process appears to be dependent upon the presence of a microsomal TAG transfer protein (MTP) which is expressed mainly in liver and intestine (Gordon et al., 1995) with a smaller amount in the heart (Bjorkegren et al., 2001). Indeed, this MTP-mediated transfer of TAG is a prerequisite for apoB translocation since individuals with abetalipoproteinemia lack MTP, and are unable to secrete apoB containing lipoproteins (Wetterau et al., 1992). The exact mechanism by which apoB acquires TAG during its translocation is not clear. An early suggestion is that the translocating apoB supported by its phospholipid framework encircles a droplet of TAG within the ER bilayer. This structure then pinches off into the lumen of the ER (Spring et al., 1992) (Fig. 2). More recently, it has been suggested that the acquisition of phospholipids and TAG are separate events (Shelness & Sellers, 2001). Whatever the mechanism, the amount of TAG encapsulated during the translocation process is insufficient to account for the much larger size, higher lipid/apoB ratio and lower density of mature VLDL. Thus, the “bulk” accretion of TAG (maturation phase) must occur by direct transfer to the nascent particle in the lumen (Fig. 2). On balance, current evidence points to the junctional complex between the smooth endoplasmic reticulum (SER) (the site of TAG synthesis) and the rough endoplasmic reticulum (RER) as the favorite candidate for this site of bulk TAG addition. This conclusion is based upon immunochemical evidence (Alexander et al., 1976), kinetic (Borchardt & Davis, 1987) and morphological (Rusinol et al., 1993) studies and upon interruption of VLDL assembly with orotic acid in which TAG droplets accumulate in “liposomes” apparently derived form this part of the secretory apparatus (Hay et al., 1988). However, some workers have provided strong evidence that the Golgi is a major site for bulk TAG transfer (Bamberger & Lane, 1990; Higgins & Hutson, 1984), and more recent work has provided support for this view (Hebbachi et al., 1999; Levy et al., 2002). In addition to the phospholipid which associates with
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GEOFFREY GIBBONS
apoB during its translocation, there seems little doubt that the maturing VLDL acquires further phospholipid in the Golgi (Higgins & Fieldsend, 1987; Vance & Vance, 1988). The biphasic pattern of phospholipid transfer observed in kinetic experiments in chicken hepatocytes (Janero & Lane, 1983) also attests to a 2-site location for addition of phospholipids to the incipient VLDL particles (Fig. 2). The site(s) of acquisition of esterified and non-esterified cholesterol have not been intensively studied. It is possible that cholesterol ester assists in the lipid nucleation of apoB at an early stage in the assembly process during translocation (Cianflone et al., 1990). Small amounts of non-esterified cholesterol could also be transferred at this stage as part of the polar lipid “belt” which the translocating apoB acquires from the inner leaflet of the ER membrane. Intracellular membranes are, however, low in cholesterol and the balance of this lipid must be added elsewhere. Finally, it should be recognized that VLDL is not secreted as particles of uniform size. Differences in the amounts of lipid acquired by each particle during its assembly results in a spectrum of particle sizes. This has important consequences for the metabolism of VLDL in the plasma, and, amongst other things, determines the likelihood of their conversion into LDL on the one hand, and clearance by the liver (as remnants) on the other (Packard & Shepherd, 1997). The metabolic heterogeneity of VLDL thus has potentially important implications for cardiovascular risk and emphasizes the essential requirement for “correct” VLDL assembly not only as an efficient, but as a safe, means of TAG transfer between organs. To summarize the main points of this section: First a small amount of TAG and phospholipid becomes associated with apoB during its translocation through the membrane of the ER. Second, the bulk of the TAG is added to the maturing particle at a later stage in the lumen of the secretory apparatus. The precise site at which this occurs yet to be been clarified. And third, a significant portion of phospholipids is added in the Golgi.
INTRACELLULAR APO B: HOW IS ITS AVAILABILITY REGULATED? The assembly of VLDL is determined by the body’s need to synthesize and transport hepatic TAG. Thus physiological changes which affect these requirements might be expected to influence VLDL assembly and secretion. Since each VLDL particle is a distinct entity containing one molecule of apoB and a variable, but limited, amount of TAG, it is theoretically possible to increase hepatic TAG output either by increasing the amount of lipid per particle (apoB output remaining constant), or by increasing the number of particles secreted (apoB output
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increases). In the former case, TAG output is varied by changes in particle size; in the latter, by changes in particle number. It is, however, important to bear in mind the following when considering the regulation of VLDL output. First, in practice, changes in TAG output are normally accompanied by changes in apoB secretion. Only in certain pathological states do these factors become uncoupled. Second, no circumstance has yet been found in which fully-made VLDL particles accumulate within the secretory apparatus. In other words, once fully-assembled, VLDL particles must be secreted. Neither does a decreased output of VLDL result in an intracellular accumulation of unassembled apoB. In general, therefore, VLDL output does not appear to be regulated by changes in the mobilization of a reservoir of intracellular apoB. The only possible alternative is that the availability of apoB changes by variations in its rate of synthesis or degradation. How is this achieved? Studies in vitro, using a variety of model systems, have shown that, in general, short-term manipulations which result in variations in apoB and VLDL output do not affect the expression of the apoB gene (Table 2). Instead, it would appear that under conditions which simulate normal physiological change, the availability of apoB for VLDL assembly is regulated post-transcriptionally. The most important of these mechanisms involves modulation of apoB-degradation (for reviews see Sparks & Sparks, 1994; Yao et al., 1997). Thus, apoB is constitutively produced in excess of the liver’s requirement for TAG secretion, the balance being channelled into degradative pathways. Changes in the proportion of newly-synthesized apoB which enter this pathway thus determine the rates of VLDL secretion. Why the hepatocyte has developed this superficially wasteful cycle of synthesis and degradation can only be guessed at but it may represent yet another of the many “futile” metabolic cycles which are common in the regulation of energy transduction at Table 2. At What Level is ApoB Secretion Regulated? Condition
(a) Short-term changes (in vitro) Simvastatin (0.5 U) Albumin (3.0%) Oleic acid (0.8 mM) Chylomicron remnants (10g) Human insulin (25 mU/ml) (b) Chronic changes (in vivo) Obesity and hyperinsulinemia
Change in ApoB Secretion (% Control)
Change in ApoB mRNA (% Control)
References
75 ( p < 0.01) 17 ( p < 0.01) 150 ( p < 0.001) 500 ( p < 0.001) 57.4 ( p < 0.01)
114 (N.S.) 95 (N.S.) 93 (N.S.) 100 (N.S.) 95 (N.S.)
Qin et al. (1992) Pullinger et al. (1989) Pullinger et al. (1989) Fazio et al. (1992) Dashti et al. (1989)
150
Inui et al. (1989), Karakash et al. (1977)
147
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GEOFFREY GIBBONS
the cellular and tissue levels of organization (Miyoshi et al., 1988). These may serve to increase sensitivity to regulation in the short-term. In addition, rates of hepatic TAG secretion need to be adjusted rapidly and frequently in response to physiological and nutritional change. With these frequent stops and starts it is, perhaps, more efficient to keep the transcriptional and translational engines running at a set rate, rather that turning them on and off. In this respect, the t1/2 of apoB mRNA is 16 h. The above considerations apply, in the main, to the normal short-term regulation of TAG secretion. By contrast, certain chronic pathological and physiological conditions result in changes in hepatic apoB mRNA. In obesity and hyperinsulinemia, the increased mRNA is associated with an increase in the secretion of apoB (see Table 2). In other cases, however, such as hypothyroidism (Green et al., 1988) and during the suckling period of development (Demmer et al., 1986), it is difficult to correlate changes which occur in apoB mRNA with corresponding changes in the output of VLDL. Nevertheless, it remains likely that, in general, the hepatocyte responds to chronic, as opposed to acute, stimuli, by changes in the rate of apoB gene transcription or by increasing the stability of apoB mRNA. The most important signal which determines the proportion of secreted apoB which is secreted and that which is degraded is the accessibility of neutral lipid which determines the conformation of apoB at all stages of VLDL assembly. The mere presence of intracellular lipid is not in itself sufficient to facilitate apoB secretion since such pools may not, as is the case with HepG2, have access to the secretory pathway (Gibbons et al., 1994). Thus, pathways which allow intracellular neutral lipids access to the secretory apparatus ensure their effective functional coupling with intracellular apoB to produce a secretion competent particle VLDL. Such a coupling is probably facilitated by the lipolysis and re-esterification cycle of cytosolic TAG (Brown et al., 1999) (see below). It seems likely that apoB-containing VLDL precursors are susceptible to proteolytic degradation at most stages of VLDL assembly. Only the final, mature particles are fully protected (Yao et al., 1997). The predominant mechanism for degradation of apoB involves ubiquitination of misfolded chains followed by chaperone-mediated delivery to the proteosome. This trafficking may occur either co-translationally, resulting from insufficient MTP-mediated transfer of lipid to the translocating apoB (Davis, 1999) or post-translationally. The latter involves anterograde translocation from the ER lumen to the cytosol via a translocation channel (Davidson & Shelness, 2000). In each case ubiquitination and proteosomal degradation is facilitated by the cytosolic chaperone Hsp 70. In an interesting development, it has been suggested that the LDL receptor in some way interacts intracellularly with incipient VLDL in a process which targets apoB to presecretory degradation (Twisk et al., 2000). This interaction would explain both the increased VLDL secretion
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in familial hypercholesterolemia (FH) and the lowered VLDL production which occurs when LDL receptors are upregulated by statin therapy. Such a relationship may provide an important regulatory link between hepatic cholesterol and TAG metabolism. As mentioned above, the effective coupling of apoB with accessible neutral lipid may well depend upon the efficient lipolysis of cytosolic TAG and the targeting of the esterified products into the secretory pathway. This latter aspect is impaired by insulin (Wiggins & Gibbons, 1992), an observation which may explain insulin’s role in the switching of apoB from secretion as VLDL to presecretory degradation (Gibbons et al., 2002; Sparks & Sparks, 1994). The main conclusions from this section are: First, short-term changes in VLDL secretion are mediated mainly by changes in the rate of apoB degradation; chronic changes by variations in apoB gene transcription. Second, TAG accessibility is a major factor in determining apoB degradation. Third, conformational changes in apoB may be important in its channelling into degradative or secretory pathways. Save for the first, the conclusions are dependent upon lipolysis-mediated transfer of cytosolic TAG into the secretory pathway.
VLDL-TRIACYLGLYCEROL: WHERE DOES IT COME FROM AND HOW DOES IT GET THERE? Hepatic VLDL is a major source of plasma TAG and, in man, is secreted at the rate of 200–400 mg/kg body mass/day. This represents a total input into the plasma of about 15–30 g/day for a person weighing 70 kg. What is the origin of the fatty acids used for the synthesis of this VLDL-TAG? In vivo, there are four potential sources: fatty acids synthesized de novo from small precursors, plasma non-esterified fatty acids (originating mainly from adipose tissue), lipoproteins entering the liver from the plasma (particularly the “remnants” of VLDL and chylomicrons), and, finally, the hepatocellular storage pool of TAG located mainly in the cytosol of the cell. This last, of course, is not a primary source and must be replenished at a rate similar to that at which it is utilized (see below). Studies in vitro, using a variety of experimental models have shown that increasing the availability of any of the three primary sources boosts the level of hepatic VLDL TAG output. In vivo, however, the situation is more complex and it is difficult to construct an ideal in vitro model in which all sources are represented in balanced proportions. This balance will obviously depend upon nutritional status and will, theoretically at least, affect both the relative availability of each of the sources, and also the proportion of the total which enters the esterification, rather that the oxidative (ketogenic) pathway (McGarry & Foster, 1980). Studies of this type have also relied heavily on the use
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of experimental animals and it should be borne in mind that the results obtained may not be invariably applicable to man. Nevertheless, it is generally agreed that both in man, and in the rat, newly-synthesized fatty acids derived from dietary carbohydrate make only a small contribution to the total TAG of secreted VLDL. In man, this contribution amounts to 0.9% in the fasted state (Hellerstein et al., 1991) compared to 0.6% in the rat (Gibbons, 1990). In the fed state, this proportion rises in man to between 1.97 and 8.5% depending upon the method used in the study (Hellerstein et al., 1996; Leitch & Jones, 1993), and to 7.7% in the rat (Table 3). In the sucrose-fed rat, in which hepatic fatty acid synthesis de novo is very high, a maximum of only 20% of the VLDL TAG is derived from this source (Table 3). As this source is derived ultimately from dietary carbohydrate, via acetyl CoA, these observations raise the question as to why hepatic VLDL output increases substantially in individuals consuming a high carbohydrate diet. Part of the answer may be found in animal experiments (Yamamoto et al., 1987) in which a diet rich in sucrose diverted a larger proportion of exogenous preformed non-esterified fatty acids (NEFA) away from the oxidative pathway (producing ketone bodies) into the esterification pathway (producing TAG). This source appears to be far more important than that arising from de novo synthesis but the exact contribution may vary from species to species and between nutritional states. In man, for instance, in the fasting state when plasma NEFA levels are high, this contribution has been reported to be as high as 100% (Table 3). On the other hand, considerable evidence exists which suggests that a significant proportion of the TAG of hepatic VLDL is Table 3. Sources of VLDL Triacylglycerol Fatty Acids. Species
Nutritional State
Experimental Model
Contribution (%) of Fatty Acids from Stated Source
References
Man Man
Fasted Fed
In vivo In vivo
Hellerstein et al. (1991) Leitch and Jones (1993)
Rat
Fasted (24 h)
In vivo
0.9 newly synthesized 2.0–8.5 newly synthesized 0.6 newly synthesized
Rat
Fed
In vivo
7.7 newly synthesized
Rat
Fasted (48 h)
In vivo
Man Rat
Fasted Chow fed (high starch) High sucrose diet
In vivo Hepatocytes
83 recycled (remnants) 17 NEFA 100 NEFA 8.2 newly synthesized
Hepatocytes
17.5 newly synthesized
Rat
Duerden and Gibbons (1988) Duerden and Gibbons (1988) Lipkin et al. (1978) Havel et al. (1970) Gibbons and Burnham (1991) Gibbons and Burnham (1991)
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not taken up by peripheral tissues but is returned to the liver as VLDL remnants (Shepherd & Packard, 1987). This represents a potentially important source of nascent VLDL TAG and in the rat, even in the starved state, this can amount to 83% of the total VLDL TAG secreted (Table 3). Although VLDL TAG must, of course, be ultimately derived from one or a combination of the above primary sources, studies in vitro suggest that exogenous, pre-formed fatty acids are not, in the main, used directly for the assembly of VLDLTAG but enter, first of all, the hepatocellular cytosolic storage pool (Gibbons et al., 1992, 2000). Fatty acids required for VLDL TAG synthesis are then mobilized from the cytosol by lipolysis followed by esterification (Lewis, 1997; Wiggins & Gibbons, 1992). This indirect route may also operate for the other primary sources of VLDL TAG. The bringing together, in this way, of fatty acids from all sources into a common pool may represent a biological “warehouse” effect in which distribution into VLDL can be more efficiently controlled. The operation of such an indirect route has other important implications for the control of VLDL assembly and secretion. These include: (i) the presence of at least two topographically distinct sites of TAG synthesis in the endoplasmic reticulum. One is responsible for the esterification of fatty acids released from intracellular TAG. Only this latter source of TAG is directly accessible for the assembly of nascent lipoprotein. These predictions are borne out by the discovery of two physically distinct sites of diacylglycerol acyl transferases, key enzymes in the synthesis of TAG in the ER (Cases et al., 2001; Owen et al., 1997) and by kinetic studies of hepatic TAG turnover (Kondrup et al., 1979). (ii) The rate of VLDL assembly can be uncoupled from the concentration of plasma NEFA, and, thus, from the overall rate of hepatic TAG synthesis. This is physiologically meaningful from a regulatory viewpoint and explains, for instance, the low rates of VLDL output in the livers of starved animals, or animals fed a high-fat diet. Both of these conditions are associated with a high plasma NEFA concentration. An interesting feature of the mobilization of cytosolic TAG is that much more is lipolysed than is actually required to meet the demands of VLDL assembly. The remaining fatty acids are re-esterified and re-cycled back to the cytosol (Wiggins & Gibbons, 1992). This apparently “futile” cycle is reminiscent of the cycle of apoB synthesis and degradation described above, and to which it may be mechanistically related. It may be worth noting here that HEP-G2 cells lack the ability to lipolyze intracellular TAG (Gibbons, 1994). This may partly explain the large intracellular accumulation of TAG when these cells are cultured in the presence of exogenous fatty acid. Lack of lipolysis may also contribute to the inability of HEP-G2 to assemble a “full-sized” VLDL particle. This results in a defective “Step 2” transfer process in which the bulk of the TAG is normally added to the nascent lipoprotein (Fig. 2). Finally, the possible contribution of hepatocellular phospholipid fatty
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GEOFFREY GIBBONS
acids to VLDL TAG should not be overlooked. Some evidence for this was first obtained over thirty years ago (Bar On et al., 1971). More recent studies have shown that a sizeable proportion of the VLDL TAG fatty acids is derived from or via cellular phospholipid (Wiggins & Gibbons, 1996). To briefly summarize the main points of this section. These are: (1) Newlysynthesized fatty acids make only a minor contribution to VLDL TAG. Extracellular fatty acids and returning “remnants” are more important. (2) Fatty acids derived from all sources are converted into cytosolic TAG prior to utilization for VLDL assembly. (3) Mobilization of cytosolic TAG for VLDL synthesis requires a lipolysis/re-esterification cycle.
VLDL ASSEMBLY: NUTRITIONAL AND HORMONAL REGULATION AT THE PHYSIOLOGICAL AND MOLECULAR LEVELS Effects of Nutritional Change: A Possible Role for Stearoyl CoA Desaturase Changes in the production of VLDL are integrated and co-ordinated with changes in other energy-related metabolic pathways. These adjustments form an essential part of the overall strategy by which the body shuffles its metabolic portfolio to deal efficiently with large fluctuations in food intake. In terms of total caloric intake, these fluctuations fall into two categories: (i) the normal, rhythmic pattern of food intake which occurs during regular consumption of relatively small-sized meals. This pattern results in cyclic diurnal variations in hepatic metabolism (e.g. bile acid synthesis, cholesterogenesis, fatty acid metabolism, glycogenesis, glucose output). In these cases the interprandial period is of regular duration and relatively short; (ii) erratic food consumption characterized by longer periods of food deprivation in which the interprandial period is of longer and indeterminate duration, e.g. starvation. Metabolically, the liver responds differently to these two types of foodintake fluctuations and this is reflected by different responses of VLDL production. In addition to these quantitative changes in the total calories consumed, there may also be marked qualitative differences (e.g. changes in the percent of fat, refined sugar, etc.) in the diet. Again, these variations are reflected by changes in VLDL production and secretion. Conditions which favor a high rate of TAG synthesis de novo from small, carbohydrate-derived precursors are usually associated with high rates of VLDL output. This association is particularly pronounced in animals fed diets rich in refined sugars such as fructose and sucrose (Boogaerts et al., 1984). As mentioned
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above, this effect appears to be due, at least in part, to an increased partitioning of exogenous fatty acids into the esterification, as opposed to the ketogenetic pathway and is accompanied by an increased output of apoB However, acute stimulation of fatty acid synthesis in vitro is not, in itself, capable of changing apoB output (Gibbons, 1990). It would thus appear that some factor present in vivo and which is responsible for co-ordinating the metabolic changes required for efficient adaptation to the chronic ingestion of a high carbohydrate diet is involved in the regulation of apoB output. It has been suggested that this may be a trans-acting factor which interacts with cis-orientated regulatory sequences, thus changing the transcription rates of the relevant genes (Sparks & Sparks, 1993). The important role of stearoyl CoA desaturase (SCD) expression in regulating VLDL output is becoming increasingly recognized (Ntambi et al., 2002). In this respect a high rate of SCD activity is associated with increased de novo lipogenesis (Bassilian et al., 2002). It is possible that this link might underlie the relationship between lipogenesis de novo and the rate of hepatic VLDL output. The above relationships between de novo fatty acid synthesis and VLDL output is further evident from the low VLDL secretion rates in starvation, insulindependent diabetes, fat-feeding, or during the suckling period (Gibbons, 1990). Although lipid and carbohydrate metabolism differ in detail between these states, they are all characterized by a chronically low conversion of carbohydrate into fat. In view of the intense interest in the role of dietary fat in lipoprotein metabolism, the relationship between fat intake and VLDL output merits further discussion. In particular, although all types of fat (saturated, monounsaturated, polyunsaturated) suppress the secretion of VLDL TAG and apoB compared to that seen with a high carbohydrate diet, the potency of each type in this respect is related to the structure of the predominant fatty acids. For instance, it is generally agreed that diets rich in –3 fatty acids (e.g. fish-oil) are the most effective and it is probable that this action contributes to the hypotriglyceridaemic effects of diets of this type (Harris, 1989). The potent suppression of SCD-1 mRNA expression by –3 PUFA (Kim et al., 2002) provides further support for the existence of a regulatory link between SCD-1 and VLDL output. It is worthy of note that despite the apparent importance of extracellular fatty acids in promoting VLDL assembly by protecting apoB from the ravages of proteolytic destruction, all the above states (starvation, insulin dependent diabetes, fatfeeding and suckling) are characterized by high plasma concentrations of NEFA. The low rates of VLDL apoB output under these conditions emphasize the importance of a mechanism by which the rate of VLDL output can be uncoupled from bulk changes in the flux of NEFA to the liver. This is achieved, in part, by an increased channelling of NEFA into the oxidative pathway. As mentioned above, however, the availability of an indirect route for transfer of fatty acids into VLDL
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Fig. 3. Indirect Pathway for the Synthesis of VLDL TAG. Source: For details see text.
(see Fig. 3) is an important contributory factor. In most of the above states, the decreased VLDL output is accompanied by an increased storage of TAG within the liver. This TAG appears not to be available for the assembly of VLDL, possibly because of a decreased TAG lipolytic activity (Gibbons et al., 2000). The above describes the response of VLDL secretion to chronic dietary change during the longer term, a response which, over time, probably becomes securely locked into place by metabolic factors operating at the level of gene transcription. There is substantial evidence, however, that rapidly reversible cyclical changes in VLDL assembly and secretion arise in the short-term in response to the normal rhythmic pattern of food intake over the diurnal period. The possible existence of this type of response was first indicated by the many observations, in man and in experimental animals, that short-term exposure of the liver to a high concentration of insulin (simulating that which occurs post-prandially) suppressed the secretion of VLDL (for reviews see Sparks & Sparks, 1994 and Gibbons et al., 2002). This results from an increase in apoB degradation (Brown & Gibbons, 2001; Sparks & Sparks, 1990). Although direct evidence for such a post-prandial change is not, at present, absolutely conclusive, this hypothesis is supported by several observations of the kinetics of VLDL metabolism in the post-prandial state (Cohen & Berger, 1990; Moir & Zammit, 1993). In particular, the fact that plasma VLDL levels remain constant in the face of a decreased peripheral removal of VLDL
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post-prandially (Potts et al., 1991) indicates a decreased hepatic output during this period. Direct exposure of the liver to a high concentration of glucose increases VLDL output (Brown et al., 1999). This effect is dependent upon glucose phosphorylation and is mediated by an increased turnover of cytosolic TAG accompanied by an increased transfer of TAG to the maturing VLDL particle (Brown et al., 1999). Consumption of meals rich in glucose, therefore, might be expected to increase VLDL output. This tendency, however, is compensated for by the concomitant increase in insulin which occurs in the post-prandial phase (Bulow et al., 1999; Gibbons et al., 2002).
Hormonal Control What is the mechanism(s) by which information about nutritional state is (are) transmitted to the liver? And how is this information translated into changes in VLDL assembly/secretion at the molecular level? The first question is relatively easy to answer since it has been recognized for a long time that changes in the absolute or relative concentrations of plasma insulin and glucagon are the most important signals which mediate the metabolic effects of nutritional change at the cellular level. More recently, leptin has joined the pancreatic hormones as a mediator of these functions (Unger, 2003). Both insulin and glucagon influence the secretion of VLDL TAG and apoB in vitro (Bjornsson et al., 1992), although not in the characteristic reciprocal manner expected from their effects on other metabolic pathways (Unger, 1975). Instead, as mentioned above, insulin acutely inhibits the secretion of VLDL TAG and apoB, a property which it shares with glucagon, although their longer-term effects are quite distinct (see below). Insulin enhances the degradation of apoB (Sparks & Sparks, 1990), as does a deficiency of lipids for VLDL assembly. These observations suggest that insulin may affect the supply of lipids to the sites of VLDL synthesis in the secretory apparatus. As mentioned above, apoB becomes associated with lipid in at least two distinct steps during this process (Olofsson et al., 1999) and a deficiency of lipid at either step results in a misfolding of apoB with consequent targeting to degradation (Yao et al., 1997). However, providing extracellular fatty acids to primary hepatocytes does not prevent insulin’s inhibitory effect on apoB secretion, implying that, under these conditions, apoB remains susceptible to insulin-mediated degradation (Bjornsson et al., 1992). Furthermore, hepatic apoB secretion is suppressed and, by implication, its degradation is increased even under physiological conditions (e.g. fat-feeding, starvation) in which the liver is exposed to a high concentration of fatty acids. Clearly, therefore, the mere presence, or availability of hepatic lipid is insufficient to prevent apoB degradation.
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GEOFFREY GIBBONS
Recent work, however, has provided strong evidence that the lipolytic mobilization of intracellular TAG is a major determinant of VLDL assembly and that newly mobilized, rather that newly synthesized, TAG, is the major determinant of apoB stability. This observation (see below) raised the possibility that insulin inhibits the lipolytic mobilization of intracellular TAG in the liver, as it does in adipose tissue. However, this was found not to be the case. Rather, insulin suppresses the proportion of newly mobilized TAG which enters the secretory apparatus and enhances the proportion which is returned to the cytosolic pool (Wiggins & Gibbons, 1992). Further work showed that the decrease in the amount of newly mobilized TAG entering the secretory pathway resulted in a decrease in the rate of maturation of VLDL from its small, apoB-containing precursor. Insulin had no inhibitory effect on the formation of the precursor itself (Brown & Gibbons, 2001). Preventing VLDL maturation resulted in an increased degradation of apoB. It has been proposed, therefore, that insulin suppresses the maturation phase of VLDL assembly by inhibiting some process involved in the lipolysis-mediated transfer of cytosolic TAG to the intracellular site responsible for the bulk lipid addition step of VLDL assembly. Recent work has suggested that this step is dependent upon ADP-ribosylation factor-1 (ARF-1) activation and its downstream product phosphatidic acid (Olofsson et al., 1999). Abnormalities of insulin action at this step leads to an unregulated transfer of TAG to apoB resulting in production of the large, VLDL particles characteristic of NIDDM (Malmstrom et al., 1997). The superficially similar effects of insulin and glucagon on the secretion of VLDL TAG and apoB are difficult to explain from a physiological viewpoint. Exposure of rat hepatocytes to glucagon produces a transient increase in the intracellular accumulation of cAMP which peaks after 20 min and returns to almost baseline after about 6 h (Bjornsson et al., 1992). Changes in the phosphorylation state of apoB are associated with changes in VLDL secretion rates (Sparks et al., 1988) but whether glucagon is involved at this level is not known. Nevertheless, a relative excess of glucagon suppresses de novo fatty acid synthesis which, as noted elsewhere, would tend to decrease VLDL output. In this respect, it is notable that hyperglucagonaemia is characteristic of insulin-dependent diabetes, of fat ingestion, and of suckling, all states in which VLDL secretion declines (Gibbons, 1990). Physiologically, the action of the two hormones in question may be explained in terms of an acute effect by insulin, in which lipid synthesis and lipid secretion are temporarily uncoupled, and a longer-term effect of glucagon. In the latter case, lipid secretion is chronically suppressed as a result of a decreased hepatic lipid synthesis. Leptin also suppresses hepatic VLDL secretion, possibly by inhibiting SCD-1 expression and fatty acid synthesis (Cohen et al., 2002).
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The Function of VLDL: A Fatty Acid Scavenger? An apparent futile cycle of fatty acid metabolism exists between the liver and adipose tissue. Thus, fatty acids originating from adipose tissue are the major precursors of hepatic VLDL TAG. These, in turn, are returned to adipose tissue by the action of lipoprotein lipase. This purely qualitative description, however, disguises the more quantitative and time-dependent aspects of the cycle, the major purpose of which may be to buffer the plasma compartment against excessive fatty acid release from adipose tissue. In this respect, the liver has a high elasticity for the storage and secretion of fatty acids as TAG. It is this property which permits its use as a “sponge,” soaking up adipose-tissue-derived fatty acids, which are produced in excess of the body’s fatty acid oxidative capacity. Impairment of this essential hepatic function would expose vulnerable tissues such as the heart and pancreas to the consequences of lipotoxity (Unger, 1995). In particular, a significant proportion of the fatty acids released by LPL during chylomicron metabolism are not re-esterified within adipose tissue, but leaks out into the plasma (Saleh et al., 1998). Any such fatty acids entering the liver in excess of the liver’s fatty acid oxidative capacity are detoxified by esterification and converted into a relatively benign, chemically inert derivative, TAG. Evidence suggests, however, that this newly synthesized TAG is not immediately secreted, but is stored temporarily and released as VLDL in the post-absorbtive state (Diraison & Beylot, 1998; Sidossis et al., 1998). Such a deliberate asynchrony of hepatic TAG synthesis, storage and secretion is facilitated by an indirect pathway of TAG recruitment for VLDL assembly described above. This temporal arrangement also ensures that most hepatic TAG is not secreted as VLDL during the immediate post-prandial phase when chylomicron concentration is high, thus providing a physiological rationale for the inhibitory effect of insulin on VLDL assembly and secretion. Interestingly, during the post-absorbtive phase, when hepatic VLDL secretion is relatively high (Bulow et al., 1999), very little of the fatty acids released from VLDL by adipose tissue LPL, actually enters adipocytes for storage. Instead, most fatty acids released enter the plasma compartment, thereby complementing the output of fatty acids resulting from hormone-sensitive lipase activity (Frayn et al., 1995). By this route the liver indirectly provides fatty acids for peripheral oxidation by high-jacking adipose tissue LPL for VLDL lipolysis. As discussed above, under most circumstances, the contribution of de novo lipogenesis (DNL) to hepatic VLDL is rather low. Thus the primary role of VLDL is not merely to participate in the overall process by which energy is transferred from dietary carbohydrate to stored fat. Instead, it has been suggested that VLDL acts primarily as part of a complex mechanism for ensuring glucose homeostasis by
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enhancement of DNL from glucose and gluconeogenic precursors (Schwarz et al., 2003). Such a mechanism would also permit the recruitment of extracellular fatty acids into the esterification pathway by inhibition of fatty acid oxidation by the FA precursor, malonyl CoA (McGarry et al., 1977).
DEFECTS IN VLDL ASSEMBLY AND SECRETION Insulin-dependent Diabetes (IDDM) There is no more dramatic illustration of the central role of insulin in the control of lipid metabolism than the profound abnormalities of plasma lipoproteins which result from the breakdown of this delicately balanced process in poorly-controlled IDDM (Gibbons, 1986; Howard, 1987). Defective control of hepatic production and peripheral clearance both contribute to diabetic dyslipidemia. Discussing the latter defect is beyond the scope of this Chapter and the present section will deal with abnormalities of production. Studies in experimental animals suggest that IDDM results in a decreased output of hepatic VLDL and that, in this state, the hypertriglyceridaemia results exclusively from a clearance defect. The decreased output associated with insulin deficiency appears paradoxical in view of the direct inhibitory effect of insulin in this process, as described above. However, although direct insulin treatment in isolated liver preparations from diabetic animals has no stimulatory effects on the secretion of VLDL, the defect can be rectified by treatment of the animals, in vivo, with insulin (Woodside & Heimberg, 1976). These observations suggest that the reduced secretion of VLDL is an indirect effect of insulin lack which is mediated by some other factor in vivo which is missing in vitro. Evidence for many other, similarly indirect effects of insulin, is slowly emerging (O’Brien & Granner, 1991). The major cause of the defective secretion of VLDL is a decreased apoB caused, at least in part, by a decreased translational efficiency of apoB mRNA (Sparks & Sparks, 1993). Changes in the phosphorylation state of apoB have also been observed (Sparks et al., 1988).
Insulin-resistance There is wide agreement that in non-insulin-dependent (NIDDM) and obesity, states characterized by tissue resistance to insulin, the production of hepatic VLDL is enhanced (Howard, 1994; Verges, 1999). It was originally thought that this resulted from the associated hyperinsulinemia in which insulin directly stimulated
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the production of VLDL (Reaven & Greenfield, 1981). This explanation, however, requires that the liver maintains normal sensitivity to the metabolic effects of insulin, which seems unlikely in view of the widespread insulin resistance in NIDDM. The idea is also inconsistent with the direct inhibitory effect of insulin on VLDL secretion as described above. A more rational interpretation is that the liver becomes insensitive to the normal inhibitory effect of insulin on the secretion of VLDL resulting in upregulation of VLDL production (Gibbons, 1986). This explanation is supported by the ineffectiveness of insulin in suppressing VLDL release in hepatocytes cultured from animal models of insulin resistance (Bourgeois et al., 1995). Further evidence has been provided by studies in human subjects in which insulin administration normally inhibited hepatic VLDL synthesis. Large (VLDL1) was inhibited to the greatest extent (Malmstrom et al., 1998), consistent with the view that insulin inhibits the maturation phase of VLDL assembly (Brown & Gibbons, 2001). However, this inhibitory effect was greatly attenuated in obese subjects (Lewis et al., 1993) and in subjects with NIDDM (Malmstrom et al., 1997). Two other factors contribute to the excessive output of VLDL in obesity and NIDDM. The first of these is an excessive release of fatty acids from adipose tissue, thus increasing substrate availability for hepatic TAG synthesis (Frayn & Coppack, 1992). The second is the chronic hyperglycaemia of NIDDM which promotes VLDL output by enhancing the maturation phase of VLDL synthesis (Brown et al., 1999). This process is dependent upon glucose phosphorylation. In summary, three factors conspire to enhance hepatic VLDL output in NIDDM. These are: resistance to the normal direct inhibitory effect of insulin on VLDL assembly; increased hepatic fatty acid flux and an increased blood glucose concentration (Gibbons et al., 2002).
Genetic Defects in ApoB As mentioned above, the genetic lesion in abetalipoproteinemia is not a defective apoB gene but the absence of expression of a microsomal triglyceride transport protein (MTP) which prevents the normal assembly of VLDL particles. In contrast to this rare disease which is characterized by a complete absence of apoB-containing particles in the plasma, several other abnormalities of VLDL metabolism result from defects in the apoB gene itself. Phenotypically, these abnormalities are of two types, depending upon whether defects in the coding for the LDL-receptor-binding domain of apoB are involved. An intact binding domain is required for the efficient clearance of LDL, the empty carrier bag of TAG transport, from the plasma via a receptor mediated mechanism (Brown & Goldstein, 1986). Structural defects in
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this region of apoB resulting from appropriate polymorphisms of the apoB gene give rise to high plasma LDL levels (Myant et al., 1991). In other cases, mutations in other parts of the apoB gene give rise to variations in the structure of apoB which, somehow, results in lower plasma apoB levels (hypobetalipoproteinemia). Many of these mutations have been identified, some of which show overt clinical symptoms and some of which do not (Pullinger et al., 1992). To summarize: Hormonal disturbances and genetic abnormalities affect the assembly, secretion and metabolism of VLDL. Some of these give rise to dyslipidemias which carry a high risk for cardiovascular disease.
SUMMARY VLDL are spherical particles secreted by the liver. They provide the means by which endogenously synthesized TAG is transported through the aqueous plasma. The structural framework of these transport vehicles is provided by the large polypeptide apolipoprotein B (apoB) which knits together an outer shell of polar lipids embracing the TAG cargo in the core of the particle. The assembly of VLDL is quantised in that it requires the successive addition of lipid to apoB at discrete steps within the hepatocyte. These occur both during its translocation through the membrane of the ER and during its subsequent passage through the secretory apparatus. Normally, apoB is synthesized constitutively and rapid changes in the body’s need to secrete hepatic TAG are achieved by varying the proportion of apoB which is degraded. Chronic changes in TAG output, however, are accompanied by pre-translational changes in apoB which affect its rate of synthesis. It is important to recognize that VLDL production makes an important contribution to energy metabolism at the whole body level. Control of its assembly is thus an essential part of the overall process by which the body rearranges its metabolic priorities to accommodate changes in dietary energy intake. Breakdown in the orchestration of this process which occurs, for instance, in diabetes and obesity interferes with the assembly of VLDL and it is now becoming possible to define the molecular basis for this defect. The assembly of VLDL is a good illustration of the means by which nutritional demands at the whole-body level are met at the cellular and molecular levels.
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Twisk, J., Gillian Daniel, D. L., Tebon, A., Wang, L., Barrett, P. H., & Attie, A. D. (2000). The role of the LDL receptor in apolipoprotein B secretion. J. Clin. Invest., 105, 521–532. Unger, R. H. (1975). Banting lecture: Diabetes and the alpha cell. Diabetes, 25, 136–151. Unger, R. H. (1995). Lipotoxicity in the pathogenesis of obesity-dependent NIDDM. Genetic and clinical implications. Diabetes, 44, 863–870. Unger, R. H. (2003). The physiology of cellular liporegulation. Ann. Rev. Physiol., 65, 333–347. Vance, J. E., & Vance, D. E. (1988). Does rat liver Golgi have the capacity to synthesize phospholipids for lipoprotein secretion? J. Biol. Chem., 263, 5898–5909. Verges, B. L. (1999). Dyslipidaemia in diabetes mellitus. Review of the main lipoprotein abnormalities and their consequences on the development of atherogenesis. Diabetes Metab., 25(Suppl. 3), 32–40. Wetterau, J. R., Aggerbeck, L. P., Bouma, M. E., Eisenberg, C., Munck, A., Hermier, M., Schmitz, J., Gay, G., Rader, D. J., & Gregg, R. E. (1992). Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia. Science, 258, 999–1001. Wiggins, D., & Gibbons, G. F. (1992). The lipolysis/esterification cycle of hepatic triacylglycerol. Its role in the secretion of very-low-density lipoprotein and its response to hormones and sulphonylureas. Biochem. J., 284, 457–462. Wiggins, D., & Gibbons, G. F. (1996). Origin of hepatic very-low-density lipoprotein triacylglycerol: The contribution of cellular phospholipid. Biochem. J., 320, 673–679. Woodside, W. F., & Heimberg, M. (1976). Effects of anti-insulin serum, and glucose on output of triglycerides and on ketogenesis by the perfused rat liver. J. Biol. Chem., 251, 13–23. Yamamoto, M., Yamamoto, I., Tanaka, Y., & Ontko, J. A. (1987). Fatty acid metabolism and lipid secretion by perfused livers from rats fed laboratory stock and sucrose-rich diets. J. Lipid Res., 28, 1156–1165. Yao, Z., Tran, K., & McLeod, R. S. (1997). Intracellular degradation of newly synthesized apolipoprotein B. J. Lipid Res., 38, 1937–1953. Zammit, V. A. (2002). Insulin stimulation of hepatic triacylglycerol secretion in the insulin-replete state: Implications for the etiology of peripheral insulin resistance. Ann. N. Y. Acad. Sci., 967, 52–65.
10.
BILIRUBIN METABOLISM
Peter L. M. Jansen and E. Edward Bittar BIOLOGICAL ASPECTS OF BILIRUBIN Bilirubin, a linear tetrapyrrole, is the product of heme following the breakdown of red blood cells by phagocytic cells. The heme is converted to bilirubin and then carried by serum albumin to the liver where most of it is conjugated with glucuronide prior to excretion into the bile. The major product of conjugation is bilirubin diglucuronide which involves the transfer of two glucuronic acid groups sequentially to the propionic acid groups of bilirubin. Conjugated bilirubin is called direct bilirubin, whilst the unconjugated fraction bound to albumin is called indirect bilirubin. Both the direct and indirect fractions equal the total serum bilirubin (total bilirubin: 0.1–1.2 mg/dL; direct bilirubin: 0.1–0.3 mg/dL). Bilirubin is considered as the main pigment of bile. When it accumulates in the blood and reaches a level exceeding 2.5 mg per deciliter, jaundice will occur. The skin and sclerae of the eyes are discolored by the yellow molecules. Jaundice (icterus) occurs because hemolysis of red blood cells is too rapid for the liver to process the bilirubin load or because of primary hepatocellular damage, or because of mechanical biliary duct obstruction. In humans, bilirubin conjugates are excreted into the bile by the liver, stored in the gall bladder, or transported directly to the small intestine and then to the colon where it is reduced by bacteria to a mixture of substances identified as urobilinogen, urobilin, stercobilin and stercobilinogen. These compounds constitute the fecal urobilinogen measured by Ehrlich’s reagent. Some of them are reabsorbed and then re-excreted either by the liver or by the kidney. However, in patients with liver disease, bilirubin conjugates are diverted to the urine, giving it a dark yellowbrown color and giving feces a light tan color. The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 257–289 © 2004 Published by Elsevier Ltd. ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15010-1
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That more than slightly elevated levels in circulating bilirubin are toxic is common knowledge. Take, for instance, the accumulation of bilirubin in neonatal conditions. The condition is often accompanied by structural brain damage (kernicterus), especially damage of the basal ganglia which is known to vary directly with plasma bilirubin levels. The brain damage seen in premature infants is attributable to the hyperbilirubinemia. These infants are not jaundiced at birth because of placental removal of bilirubin. The accumulation of bilirubin in the blood in neonates may well exceed that found in adults with jaundice especially when caused by severe hemolysis e.g. Rh factor incompatibility. Nonetheless, we are left with the more difficult question as to why the relation between slightly elevated levels in serum bilirubin are cytoprotective. Examples of this include the lower risk for coronary artery disease (Mayer, 2000) with cardioprotection comparable to that of HDL-cholesterol (Hopkins et al., 1996), and cancer mortality in adults as found in a 10-year prospective cohort study of the Belgian population, that is inversely related to serum bilirubin concentrations. And in animal models, such as mice, the elimination of bilirubin doubles the stroke damage. It is also well to remember that infants are healthier when breast fed and more disease-resistant. And premature infants are protected against the retinopathy of prematurity (Dani et al., 2003). Studies by Hammerman and her colleagues (1998) of bilirubin in prematures have shown that the concentrations of most antioxidant enzymes are reduced, more particularly during the early neonatal period. A strong case can be made for an active clinical role of bilirubin as an antioxidant on the grounds that it is used-up in response to the generation of oxygen-derived free radicals. However, this argument also is rendered somewhat weak if oxidative stress induces antioxidant release. Thus, the positive correlation is not with clinical disease but rather with a biochemical index of the antioxidant status.
The Heme-Heme Oxygenase System There are a number of good reviews of the subject of the heme-heme oxygen system, amongst which is that by Maines (1997). That by Wagener et al. (2003) considers inflammatory processes as being triggered by the release of heme locally. The antagonist is the HO-1 isoform. Heme derived from the hemoglobin of aged red blood cells e.g. in the spleen, is converted to biliverdin by the enzyme heme oxygenase (HO), and subsequently reduced by biliverdin reductase (BVR) to the water insoluble bilirubin. As shown in Fig. 1, heme oxygenase cleaves the tetrapyrrole ring of heme at the ␣-methene
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Fig. 1. Conversion of Heme (Ferriprotoporphyrin IX) into Bilirubin IX␣.
bridge to form the linear tetrapyrrole bilirubin IX␣ with an oxygen atom at either end. During this conversion step, one C-atom is removed, and released as carbon monoxide, CO. Note that CO binds the C = O axis perpendicular to the heme plane but fails to form a corresponding hydrogen bond. Thus, the result is a decrease in affinity for CO (Oldfield). Free ferrous iron (Fe2+ ) is also a byproduct of the catalytic conversion of heme by HO. Although this increases the internal free iron initially, the concentration decreases on account of the action of ferritin as an antioxidant by sequestering iron (Otterbein & Choi, 2000). Both CO and Fe2+ are biologically active. CO, for example, behaves as a neuronal signaling molecule since it activates guanylyl cyclase, thus generating cGMP, while Fe2+ induces the expression of ferritin. It also is widely recognized that free iron as Fe2+ is a potent pro-oxidant.
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To date, three isoforms of heme oxygenase have been identified: HO-1, HO-2 and HO-3. HO-1 is the inducible isoform (heat shock-protein-32) which is ubiquitously distributed in mammalian tissues, and occurs in abundance in the spleen and liver. HO-2 which is constitutively expressed, occurs in high concentrations in brain and testis. Expression is found in both neuronal and glial cells (Kitamura et al., 1998). In the case of HO-3, which is constitutively expressed, it is relatively inactive in heme catabolism. HO-1 is a typical example of a stress protein which responds to acute cell injury. Heme, its natural substrate, is a powerful inducer of the enzyme. So is heat shock. Other inducers include heavy metals, hormones, and oxidative stressinducing agents, e.g. cytokines, pyrrolidine dithiocarbamate (PDTC) (Hartsfield et al., 1998) and H2 O2 . Mice missing the HO-1 gene are particularly vulnerable to oxidative stress caused by H2 O2 and heme. Such knock-out mice are also found to undergo a doubling in stroke damage, compared with controls. Without HO-1 and, hence, without bilirubin formation, the cellular damage inflicted by highly reactive oxygen species (ROS) is considerably greater than that seen by depleting the cells of the primary antioxidant glutathione (see later). Since it is known that HO-1 is the rate-limiting enzyme in bilirubin formation, the effects of human deficiency in heme oxygenase have been investigated and found to be associated with hemolytic anemia, growth retardation, chronic endothelial cell damage, iron deposition, and increased vulnerability to hemin-induced cell injury. Recall that hemin is Fe3+ protoporphyrin IX, while the Fe2+ complex with protoporphyrin IX is protoheme. As we shall show later, upregulation of HO-1 by means of different inducers, or modulators, or by gene transfer has led to convincing evidence supporting the notion that induction is an adaptive response and that the enzyme participates in the defense of the cell against acute oxidative stress injury. For example, overexpression of HO-1 in human corneal endothelial cells promotes angiogenesis and protects against oxidative stress; it also protects the endothelial cells of the coronary arteries from injury caused by heme/hemoglobin What has more recently emerged is an important relationship between CO (from HO-1) and NO (from iNOS) as a mechanism limiting cell injury e.g. liver injury. NO, being a free radical can interact with other free radicals, e.g. superoxide anion. This bears out the work of Choi who drew the conclusion that HO-1 is probably cytoprotective in response to the production of CO which acts as a vasodilator. Before touching upon HO-2, it is noteworthy that transcriptional control of HO1 is related to several factors such as NFB and activator protein-1 (AP-1). Both are activated by free radicals generated by heme and free Fe2+ which is a powerful pro-oxidant. NFB exists in the cytoplasm as a dimer bound to the inhibitory protein IB whose phosphorylation and ubiquitination leads to the release of
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NFB. Thence, NFB moves to the nucleus where it binds to the promoter region of genes with a B motif and hence, induces gene transcription. As has been shown by Dor´e and his colleagues (1999), the activity of HO2 in the brain is modulated by phosphorylation. There also is evidence that neuronal cell cultures isolated from rat brain cells deficient in HO-2 show increased neurotoxicity that is reversed by bilirubin in low concentration. To check whether the brain is neuroprotective, a model of vascular ischemia (and reperfusion) was used. This demonstrated a worsening of focal ischemia in mice deficient in HO-2. However, worsening failed to occur in HO-1 deficient mice. Injection of N-methyld-aspartate (NMDA) into these mice led to a worsening in the neural damage. Taken together, these observations mean that HO-2 in the brain is a neuroprotective system (see Dor´e, Sampei et al., 1999; Dor´e, Takahashi et al., 1999; Dor´e et al., 2000). Intracellular Bilirubin Concentration is ∼50 nM Some of the data obtained by Snyder and his colleagues sufficiently indicate that intracellular bilirubin levels are in the low nanoMolar range e.g. ∼50 nM (Bara˜nano et al., 2002); that is, far more than a thousand fold less than the amount of oxidants which lies in the M range for glutathione. If this be true, it would then be a matter of some importance to elucidate the nature of the mechanism responsible for the rather small bilirubin gradient prevailing across the cell membrane, thus favoring the inward movement of bilirubin. The possibility that this gradient is altered in hyperbilirubinemia and disorders in infants that are closely associated with oxygen radical-mediated cell injury such as necrotizing enterocolitis in which lower circulating bilirubin is found should not be ignored since this is an important point, and obviously a large clinical subject in which difficult issues are certain to arise. It will clarify our ideas considerably if the observations of Snyder and his colleagues are first confirmed in other laboratories elsewhere. Its biologic significance is underlined by the fact that with the exception of birds, all animals form bilirubin.
The Important Role of Biliverdin Reductase Although knowledge of biliverdin reductase (BVR) remains somewhat scanty, it is now recognized that the autophosphorylated enzyme catalyzes the last step in heme catabolism (Salim et al., 2001). That is, biliverdin, the product of heme oxygenase action, is converted by biliverdin reductase in a phosphorylationdependent reaction to bilirubin, and thereby recycled. The reaction itself is unique
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in the sense that it has dual pH/dual cofactor requirements. Currently, there is ongoing investigation into the specificity of tetrapyrrole substrate for human biliverdin 1X␣ reductase and biliverdin 1X reductase against a backdrop of basic information concerning the 1X␣ isomers of biliverdin and bilirubin as both heme oxygenases HO-1 and HO-2, and biliverdin-1X␣ reductase. High levels of the 1X␣ isomer of bilirubin are found at birth, this being physiologyical jaundice of the newborn. If, however, the protective binding capacity of serum albumin is exceeded, cytotoxicity may occur. Evidence has been obtained that bilirubin-1X␣ functions as an antioxidant, both in its free form and bound form (Stocker). Both biliverdin-1X␣ and bilirubin-1X␣ are thought to act as modulators of the immune system (Nakagami). It is particularly interesting to note Snyder’s astute comments concerning the evolution of the mechanism of bilirubin formation when water-soluble biliverdin per se could have been the perfect substitute. One plausible explanation lies in the lipid solubility of bilirubin. This allows bilirubin to cross lipid barriers freely and rapidly. However, this is not the case with bilirubin mono-and diglucuronide both of which are excreted into bile because of increased water solubility due to glucuronidation and because of reduced lipid solubility. BVR activity is reduced by SH reactive agents and toxic metals such as cobalt, and tin protoporphyrin IX (SnPP). Reducing and tuning its cellular activity by using the new method of RNA interference is strikingly demonstrated by the work of Bara˜nano, Rao et al. (2002). RNAi interference silences post-transcriptionally those genes selected by targeting and destroying specific mRNAs. To grasp and appreciate the extraordinary significance of this method, a few more words are warranted.
RNA Interference: A New and Powerful Tool This is a gene silencing technique based on the introduction of multiple short double-stranded RNA duplexes into the cell (see Bass, 2001; Hannon, 2002). In this way, expression of the homologous gene – the target – is blocked. This also is a technique with which to produce gene knockouts. SiRNAs are prepared in respect of each target gene and then transfected, injected or electroporated into cells following which dsRNA is reduced to small interfering RNAs (SiRNA) that are 21–23 nucleotides by the action of an endogenous ribonuclease called Dicer. Once formed, the SiRNA associate with a multiprotein effector complex called RISC which is the RNA-induced silencing complex. Finally, the complex targets the homologous RNA by base-pairing for degradation. Specialized SiRNAdelivery reagents, expression constructs or lentiviral-systems (Verma) are now
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available commercially. For example, by infecting zygotes, Verma has been able to produce transgenic mice in which RNAi-directed gene down-regulation develops throughout the animal. Among the various procedures widely in use at present is that of Transilent SiRNA vectors which reduce gene expression by 80–90%. The principle is to use plasmids that target the mRNA of a specific transcriptional factor. The plasmids encode RNA sequences that are converted into SiRNA transcripts. The vectors are transfected into the cells. The magnitude of the gene silencing effect is estimated at the level of mRNA and protein for each target gene. Each SiRNA is known to suppress its target without influencing the expression of the neighboring non-target genes. Silencing is analyzed by Northern blot analysis. Real-time RT-PCR (polymerase chain reaction) has been considerably improved by priming cDNA synthesis with high stringency oligo (dT). This modified method gives 90–96% reduction (gene knock down) in specific mRNA in cells transfected with SiRNAs. This result agrees with immunofluorescence data. Studies show that chemically synthesized SiRNA is more effective for transfection than that enzymatically prepared. Apparently there is an intrinsic difference between SiRNA produced by chemical and enzymatic means. Additional studies show that in vitro transcription of SiRNA is tenfold more potent for silencing than the chemically synthesized SiRNA. It should also be noted that the use of a 5 leader sequence in the pre-SiRNA molecules and removed after transcription, allows SiRNA of any sequence to be prepared. Only a few molecules per cell for mammalian cell cultures and various invertebrate systems such as C. elegans, Drosophila or trypanosomes are enough to silence gene activity, thereby revealing the gene’s null phenotype (Hannon, 2002; Zamore, 2001) which is obviously a matter of great biological importance.
Bilirubin: More Powerful than Glutathione Among a host of questions addressed by Snyder and his colleagues is whether bilirubin is more powerful as an anti-oxidant than glutathione (GSH). Increasing interest attaches to this inquiry in part because the current dogma holds that glutathione is the primary water-soluble intracellular antioxidant (Meister). One of the characteristics of glutathione (G-SH) is the presence of a free SH group which if oxidized leads to the formation of a disulfide bridge. It is this redox reaction and GSH reductase that maintain GSH in the reduced form. Though GSH acts primarily intracellularly, it also regulates the redox status of proteins in plasma and extracellular fluids e.g. CSF. Within most mammalian cells, the concentration
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of GSH lies in the 3–5 mM range. More recent studies show that GSH is secreted by epithelial and liver cells. Insofar as its protective actions are concerned, GSH reduces peroxides via glutathione peroxidase which contains selenium. This is part of a defense system for trapping and detoxifying reactive oxygen species. Selenium destroys the peroxides that are formed. Selenium-containing proteins in humans are fairly numerous, among which is thioredoxin reductase. This is a 55-kD protein that has one selenocysteine (see later). Now, what does an excess of H2 O2 do in terms of membrane damage? First, it attacks the double bonds of unsaturated fatty acids of phospholipids in cellular membranes. And second, the fatty acid hydroperoxides formed react with C-C chain cleavage and disruption of the membrane. What is plain at once is that these rough calculations take no account of the thioredoxin system. If the argument that the glutathione system is more powerful than the thioredoxin system has validity, it is then not difficult to see that the turnover rate of bilirubin falls within the limits of a redox cycle driven by NADPH. Something should be said about the experiments Snyder and his colleagues performed to determine whether bilirubin protects brain cells, cancer cells and HeLa cells from damage by peroxyl radicals. Bilirubin was found to have the power to scavenge peroxyl radicals that exceed the bilirubin concentration 10,000-fold. The data obtained by Bara˜nano and his colleagues (2002) show a tripling of ROS levels in HeLa cells depleted of BVR protein and catalytic activity. Measurements of ROS were performed with H2 DCFR, a fluoroscein compound which in loaded cells is oxidized to a fluorescent product by ROS. This type of experiment was also performed on neurons from neuronal cultures. In order to draw a comparison between HeLa cells depleted of BVR and cells depleted of GSH, the -glutamyl cysteine synthase was inhibited by applying buthionine sulfoxine (BSO). Such treatment reduces GSH levels in HeLa cells by >95% and raises ROS levels by not more than 50%. That is to say, ROS levels are higher in HeLa cells rendered deficient in BVR than in cells depleted of glutathione. ROS levels are also elevated in neurons silenced with RNAi. All told, depletion of BVR is accompanied by raised ROS levels suggesting the development of oxidative stress. As might be expected, the maneuver depleting the cell of BVR practically stops the formation of bilirubin. The data obtained show a substantial increase in the vulnerability of HeLa cells to treatment with hydrogen peroxide monitored by following apoptotic activity using caspase. Controls, however, show cell survival despite a 10,000-fold excess of H2 O2 . It is interesting to note that HeLa cells depleted of GSH show no more than a slight increase in apoptotic cell death. Such
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a result suggests that bilirubin is a sufficient substitute for GSH. Whether HeLa cells depleted of both bilirubin and GSH can remain viable in the presence of a similar concentration of H2 O2 is not yet known.
Microarray Expression and Analysis A few comments concerning the principles underlying micro-array studies would seem appropriate here. To the extent that the investigator seeks global and high throughput analysis of genes and gene products, microarray technology is the appropriate tool for such studies (Tefferi et al., 2002). Affymetrix, for example, offers a high density oligo chip – the so-called Gene Chip – that carries several hundred thousand spots, representing more than 15,000 human genes. Hierarchical aggregative clustering is the most commonly used clustering method in gene expression analysis. The results are expressed in the form of a binary tree. An agglomerative hierarchical clustering algorithm is then applied to those genes of interest to generate gene expression profiles. This foregoing work was extended to include microarray expression analysis of BVR-deficient cells. The observations made show upregulation in BVR-deficient cells of two main classes of genes. In one class, several antioxidant genes are induced, including heme oxygenase-1, thioreductase and metallothioneins. These are genes known to be induced by oxidative stress, and known to participate in cytoprotection. In the other class, the genes sharing increased expression are those involved in heme metabolism; for example, heme oxygenases. Cells with reduced BVR respond by increasing the synthesis of their own substrate, biliverdin. Microarray analysis also reveals increased expression of antioxidant genes. Both heme synthesis and degradation are upregulated. However, of the 4,368 genes that were detected, 645 show a significant increase in expression, while 550 show a significant decrease. This bimodal behavior is unaccounted for.
The Thioredoxin Reductase Defense System This section is concerned with thioredoxins, a subject that falls within the scope of cell redox balance. The subject is reviewed by Arner and Holmgren (2000) and Nordberg and Arner (2001). Very briefly, thioredoxins (TRXs) constitute a family of proteins that function as general protein-disulfide reductases, and behave as an integral part of the cellular thiol reducing systems. Present evidence strongly suggests that the two systems
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of thioredoxin and glutathione (GSH) are coupled to the peroxidases (TPx and GPx) and engaged in cross-talk (Casagrande et al., 2002; Das & White, 2002). Besides its antioxidant function which is directly attributable to TRX peroxidases, TRX acts as a regulator of other antioxidants e.g. Mn-dependent superoxide dismutase. That is to say, TRX reduces the oxidized form of TRX peroxidase before reduced peroxidase can scavenge ROS e.g. H2 O2 . And in the case of the induction of MnSOD by TRX, it is specific in the sense that Cu/Zn SOD is not induced. The TRX defense system against reactive oxygen species, e.g. H2 O2 includes TRX, thioredoxin reductase (TR) and NADPH which is a source of reducing equivalents. As for TRX peroxidase, it is a 25 kD protein that reduces H2 O2 to water by using electrons (reducing equivalents) coming from TRX. In the presence of oxidative stress, TRX dimerizes with Cys-72 forming the disulfide bridge. The glutathionylation site is located at Cys-72. Whereas TRX contains five cysteine residues, two of which are catalytic that respond to oxidative stress, GSH is a small tripeptide with a single cysteine residue which readily undergoes oxidation reductions. Oxidative stress is accompanied by the movement of TRX from the cytosol to the nucleus. Whether TRX-2 which is found in mitochondria (Tanaka et al., 2002) is able to reach the nucleus is not yet known. TRX is known to stimulate the binding of transcriptional factors to DNA. For example, the binding of NFB to DNA is increased via the reduction of Cys-62 in the NFB subunit. Furthermore, TRX increases AP-1 activity via binding to redox factor-1 (Ref-1). Such studies may eventually be greatly advanced by work along the lines pursued by Jurado and coworkers (2003) who have already attempted to focus on the absolute expression patterns of gene coding for the recognized components of TRX and GRX. GRX is a member of the thiol-disulfide oxido reductases family. It is a cytosolic protein that acts as a cyto-protective antioxidant. H2 O2 is known to increase the expression of this protein. It should be added in passing that TRX expression is found in vascular endothelial cells. Induction by TRX is essentially a protective mechanism against oxidative and nitrosative stress. Coronary atherosclerosis studies in humans show that TRX expression is increased. This finding is in accord with the view that inflammatory stimuli increase the expression of TRX. Thus it is not surprising to find that TRX levels are increased in myocytes and serum during heart failure. What, then, we may ask is there to be learnt here. With bilirubin on the one hand, and thioredoxin on the other, it would seem essential to return to the study of rat brain tissue, cancer cells and HeLa cells in addition to rat cardiomyocytes to elucidate the role of thioredoxin as an antioxidant and determine whether there is cross-talk between the redox proteins.
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A Redox Cycle Model Bara˜nano and Snyder (2001) advocate the view that bilirubin cycles in cells at a considerably higher rate than GSH, and that the cycle is redox in nature requiring energy in the form of electrons supplied by the NADPH → NADP+ reaction which drives and amplifies the cycle. It may be well to re-emphasize Snyder’s viewpoint that bilirubin’s site of action as a cytoprotective antioxidant is the cell membrane. However, this is not yet acceptable in the absence of any proof; presumably, the plasmalemma is its site of action. It is thus for this reason that we may have to reconsider the question whether the lipid-soluble vitamin E, ␣-tocopherol, is the only major membrane bound antioxidant. As will be recalled, ␣-tocopherol is converted to a radical by donating available hydrogen to lipid or a lipid peroxyl radical (Van Acker). Oxidized ␣-tocopherol can then be reduced to its original form by cytosolic ascorbic acid. Methyl substituents can render the radicals more stable (Metzler). The nature of the interaction between bilirubin and the oxidant (reactive oxygen or nitrogen radical) and how this leads to the formation of BV is not yet clear. Whether the ratio of one bilirubin molecule to 10,000 oxidant molecules (meaning free radicals) has any general validity remains to be determined.
Oxidative Degradation Products of Bilirubin Attempts were made by Kranc and his colleagues (2000) to explain the question as to whether one or more of the oxidative degradation products (termed biotripyrrins) of biliverdin and/or bilirubin formed by reaction with H2 O2 are responsible for some of the complications such as cerebral vasospasm or pathological vasoconstriction found in patients with subarachnoid hemorrhage. Increased levels of bilirubin and peroxidation are known to occur in the CSF in patients following subarachnoid hemorrhage. Bilirubin alone, however, fails to produce vasospasm in vitro. These investigators were able to isolate and characterize three vasoactive fragments of bilirubin. Two are isomeric monopyrrole derivatives, and the third is 4methyl-3-vinylmaleimide which is a previously isolated photodegradation product of biliverdin. These three molecules are present in the CSF from subarachnoid hemorrhage patients and produce vasospasm in vitro.
Total Antioxidant Status The antioxidant defense system comprises three main groups of antioxidants: primary, secondary and tertiary defense. By definition, the total antioxidant status
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(TAS), capacity (TAC) or power refers to the total antioxidant effect of these three defense systems in the plasma and extracellular fluids. The three systems are: (1) Primary antioxidants that stop the formation of new free radical species. These include superoxide dismutase (SOD), glutathione peroxidase, and metal-binding proteins e.g. ferritin. (2) Secondary antioxidants that trap radicals, and hence, prevent chain reactions. Examples include vitamin E, vitamin C, -carotene, uric acid, albumin and bilirubin. And (3) Tertiary antioxidants that repair biomolecules damaged by free radicals e.g. DNA repair enzymes. As has been contended in the published literature, it is of conceptual importance to distinguish between the two terms capacity and activity of the antioxidant. Total or over-all capacity represents the cumulative effect of all antioxidants, whereas activity refers to the rate constant of a single antioxidant. On the new view that bilirubin is a considerably more powerful antioxidant than GSH, it would seem reasonable to regard bilirubin as the primary antioxidant. Both in vitro and in vivo observations indicate that bilirubin is a powerful antioxidant and scavenger of reactive oxygen radicals. The various circulating forms of bilirubin are free bilirubin, albumin-bound bilirubin, conjugated and unconjugated bilirubin, all of which are powerful antioxidants. They also are scavengers of peroxyl radicals viz. singlet oxygen, O2 , combined with unsaturated fatty acids to form a lipid peroxyl radical LOO• , and protect human low density lipoprotein (LDL) from peroxidation. At this late point, brief digression is desirable in order to say a few words about common free radicals and other reactive oxygen species (ROS) The simplest free radical is the hydrogen atom. It is written as H• . By definition, a free radical has an odd number of electrons which is indicated by a dot (• ). In the case of the oxygen molecule, it lacks an odd number of electrons but in the ground state the oxygen molecule is bi-radical and does have two “lone” non-paired electrons (O• –O• ), each located in separate orbitals. • Another common free radical is the superoxide radical (O− 2 ) which is formed by one electron reduction of a di-oxygen molecule. It is formed in the internal mitochondrial membrane (NADH ubiquinone reductase and ubiquinone cytochrome C reductase). When reduced, this species forms H2 O2 . In most tissues, a second source of superoxide radicals is cytosolic xanthine oxidase which generates this radical from hypoxanthine and oxygen. Both the liver and jejenum contain appreciable amounts of xanthine oxidase. In liver disease the enzyme is released into the circulation. Hydrogen peroxide, H2 O2 , is produced by enzymatic reaction in all eukaryotes. These enzymes are present in mitochondria, peroxysomes and microsomes. For example, it is released by mitochondria following the reduction of the superoxide • radical, O− 2 . Superoxide dismutase (SOD) dismutates two superoxide molecules
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2− 2− • by adding an electron, it goes from O− 2 to O2 . O2 then picks up 2 protons, to become H2 O2 . Catalase can convert a fraction of the H2 O2 into water. Another step in the O2 cycle is the conversion of H2 O2 into 2 highly reactive hydroxyl radicals. Under physiological conditions, H2 O2 production by the cell results in a relatively constant concentration in the range of 10−9 –10−7 M. Note that cellular membranes are readily permeable to H2 O2 and that during hypoxia its outflux is increased. In the presence of Fe2+ , H2 O2 produces the very active species OH• by the Fenton reaction, first described in 1894
Fe2+ + H2 O2 → Fe3+ + OH• + OH− The free hydroxyl radical is highly reactive; it pulls off a hydrogen atom and produces water. The reaction is considered as the most widely prevalent reaction in biological systems and the cause of a variety of harmful lipid peroxidation products. Here may lie a problem: it is that in addition to HO• the reaction of H2 O2 with Fe2+ does generate Fe2+ /Fe3+ ratios that increase lipid oxidation. In fact, it appears that a 1:1 ratio maximizes lipid peroxidation. According to Hempel and his colleagues (1996), who used human umbilical vein endothelial cells, 100 M H2 O2 is not cytolytic; whereas, 250 M is. Fe2+ protects against this cytoxicity and prevents the rise in intracellular Ca2+ caused by H2 O2 . Extracellular Fe2+ preserves internal GSH in H2 O2 -exposed cells, and it also generates HO• outside the cell, as indicated by electron paramagnetic resonance (EPR) spin trapping. Clearly, this rather intriguing study based in part upon the use of the Fenton reaction has a direct bearing on certain sets of experiments performed by Snyder and his colleagues. More to the point perhaps, is the finding by Hempel and coworkers that 250 M H2 O2 plus 500 M Fe2+ cause cytolysis from H2 O2 alone but no cytolysis from H2 O2 plus Fe2+ . These observations raise the possibility that extracellular Fe2+ protects the cell interior from H2 O2 by initiating the Fenton reaction outside the cell. Conceivably this is the reason why oxidant injury fails to take place. That is, external Fe2+ detoxifies H2 O2 . It is not without some interest that Snyder and his colleagues had drawn the conclusion that OH-1 prevents apoptosis by regulating cellular iron (Ferris et al., 1999). It seems as if Snyder’s group was unaware of the work of Hempel and his group. It has long been known that univalent reduction of oxygen generates highly • reactive intermediates including H2 O2 , O2 , superoxide anion radical (O− 2 ) and • HO . Lipid peroxidation is initiated by the removal of an allylic hydrogen from a polyunsaturated fatty acid (PuFA). Lipid peroxidation is likewise initiated by
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using a transitional metal e.g. iron, which as we have already seen, exacerbates the toxicity of H2 O2 resulting from the production of the hydroxyl radical. A lipid radical L is formed after the removal of allylic hydrogen from PuFA, leading to an immediate rearrangement that involves the formation of a more stable lipid radical. If the radical is in an aerobic environment, it then reacts with oxygen, thus forming a lipid peroxyl radical (LOO• ). This process of lipid peroxyl radical removal of allylic hydrogen atoms from PuFA is of a sustained nature, leading to the formation of lipid hydroperoxidation (LOOH) and a second lipid radical, L. Thus, more lipid hydroperoxide formation follows as the second lipid radical proceeds through the same reaction as the first. After eight or so rounds, the so-called propagation stops. This is then considered as a termination event, presumably caused by interaction with another protein or radical or compound that renders it to act as a free radical trap. The result is the formation of a stable end product. Although ozone (O3 ) is not recognized as being a free radical, it is able to stimulate lipid peroxidation, thereby bringing about membrane damage largely in the airways. The mechanism by which ozone stimulates peroxidation is unclear. It would certainly be rash to overlook nitric oxide (NO) that is not a very reactive species. However, it does react rapidly with O2 , yielding the highly reactive peroxinitrite (ONOO− ).
Biomarkers of Lipid Peroxidation The problem of lipid peroxidation studies is complicated by the fact that robust biomarkers of lipid peroxidation in the human body are not yet available. The best available method is the measurement of F2 -isoprostane levels in human body fluids and urine by mass spectrometry. Halliwell (2000) has laid down ten criteria that need to be met by an ideal assay. No existing assay is ideal. As a biomarker, however, isoprostane meets most of these criteria. Isoprostanes are specific products resulting from peroxidation of unsaturated fatty acid residues in lipids. They are metabolized and excreted. Whether F2 -isoprostanes levels in human body fluids and urine are altered in such conditions as hyperbilirubinemia is completely unknown.
Does HO-1-Bilirubin Promote Cardioprotection? It is well-established that reoxygenation of hypoxic cardiomyocytes results in substantial injury. It therefore seemed important to ascertain whether the addition
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of bilirubin or hemin during hypoxia would attenuate this harmful effect of reperfusion. Studies by Foresti and his colleagues (2001) using cultured rat cardiomyocytes reveal that hypoxia causes a time-dependent increase in both HO1 expression and heme oxygenase activity which decline during reoxygenation. Reoxygenation of hypoxic cardiomyocytes produces marked injury but its severity is reduced when the incubation medium contains hemin or bilirubin during hypoxia. These studies also show that the generation of reactive oxygen species (ROS) is enhanced after hypoxia and, once again, both hemin and bilirubin attenuate this effect. The conclusion thus reached is that hypoxic cardiomyocytes are protected from reoxygenation injury by the HO-1-bilirubin pathway.
CLINICAL CHEMISTRY OF BILIRUBIN In the circulation bilirubin is firmly bound to albumin, whereas in the liver, it is efficiently removed from its albumin bond. It is thus the free unbound bilirubin that is taken up in the liver (Fig. 2). The exact mechanism of hepatic bilirubin uptake is incompletely understood. The sinusoidal membrane of hepatocytes contains a transport protein, OATP2 (organic anion transport protein 2 or SLC21A6), that is responsible for the hepatic uptake of bilirubin (Cui et al., 2001). This transport protein has a broad substrate specificity and substrates include bile salts, estroneand thyroid hormones and a number of drugs (Faber, Muller & Jansen, 2003). As mentioned earlier, bilirubin is conjugated to glucuronic acid and secreted mainly as a diglucuronide into the bile with both carboxyls conjugated to a
Fig. 2. Uptake, Conjugation and Secretion of Bilirubin.
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glucuronic acid molecule. Bilirubin conjugates are substrates for MRP2 (ABCC2), a canalicular protein of the ATP-binding cassette superfamily of transport proteins. MRP2 accepts a large number of glucuronide- and glutathione-conjugated drugs and endogenous metabolites as its substrates (Faber, Muller & Jansen, 2003). Interactions between the various substrates at this level are likely and drug-induced hyperbilirubinemia may be the result of these interactions. Bile salts have their own transporter, BSEP, the bile salt export pump (ABCB11). Therefore, bile salt transport is not likely to interfere with bilirubin secretion.
Detection of Bilirubin and Conjugates In the diagnostic work-up of patients with jaundice, a useful differentiation is that between unconjugated and conjugated jaundice. Unconjugated and conjugated bilirubin in serum can easily be differentiated by chemical methods involving the diazo reaction. Most automated methods are based on this reaction. Quantification of serum bilirubin by these methods is not absolute and so-called “direct-reacting” bilirubin is not an accurate indicator of conjugated bilirubin. At elevated total bilirubin levels, “direct-reacting” bilirubin may be falsely positive. If one wants to differentiate between conjugated and unconjugated bilirubin, high-pressure liquid chromatography should be used (Jansen, Peters & Jansen, 1986; Pieper-Bigelow, Eckfeldt & Levitt, 1995). Small amounts of conjugated bilirubin in serum isolated by HPLC are an indication of early cholestasis. The clinical use of HPLC is both cumbersome and expensive, and therefore, has never gained much popularity for this purpose. For use in neonates, however, several non-invasive transcutaneous devices for determination of bilirubin are available (Bertini & Rubaltelli, 2002).
Unconjugated Hyperbilirubinemia Unconjugated jaundice is due mainly to overproduction of bilirubin from excessive hemolysis and occurs in patients with conjugation defects. Theoretically, unconjugated jaundice could also be the result of disturbed hepatic uptake but there is no convincing evidence that these defects really exist. Conjugated jaundice usually is the consequence of a secretion defect, based on either intrahepatic or extrahepatic causes. Autoimmune, alcoholic or viral hepatitis, is associated with elevations of both unconjugated and conjugated bilirubin. Normal serum bilirubin levels, measured by most methods, do not exceed 1 mg/dl or 17 micromol/l. When bilirubin is overproduced, as in hemolysis, hepatic
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uptake and/or conjugation is overwhelmed, and the serum level of unconjugated bilirubin increases. However, during hemolysis serum bilirubin levels usually stay below 80–100 micromol/l. Higher levels indicate impairment of bilirubin clearance by the liver as occurs in liver disease (Berk et al., 1975). Expressed in a different way, extensive hemolysis may occur without jaundice as exemplified by hemoglobinuria but not if liver cell damage is also present. In the endoplasmic reticulum of hepatocytes, unconjugated bilirubin is converted to bilirubin mono- and diglucuronide by the enzyme UDPglucuronosyltransferase UGT1A1. There are a great number of glucuronyltransferases but UGT1A1 is dedicated to the conjugation of bilirubin. Several conjugation defects are known to exist varying from the common Gilbert syndrome to the rare Crigler Najjar syndromes type I and II. These are due to genetic defects of UGT1A1.
Conjugated Hyperbilirubinemia After conjugation, bilirubin-glucuronides are secreted into the bile. Secretion from hepatocyte to bile is mediated by the ATP-dependent transport protein MRP2 (Multidrug Resistance-associated Protein) (Buchler et al., 1996; Palusma et al., 1986). Transport by this protein is directly coupled to hydrolysis of ATP. The protein is able to transport substrates against considerable concentration gradients and is subject to regulation. Expression of the protein is greatly reduced in sepsis and cholestasis, at least in rats. In humans, however, the protein is less sensitive to regulatory changes in expression. Most causes of jaundice are due to bile secretion problems. Possible causes include bile duct obstruction caused by stones or tumors, by viral or autoimmune hepatitis, by drug-induced cholestasis, by advanced stages of primary biliary cirrhosis, by primary sclerosing cholangitis and by genetic or acquired diseases. When bilirubin cannot be secreted by hepatocytes into bile, it leaves the hepatocyte via the sinusoidal membrane and thus returns to the bloodstream. There is an interesting mechanism at work under these conditions: MRP3, a transport protein located in the sinusoidal membrane that is lowly expressed under normal conditions, is greatly up-regulated during cholestasis or other causes of reduced bilirubin clearance (Ogawa et al., 2000; Zollner et al., 2003). This protein helps the transport of bilirubin conjugates across the sinusoidal membrane. During cholestasis, for example, tubular MRP2 expression in the kidneys is maintained, or even elevated, and this protein actively participates in the removal of conjugated bilirubin via the urine (Denson et al., 2002; Tanaka et al., 2002). This is shown in Fig. 3.
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Fig. 3. Renal Bilirubin-glucuronide Secretion During Cholestasis.
Neonatal Jaundice About 60% of normal term newborns develop neonatal jaundice, for which the expression “physiological jaundice of the newborn” applies. The jaundice occurs between the 2nd and 8th day of life and serum levels of unconjugated bilirubin do not exceed 100 micromol/l. Bilirubin production in the neonate is increased because of a short red cell life-span (Kaplan et al., 2002) but, in contradistinction to current dogma, no relation exists between fetal hemoglobin and neonatal jaundice (Kanai et al., 2003). During this period the capacity of the liver to conjugate bilirubin is not yet fully developed. Entero-hepatic bilirubin cycling may also play a role. In the gut, conjugated bilirubin is hydrolyzed by intestinal glucuronidase but, due to the lack of microflora, bilirubin is not further metabolized to urobilinogen. Part of the deconjugated bilirubin is reabsorbed and added to the serum pool of unconjugated bilirubin. Bilirubin is conjugated in the liver by the enzyme UGT1A1, a member of the UDP-glucuronosyltransferase family 1. These enzymes are under transcriptional control of the nuclear hormone receptors, CAR (constitutive androstane receptor) and PXR (nuclear pregnane X-receptor) (Huang et al., 2003; Xie et al., 2003). Interestingly CAR, has a low expression in neonatal mice. It is only fully expressed 2 weeks after birth (Huang et al., 2003). CAR deficiency may thus be a factor in the development of neonatal jaundice. This would also explain why the CAR-
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ligand phenobarbital is not useful in the treatment of neonatal jaundice. Moreover, phenobarbital is only a weak CAR agonist. The factors contributing to the elevated serum bilirubin levels are exaggerated in the premature newborn, and, therefore, in these infants, serum bilirubin rises faster and to a higher level than in full-term infants. Hemolysis, due to ABOor rhesus-antagonism between mother and child, will cause an increased rate and level of serum bilirubin. Infants with the Gilbert syndrome, either due to a TATAA box defect in the promotor region of the UGT1A1 gene, or a point mutation in the coding sequence of the gene (Gly71Arg at nucleotide 211 in Japanese infants), have increased neonatal bilirubin levels (Huang et al., 2002; RoyChowdhury et al., 2002; Yamamoto et al., 2002). The combination of the Gilbert syndrome, ABO antagonism or hemoglobinopathies, like glucose-6-phosphate dehydrogenase deficiency, is associated with prolonged and elevated levels of serum bilirubin (Kaplan et al., 2000). Infants with the Gilbert syndrome may develop prolonged jaundice (>14 days of life) while being breast-fed (Monaghan et al., 1999). Unbound unconjugated bilirubin is neurotoxic and may cause kernicterus. Certain drugs (e.g. sulfonamides) that displace bilirubin from albumin increase the risk for kernicterus. There are no accurate tests to measure bound and unbound unconjugated bilirubin in serum. Values for the total serum bilirubin concentration are not absolutely predictive for kernicterus risk. A child on antibiotics may be more at risk than a child with a similar total serum bilirubin level but without drugs. Phototherapy is the first treatment of choice. In healthy term newborns, phototherapy is initiated if serum bilirubin rises above 260 micromol/l at 24–48 hrs after birth; above 310 micromol/l at 48–72 hrs, and above 340 micromol/l at more than 72 hrs after birth. Exchange transfusion should be given if phototherapy fails and the serum bilirubin is above 340 micromol/l, 24–48 hrs after birth; above 430 micromol/l, 48–72 hrs and more than 72 hrs after birth. If the serum bilirubin is above 430 micromol/l, 24–48 hrs after birth or above 510 micromol/l, 48 hrs after birth, and thereafter, exchange transfusion should be combined with intense phototherapy. These are the recommendations made in 1994 by the American Academy of Pediatrics (www.aap.org). Epidemiological studies revealed that the administration of phototherapy in the newborn is not optimal; nor is it used when it should be, and sometimes used inappropriately (Atkinson et al., 2003). Tin-mesoporphyrin, a heme-oxygenase inhibitor, has been used to prevent jaundice in glucose-6-phosphate dehydrogenase-deficient infants (Kappas, Drummond & Valaes, 2001). Agar, cholestyramine and calcium phosphate interrupt the enterohepatic cycle of unconjugated bilirubin but the effect obtained has not been impressive enough.
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Fig. 4. Genetic Organization and Transcription of the UGT1A1 Locus. Gilbert Syndrome Mutations are Indicated.
PATHOBIOLOGICAL ASPECTS Gilbert Syndrome Gilbert(‘s) or the Meulengracht syndrome is characterized by a mild elevation in the serum of unconjugated bilirubin (30–80 micromol/liter). It occurs in ∼6% of the Caucasian population and more commonly in males than in females. It is caused by a reduction of the activity of the bilirubin-conjugating enzyme in the liver, uridine-diphosphoglucuronate glucuronosyltransferase or UGT1A1. UGT1A1 is encoded by the UGT1 gene locus on chromosome 2q37. Figure 4 shows the organization of the UGT1 locus. Four exons (exons 2–5) at the 3 end encode the common carboxyterminal domain of all UGT isoforms encoded by this locus. This carboxyterminal domain contains the membrane spanning region and the UDPglucuronic acid binding site. Upstream of these four exons are a number of exons that encode the substrate binding site. Each unique exon 1 is spliced to exon 2 and the intervening mRNA segment is spliced out. Each of the exon 1’s is preceded by a different promotor, allowing differential regulation of the various UGT1 enzymes. The promotors contain a TATAA box that is important for transcription by RNA polymerase II. Bilirubin UGT1A1 contains a TATAA box with six TA dinucleotide repeats. A prolonged TATAA box is associated with a decreased transcription rate and decreased UGT1A1 activity (Bosma et al., 1995; Monaghan et al., 1996).
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Caucasian patients with the Gilbert syndrome almost invariably are homozygous for a TATAA box with seven instead of six TA repeats. The prolonged TATAA box with seven TA repeats has been named UGT1A1*28 allele. Among Caucasians the frequency of the UGT1A1*28 allele is approximately 35–40% (Bosma et al., 1995; Monaghan et al., 1996). Thus, homozygosity for UGT1A1*28 among Caucasians may be in the order of 12–16%, and in Asians in the order of 2–3%. Not all of these persons have the clinical manifestations of the Gilbert syndrome. Additional factors, such as increased erythrocyte turnover (but not necessarily overt hemolysis) or impaired hepatic uptake play a role in the expression of the syndrome (Berk & Blaschke, 1972; Berk, Blaschke & Waggoner, 1972; Kaplan et al., 2001; Persico et al., 2001). Among Asian populations the prevalence of UGT1A1*28 is low. Asian patients with the the Gilbert syndrome often have missense mutations of the UGT1A1 coding region, like the G71R and the Y486D mutation (Aono et al., 1995; Koiwai et al., 1995; Maruo et al., 1998). The serum bilirubin level is the result of bilirubin synthesis and removal. A combination of UGT1A1*28 homozygosity and overt hemolysis will cause increased serum bilirubin levels. This is particularly seen in the Mediterranean area where glucose-6-phosphate dehydrogenase and -thalassemia are prevalent. In neonates this combination may lead to kernicterus and/or prolonged neonatal jaundice (Kaplan et al., 1997; Tzetis et al., 2001). Also without the mentioned hemolytic syndromes, the Gilbert syndrome may cause prolonged neonatal jaundice (Bancroft, Kreamer & Gowley, 1998; Maruo et al., 2000). Hypersplenism and congenital spherocytosis in combination with UGT1A1*28 will also lead to increased serum bilirubin levels. In patients with congenital spherocytosis and Gilbert syndrome, incidence of gallstones is high (del Giudice et al., 1999). The Gilbert syndrome is diagnosed by measuring an elevated unconjugated bilirubin level in the absence of hemolysis and with normal liver function tests and normal serum liver enzymes. Upon fasting (400 kcal for 48 hrs) a doubling of serum bilirubin levels is seen in patients with the Gilbert syndrome (Felsher, Rickard & Redeker, 1970). However, this test lacks specificity. Also nicotinic acid administration leads to an increase in serum bilirubin levels but this test lacks specificity, as well (Davidson et al., 1975). A liver biopsy to test bilirubin UDPglucuronosyltransferase activity is not recommended. The UGT1A1*28 gene can be tested by a rapid RT-PCR test. The Gilbert syndrome, in combination with hemolysis, may lead to considerably elevated serum bilirubin levels. Treatment is not necessary. These patients often complain of fatigue. The etiology of this symptom is unknown. Patients with Gilbert’s syndrome more often experience side effects such as diarrhea upon administration of the topomerase I inhibitor irinotecan (CPT-11) (Ando et al., 2000). There appears to be a linkage between UGT1A1*28 and a variant UGT1A6 (UGT1A6*02) with reduced enzymatic activity. Therefore,
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patients with the Gilbert syndrome may have reduced glucuronidation of aspirin, paracetamol, coumarin- and dopamine-derivatives, all substrates of UGT1A6 (Peters, te Morsche & Roelofs, 2003).
Crigler-Najjar Syndrome The Crigler Najjar syndrome is characterized by a pronounced elevation of serum unconjugated bilirubin (in excess of 100 micromol/l but usually higher: 150–400 micromol/l)). It is also called hereditary nonhemolytic unconjugated hyperbilirubinemia. There are two types, type 1 in which serum bilirubin levels do not respond to phenobarital treatment, and type 2 or Arias syndrome in which serum bilirubin decreases upon treatment of the patient with phenobarbital. It is a very rare disease (∼1 per million). In patients with Crigler-Najjar syndrome type 1 (CNS-1), bilirubin glucuronidation is completely absent and in patients with CNS-2, bilirubin glucuronidation is partially deficient (Sinaasappel & Jansen, 1991). Likewise serum levels of unconjugated bilirubin in these patients are elevated to levels above ∼340 micromol/l (CNS-1) and to levels between 150 and 340 micromol/l (CNS-2). Bile of patients with CNS-1 contains only trace amounts of bilirubin whilst in CNS-2 patients bile contains bilirubin mono-and diglucuronide in low concentrations, with an increased proportion of monoglucuronide (Fevery et al., 1977; Sinaasappel & Jansen, 1991). Another difference between CNS-1 and CNS2 is that CNS-1 patients do not respond to phenobarbital treatment while in CNS-2 serum bilirubin levels can be lowered by phenobarbital by more than 30% (Arias et al., 1969). Neonates with CNS-1 are at risk to die of kernicterus unless treated by exchange transfusion immediately after birth and phototherapy (van der Veere et al., 1996). In these patients phototherapy has to be continued for many years until transplantation. Also adult patients with CNS-1 can develop kernicterus (Blaschke et al., 1974); for CNS-2 this is rare but has been reported (Chalasani et al., 1997). The bilirubin glucuronidation deficiency in CNS is due to mutations in the UGT1A1 gene. At least 50 different mutations have been described to date (Kadakol et al., 2000) (Fig. 5). Many of these mutations cause single amino acid substitutions that completely (CNS-1) or partially (CNS-2) inactivate the enzyme, others produce stop codons or are frame shift mutations resulting in a truncated protein. In CNS-2 patients most mutations cause single amino acid substitutions, resulting in an enzyme with reduced activity. Genetic lesions in both CNS-1 and CNS-2 occur in any of the five exons of the UGT1A1 gene (Aono et al., 1993; Bosma et al., 1993; Gantla et al., 1998; Kadakol et al., 2000; Ritter et al., 1992; Seppen et al., 1996).
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Fig. 5. UGT1A1 Locus with Crigler Najjar Syndrome Mutations. CNS-1, Crigler Najjar Type 1; CNS-2, Crigler Najjar Type 2.
CNS-1 and CNS-2 are autosomal recessive diseases. There is debate whether CNS-2 can also be inherited in an autosomal dominant fashion. The caveat here may be unidentified mutations in the so-called normal allele. A dominant negative effect may be another possibility: UGT1 functions as a dimer and a dimer of a non-affected and an affected protein may be less active than a homodimer of two non-affected proteins (Koiwai et al., 1996). The height of the serum unconjugated bilirubin level, the response to phenobarbital, bile analysis, and possibly an enzyme-activity test on a liver biopsy, will lead to the diagnosis (Sinaasappel & Jansen, 1991). However, as in any other genetic disease, mutation analysis will provide the final proof. Prenatal diagnosis, using chorionic villus samples, can be performed. This should preferably be done when the mutation has already been established in an older child with the disease (Ciotti et al., 1997; Francoual et al., 2002). Treatment consists of liver transplantation in patients with CNS-1 and life-long phenobarbital treatment in patients with CNS-2. In CNS-1, intensive phototherapy for many years is helpful in postponing liver transplantation until adolescence. At this age phototherapy becomes less efficient while adults still can develop kernicterus (Blaschke et al., 1974; Chalasani et al., 1997). Future modes of treatment include hepatocyte transplantation (Fox et al., 1998) or gene therapy.
Dubin-Johnson Syndrome The Dubin-Johnson syndrome is characterized by an elevation of conjugated bilirubin in the serum without other liver function abnormalities. In the liver biopsy, one sees a black lysosomal pigment. The total urinary coproporphyrin
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Fig. 6. Dubin-Johnson Syndrome. Note: Due to defective canalicular MRP2, bilirubin glucuronides cannot be secreted into bile. MRP3 in the basolateral membrane of hepatocytes is induced, and this protein mediates the secretion of bilirubin glucuronides across the basolateral membrane.
excretion in the urine is normal but coproporphyrin isomer I excretion is elevated. It is an autosomal recessive disease and rare with a gene frequency (in Japan) of ∼1:800,000. In the Dubin-Johnson syndrome, the hepatobiliary secretion of conjugated bilirubin is impaired due to a deficiency of the transporter protein MRP2 (ABCC2; canalicular Multispecific Organic Anion Transporter, cMOAT). This protein is located in the canalicular membrane of hepatocytes (Fig. 6). The MRP2 gene is located on chromosome 10q24 (van Kuijck et al., 1997). The described MRP2 gene mutations (Fig. 7) either preclude synthesis of an intact active protein, causes the synthesis of a protein that cannot be targeted correctly from the endoplasmic reticulum to the canalicular membrane or produces a protein that is correctly targeted but is inactive or less active (Hashimoto et al., 2002; Kartenbeck et al., 1996; Keitel et al., 2000; Mor-Cohen et al., 2001; Tate et al., 2002; Toh et al., 1999; Tsujii et al., 1999; Wada et al., 1998). As a result, conjugated bilirubin cannot be secreted into bile. Serum bile salt levels are normal. MRP3 (ABCC3) is a protein that is able to transport many of the MRP2 substrates (Konig et al., 1999). Under normal conditions, MRP3 is lowly expressed in the liver but in patients with Dubin-Johnson syndrome its expression is increased. As a basolateral protein, it transports conjugated bilirubin from hepatocytes to blood. Conjugated bilirubin is removed from the blood via renal secretion. However, this is less efficient than removal via the hepatobiliary route and, therefore, patients with the Dubin-Johnson
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Fig. 7. Dubin-Johnson Syndrome Mutations in the Canalicular Transport Protein MRP2.
syndrome are permanently jaundiced. The liver of these patients looks grossly black. Liver histology shows a normal liver architecture but a pathognomic blackbrown lysosomal pigment. The pigment is an oxidative metabolite of tyrosine and tryptophane, which normally are secreted into bile as organic anions (Arias & Blumberg, 1979; Kitamura et al., 1992). In the Dubin-Johnson syndrome the serum conjugated bilirubin concentration is usually between 50 and 85 micromol/l but can be elevated up to 385 micromol/l. It increases during pregnancy (Cohen, Lewis & Arias, 1972). Total urinary coproporphyrin is normal, and coproporphyrin isomer I is relatively elevated (>80%; normal <27%) (Wolkoff, Cohen & Arias, 1973). Hepatobiliary secretion of the scintigraphic agent HIDA is delayed. The initial plasma disappearance of sulfobromophthalein is normal, with a normal 45 minute retention value, but there is a secondary rise after 90 minutes (Gutstein, Alpert & Arias, 1968). The liver is grossly black and this is due to a black lysosomal pigment in hepatocytes. Routine liver function tests are normal with normal bile acid levels. The disease has an excellent prognosis and therapy is not needed.
Rotor Syndrome Like the Dubin-Johnson syndrome, patients with Rotor syndrome have elevated serum levels of both conjugated and unconjugated bilirubin (50–100 micromol/l). In contrast to the Dubin-Johnson syndrome, liver histology is entirely normal.
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The total urinary coproporphyrin excretion is elevated while in the DubinJohnson syndrome it is normal. The disease is extremely rare. A possible causative gene defect has not yet been identified. Kinetic analysis of intravenous sulfobromophthalein disappearance curves has suggested that this disease is caused by defective storage and/or hepatic uptake of the dye and not by defective hepatic secretion (Dubin-Johnson syndrome) (Wolkoff et al., 1976; Wolpert et al., 1977). Since serum bilirubin is partly conjugated, defective storage after conjugation, with reflux of the conjugate to the serum is the more likely mechanism (Berthelot & Dhumeaux, 1978). An alternative mechanism might be an inappropriate expression of an active transporter, transporting conjugated bilirubin back to the serum rather than to the bile. However, this is speculation. The characteristic lysosomal pigment of the Dubin-Johnson syndrome is missing in Rotor syndrome. Total urinary coproporphyrin excretion is elevated, with a relatively increased proportion of coproporphyrin isomer I (Wolkoff et al., 1976). In the Dubin-Johnson syndrome, total urinary coproporphyrin excretion is normal. The diagnosis is suspected in patients with a mildly elevated serum bilirubin that is partly of the conjugated type. Liver enzymes and serum indicators of liver function are normal. Liver histology is also normal. The liver does not contain abnormal pigment as in the Dubin-Johnson syndrome. Abnormal 45 minute retention after intravenous injection of sulfobromophthalein (34 ± 2% vs. 4 ± 0.6% in controls). Serum bile salt levels are normal. Urinary total coproporhyrin is elevated, 50–70% of which is coproporphyrin isomer I. Treatment is unnecessary.
CONCLUSION I. Apart from confirming the conclusion reached by Stocker et al. (1987) that bilirubin is a physiologic antioxidant, Snyder and coworkers obtained compelling evidence that bilirubin is also considerably more powerful than glutathione as an antioxidant. However, nothing is said about the role of thioredoxin in cell redox balance. Furthermore, bilirubin is found to behave as an effective ROS scavenger. Bilirubin levels in several types of cells were estimated for the first time as being in the low nanoMolar range e.g. ∼50 nM. Whether the bilirubin gradients across the cell membrane are appreciably altered in conditions such as hyperbilirubinemia is not yet known. Using the RNA interference method to abolish biliverdin reductase activity, Snyder and coworkers were able to show that cellular depletion of bilirubin leads to increased levels of ROS and apoptotic cell death. By contrast, cellular depletion of glutathione causes lesser increases in ROS levels and apoptosis. They also sought to monitor gene expression using microarray expression analysis and found two classes of genes. However, this line of work appears incomplete.
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The general conclusion arrived at by Snyder and coworkers concerning the main physiologic function of bilirubin is best described by a redox cycle in which bilirubin is oxidized to biliverdin, and immediately acted upon catalytically by biliverdin reductase back to bilirubin. This is an NADPH-dependent cycle that can be drastically amplified to protect cells from a 10,000-fold excess of H2 O2 . Attention is also drawn to the work of Foresti and coworkers whose studies on rat cardiomyocytes clearly show that hypoxia stimulates HO-1 expression and that this pathway provides significant protection from re-oxygenation mediated damage of cardiomyocytes. Cardioprotection is accounted for by bilirubin’s ability of reducing oxidant stress and the ability of heme oxygenase to increase the supply of bilirubin. The role of iron is far from clear. II. Jaundice is a hallmark of liver disease and is sometimes mentioned as a synonym of liver disease. The causes of jaundice are mostly acquired and include viral or autoimmune liver disease, drug interactions and extrahepatic bile duct obstruction. In addition, there are genetic causes that with the exception of the Gilbert syndrome, are very rare. The Gilbert syndrome, though not rare, is often misdiagnosed. Jaundice in these patients is harmless, whilst in patients with liver disease it may often be a sign of severe liver dysfunction.
REFERENCES Ando, Y., Saka, H., Ando, M., Sawa, T., Muro, K., Ueoka, H. et al. (2000). Polymorphisms of UDPglucuronosyltransferase gene and irinotecan toxicity: A pharmacogenetic analysis. Cancer Res., 60, 6921–6926. Aono, S., Adachi, Y., Uyama, E., Yamada, Y., Keino, H., Nanno, T. et al. (1995). Analysis of genes for bilirubin UDP-glucuronosyltransferase in Gilbert’s syndrome. Lancet, 345, 958–959. Aono, S., Yamada, Y., Keino, H., Hanada, N., Nakagawa, T., Sasaoka, Y. et al. (1993). Identification of defect in the genes for bilirubin UDP-glucuronosyl-transferase in a patient with Crigler-Najjar syndrome type II. Biochem. Biophys. Res. Commun., 197, 1239–1244. Arias, I. M., & Blumberg, W. (1979). The pigment in Dubin-Johnson syndrome. Gastroenterology, 77, 820–821. Arias, I. M., Gartner, L. M., Cohen, M., Ezzer, J. B., & Levi, A. J. (1969). Chronic nonhemolytic unconjugated hyperbilirubinemia with glucuronyl transferase deficiency. Clinical, biochemical, pharmacologic and genetic evidence for heterogeneity. Am. J. Med., 47, 395–409. Arner, E. S., & Holmgren, A. (2000). Physiological functions of thioredoxin and thioredoxin reductase. Eur. J. Biochem., 276, 6102–6109. Atkinson, L. R., Escobar, G. J., Takayama, J. I., & Newman, T. B. (2003). Phototherapy use in jaundiced newborns in a large managed care organization: Do clinicians adhere to the guideline? Pediatrics, 111, e555–e561. Bancroft, J. D., Kreamer, B., & Gourley, G. R. (1998). Gilbert syndrome accelerates development of neonatal jaundice. J. Pediatr., 132, 656–660.
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Hartsfield, C. L., Alam, J., & Choi, A. M. K. (1998). Transcriptional regulation of the heme oxygenase 1 gene by pyrrolidine dithiocarbamate. FASEB J., 12, 1675–1682. Hashimoto, K., Uchiumi, T., Konno, T., Ebihara, T., Nakamura, T., Wada, M. et al. (2002). Trafficking and functional defects by mutations of the ATP-binding domains in MRP2 in patients with Dubin-Johnson syndrome. Hepatology, 36, 1236–1245. Hempel, S. L., Buetnner, G. R., Wessel, D. A., Galvan, G. M., & O’Malley, Y. Q. (1996). Extracellular iron can protect cells from hydrogen peroxide. Arch. Biochem. Biophys., 330, 401–408. Hopkins, P. M., Wu, L. L., Hunt, S. C., James, B. C., Vincent, G. M., & Williams, R. R. (1996). Higher serum bilirubin is associated with decreased risk for early familial coronary artery disease. Arteriosclr. Thromb. Vasc. Biol., 16, 250–255. Huang, C. S., Chang, P. F., Huang, M. J., Chen, E. S., Hung, K. L., & Tsou, K. I. (2002). Relationship between bilirubin UDP-glucuronosyl transferase 1A1 gene and neonatal hyperbilirubinemia. Pediatr. Res., 52, 601–605. Huang, W., Zhang, J., Chua, S. S., Qatanani, M., Han, Y., Granata, R. et al. (2003). Induction of bilirubin clearance by the constitutive androstane receptor (CAR). Proc. Natl. Acad. Sci. USA, 100, 4156–4161. Jansen, P. L., Peters, W. H., & Janssens, A. R. (1986). Clinical value of serum bilirubin subfractionation by high-performance liquid chromatography and conventional methods in patients with primary biliary cirrhosis. J. Hepatol., 2, 485–494. Kadakol, A., Ghosh, S. S., Sappal, B. S., Sharma, G., Chowdhury, J. R., & Chowdhury, N. R. (2000). Genetic lesions of bilirubin uridine-diphosphoglucuronate glucuronosyltransferase (UGT1A1) causing Crigler-Najjar and Gilbert syndromes: Correlation of genotype to phenotype. Hum. Mutat., 16, 297–306. Kanai, M., Akaba, K., Sasaki, A., Sato, M., Harano, T., & Shibahara, S. et al. (2003). Neonatal Hyperbilirubinemia in Japanese neonates: Analysis of the heme oxygenase-1 gene and fetal hemoglobin composition in cord blood. Pediatr. Res. (in press). Kaplan, M., Hammerman, C., Renbaum, P., Klein, G., & Levy-Lahad, E. (2000). Gilbert’s syndrome and hyperbilirubinaemia in ABO-incompatible neonates. Lancet, 356, 652–653. Kaplan, M., Hammerman, C., Rubaltelli, F. F., Vilei, M. T., Levy-Lahad, E., Renbaum, P. et al. (2002). Hemolysis and bilirubin conjugation in association with UDP-glucuronosyltransferase 1A1 promoter polymorphism. Hepatology, 35, 905–911. Kaplan, M., Muraca, M., Hammerman, C., Rubaltelli, F. F., Vilei, M. T., Vreman, H. J. et al. (2002). Imbalance between production and conjugation of bilirubin: A fundamental concept in the mechanism of neonatal jaundice. Pediatrics, 110, e47. Kaplan, M., Renbaum, P., Levy-Lahad, E., Hammerman, C., Lahad, A., & Beutler, E. (1997). Gilbert syndrome and glucose-6-phosphate dehydrogenase deficiency: A dose-dependent genetic interaction crucial to neonatal hyperbilirubinemia. Proc. Natl. Acad. Sci. USA, 94, 12128–12132. Kappas, A., Drummond, G. S., & Valaes, T. (2001). A single dose of Sn-mesoporphyrin prevents development of severe hyperbilirubinemia in glucose-6-phosphate dehydrogenase-deficient newborns. Pediatrics, 108, 25–30. Kartenbeck, J., Leuschner, U., Mayer, R., & Keppler, D. (1996). Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome. Hepatology, 23, 1061–1066. Keitel, V., Kartenbeck, J., Nies, A. T., Spring, H., Brom, M., & Keppler, D. (2000). Impaired protein maturation of the conjugate export pump multidrug resistance protein 2 as a consequence of a deletion mutation in Dubin-Johnson syndrome. Hepatology, 32, 1317–1328.
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Kitamura, T., Alroy, J., Gatmaitan, Z., Inoue, M., Mikami, T., Jansen, P. et al. (1992). Defective biliary excretion of epinephrine metabolites in mutant (TR-) rats: Relation to the pathogenesis of black liver in the Dubin-Johnson syndrome and Corriedale sheep with an analogous excretory defect. Hepatology, 15, 1154–1159. Kitamura, Y., Matsuoka, Y., Nomura, Y., & Tamiguchi, T. (1998). Induction of inducible nitric oxide synthase and heme oxygenase-1 in rat glial cells. Life Sci., 62, 1717–1721. Koiwai, O., Aono, S., Adachi, Y., Kamisako, T., Yasui, Y., Nishizawa, M. et al. (1996). Crigler-Najjar syndrome type II is inherited both as a dominant and as a recessive trait. Hum. Mol. Genet., 5, 645–647. Koiwai, O., Nishizawa, M., Hasada, K., Aono, S., Adachi, Y., Mamiya, N. et al. (1995). Gilbert’s syndrome is caused by a heterozygous missense mutation in the gene for bilirubin UDPglucuronosyltransferase. Hum. Mol. Genet., 4, 1183–1186. Konig, J., Rost, D., Cui, Y., & Keppler, D. (1999). Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane. Hepatology, 29, 1156–1163. Kranc, K. R., Prne, G. J., Tao, L., Claridge, D. W., Harris, D. A., Cadoux-Hudson, T. A. D., Turnbull, J. J., Schoffield, C. J., & Clark, J. F. (2000). Eur. J. Biochem., 267, 7094–7101. Maines, M. D. (1997). The heme oxygenase system: A regulator of second messenger gases. Annu. Rev. Pharmacol. Toxicol., 37, 517–554. Maruo, Y., Nishizawa, K., Sato, H., Sawa, H., & Shimada, M. (2000). Prolonged unconjugated hyperbilirubinemia associated with breast milk and mutations of the bilirubin uridine diphosphate- glucuronosyltransferase gene. Pediatrics, 106, E59. Maruo, Y., Sato, H., Yamano, T., Doida, Y., & Shimada, M. (1998). Gilbert syndrome caused by a homozygous missense mutation (Tyr486Asp) of bilirubin UDP-glucuronosyltransferase gene. J. Pediatr., 132, 1045–1047. Mayer, M. (2000). Association of serum bilirubin concentration with risk of coronary artery disease. Clin. Chem., 46, 1723–1727. Monaghan, G., McLellan, A., McGeehan, A., Li, V. S., Mollica, F., Salemi, I. et al. (1999). Gilbert’s syndrome is a contributory factor in prolonged unconjugated hyperbilirubinemia of the newborn. J. Pediatr., 134, 441–446. Monaghan, G., Ryan, M., Seddon, R., Hume, R., & Burchell, B. (1996). Genetic variation in bilirubin UDP-glucuronosyltransferase gene promoter and Gilbert’s syndrome. Lancet, 347, 578–581. Mor-Cohen, R., Zivelin, A., Rosenberg, N., Shani, M., Muallem, S., & Seligsohn, U. (2001). Identification and functional analysis of two novel mutations in the multidrug resistance protein 2 gene in Israeli patients with Dubin-Johnson syndrome. J. Biol. Chem., 276, 36923–36930. Nordberg, J., & Arner, E. S. (2001). Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med., 31, 1287–1312. Ogawa, K., Suzuki, H., Hirohashi, T., Ishikawa, T., Meier, P. J., Hirose, K. et al. (2000). Characterization of inducible nature of MRP3 in rat liver. Am. J. Physiol. Gastrointest. Liver Physiol., 278, G438–G446. Otterbein, L. E., & Choi, A. M. (2000). Heme oxygenase: Colors of defense against cellular stress. Am. J. Physiol., 279, 1029–1037. Persico, M., Persico, E., Bakker, C. T., Rigato, I., Amoroso, A., Torella, R. et al. (2001). Hepatic uptake of organic anions affects the plasma bilirubin level in subjects with Gilbert’s syndrome mutations in UGT1A1. Hepatology, 33, 627–632.
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Peters, W. H., te Morsche, R. H., & Roelofs, H. M. (2003). Combined polymorphisms in UDPglucuronosyltransferases 1A1 and 1A6: Implications for patients with Gilbert’s syndrome. J. Hepatol., 38, 3–8. Pieper-Bigelow, C., Eckfeldt, J., & Levitt, M. D. (1995). Sensitivity of HPLC and conventional bilirubin measurements in the detection of early cholestasis. J. Lab. Clin. Med., 125, 654–661. Ritter, J. K., Yeatman, M. T., Ferreira, P., & Owens, I. S. (1992). Identification of a genetic alteration in the code for bilirubin UDP-glucuronosyltransferase in the UGT1 gene complex of a CriglerNajjar type I patient. J. Clin. Invest., 90, 150–155. Roy-Chowdhury, N., Deocharan, B., Bejjanki, H. R., Roy-Chowdhury, J., Koliopoulos, C., Petmezaki, S. et al. (2002). Presence of the genetic marker for Gilbert syndrome is associated with increased level and duration of neonatal jaundice. Acta. Paediatr., 91, 100–101. Salim, M., Brown-Kipphut, B. A., & Maines, M. D. (2001). Human biliverdin reductase is autophosphorylated, and phosphorylation is required for bilirubin formation. J. Biol. Chem., 276, 10929–10934. Seppen, J., Steenken, E., Lindhout, D., Bosma, P. J., & Elferink, R. P. (1996). A mutation which disrupts the hydrophobic core of the signal peptide of bilirubin UDP-glucuronosyltransferase, an endoplasmic reticulum membrane protein, causes Crigler-Najjar type II. FEBS Lett., 390, 294–298. Sinaasappel, M., & Jansen, P. L. (1991). The differential diagnosis of Crigler-Najjar disease, types 1 and 2, by bile pigment analysis. Gastroenterology, 100, 783–789. Stocker, R., Yamamoto, Y., McDonagh, A. F., Glazer, A. N., & Ames, B. N. (1987). Bilirubin is an antioxidant of possible physiological importance. Science, 235, 1043–1046. Tanaka, T., Hosoi, F., Yamaguchi-Iwai, Y., Nakamura, H., Masutani, H., Meda, S., Nishiyama, A., Takeda, S., Wada, H., Spyrou, G., & Yodoi, J. (2002). Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis. EMBO J., 21, 1695–1703. Tanaka, Y., Kobayashi, Y., Gabazza, E. C., Higuchi, K., Kamisako, T., Kuroda, M. et al. (2002). Increased renal expression of bilirubin glucuronide transporters in a rat model of obstructive jaundice. Am. J. Physiol. Gastrointest. Liver Physiol., 282, G656–G662. Tate, G., Li, M., Suzuki, T., & Mitsuya, T. (2002). A new mutation of the ATP-binding cassette, subfamily C, member 2 (ABCC2) gene in a Japanese patient with Dubin-Johnson syndrome. Genes Genet. Syst., 77, 117–121. Toh, S., Wada, M., Uchiumi, T., Inokuchi, A., Makino, Y., Horie, Y. et al. (1999). Genomic structure of the canalicular multispecific organic anion-transporter gene (MRP2/cMOAT) and mutations in the ATP-binding-cassette region in Dubin-Johnson syndrome. Am. J. Hum. Genet., 64, 739–746. Tsujii, H., Konig, J., Rost, D., Stockel, B., Leuschner, U., & Keppler, D. (1999). Exon-intron organization of the human multidrug-resistance protein 2 (MRP2) gene mutated in DubinJohnson syndrome. Gastroenterology, 117, 653–660. Tzetis, M., Kanavakis, E., Tsezou, A., Ladis, V., Pateraki, E., Georgakopoulou, T. et al. (2001). Gilbert syndrome associated with beta-thalassemia. Pediatr. Hematol. Oncol., 18, 477–484. van der Veere, C. N., Sinaasappel, M., McDonagh, A. F., Rosenthal, P., Labrune, P., Odievre, M. et al. (1996). Current therapy for Crigler-Najjar syndrome type 1: Report of a world registry. Hepatology, 24, 311–315. van Kuijck, M. A., Kool, M., Merkx, G. F., Geurts, vK., Bindels, R. J., Deen, P. M. et al. (1997). Assignment of the canalicular multispecific organic anion transporter gene (CMOAT) to human chromosome 10q24 and mouse chromosome 19D2 by fluorescent in situ hybridization. Cytogenet. Cell Genet., 77, 285–287.
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11.
CLINICAL BIOCHEMISTRY OF THE LIVER
Neil McIntyre INTRODUCTION All clinical biochemical laboratories use a small battery of blood tests to detect and manage liver disease. It usually includes total bilirubin, aspartate and/or alanine aminotransferase, alkaline phosphatase and albumin; other tests such as gamma glutamyltransferase (GGT) may be added (Table 1). Although called “liver function tests” they are not specific for liver disease and are of little use for assessing liver function. Some functions of the liver, such as galactose elimination, bile acid clearance and removal of some dyes, can be quantified but the methods, discussed later, are inconvenient and costly. Urine tests for bilirubin and urobilinogen are easily performed in the ward or clinic. Prothrombin and partial thromboplastin times, useful markers of the severity of liver disease, are measured in the hematology laboratory; although valuable in managing liver disease they are not usually considered as “liver function tests” and will not be considered in this chapter.
SERUM BILIRUBIN (McIntyre & Rosalki, 1999; Ostrow, 1986) Bilirubin is formed from the heme of hemoglobin, myoglobin and other heme proteins. Insoluble in water, it binds strongly to albumin before uptake by the The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 291–316 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15011-3
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Table 1. Liver “Function” Tests.a Serum bilirubin – total – unconjugated, conjugated and bili-proteins Urinary bilirubin and urobilinogen Serum aminotransferases – aspartate, alanine Glutathione-S-transferase Lactate dehydrogenase Serum alkaline phosphatase 5 -Nucleotidase Leucine aminopeptidase Gammaglutamyl transferase Serum albumin Pre-albumin Ceruloplasmin ␣-1-antitrypsin ␣-fetoprotein Cholinesterase Serum bile acids Serum cholesterol Triglyceride Lecithin-cholesterol acyltransferase a Commonly
used tests are in bold face.
liver where it is esterified with glucuronic acid. The water soluble mono- and di-glucuronides are efficiently excreted in bile, little entering the blood in healthy people. They are hydrolyzed by ileal and colonic bacteria; the bilirubin is degraded to “urobilinogen” (a mixture of isomers), most of which is excreted in the feces. Some urobilinogen is absorbed, removed from portal blood by the liver, and excreted in bile; a small amount escapes hepatic uptake and is lost in the urine. Bilirubin esters react directly with Ehrlich’s diazo reagent to give a violet color; unesterified bilirubin requires ethanol or another accelerator for color development. Total bilirubin is measured by the depth of color produced when diazo reagent and an accelerator are added to serum; “direct” bilirubin is the result in the absence of accelerator. “Indirect” bilirubin is the difference between total and “direct” measurements. “Direct” and “indirect” bilirubin are inaccurate measures of esterified and unesterified bilirubin, as some of the latter reacts “directly.” “Direct” bilirubin therefore overestimates esters at relatively low bilirubin levels, but still allows identification of the “unconjugated hyperbilirubinemia” of Gilbert’s syndrome and hemolysis. “Direct” bilirubin is of little value at high bilirubin levels when it tends to underestimate esters.
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The “normal” range for total serum bilirubin is usually taken as 3–17 umol/l. The mean level is 3–4 umol/l higher in men than in women; as Gilbert’s syndrome, a completely benign condition, is often suspected when the serum bilirubin is just above 17 umol/l this explains, in part, why it appears more prevalent in men. The upper limit for “direct reacting” bilirubin is usually taken as about 3 umol/l (about 20% of the total); levels above 5 umol/l, with a normal total bilirubin, suggest a conjugated rather than an unconjugated hyperbilirubinemia, but only if measured in a good laboratory (on a good day!). Accurate methods for measuring bilirubin and its conjugates (e.g. alkaline methanolysis and high performance liquid chromatography), rarely used routinely, show that conjugated bilirubin normally accounts for only 4–5% (<1 umol/l) of total bilirubin. They are the most sensitive marker of liver disease, which causes an increase in bilirubin esters; they also allow discrimination between the “unconjugated hyperbilirubinemia” of Gilbert’s syndrome and that of hemolysis, as the proportion of esters (as a percent of total bilirubin) is normal with hemolysis but low (<1.7%) in Gilbert’s syndrome, a disorder of conjugation. Total bilirubin increases with fasting or a low fat intake; this is an important factor when interpreting bilirubin results. Bilirubin is affected by light; serum or plasma samples should therefore be kept in the dark, preferably in a refrigerator, before measurements are made. An elevated serum total bilirubin reflects increased production, reduced hepatic uptake and/or conjugation, impaired transport of bilirubin esters into bile (with parenchymal liver diseases), or their regurgitation into the blood from biliary canaliculi (with biliary obstruction). Bilirubin itself is insoluble in water and is not excreted in urine. Bilirubin esters are excreted in urine when their plasma level increases; the urine becomes brown in color. Bilirubinuria establishes the presence of a liver disorder. Other compounds cause dark urine. Bilirubinuria should be confirmed using test strips impregnated with a diazo reagent; these detect as little as 1–2 umol of bilirubin per liter; they are underused. Bilirubinuria usually precedes jaundice and may be found when the total serum bilirubin is normal or only slightly elevated. When frank jaundice is present bilirubinuria simply confirms an increase in plasma bilirubin esters. When it is absent in a jaundiced patient, two possibilities are suggested. A simple unconjugated hyperbilirubinemia (due to hemolysis, Gilbert’s syndrome or the rare Crigler-Najjar syndromes) is likely if other liver tests are normal (see Table 2). However, conjugated pigment (detectable as “direct” hyperbilirubinemia) may become covalently bound to serum albumin and other proteins, forming bili-alb or bili-proteins, thus escaping urinary excretion. This fraction may constitute a major fraction of serum total bilirubin, particularly during recovery from jaundice. In
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Table 2. Causes of an Isolated Increase in Serum Unconjugated (“Indirect”) Bilirubin. Hereditary disturbances of bilirubin conjugation Gilbert’s syndrome Crigler-Najjar syndromes (types 1 and 2) Hemolytic disorders Congenital Hereditary spherocytosis, elliptocytosis, G-6-PD deficiency and other enzyme disorders, and sickle cell disease. Acquired Erythroctye fragmentation syndromes, drugs and toxins, and autoimmune hemolytic anemias.
the late stage of an acute hepatitis bilirubin may be absent from the urine even at serum bilirubin levels as high as 170 umol/liter; at the onset bilirubinuria may be found before jaundice appears. When other liver function tests are abnormal serum bilirubin levels above 17 umol/l usually indicate liver disease of some kind and bilirubin esters are elevated. The actual level of bilirubin is rarely of diagnostic value. In acute liver diseases, the serum level is also of little prognostic value; complete recovery usually occurs, even after deep jaundice, with resolution of conditions such as acute viral hepatitis or biliary obstruction. With chronic liver diseases, however, a gradual and pronounced increase in serum bilirubin without obvious cause (such as blood transfusion, which increases the heme load, or the administration of certain drugs) is an ominous prognostic sign. In primary biliary cirrhosis a level of 100 umol/l has been used to trigger consideration of liver transplantation. In bile duct obstruction, even if complete, serum bilirubin tends to plateau between 170 and 500 umol/l; the major pathway for removal of bilirubin in this situation is urinary excretion, but bilirubin also breaks down to unidentified compounds. Extreme hyperbilirubinuria (up to 1500 umol/l or greater) only occurs in severe parenchymal liver disease associated with renal failure and/or with hemolysis (due to sickle cell disease, G-6-PD deficiency or blood transfusion).
URINARY UROBILINOGEN (McIntyre & Rosalki, 1999) Freshly voided urine containing urobilinogen gives a purple reaction with Ehrlich’s aldehyde reagent. A dipstick containing this reagent allows rough quantification.
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Urinary excretion of urobilinogen is affected by urine pH; its tubular reabsorption increases with increasing urinary acidity which also renders it less stable. Its estimation in acid urine is thus an unreliable index of plasma levels. The peak urinary output of urobilinogen tends to occur between 12 noon and 1600 hours, probably due to the urinary “alkaline tide.” With liver damage hepatic uptake and biliary excretion of urobilinogen falls, and more is excreted in urine. With severe biliary obstruction less bilirubin enters the intestine, less urobilinogen is made and absorbed, and urinary levels fall. However, changes in urinary urobilinogen occur which are unrelated to changes in hepatic function. Intestinal production of urobilinogen increases with constipation or bacterial contamination of the small bowel, or with over-production of bilirubin due to hemolysis. Urobilinogen production and urinary excretion falls with diarrheal states and treatment with antibiotics. Although much emphasis is placed on urobilinogen metabolism in undergraduate texts, the detection of urinary urobilinogen is of little clinical value.
ASPARTATE AND ALANINE AMINOTRANSFERASES (McIntyre & Rosalki, 1999; Moss & Henderson, 1994) Over forty years ago, marked elevations of serum aspartate aminotransferase (AST, glutamic-oxaloacetic transaminase, SGOT) and alanine aminotransferase (ALT, glutamic-pyruvate transaminase, SGPT) were found in viral hepatitis and other hepatic disorders; they were considered an index of liver cell injury. Aspartate aminotransferase catalyzes the reaction: aspartate + alpha-ketoglutarate = oxaloacetate + glutamate Alanine aminotransferase catalyzes the reaction: alanine + alpha-ketoglutarate = pyruvate + glutamate The co-enzyme for both is pyridoxal phosphate, which binds to a lysine residue in the enzyme, forms a transient Schiff base with the relevant aminoacid, receives the amino group and transfers it to the oxoacid. Both enzymes leak from damaged cells, due to increased membrane permeability or cell necrosis. Measurement of these enzymes in serum is done mainly to identify or confirm the presence of liver disorder. Large amounts of AST are found in liver, but also in cardiac and skeletal muscle, kidney, pancreas and red cells, and serum levels may also rise with damage to these tissues. Because ALT was originally found in low concentration in tissues other than liver, a high serum ALT
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was considered relatively specific for hepatic damage but there may be a marked increase in some skeletal muscle disorders. When hepatocytes are damaged there is usually an increased serum level of both AST and ALT. As this occurs in many liver diseases the finding is of limited value for differential diagnosis. We cannot identify the organ source of serum AST but two isoenzymes, one cytoplasmic, the other mitochondrial, can be measured individually. Release of mitochondrial AST from hepatocytes probably implies more severe cellular damage than release of the cytoplasmic isoenzyme or of ALT, which is also confined to the cytoplasm. The ratio of mitochondrial AST to cytoplasmic or total AST has been proposed as a diagnostic test, as it increases with severe hepatocellular necrosis and in alcoholic liver disease, but few, if any, routine laboratories measure mitochondrial isoenzyme activity. Plasma or serum should be separated soon after blood collection to avoid release of erythrocyte AST. Hemolysis causes a small increase in serum AST levels, and gross hemolysis may give misleading results in the measurement of AST. Aminotransferase levels may be spuriously low in uremia, possibly as a result of vitamin B6 deficiency, but this does not appear to cause the low serum levels often seen in patients on long-term hemodialysis. The main value of aminotransferase measurements is to detect hepatocellular damage, and to monitor the patient’s progress; return to normal suggests resolution of the factors causing hepatocellular damage. Liver disease does not always result in aminotransferase elevation; levels may be normal in patients with established but well compensated cirrhosis, with chronic hepatitis C, and in patients with chronic and incomplete biliary obstruction. Furthermore, relatively small increases are sometimes encountered even in severe hepatitis (although the initial increase may then have been missed). Some laboratories measure both enzymes in their battery of liver tests, others only one; there has been debate over their relative value. Those advocating the use of ALT alone do so because they consider it (mistakenly) to be specific for hepatic damage. Those using AST alone do so because it can also be used to detect damage to cardiac and skeletal muscle, and because it is rarely difficult on clinical grounds to decide whether a high AST is due to liver disease. We now realize that both enzymes need to be measured in hepatological practice. With fatty liver or hepatitis C ALT may be high when AST is in the normal range; in alcoholic hepatitis and cirrhosis, AST may rise without an increase in ALT (probably due to a reduced hepatic content of ALT). These conditions may be therefore missed if only one aminotransferase is measured. An obvious increase in the AST:ALT ratio (from <1 to >1) is also a useful indicator of the development of cirrhosis in patients with chronic viral hepatitis (B and C) or primary biliary cirrhosis, but there is overlap in individual cases.
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Table 3. Causes of a Marked Elevation of Serum Aminotransferases. Acute hepatitis Viral and drug induced Autoimmune hepatitis Shock liver Hypotension, acute heart failure Acute biliary obstruction Gallstone obstruction, acute cholangitis
Because they increase in so many conditions even quite marked elevations of aminotransferase levels (up to 500 u/l) are of limited value in differential diagnosis. They have been used to differentiate “hepatocellular” jaundice (when they tend to be high) from “obstructive” jaundice (when they tend to be low). But there are many exceptions to this “rule,” and the classification of jaundice into hepatocellular and obstructive causes on this basis may be misleading. Very high AST and ALT levels are of diagnostic value. Levels more than 20x the upper reference limit (about 1000 units/l) suggest acute hepatitis (due to a virus or drug), “shock liver” (due to hypotension or acute heart failure), or acute extrahepatic biliary obstruction, ascending cholangitis or autoimmune hepatitis (Table 3). Blood should be taken at presentation because in most of these conditions AST and ALT levels may fall rapidly, and are then of little diagnostic value. If previous AST/ALT levels are available they help to establish the cause of a very high aminotransferase. In viral hepatitis, and with most drugs causing acute hepatitis, aminotransferase levels rise gradually for a week or two before the onset of jaundice; normal or only modest elevations of AST/ALT at the onset of symptoms virtually exclude acute hepatitis as a cause. With acute hypotension and heart failure, and with acute biliary obstruction, there is usually an abrupt rise and a rapid fall if the underlying problem can be treated effectively. In rare cases an elevation of serum AST, often marked, results from the presence of “macro AST,” a complex of the enzyme with large molecular weight immunoglobulin (usually IgG, rarely an IgM). This finding, analogous to macroamylasemia, may cause diagnostic confusion. In about 75% of cases of uncomplicated acute viral hepatitis (A and E, and many cases of B and B + D hepatitis) aminotransferase levels fall to normal within 8 weeks. Chronic liver disease often results from infections with hepatitis B and C, and there is usually a persistent (but often modest) increase in AST and ALT levels. In acute hepatitis C, initial aminotransferase levels are much lower than with other types of viral hepatitis (usually less than 1000 iu/l) but many patients
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develop chronic hepatitis. With hepatitis A, which has no long term sequelae, aminotransferase levels may take many months to return to normal, and during this time levels may rise again due to exacerbation of the disease. More modest aminotransferase increases, up to about 10 × normal, occur in many liver diseases, e.g. chronic hepatitis, cirrhosis, biliary obstruction, hepatic infiltration and neoplasia. ALT is more frequently increased, except in cirrhosis and infiltrative disease. In developed countries the most common cause of ALT elevation in the general population is fatty liver (associated with obesity, diabetes, hyperlipidemia and alcohol abuse); in endemic areas it is hepatitis C. In both conditions a modest elevation of ALT is usually accompanied by a similar elevation of gammaglutamyltransferase. Minor increases in AST and ALT are occasionally found when there is no evidence of significant liver disease on liver biopsy. This is particularly true for ALT, which rises in many conditions (and with many drugs), and for AST after short periods of binge drinking in healthy subjects. Serum AST activity increases in diseases other than those affecting the liver, e.g. myocardial infarction, myocarditis and pulmonary embolism. AST and ALT both rise with some myopathies (e.g. Duchene dystrophy, active polymyositis and hypothyroidism) and with trauma (even intramuscular injections). Serum creatine kinase (CK) levels help to identify AST and ALT elevations due to muscle disease, but quite marked rises of CK may occur without obvious cause in Afro-Caribbean patients; the normal upper limit for CK in this population is twice that for Caucasians. There may be confusion when diseases of skeletal muscle occur with liver disease. In chronic alcoholics painless chronic myopathy is frequent, and acute myopathy can follow a drinking bout; these conditions may contribute to the serum aminotransferase elevations seen in such patients.
OTHER TESTS Glutathione-S-Transferase (Beckett & Hayes, 1987) Glutathione-S-transferases (GST) are widely distributed detoxification enzymes. The bilirubin binding protein, ligandin, is a GST found in liver; its serum activity is a highly sensitive index of hepatocellular damage but is difficult to measure. It has a short plasma half life, allowing early recognition of cessation of active cellular damage. It rises with acute hepatitis of viral or drug origin, massive increases being found with paracetamol toxicity and fulminant hepatitis, with primary and secondary hepatic malignancies, and in untreated hyperthyroidism (presumably
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due to sub-clinical liver damage). It may be high in chronic hepatitis C when AST and ALT levels are normal, and increases occur with alcoholic liver disease, particularly after binge drinking.
Lactate Dehydrogenase and the LD5 Iso-Enzyme Modest elevations of serum lactate dehydrogenase (LD) activity are found early in acute viral hepatitis; LD activity seems to be greater in hepatitis due to paracetamol and ischemia. In most other liver diseases normal (or near normal) values of LD are usual. With hepatic malignancy LD increases in up to 80% of patients, the frequency depending on the extent of metastatic disease; high levels may be found. Although total serum LD is raised in many conditions, a preferential increase in the LD5 iso-enzyme is found only with liver disease, malignancy and muscle disorders. The plasma concentrations of other substances rise with hepatocellular damage. Some are enzymes, presumably released from damaged hepatocytes. The pattern of response does not necessarily mirror that of aminotransferases; in acute viral hepatitis, lactate dehydrogenase and sorbitol dehydrogenase return to normal much more quickly. Serum glutamate dehydrogenase tends to rise with large duct obstruction; its activity in liver tissue, where it is located centrizonally, may also rise with biliary obstruction. Not surprisingly, many of these enzyme measurements have been advocated as useful liver function tests in their own right, or as contributors to patterns of abnormality that might help in differential diagnosis. They have not been widely employed and so it is difficult to assess their relative merits; they seem to have little advantage over aminotransferase estimations.
ALKALINE PHOSPHATASES (McIntyre & Rosalki, 1999; Moss & Henderson, 1994) Alkaline phosphatases, a family of zinc metallo-enzymes with a serine at the active centre, release inorganic phosphate from organic orthophosphates and are present in nearly all tissues. In liver, alkaline phosphatase is found histochemically in the microvilli of bile canaliculi and on the sinusoidal surface of hepatocytes. Alkaline phosphatases from liver, bone and kidney are transcribed from the same gene; alkaline phosphatases from intestine and placenta are coded by other, different genes.
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Total alkaline phosphatase activity should be measured in fresh, unhemolyzed serum or heparinized plasma. Citrate, oxalate and EDTA complex with the zinc in the phosphatase and inactivate it. The international “reference” method uses p-nitrophenol phosphate as substrate in an alkaline transphosphorylating buffer such as 2-amino-2-methyl-1-propanol. The various isoenzymes in serum can be identified and measured by several methods. For routine analysis electrophoresis, which depends mainly on charge, is the method of choice. This allows identification of placental and intestinal isoenzymes, tumor isoenzymes, a high molecular mass “biliary” (or fast liver) isoenzyme, and one or more unusual hepatic isoenzymes. Liver (“slow” liver band) and bone isoenzymes, found in similar proportions in normal sera, are difficult to distinguish with this method; this is unfortunate as the usual question posed by clinicians is whether an increased alkaline phosphatase level is due to liver or bone isoenzyme. These can be identified and quantified using wheat-germ lectin affinity electrophoresis. Other methods can also be used to distinguish between liver and bone alkaline phosphatases. Urea and guanidine inhibit them differently, but the differences are small, determination errors large, and inhibitors of intestinal alkaline phosphatase must be used to compensate for a possible contribution from this fraction. Assay based on heat stability is tedious and subject to considerable inaccuracy. Monoclonal antibodies can identify bone and liver isoenzymes, with some crossreactions but without interference from alkaline phosphatase from other tissues; convenient clinical assays are not yet available. The reference range (and units) for serum total alkaline phosphatase activity varies with the method used, the age and gender of the patient, and with other factors. Any one isoenzyme may be increased even when total alkaline phosphatase is normal. Alkaline phosphatase elevation occurs in many diseases other than those involving the liver. Bone and liver isoenzymes account for most of the activity in normal serum. Alkaline phosphatase activity may be above the normal adult range until about 20 years of age, with peaks in the neonatal period and in adolescence (periods of rapid bone growth). Liver phosphatase tends to increase in elderly males, bone phosphatase in post-menopausal females. Intestinal phosphatase may contribute up to 20% of total activity in about 20% of normal subjects; it is associated with blood groups A and O, the ABH red cell antigen and absence of the Lewis antigen, and its serum level tends to rise after a fatty meal. In normal pregnancy alkaline phosphatase activity increases during the third month, rises to twice the usual adult female level in late pregnancy, and can remain high for weeks after delivery. The main source is the placenta. Serum bone-specific alkaline phosphatase often remains higher (for up to six
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Table 4. Causes of an Increase in Serum Alkaline Phosphatase Activity. Liver alkaline phosphatase Obstructive liver disease; extra-hepatic and intra-hepatic Minor increase with age Bone alkaline phosphatase Physiological – childhood, puberty, post-menopausal Paget’s disease, osteomalacia, bone metastases Intestinal alkaline phosphatase Cirrhosis Physiological after fat ingestion in secretors of blood groups O and B Placental alkaline phosphatase Pregnancy Indian childhood cirrhosis Tumor alkaline phosphatase Ovarian, testicular, hepatocellular carcinoma Macro alkaline phosphatase – immunoglobulin bound
months) in women who breast-feed than in those who bottle feed their babies (see Table 4).
Alkaline Phosphatase and Liver Disease It was recognized over 60 years ago that serum alkaline phosphatase activity rose with bile duct obstruction, and that lower but still high levels were found in “toxic, infective, and catarrhal” jaundice; the increase was attributed to regurgitation of bile phosphatase. With the introduction of the King-Armstrong method of measurement in 1934, a phosphatase level over 30 KA units became accepted, erroneously, as a diagnostic criterion for an extrahepatic block. Serum alkaline phosphatase activity goes up in many types of liver disease; the highest levels are seen with either intra- or extra-hepatic obstruction to bile flow, and with intrahepatic space occupying lesions such as primary or metastatic liver tumors. The high phosphatase of liver disease is not simply due to retention of the biliary enzyme. The phosphatase accumulating in plasma is made in the liver, and animal studies show increased hepatic synthesis of alkaline phosphatase after biliary ligation. There are at least two hepatic isoenzymes, one from hepatocytes (“slow” liver), and a high molecular mass biliary phosphatase (“fast” liver) from the canalicular membrane. With biliary obstruction the membrane phosphatase, solubilized by retained bile salts or shed as fragments, reaches plasma by
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paracellular regurgitation or transcellular endocytosis. “Biliary” enzyme is a better marker of biliary obstruction than total serum alkaline phosphatase activity, but is rarely sought in clinical practice. A high serum alkaline phosphatase is not always found in “cholestatic” liver disease. It may be normal or only slightly raised in patients with primary biliary cirrhosis or primary sclerosing cholangitis, with a confirmed extrahepatic block due to tumor or stones, or with bacterial cholangitis, particularly in the early stage. This may cause diagnostic confusion. In acute viral hepatitis, serum alkaline phosphatase is usually either normal or only moderately raised, but up to 40% of patients have levels two and a half times the upper reference limit. Hepatitis A may present an obstructive picture with prolonged itching and marked elevation of alkaline phosphatase, and a very high alkaline phosphatase has been reported in Epstein-Barr virus infection, even with normal bilirubin levels. Serum alkaline phosphatase is increased by drugs which cause cholestatic liver disease; an elevation has also been found with cimetidine, furosemide, phenobarbital and phenytoin. In about 25% of patients with cirrhosis, an intestinal band may be the major alkaline phosphatase in serum (normally it does not exceed 20% of total phosphatase activity). This may be due to destruction of receptors for intestinal alkaline phosphatase on the liver cell surface (causing reduced hepatic uptake), or to diminished hepatic excretion or catabolism. When osteomalacia complicates liver disease, the bone isoenzyme may increase in association with increased osteoblastic activity. An increase in serum alkaline phosphatase with the properties of hepatic phosphatase has been found in Hodgkin’s disease, congestive heart failure, and in infectious and inflammatory diseases not primarily involving the liver (e.g. polymyalgia rheumatica); its origin is not clear. Some tumors secrete specific isoenzymes into plasma. The Regan isoenzyme (a heat-stable placental type isoenzyme) is found with bile duct carcinoma, the Kasahara isoenzyme (a fetal-intestinal type phosphatase) in about 30% of patients with hepatocellular carcinoma; in the latter condition another distinct alkaline phosphatase (heat labile) may be found. Unfortunately, such isoenzymes are of little diagnostic value; they are found in few patients with tumors, and generally at low activity so that sensitive immunological methods need to be used. They help in monitoring anti-tumor therapy; successful treatment results in a fall or disappearance of isoenzyme from plasma. Identification of alkaline phosphatase isoenzymes is of limited value in distinguishing between various kinds of liver disease; “biliary” enzyme is more frequently raised in cholestatic and neoplastic disorders, and intestinal
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alkaline phosphatase in parenchymal disease, but there is considerable overlap. However, isoenzyme studies certainly help to decide whether an elevated alkaline phosphatase activity is due to liver disease or bone disease. When alkaline phosphatase isoenzyme results are not available, clinicians tend to use other markers of biliary obstruction (gamma-glutamyl-transferase or 5 -nucleotidase) in order to confirm the hepatic origin of raised phosphatase levels. Gammaglutamyltransferase has 90% sensitivity and specificity for this purpose, as it is normal with bone disease. When a marked alkaline phosphatase elevation is accompanied by a modest gamma-glutamyltransferase elevation, the possibility of concomitant bone and liver disease should be considered. Occasionally serum liver phosphatase increases due to its binding to serum immunoglobulins which interferes with plasma clearance of the enzyme. This has been reported with auto-immune hepatitis and ulcerative colitis. In rare families serum liver phosphatase activity is increased for no obvious reason.
5 -NUCLEOTIDASE AND LEUCINE AMINOPEPTIDASE (McIntyre & Rosalki, 1999; Moss & Henderson, 1994) 5 -nucleotidase is an alkaline phosphatase acting on nucleotides with a phosphate at the 5 position of the pentose. Although present in all tissues only liver disease appears to cause an increase in its serum activity (normal range 1–15 i.u./l). The highest levels occur with obstruction to bile flow (intra- or extra-hepatic, but moderate elevations are found with chronic hepatocellular disorders. Leucine aminopeptidase hydrolyzes peptides in which an l-leucine residue contains the free amino group. Widely distributed in the body, it is present in bile, bile ducts and canaliculi. Blood levels are highest with intra- or extra-hepatic obstruction to bile flow, but also increase in acute hepatitis, cirrhosis, hepatic malignancy, and in the last trimester of pregnancy. These tests are rarely used in routine practice. Both were used to confirm a hepatic cause of an elevated alkaline phosphatase, but have been superseded by measurement of GGT. However, alkaline phosphatase iso-enzyme studies are better than 5 -nucleotidase, leucine aminopeptidase or GGT for establishing liver disease as the cause of an elevated alkaline phosphatase.
GAMMA-GLUTAMYL TRANSFERASE (GGT) (Moss & Henderson, 1994; Nemezansky, 1986) Gamma-glutamyl transferase is a membrane bound glycoprotein which catalyses the transfer of ␥-glutamyl groups from ␥-glutamyl peptides, particularly
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glutathione, to other peptides, to amino acids and to water. Its gene on chromosome 22 codes for a precursor protein of about 61 kD. The enzyme is found mainly in cells with a high rate of secretory or absorptive activity, but also in many other tissues. Large amounts are found in kidneys, pancreas, liver, intestine and prostate. The GGT activity of bile is approximately 100 times greater than that of normal serum. There are several GGT isoenzymes, but without clear evidence of tissue specificity. The heterogeneity appears to be related to the number of sialic acid residues, to the degree of glycosylation, and to binding to lipoproteins. The reference interval for serum GGT is higher in men than in women. Activity is high in neonates and infants up to 1 year, and in subjects above the age of 60. Because about 15% of those screened in some centers have levels over the usually accepted upper limit of normal (50 mU/liter in men, 30 mU/liter in women) a higher upper limit is sometimes used (e.g. 80 in men, and 50 in women). About 4% of men have a level more than 100 mU/liter. Reference levels are lower in lifelong abstainers from alcohol than in the general population. Serum GGT activity rises in most kinds of liver disease, and is high in about 90% of patients with hepatobiliary disease. It is therefore of little value in differentiating between them. It is a sensitive indicator of the presence of liver disease (0.87–0.95) but has limited specificity, because many other conditions, not primarily hepatic, increase serum GGT, possibly as a consequence of mild, clinically insignificant hepatic involvement. The highest levels are found with intrahepatic biliary obstruction or with primary or secondary hepatic malignancies. However, a normal or low GGT is seen occasionally in patients with intrahepatic cholestasis, even with a very high bilirubin and alkaline phosphatase. In infants with idiopathic cholestasis a normal GGT is considered to be of poor prognostic significance. In acute viral hepatitis serum GGT levels reach a peak in the second or third week of the illness but in some patients levels are still up at six weeks. Levels remain high with the development of chronic hepatitis or cirrhosis, and are usually elevated in chronic hepatitis C. GGT levels are of definite, though limited, value in managing alcoholic patients. They may rise, presumable due to enzyme induction, even in the absence of significant liver damage, but there is a poor correlation between alcohol intake and GGT activity. With abstinence GGT falls to normal over 2–5 weeks; if it does not, there may be continued alcohol intake, underlying liver damage, or another reason for the high GGT. Unfortunately, about a third to a half of heavy drinkers show no elevation of GGT in the absence of liver disease, so it is not a sensitive screening test for alcohol abuse. GGT levels do not rise as the result of an alcoholic binge in healthy subjects, but may do so in alcoholics and patients with other liver disorders. An increased GGT level is found in many patients who drink no alcohol or only modest amounts. It is important that such patients are not labeled as
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alcoholics. The most common cause is fatty liver associated with obesity, diabetes or hypertriglyceridemia. About 20% of patients with uncomplicated diabetes mellitus have a high serum GGT activity, but rarely more than three times the upper limit of normal. The enzyme probably comes from the fatty liver often found in diabetics. Activity is also high in acute pancreatitis, and most cases of acute myocardial infarction; as there is no measurable GGT activity in cardiac or skeletal muscle, the rise in the latter may be due to secondary effects on the liver, as high levels are also found in congestive heart failure. Modest increases in serum GGT activity occur with enzyme inducing drugs such as phenobarbitone, phenytoin and other anticonvulsant drugs, paracetamol (aminopyrine), tricyclic antidepressants and glutethimide. Smaller increases occur with anticoagulants, oral contraceptives and antihyperlipidemic drugs. Sometimes very high levels of serum ␥-glutamyl transferase are found (up to 1000 u/l) when there is no detectable cause.
PLASMA PROTEINS The liver makes many circulating plasma proteins. Liver disease affects the plasma concentration of many of them. The effects are complex, depending on changes in protein synthesis, catabolism by various tissues, and the effects of liver disease on the volume and distribution of extra-cellular fluids. There may also be changes in the metabolism of plasma proteins produced outside the liver. Estimating “total plasma protein” is of relatively little value as it may be normal even with marked disturbances of individual components. However, an abnormal result may suggest the need to measure the different fractions. Serum protein electrophoresis gives patterns characteristic of, but not diagnostic for, certain types of liver disease. A low albumin and high gamma globulin are found with non-biliary cirrhosis, a marked rise in gamma globulin with autoimmune hepatitis. With biliary obstruction ␣- and -globulins may increase with accumulation of abnormal lipoproteins; a fall in haptoglobin due to intravascular hemolysis causes a low ␣-2 band. A reduced ␣-1 band suggests ␣1-antitrypsin deficiency. Unfortunately the various electrophoretic abnormalities occur in conditions other than liver disease and are of little diagnostic help in jaundiced patients. Serum or Plasma Albumin (Rothschild, Oratz & Schreiber, 1988) Albumin is the most abundant circulating protein. Its total exchangeable pool is about 3.5–5.0 g/kg body weight, 38–45% being present in plasma in which levels
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are normally between 35 and 50 g/l. Albumin accounts for most of the colloid osmotic pressure of plasma. It has high affinity binding sites for many naturally occurring compounds (including bilirubin) and many drugs. Liver is the only site of synthesis (about 15 g/day in a “normal” 70 kg person). About 1 g is lost daily via the gut; the rest is degraded by an unknown mechanism. Serum albumin is widely used as a test of liver function, because it falls with reduction in hepatic protein synthesis. Unfortunately it also falls with gastrointestinal and renal loss, increased catabolism, increased vascular permeability and over-hydration. Changes in serum albumin concentration should therefore be interpreted with caution. It may take many days before reduced synthesis causes an obvious change in serum albumin because of its long half-life (about 20 days), and because when synthesis falls there is a reduction in the fractional catabolic rate. However, with fever or trauma serum albumin levels tend to drop rapidly, often below 30 g/l, even if there is no liver disease; the albumin half-life may fall to about seven days, suggesting increased albumin removal as well as impaired albumin synthesis. The low serum albumin level often found with severe chronic liver disease is due mainly to reduced synthesis, but also results from expansion of the extra-cellular space, which may contain more albumin than normal despite the low concentration. Although the fractional catabolic rate is low, the absolute rate of degradation and synthesis may be normal or even high. In cirrhotics with ascites, hepatic secretion of albumin is disturbed; some enters the blood stream normally via the sinusoids, but much is released directly into the ascites.
Specific Protein Measurements (McIntyre & Rosalki, 1999) Alpha-1-globulin is composed mainly of ␣-1-antitrypsin and ␣-1 acid glycoprotein (orosomucoid), two acute phase proteins which increase in many inflammatory disorders, orosomucoid being especially responsive in liver disease. Haptoglobin, another acute phase protein, runs as an ␣-2 globulin. Increased clearance of haptoglobin-hemoglobin complexes causes the low haptoglobin level seen with intravascular or severe extravascular hemolysis. In non-biliary cirrhosis the -globulin iron binding protein, transferrin, may be reduced; it is considered a negative acute phase protein because it falls non-specifically in many inflammatory disorders. The immunoglobulins (principally IgG, IgA and IgM) are located within the ␥-globulin fraction. Diffuse (polyclonal) increase of staining in this region is common in chronic liver disease (especially autoimmune hepatitis) and in chronic inflammatory and other auto-immune disorders. IgA runs in the -␥ region; an increase in non-biliary cirrhosis (especially alcoholic) causes beta-gamma “fusion” or “bridging” on electrophoresis.
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Although changes in these proteins may be inferred from electrophoretic appearances, they are better determined by quantitative immunological measurement. Specific proteins whose concentration is of particular interest in liver disease include pre-albumin, ␣-1 anti-trypsin, ␣-fetoprotein, ceruloplasmin, procollagen-III-peptide, and the immunoglobulins.
Pre-Albumin (Transthyretin; Hutchinson et al., 1981) Pre-albumin, a tetramer of four identical subunits, binds iodothyronines; it also binds one molecule of retinol binding protein (RBP) which serves to minimize urinary loss of RBP. The serum pre-albumin level is 0.2–0.3 g/l; that of RBP 0.04–0.05 g/l. Measurement of pre-albumin has been proposed as a liver function test as it often falls in liver disease due to reduced synthesis. Because of its short half-life (1.9 days), changes precede alterations in serum albumin. Reduction in plasma RBP in chronic liver disease may be associated with impaired dark adaptation.
Serum Ceruloplasmin Ceruloplasmin, an intensely blue ␣-2-globulin normally present in plasma at a level of 0.2–0.4 g/l, is synthesized in liver. Its gene is on chromosome 3. It is an oxidase for certain aromatic amines and phenols, for cysteine, ascorbic acid and ferrous ions. Its physiological function is still not entirely clear. However, with hereditary absence of ceruloplasmin there is marked iron overload; this suggests that oxidation of ferrous to ferric ions plays a key role in iron metabolism. It is an acute phase protein, its serum concentration rising in pregnancy, with estrogens, infections, rheumatoid arthritis, some malignancies, active non-Wilson’s liver disease and obstructive jaundice. Serum ceruloplasmin is an important diagnostic marker in Wilson’s disease in which the serum level is usually low. A low ceruloplasmin is also found in neonates, Menkes’ disease, kwashiorkor and marasmus, protein losing enteropathy, nephrotic syndrome, severe hepatic insufficiency, copper deficiency, and in hereditary hypo-ceruloplasminemia and a-ceruloplasminemia.
Procollagen-III-Peptide Procollagen-III-peptide is removed from the N-terminal end of procollagen-III in the production of type III collagen. Its serum concentration increases with hepatic
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fibrosis, but also with inflammation and necrosis. The value of serum procollagenIII-peptide measurements is uncertain, but they may help in monitoring chronic liver disease.
Alpha-1 Antitrypsin Alpha-1-antitrypsin, a glycoprotein of about 54 kD, is synthesized by the liver. It inhibits serine proteinases, especially elastase. Its serum concentration is normally 1–1.6 g/l. An acute phase protein, it increases with inflammatory disorders, pregnancy and with oral contraceptives. Alpha-1-antitrypsin shows genetic polymorphism. Approximately 90% of Caucasian populations are homozygous for the M allele (i.e. MM phenotype); other alleles include F, S, Z and null forms. The phenotype is best determined by iso-electric focusing; allelic variation may be associated with a low plasma concentration and deficient functional (inhibitory) capacity. Plasma levels of alpha-1-antitrypsin are approximately 15% of normal with the ZZ phenotype; 38% with SZ; about 60% with MZ and FZ. The presence of the Z allele, particularly in homozygotes, is associated with defective processing of the protein in the liver. The precursor protein, deficient in sialic acid, is poorly secreted by hepatocytes; its intrahepatic accumulation, which can be demonstrated histochemically, may cause liver damage. Neonatal hepatitis occurs in Pi ZZ homozygotes, less often with MZ and SZ phenotypes. Cirrhosis in adults is found with ZZ, less commonly with MZ, SZ and FZ phenotypes. Hereditary ␣-1-antitrypsin deficiency is often suspected because of a reduced ␣-1 band on electrophoresis; deficiency should be confirmed by quantitative measurement.
Alpha Fetoprotein This protein is measured by radio- or enzyme immunoassay. It is the major protein of fetal plasma in early gestation, but levels are very low subsequently (reference limit 25 ug/l). More than 90% of patients with hepatocellular carcinoma have an increased serum level. This is also found with other liver diseases including chronic hepatitis, and the regeneration phase of viral hepatitis, and in up to 15% of cirrhotics without hepatocellular carcinoma; the increase is generally minor compared with that seen with hepatocellular carcinoma. To improve specificity for hepatocellular carcinoma (but with loss of sensitivity) levels above 400 ug/l have been regarded as a diagnostic prerequisite. At such levels the positive predictive value is 70% or more for hepatocellular carcinoma; less than 5% are false positives, and these are often
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transient elevations. High ␣-fetoprotein levels usually accompany hepatocellular carcinoma in blacks and Chinese. They are less common in white Europeans, and with hepatocellular carcinoma arising in non-cirrhotic liver; ␣-fetoprotein may be undetectable when the tumor is associated with oral contraceptive therapy. Serial determination of ␣-fetoprotein is of particular value in monitoring patients with cirrhosis; a progressive increase one should lead to a rigorous search for hepatocellular carcinoma. With successful therapy there is a fall in ␣-fetoprotein levels.
Cholinesterase (McIntyre & Rosalki, 1999; Moss & Henderson, 1994) Cholinesterase, which hydrolyzes many choline esters, is made in the liver. Serum levels fall with decreased hepatic protein synthesis, the changes paralleling those of serum albumin. Low values due to genetic polymorphism are recognizable by the altered inhibition characteristics of the serum enzyme. In acute hepatitis of infective or toxic origin, plasma cholinesterase falls within days, returning gradually to normal with recovery. Low levels are also found with chronic hepatitis and cirrhosis, and with neoplastic and other infiltrative diseases of the liver. In obstructive jaundice values are normal unless there is concomitant liver disease, but if the obstruction is due to a tumor reduced values may be found. Low enzyme levels, due to impaired enzyme synthesis, can occur with malignant disease, even if it is localized and does not involve the liver. With steatosis levels are normal or somewhat increased. Many drugs appear to cause a reduction in cholinesterase activity. Cholinesterase has been used to assess liver function before and after hepatic transplantation. It is best studied serially and is of greatest value as a prognostic tool. A sudden or marked fall to a quarter of the usual activity is an ominous sign.
SERUM BILE ACIDS (Hoffman, 1989) Bile acids are derived from catabolism of cholesterol. The two main primary bile acids, cholic and chenodeoxycholic acids, are made in the liver, conjugated with glycine or taurine, and excreted in bile where they play a key role in the digestion and absorption of fat and fat-soluble compounds. Bile acids are reabsorbed from the terminal ileum by an efficient active transport mechanism, but some reach the colon where they undergo bacterial deconjugation. Bacteria also dehydroxylate them at the 7-␣-position, and so convert cholic acid to deoxycholic acid, and
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chenodeoxycholic to lithocholic acid. The resulting secondary bile acids are absorbed from the colon. Normal liver removes cholic, chenodeoxycholic and deoxycholic acids very efficiently from portal blood and excretes them rapidly into bile, thus establishing an entero-hepatic circulation (i.e. intestine – portal vein – liver – bile – intestine). Lithocholate is sulphated by the liver and lithocholate sulphate is removed in the feces. Liver disease affects bile salt metabolism in several ways. It may impair primary bile acid synthesis, change the relative proportions of the different bile acids, affect the amount of bile acid which is conjugated, change the taurine:glycine ratio, or lead to the production of unusual bile acids. If less primary bile acid enters the intestine, there will be reduced synthesis of secondary bile acids and a fall in plasma levels of deoxycholate. Impaired liver function, or diversion of portal blood, reduces bile acid removal from portal blood causing an increased level of plasma bile acids, particularly after meals. Plasma bile acids rise with biliary obstruction because they regurgitate from the biliary tree into the blood stream. When plasma bile acid levels are high their urinary excretion increases. For clinical purposes the simplest bile acid test is the total serum bile acid concentration, either fasting or after a meal, but it is rarely available in routine laboratories. The fasting level, normally up to 15 umol/l, is increased in only about two-thirds of patients with a variety of types of liver disease; it is therefore of limited value in screening for liver disease. Levels remain high after eating in almost all patients with significant liver disease; the bile acid level two hours after a meal can thus be used to screen for liver disease. The finding of a high serum bile acid level has high specificity for the detection of liver disease (compared with other individual tests), but its sensitivity is limited. Addition of a bile acid test would not improve the specificity already obtained with the usual batteries of tests.
AMMONIA Arterial ammonia levels tend to be high in chronic liver disease, particularly if there is hepatic encephalopathy or a large amount of portal systemic shunting. They also increase in severe acute hepatitis and fulminant hepatic failure. There is a poor correlation between ammonia levels and the degree of encephalopathy. Measurement of blood ammonia is of little clinical value in liver disease. A high blood ammonia is also seen in hereditary deficiencies of urea cycle enzymes, when the blood ammonia is usually higher than in acquired liver diseases; other liver function tests tend to be normal in these conditions.
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OTHER TESTS WHICH MAY BE USEFUL IN MANAGING LIVER DISEASE Glucose Glucose intolerance (due to insulin resistance) and frank diabetes mellitus (due to an added impairment of insulin secretion) are common in patients with cirrhosis. With fulminant hepatic failure hypoglycemia is common and may persist despite intravenous administration of relatively large amounts of glucose; frequent blood glucose measurements are therefore mandatory in this condition. Hypoglycemia also occurs in acute fatty liver of pregnancy. Cholesterol, Triglyceride, and Lipoproteins (Harry & McIntyre, 1999) Serum total cholesterol level is often elevated in patients with biliary obstruction, due to a rise in free cholesterol, and may reach very high levels in some patients with primary biliary cirrhosis. Lipoprotein electrophoresis usually shows an abnormal pattern, with loss of alpha (HDL) and pre- (VLDL) bands and a prominent beta band. Much of the cholesterol is carried in an abnormal lipoprotein, LP-X, which is rich in free cholesterol and phospholipid (lecithin). In severe parenchymal liver disease, acute or chronic, total cholesterol tends to fall due to a reduction in cholesteryl ester. Electrophoresis reveals a loss of alpha (HDL) and pre-beta (VLDL) bands. In acute hepatitis, there may be a rebound hypercholesterolemia during the recovery phase. The abnormalities underlying the lipoprotein changes of liver disease are complex. Lecithin cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transfer of an acyl group from lecithin to cholesterol with the formation of cholesteryl ester. It is produced in the liver, has a short half life in plasma, and falls in many types of liver disease. Some workers hold that determination of LCAT is the single most sensitive test of hepatocellular dysfunction. The changes in plasma lipids and lipoproteins in liver disease seem to depend mainly on plasma LCAT activity, although regurgitation of biliary lipid contributes to the high cholesterol and phospholipid levels found with biliary obstruction. Serum triglyceride levels increase in various types of liver disease; they tend to show reciprocal changes with total serum cholesterol because when LCAT activity falls insufficient cholesteryl ester is produced to occupy the core of the LDL particles derived from triglyceride rich VLDL.
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QUANTITATIVE TESTS OF LIVER FUNCTION Galactose Elimination Capacity (Tygstrup, 1990) Galactose, a naturally occurring monosaccharide, is removed from plasma only by liver and kidneys. Conversion to galactose-1-phosphate by hepatic galactokinase is the rate-limiting step in its metabolism. At high plasma levels the reaction is saturated and so liver removes galactose at a constant rate (i.e. zero order kinetics). To calculate the hepatic removal rate plasma levels are measured every 5 min for an hour after rapid intravenous injection of a bolus of galactose; urinary galactose is also measured. From 20 to 40 min after injection galactose levels fall linearly with time; with several assumptions, and taking urinary excretion into account, the “galactose elimination capacity” rate of the liver can be calculated. In normal subjects it is about 270+/−40 (S. D.) mg/min per m2 of body surface area, or 6.7+/−1.0 mg/min per kg body mass. Galactose elimination capacity correlates well with other indices of hepatocellular function, like prothrombin time, serum albumin and antipyrine removal rate, but with the advantage of measuring just one aspect of hepatocellular function. There is considerable overlap between normal subjects and patients with a variety of liver diseases. Repeat estimations in the same subject after a short time interval vary by only about 10%. The test is most useful for assessing improvement or deterioration in liver function over a longer period of time.
Aminopyrine Removal and Breath Test (Schoeller et al., 1982; Tygstrup, 1990) Removal and catabolism of drugs are functions of the liver which can be assessed in several ways. After a single injection we can follow plasma levels, or the decay in plasma radioactivity of radio-labeled drug. Production of drug metabolites can be assessed from blood or urine measurements. Several groups of drugs are Ndemethylated; the methyl groups removed are oxidized and their carbon atoms appear in the breath as CO2 . The disappearance rate from plasma of labeled aminopyrine (14 C-dimethyl amino-antipyrine) is easily measured. This compound can also be used in noninvasive breath tests. After oral administration of 14 C-aminopyrine breath CO2 is collected by asking the subject to blow into scintillation vials containing a trap for CO2 ; an indicator changes color when 2 mmol of CO2 has been taken up. From the specific activity of breath CO2 one can calculate the amount of labeled CO2 produced by demethylation, assuming constant endogenous production of CO2 . If the specific activity of CO2 samples is plotted against time the disappearance curve
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over about 12 hours is roughly exponential, and so a decay constant, Kb, can be estimated. This correlates well with the decay constant for plasma disappearance (Kp), with BSP disappearance, and with the galactose elimination capacity. Sampling of breath over many hours is tedious. The test has been modified to last only two hours, either by taking the mean value for CO2 specific activity during the first two hours after ingestion of the drug, or by sampling breath only at two hours; results in patients can be compared with those obtained from normal subjects. Values for patients with hepatocellular dysfunction are generally lower than in normal subjects; normal results may be found with biliary obstruction. The aminopyrine two-hour breath test is not a useful diagnostic test but it allows sequential measurements of one aspect of hepatic function to be made in the same subject over time; the coefficient of variation of duplicate tests is about 6%. There is reluctance to use radioactive material with a long half-life for repeated studies in man, but the radiation dosage of the test is small. 13 C aminopyrine can be used but then mass spectrometry is required for measurement of the isotope.
Hepatic Removal of Bromosulphthalein, Dibromosulphthalein and Indocyanine Green (Schoeller et al., 1982; Tygstrup, 1990) Bromosulphthalein (BSP) was widely used to measure certain aspects of hepatic function because it is easy to measure in blood and bile. It is removed from blood by hepatocytes, bound by intracellular proteins, and excreted in bile either unconjugated (approximately 30%) or conjugated with glutathione. In the USA a simple BSP test was widely used to detect liver disease in non-jaundiced subjects. After an IV bolus of BSP its plasma level was measured on a single blood specimen taken at 45 minutes. Assuming an initial distribution in a plasma volume of 50 ml/kg body mass, and thus a known initial concentration of BSP, retention at 45 minutes is easily calculated. In normal subjects, it is up to 7% of the original dose. Higher values are found with many types of liver disease. This test was not popular in Britain, because it provides limited information and is potentially dangerous. Tissue damage results if BSP extravasates at the site of injection: anaphylaxis has been reported on numerous occasions; and if the BSP is not properly prepared for injection, neurological problems may result from injection of microcrystals. More complex studies of BSP removal have also been used to explore different aspects of the hepatic handling of BSP. After a single IV injection the plasma BSP disappearance curve can be represented by two exponentials. If a two compartment model is assumed (plasma and liver), with loss of BSP only in urine and bile, we can calculate the size of the plasma compartment, hepatic storage, the transfer of
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dye between compartments, urinary excretion, and the rate of biliary excretion – which has a theoretical maximum level, the Tm. Unfortunately, BSP conjugation is neglected and other problems also affect interpretation. Even so, it is a useful method of evaluating changes in a fairly clearly defined function of the liver. Attempts were also made to estimate the Tm and hepatic storage capacity (S) for BSP from plasma levels found during constant infusion of the dye at different rates. Unfortunately, the method was based on false assumptions. It is easily shown that the resulting values for S must be wrong; they depend not only on the liver’s capacity to concentrate BSP but also on blood flow (McIntyre, Mulligan & Carson, 1973). Dibromosulphthalein (DBSP) is handled by the liver like BSP, but has the advantage that it is not conjugated with glutathione; this simplifies the assumptions involved in analysis of plasma disappearance. Indocyanine green (ICG) is also rapidly removed by the liver and excreted into bile without conjugation. It is much safer than BSP (but may be contaminated by small amounts of sodium iodide), is simple to measure, and can be used to study hepatic uptake, storage and transport of an exogenous dye. ICG retention at 20 minutes can be measured and plasma disappearance curves analyzed like BSP disappearance curves (Keiding & Skak, 1988). It is more expensive than BSP, and as it is unstable in plasma levels must be measured soon after withdrawal of the sample. Both BSP and ICG have been used to measure hepatic blood flow by the Fick principle. The plasma level is held constant by continuous infusion of the dye; hepatic blood flow can then be calculated by dividing the rate of infusion of dye by the difference in concentration between arterial and hepatic venous plasma. Unfortunately this measurement calls for hepatic venous catheterization.
Dynamic Studies of Bilirubin Metabolism (Ostrow, 1986) Following injection of radioactive unconjugated bilirubin the label is rapidly removed from plasma; its disappearance curve can be represented as the product of three exponentials. Estimates that can be made from analysis of this curve include the mass of unconjugated bilirubin in the initial pool, its initial volume of distribution, the appearance rate of unlabeled bilirubin, hepatic clearance of bilirubin, and the plasma bilirubin turnover rate. Assuming a three-compartment model one can estimate the size of the compartments and the rate of transfer between them, but the calculations depend on the nature of the assumptions made in constructing the model. Such analyses have helped in the study of disorders of unconjugated bilirubin metabolism, like Gilbert’s syndrome and the Crigler-
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Table 5. Quantitative Tests of Liver Function. Galactose elimination capacity Aminopyrine disappearance and demethylation Bromosulphthalein, Di-bromosulphthalein and indocyanine removal Bilirubin kinetics Bile salt removal and metabolism
Najjar syndrome. The method is expensive and time consuming and does not allow interpretation of findings in conjugated hyperbilirubinemias; it is therefore of little value for studying patients with most types of liver disease.
Dynamic Studies of Bile Acid Metabolism (Hoffman, 1989) Measurements have been made of the fractional disappearance rate of bile acids from plasma after injection of radio-labeled bile acids. They disappear rapidly (within minutes) from plasma; data interpretation is complicated because there may be considerable removal of the label before adequate mixing. Although a reduced removal rate can be demonstrated when there is impaired hepatic function, there are problems in deciding the physiological significance of the changes. The fate of a labeled bile acid can be followed through the various compartments of the total bile acid pool. Mathematical interpretation of the results is extremely complicated because of entero-hepatic re-circulation, conversion of primary to secondary bile acids, and the occurrence of both conjugation and de-conjugation of bile acids. This method is of little value for repeated study of patients with liver disease. Table 5 lists the quantitative tests of liver function.
SUMMARY Biochemical investigations are widely used in the management of patients with disorders of the liver and/or biliary tree. A small number of them are cheap to perform and are therefore used routinely as a small battery of so-called “liver function tests” (LFTs); this usually includes serum bilirubin, aspartate and alanine aminotransferases, alkaline phosphatase, ␥-glutamyl transferase and albumin. LFTs help to detect liver disease and to monitor its course, but are of relatively little use for precise diagnosis. Despite their name, they are also of little value for assessing hepatic function.
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Some biochemical tests are of diagnostic value for certain conditions, but the results may be difficult to interpret and immunological tests, liver biopsy and appropriate imaging techniques remain important tools for the hepatologist. Few methods are available for the assessment of individual functions of the liver. Those that are tend to be relatively complicated to perform and are usually costly, in terms of the materials used, laboratory time and in inconvenience to the patient. They help our understanding of the pathophysiology of liver disease, and are of particular value for assessing the response to new therapies.
REFERENCES Beckett, G. J., & Hayes, J. D. (1987). Plasma glutathione-S-transferase measurements and liver disease in man. Journal of Clinical Biochemistry and Nutrition, 2, 1–24. Harry, D. S., & McIntyre, N. (1999). Plasma lipids and lipoproteins (Chapter 2.12). In: J. Bircher, J.-P. Benhamou, N. McIntyre, M. Rizzetto & J. Rodes (Eds), Oxford Textbook of Clinical Hepatology (2nd ed., pp. 287–302). Oxford: Oxford University Press. Hoffman, A. F. (1989). Enterohepatic circulation of bile acids. In: Handbook of Physiology – The Gastrointestinal System (Vol. III). Baltimore: American Physiological Society. Hutchinson, D. R., Halliwell, R. P., Smith, M. G., & Parke, D. V. (1981). Serum “prealbumin” as an index of liver function in human hepatobiliary disease. Clinica Chimica Acta, 114, 69–74. Keiding, S., & Skak, C. (1988). Methodological limitations of the use of intrinsic hepatic clearance of ICG as a measure of liver cell function. European Journal of Clinical Investigation, 18, 507–511. McIntyre, N., Mulligan, R., & Carson, E. R. (1973). BSP Tm and S: A critical re-evaluation. In: G. Paumgartner & R. Preisig (Eds), The Liver: Quantitative Aspects of Structure and Function (pp. 417–427). Basel: Karger. McIntyre, N., & Rosalki, S. B. (1999). Biochemical investigations in the management of liver disease (Chapter 5.1). In: J. Bircher, J.-P. Benhamou, N. McIntyre, M. Rizzetto & J. Rodes (Eds), Oxford Textbook of Clinical Hepatology (2nd ed., pp. 503–521). Oxford: Oxford University Press. Moss, D. W., & Henderson, A. R. (1994). Enzymes (Chapter 20). In: C. A. Burtis & E. R. Ashwood (Eds), Tietz Textbook of Clinical Chemistry (2nd ed., pp. 735–896). Philadelphia: W. B. Saunders. Nemezansky, E. (1986). Gamma-glutamyltransferase (GGT) (Chapter 9). In: J. A. Lott & P. L. Wolf (Eds), Clinical Enzymology. New York: Field Rich and Assoc, distributed by Year Book Publishers. (NB 1986 NOT 1968!!) Ostrow, J. D. (Ed.) (1986). Bile pigments and jaundice. New York: Marcel Decker. Rothschild, M. A., Oratz, M., & Schreiber, S. S. (1988). Serum albumin. Hepatology, 8, 385–401. Schoeller, D. A., Baker, A. L., Monroe, P. S., Krager, P. S., & Schneider, J. F. (1982). Comparison of different methods of expressing results of the aminopyrine breath test. Hepatology, 2, 455–462. Tygstrup, N. (1990). Assessment of liver function: Principles and practice. Journal of Gastroenterology and Hepatology, 5, 468–482.
12.
ALCOHOLIC LIVER DISEASE
S. F. Stewart and C. P. Day INTRODUCTION Alcohol was recognized to be a cause of liver damage by the ancient Greeks, and it is currently the most common cause of liver disease in the Western World. The magnitude and range of the health and socio-economic problems attributable to alcohol abuse are enormous. Cirrhosis, predominantly alcoholic, is now the fourth commonest cause of death between the ages 25–64 in the USA and alcohol may also make a significant contribution to cardiovascular-related mortality. The overall socio-economic cost of alcohol abuse in the USA, in terms of healthcare, crime and loss of work capacity, has been estimated at over $160,000 million per year. Several independent studies have demonstrated a close correlation between deaths from cirrhosis and per capita alcohol consumption. Perhaps the best example of this is the effect of wine rationing in France during the Second World War which was associated with an 80% reduction in cirrhosis deaths, followed by a return to pre-war levels when restrictions were removed (Lelbach, 1985). The worldwide increase in mortality from cirrhosis observed during the 1950s and 1960s was associated with a similar rise in alcohol consumption, attributed largely to the falling price of alcohol relative to income (Masse et al., 1976). Conversely, the reduction in per capita alcohol intake that has occurred in several countries since the late 1970s (including the USA) has recently been reflected in some reduction of deaths due to cirrhosis. Despite the striking association between alcohol abuse and liver disease, the precise mechanisms of alcohol-induced liver damage are not well understood. This applies across the full spectrum of alcohol-associated liver pathology from simple
The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 317–359 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15012-5
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fatty liver through alcoholic hepatitis and hepatic fibrosis to established cirrhosis. Many of the pathogenic mechanisms proposed have been related to the metabolism of alcohol and the subsequent generation of its major metabolite, acetaldehyde. Considerable advances have, however, been made towards understanding the various biochemical pathways involved in the oxidation of alcohol and how they might be involved in determining the severity of tissue injury following alcohol intake. Evidence has also been provided supporting a role for several other mechanisms unrelated to alcohol metabolism, and at present, it remains unclear which, if any, of these are important. A better understanding of the exact mechanisms underlying alcohol-induced liver injury may eventually lead to unraveling perhaps the greatest mystery in the field of alcoholic liver disease, namely, the enormous between-individual variation in the range of disease produced in response to the ingestion of apparently similar amounts of alcohol. Very little is known of the factors determining why the majority of alcoholic patients remain at the stage of fatty liver while less than 30% progress to alcoholic hepatitis, and only approximately 20% develop cirrhosis. Moreover, up to a third of confirmed alcoholics have completely normal liver histology. Clearly, factors other than cumulative amount of alcohol consumed are important in determining the hepatic response to excessive intake in a particular individual. Factors considered important in this respect include both other exogenous (environmental) influences, such as nutrition and hepatotrophic viruses, and endogenous (genetic) determinants (Day & Bassendine, 1992). Elucidating these factors would have obvious benefits for both prevention and treatment of alcoholic liver disease and clues towards understanding the genetic basis for individual differences in susceptibility to alcoholic liver damage are now emerging. This chapter will concentrate initially on the metabolism of alcohol and the pathogenic mechanisms considered to play a role in producing the range of alcohol-related liver pathology. This will be followed by consideration of environmental and genetic factors that may determine whether a particular individual develops mild or severe liver disease or no disease at all. Finally, the clinicopathological aspects of alcoholic liver disease will be reviewed, including the early recognition of patients with disease, histological classification of disease, clinical presentation, factors determining prognosis and consideration of the various treatment modalities available.
METABOLISM OF ALCOHOL An appreciation of the absorption, distribution, metabolism and elimination of alcohol is critical to understanding the variety of biochemical disturbances that are
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commonly observed in alcoholic patients such as hyperuricemia, hyperlipidemia and hypoglycemia. In addition, alcohol metabolism underlies several of the mechanisms considered to play a role in the pathogenesis of alcohol-related liver damage and some of the more recent explanations for inter-individual susceptibility to alcoholic liver disease. Alcohol metabolism will be discussed in terms of the fate of a unit of alcohol following ingestion. One unit is equivalent to 10 grams or 12.5 mls of absolute alcohol which is present in approximately half a pint (284 ml) of beer, and 1 standard measure of wine (114 ml) or spirits (24 ml). In this chapter “alcohol” refers exclusively to ethanol, the only alcohol present in commercially available alcoholic beverages.
Absorption, Distribution and Excretion The typical time course of blood alcohol concentration following the ingestion of one unit is shown in Fig. 1. The peak level occurs approximately 20 min after ingestion and reaches between 10 and 15 mg/100 ml. The rate of rise and height of peak is a function of alcohol absorption and tissue distribution. In addition, it has recently been suggested that the peak value may be influenced by firstpass metabolism of alcohol by alcohol dehydrogenase activity within the gastric mucosa. However, the biological importance of this effect is controversial (vid´e infra). Alcohol is absorbed from the gastrointestinal tract by simple diffusion (Berggren & Goldberg, 1940). Between 20 and 50% of absorption occurs in the stomach and the remainder in the duodenum and upper jejunum. The rate
Fig. 1. The Typical Time Course of Blood Alcohol Concentration Following the Ingestion of One Unit Either Following a Meal or on an Empty Stomach.
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of absorption is delayed following a meal (Fig. 1) and increases in proportion to the alcohol concentration of the drink consumed. Since absorption is more rapid from the intestine than the stomach, any pathological condition, drug or surgical intervention that delays or increases gastric emptying will also affect alcohol absorption accordingly. Following absorption, the tissue distribution of alcohol is determined principally by blood flow and water content. Thus, in organs with a rich vasculature such as brain, lungs and liver, alcohol levels rapidly equilibrate with the blood. Alcohol is poorly soluble in lipids and therefore tissues with a high fat/water ratio attain much lower levels than organs such as kidney, where the high water content results in urinary alcohol levels 1.3 times higher than those in blood. The low lipid solubility of alcohol also explains why, following the ingestion of the same amount of alcohol per unit weight, an obese person attains a higher level of blood alcohol than a thin person. Furthermore, the higher fat content of female body composition compared to male has been invoked as part of the explanation for their higher alcohol levels following the ingestion of similar amounts of alcohol per unit weight (Marshall et al., 1983). Over 90% of circulating alcohol is oxidatively metabolized, primarily in the liver, and excreted as carbon dioxide and water. The remainder is eliminated unchanged in the urine (<1%) and breath (1–5%).
Metabolism of Alcohol In view of the negligible renal and pulmonary excretion, the rate of alcohol elimination is largely determined by the body’s capacity for alcohol oxidation. The rate of alcohol metabolism does not vary widely in the population and above a concentration of 10 mg/100 ml occurs at a constant rate of approximately 100 mg/kg body weight per hour; so-called “zero-order” kinetics. A 70 kg man, therefore, eliminates one unit of alcohol in about 90 minutes. An important implication of this type of kinetics is the absence of a rapid feedback mechanism to increase the rate of alcohol oxidation in response to its concentration. Heavy, repeated alcohol consumption can, however, increase the rate of elimination by up to 100%.
Site of Alcohol Oxidation Alcohol metabolism is performed almost entirely by the liver, which contains several different high affinity (low Km ) enzyme systems capable of oxidizing alcohol. Other organs, including kidney, intestine and bone marrow, also possess alcohol oxidizing capacity, but because of the low affinity of the alcohol
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dehydrogenase (ADH) activities present in these tissues, they make an insignificant contribution to overall alcohol oxidation at the concentrations attained following normal “social” drinking. The possible exception is the ADH activity present in the gastric mucosa, where the very high gastric levels of alcohol following ingestion may render the affinity of the enzyme(s) present less critical and the resulting alcohol oxidation significant. This effect has been claimed to contribute to a significant first-pass metabolism of alcohol determining both its bioavailability and its toxic effects (Caballeria et al., 1987). This gastric “barrier” may be lower in females and further contribute to their increased susceptibility to alcohol (Frezza et al., 1990), and commonly used anti-ulcer drugs, such as H2 -receptor antagonists, may also influence the activity of gastric ADH (Caballeria et al., 1989). However, many of the assumptions on which this gastric first-pass effect are based have been questioned (Levitt, 1993) and further studies are awaited.
Oxidation of Alcohol to Acetaldehyde Alcohol oxidation in the liver takes place via three steps (Fig. 2). First, alcohol is oxidized, principally within the cytosol, to acetaldehyde. Then acetaldehyde is further oxidized to acetate, primarily within the mitochondria, and finally, acetate is released into the blood and oxidized to carbon dioxide and water in peripheral tissues. At least three enzyme systems with the capacity to oxidize alcohol to acetaldehyde are present within the liver, although in normal individuals only the alcohol dehydrogenase enzymes are important.
The Alcohol Dehydrogenase (ADH) Pathway ADH catalyzes the oxidation of alcohol to acetaldehyde, transferring hydrogen to the cofactor, nicotinamide adenine dinucleotide (NAD), which is converted to its reduced form, NADH. The resulting increase in the ratio of NADH/NAD, which is further increased by acetaldehyde oxidation, is responsible for the majority of the metabolic imbalances that occur following alcohol ingestion and plays a major role in the initial pathogenesis of alcohol-induced fatty liver (Section C-l). Human ADH exhibits multiple isoenzymes that have been divided into five major classes on the basis of their electrophoretic mobility, kinetic properties and inhibition by pyrazole (Vallee & Bazzone, 1983). They are encoded by at least seven different gene loci, ADHI to ADH7, encoding the ␣-, -, ␥-, -, -, - and -subunits respectively. The class I isoenzymes are formed by random
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Fig. 2. The Three Pathways of Alcohol Oxidation.
association of the ␣-, - and ␥-subunits to form the active homo- or heterodimeric isoenzymes, whereas the others are all homodimers. The ADHI and ADH3 genes are polymorphic, encoding three different  and two different ␥ subunits with different kinetic properties resulting in isoenzymes with different rates of alcohol oxidation in vitro (Bosron & Li, 1986). Expression of the ADH genes is tissue specific. The liver contains the highest levels of class I activity, while class III activity is present equally in all tissues. In humans, the class II isoenzyme, ADH has been found only in the liver while the class IV enzyme, -ADH is present only in the stomach (Moreno & Pares, 1991). The class I isoenzymes have by far the lowest Km and highest Vmax for alcohol and accordingly are thought to be responsible for the major part of hepatic alcohol oxidation. The overall Km of liver ADH activity is in the order of 1 mmol (4 mg/100 ml), which explains why alcohol follows zero-order kinetics at anything above very low blood levels. Experiments performed both in vivo and in vitro suggest that the principal regulatory mechanism for the ADH pathway is the capacity of the mitochondria to reoxidize NADH back to NAD (Lester & Keokosky, 1967).
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The Microsomal Ethanol-Oxidizing System (MEOS) Pathway In addition to ADH, alcohol is metabolized by the MEOS; an accessory pathway that principally involves a specific alcohol-inducible form of cytochrome P450 designated CYP2E1 (Lieber, 1988). The enzyme is located on the endoplasmic reticulum, is present in greater amounts in perivenular than periportal hepatocytes and requires oxygen and NADPH (see Fig. 2). The CYP2E1 protein has been purified and the rat gene cloned, sequenced and localized to chromosome 7 (Umeno et al., 1988). The overall contribution of MEOS to alcohol oxidation in vivo is unclear, particularly as its Km for alcohol is in the order of 50–80 mg/100 ml. However, it may play an important role at high blood alcohol levels or following chronic alcohol abuse, in view of its inducibility. Alcohol induction of CYP2E1, associated with microsomal enzyme systems in general, has also been implicated in the tolerance to various drugs commonly observed in alcoholics and may explain their increased susceptibility to hepatotoxicity by other drugs and xenobiotics which are converted to toxic metabolites by microsomal enzyme systems. An important example of this phenomenon is the increased susceptibility of the alcoholic to the toxic effects of acetaminophen, whereby severe liver damage has been reported in alcoholics taking large, but previously considered safe, doses (Seeff et al., 1986). Enhanced microsomal enzyme activity may also lead to an increased rate of testosterone breakdown, contributing to low blood levels of hormone already decreased due to inhibition of testosterone production by the direct toxic effects of alcohol on the testes (Gordon et al., 1976).
The Catalase Pathway The third pathway for alcohol oxidation is catalyzed by the enzyme, catalase. This enzyme is located in the peroxisomes of most tissues and requires the presence of hydrogen peroxide (Fig. 2). The reaction is limited by the availability of hydrogen peroxide which in normal circumstances is low and suggests that the catalase pathway accounts for less than 2% of overall in vivo alcohol oxidation (Boveris et al., 1972).
Oxidation of Acetaldehyde to Acetate Over 90% of the acetaldehyde formed from alcohol oxidation is further oxidized in the liver to acetate by aldehyde dehydrogenase (ALDH). ALDH, like ADH, uses NAD as a cofactor and further increases the NADH/NAD ratio. Human ALDHs
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are encoded at four independent loci on four different chromosomes (Smith, 1986). ALDH2 on chromosome 12 encodes the major mitochondrial enzyme, which has a low Km for acetaldehyde, and is responsible for the majority of acetaldehyde oxidation. The ALDH2 gene exists in at least two allelic forms, ALDH2∗1 and ALDH2∗2 (Goedde & Agarwal, 1987). Isoenzymes present in individuals homozygous for the ALDH2∗2 allele have little or no catalytic activity, while those present in heterozygotes have measurable although reduced activity compared to the isoenzymes present in ALDH2∗l homozygotes (Crabb et al., 1989). Interestingly, the inactive form of ALDH2 is present in about 50% of Orientals but has not been found in Caucasian populations (Bosron & Li, 1987). The implications of this polymorphism, and those of the ADH2 and ADH3 genes, for the risk of alcoholism and alcohol-related disease will be discussed later. The cytosolic form of ALDH, ALDH1 has a higher Km for acetaldehyde than ALDH2 and may play a role following the ingestion of large doses of alcohol. ALDH inhibitors such as disulfuram (Antabuse) have been used in the treatment of alcoholism to sensitize alcoholics to the unpleasant effects of alcohol intake secondary to high levels of acetaldehyde.
Alterations in Alcohol Metabolism Following Chronic Consumption Many studies have shown that chronic alcohol consumption increases the rate of alcohol elimination except in the presence of severe liver damage. This increase is due both to alcohol induction of the MEOS and to adaptive changes in the ADH pathway. The basis for the increased activity of the ADH pathway is probably increased mitochondrial reoxidation of NADH to NAD, which, as discussed already, is the important rate-limiting step. It has been suggested that the increased mitochondrial NADH-reoxidation rate is secondary to alcohol-induced stimulation of Na+ -K+ -ATPase activity, leading to enhanced ATP and oxygen consumption (Bernstein et al., 1975). This so-called “hypermetabolic state” of the liver has also been implicated in the pathogenesis of alcohol-related liver injury. Alcohol elimination is decreased in jaundiced patients with alcoholic cirrhosis and animals with non alcohol-related liver disease (Lieberman, 1963); this probably reflects decreased ADH activity (Figueroa & Klatz, 1962). An important consequence of the increased rate of alcohol oxidation in alcoholics is that, following alcohol ingestion, levels of acetaldehyde in both blood and tissues are higher than those seen after similar ingestion in non-alcoholic controls (Korsten et al., 1975; Lindros et al., 1980). This increase is potentiated by a reduction in the capacity of the mitochondria to oxidize acetaldehyde, at least in alcoholfed rats, and a reduction in total hepatic ALDH activity, observed in chronic
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alcoholic patients with and without liver disease (Nuutinen et al., 1983). The increased hepatic levels of acetaldehyde in alcoholics compared to controls may have important implications for disease pathogenesis, since acetaldehyde is widely considered to be one of the main candidates for the initiation of alcohol-related liver injury.
PATHOGENESIS OF ALCOHOLIC LIVER DISEASE Although several mechanisms have been proposed to explain alcohol-induced liver injury, it is still unclear which are the most important in humans, since most of the information has, of necessity, come from animal studies. Our understanding of the pathogenic mechanisms varies with the stage of disease. Thus, the mechanisms underlying alcoholic fatty liver are reasonably well understood, but are less clear for alcoholic hepatitis and cirrhosis. This may partly reflect the difficulty in developing reliable animal models for the latter conditions compared with fatty liver, although this problem seems to have been partly resolved by the development of a rat model employing the continuous intragastric infusion of alcohol (French et al., 1986). This section will review separately the mechanisms considered to play a role in the pathogenesis of fatty liver, hepatocyte necrosis and liver fibrosis.
Alcoholic Fatty Liver Fatty change, characterized by the accumulation of triglyceride in hepatocytes, is the initial and most common finding in patients chronically abusing alcohol. Its pathogenesis is multifactorial (Fig. 3) and controversy exists as to which mechanism is the most important. Furthermore, the mechanisms change with the stage of disease. In the early stages of alcohol-induced damage, most of the mechanisms considered to be important are secondary to the shift in the hepatic redox state due to the increase in NADH/NAD ratio accompanying the oxidation of alcohol by alcohol and acetaldehyde dehydrogenases. Following chronic alcohol consumption, this redox change attenuates (Salaspuro et al., 1981), and the persistence of fatty liver probably reflects the chronic toxicity of alcohol on the mitochondrial system. Three main mechanisms have been proposed to play a role in the development of alcoholic fatty liver: (i) increased substrate supply for fatty acid esterification; (ii) direct stimulation of the esterification pathway; and (iii) decreased export from the liver of triglyceride as very-low-density lipoproteins (VLDL).
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Fig. 3. The Multiple Mechanisms through which Alcohol Can Lead to Fatty Liver.
Increased Substrate Supply The two substrates for triglyceride synthesis are glycerol-3-phosphate and nonesterified fatty acids. Hepatic levels of glycerol-3-phosphate are increased by the alcohol-induced redox shift, which leads to the conversion of dihydroxyacetonephosphate, generated via the glycolytic pathway, to glycerol-3-phosphate. Several different mechanisms have been suggested to produce increases in hepatic levels of free fatty acids.
Increased Uptake from the Plasma Uptake of fatty acids by the liver occurs in a concentration-dependent manner. Increases in plasma fatty acid concentrations occur following the acute ingestion of very large amounts of alcohol due to catecholamine-mediated mobilization of adipose tissue fat (Brodie et al., 1961). During chronic alcohol intake, however, hepatic fatty acids are mainly dietary in origin (Lieber et al., 1966) and it is interesting that in animal models of alcoholic fatty liver, the administration of
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a high fat diet increases its severity (French et al., 1986). There is also some evidence that the hepatic uptake of fatty acids is increased secondary to increased hepatic blood flow in chronically alcohol-fed animals.
Inhibition of Fatty Acid Oxidation Inhibition of fatty acid oxidation by alcohol has been convincingly demonstrated both in vivo and in vitro (Blomstrand et al., 1973; Ontko, 1973). This was initially thought to be due to the redox shift, which directly suppresses both the -oxidation spiral and the citric acid cycle that utilizes the acetyl CoA product of -oxidation. The inhibition of fatty acid oxidation at the level of the citric acid cycle is compounded by acetate derived from alcohol oxidation, which competes with fatty acid-derived acetyl CoA for entry to the cycle. More recently, however, it has been shown that a defect in fatty acid oxidation can occur independently of any redox change and after a period of abstinence (Leung & Peters, 1986). Reversal of this defect by the addition of malate suggested that it might be attributed to a deficiency of citric acid cycle intermediates occurring secondary to the relative carbohydrate deficiency known to be associated with chronic alcohol abuse (Morgan, 1982). The abnormalities in mitochondrial morphology and overall function that occur following alcohol intake may also contribute to a non-redox-related defect in fatty acid oxidation (Arai et al., 1984).
Increased Endogenous Synthesis of Fatty Acids This was initially considered to be the principal mechanism responsible for alcoholic fatty liver, but studies in both experimental animals and humans have shown that this is not the case, for fatty acid synthesis was actually reduced due to feedback inhibition by triglyceride accumulation (Venkatesan et al., 1987).
Direct Stimulation of Esterification In view of the variety of mechanisms leading to increased substrate supply, it is not surprising that increased fatty acid esterification has been observed in liver biopsies from humans with alcoholic fatty liver. However, recent studies have provided evidence that, in addition to any effect through increased substrate supply, there is direct stimulation of the enzymes of the esterification pathway following alcohol intake. In this respect, the enzyme phosphatidate phosphohydrolase (PAP), which
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catalyses the rate-limiting step in esterification, has received the most attention. Evidence suggesting that increased PAP activity is important in the pathogenesis of alcoholic fatty liver was initially provided by animal studies in rats and baboons, and a study in humans has shown a close correlation between the severity of fatty liver and activity of PAP suggesting that this enzyme may indeed play an important role in the pathogenesis of alcoholic fatty liver (Day et al., 1993a).
Decreased Export of Triglyceride as VLDL Triglyceride is normally exported from the liver packaged with phospholipids as VLDL particles, a process that, in healthy livers, increases in proportion to triglyceride synthesis to prevent triglyceride accumulation. In the early stages of alcoholic liver damage the increase in triglyceride synthesis is matched by an increased production of VLDL resulting in the hypertriglyceridemia commonly observed in such patients. However, as the disease progresses, alcohol impairs the VLDL packaging process by damaging the hepatocyte microtubular system, resulting in a lowering of plasma VLDL levels and contributing to the pathogenesis of triglyceride accumulation (Venkatesan et al., 1988).
Alcohol-Induced Hepatocyte Necrosis The mechanisms by which alcohol consumption leads to irreversible hepatocyte necrosis are less well understood than those leading to fatty liver. Over the past three decades, at least eight hypotheses have been proposed with no clear consensus emerging as to which, if any of these is important. This debate is of more than academic interest, since determining pathogenic mechanisms is necessary for the rational design of new treatment modalities for patients with alcoholic liver disease. The following review is largely restricted to those mechanisms that have led to recent therapeutic developments.
Induction of Hypermetabolic State The “hypermetabolic” hypothesis, which is based largely on the redox shifts occurring secondary to alcohol oxidation, has been derived mainly from studies on rats. It is postulated that catecholamine-driven increased activity of Na+ -K+ ATPase leads to increased oxygen and ATP consumption, and an adaptive increase in the reoxidation of NADH to NAD. The induction of this state requires thyroxine
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and can be abolished by thyroidectomy. The increased oxygen requirement leads to hypoxic damage in the perivenous zones of the liver where the oxygen tension is lowest (Israel et al., 1975). This model of alcohol toxicity forms the rationale for clinical trials with the antithyroid drug, propylthiouracil (Orrego et al., 1987a).
Free Radicals and Oxidative Damage Free radicals are highly reactive and unique chemical species consisting of atoms or molecules in a particular form characterized by an unpaired electron in the outer orbital. They are potentially very reactive and can damage a wide range of cellular components; including membrane phospholipids via lipid peroxidation, proteins and nucleic acids (Cross et al., 1987). Aerobic organisms defend themselves against free radicals by possessing both protective enzymes, such as superoxide dismutase, and free radical scavengers including the tripeptide, glutathione (GSH). This type of free radical-generated oxidative stress is probably the basis for the hepatotoxicity of acetaminophen which is metabolized into a highly reactive electrophilic metabolite by the cytochrome CYP2E1. The therapeutic benefit of N-acetylcysteine in acetaminophen overdose is due, in part, to repletion of GSH stores. Recently both free radical generation and lipid peroxidation have been observed following chronic alcohol intake in animals and humans, although their precise role in the hepatotoxicity of alcohol remains controversial (Cederbaum, 1989). Alcohol induces the formation of free radicals firstly by increasing the concentration of reduced NAD and NADP in the cell, and secondly, by inducing the microsomal enzymes, NADPH oxidase and NADPH-cytochrome P450 reductase. The first enzyme catalyzes the production of the free radicals, superoxide and hydrogen peroxide from NADPH, and the second enzyme catalyzes their further conversion to the highly reactive hydroxyl radical, a reaction requiring the presence of free iron. The release of iron from ferritin stores is favored by the increased NADH/NAD ratio (Shaw, 1989). Hydroxyl radicals can remove electrons from the polyunsaturated fatty acids that are present in membrane phospholipid side-chains, generating lipid radicals, reactive aldehydes and initiating lipid peroxidation. The lipid radicals ultimately decompose cleaving the fatty acid chain. Alcohol further promotes lipid peroxidation by depleting liver GSH stores which are already lower in perivenular compared to periportal cells, thus contributing further to the predilection of alcohol-related damage for the perivenous zones (Altomare et al., 1988; Lieber et al., 1966). Despite the uncertainty surrounding the importance of oxidative stress in the pathogenesis of alcoholic liver disease, this mechanism
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has already had therapeutic implications. Administration of the phospholipid lecithin to reverse the membrane damage due to lipid peroxidation, prevented the development of hepatic fibrosis in alcohol-fed baboons (Lieber et al., 1990b). Although this result was not repeated in a single human study. Repletion of GSH stores by feeding its precursor, 5-adenosylmethionone (SAMe) to alcoholic baboons attenuated the liver injury (Lieber et al., 1990a). SAMes also increased GSH levels in human cirrhotics (Vendemiale et al., 1989) and show promise in some patient groups (Mato et al., 1999).
Acetaldehyde Toxicity Acetaldehyde, the product of all pathways of alcohol oxidation, is widely considered to be a factor in the hepatic injury associated with alcohol abuse. As discussed, following alcohol consumption, acetaldehyde levels are higher in alcoholics than controls due both to increased alcohol oxidation and decreased acetaldehyde oxidation. Acetaldehyde is highly reactive and can chemically modify membrane phospholipids, proteins and glutathione and can also damage the microtubular system. Its three main potential hepatotoxic mechanisms can be summarized as follows:
Formation of Protein-Acetaldehyde Adducts Acetaldehyde binds covalently to the lysine residues of proteins to form stable protein-acetaldehyde adducts (protein-AAs) (Jennett et al., 1990). These adducts have been detected in vivo in the liver of alcohol-fed rats (Lin et al., 1988), and in patients with alcoholic liver disease (Niemela et al., 1991). Two different mechanisms have been proposed implicating the formation of protein-AAs in alcohol-induced damage. First, acetaldehyde-modified proteins may have altered biological function, including receptor function and enzymic activity. Secondly, protein-AAs may be immunogenic (Israel et al., 1988). Certainly, circulating antibodies against protein-AAs have been detected in mice fed alcohol and in alcoholic patients. More recently acetaldehyde has been implicated in the generation of more complex adducts incorporating one of the aldehyde endproducts of lipid peroxidation, malondialdehyde (Tuma et al., 1996). These so called MAA adducts are also immunogenic, but are more likely than acetaldehyde adducts to have a significant role under normal pathophysiological conditions. However, the role of this immune response in the pathogenesis of alcohol-induced liver damage is still, at this time, speculative.
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Inhibition of Microtubular Function Acetaldehyde-induced damage to the microtubules of the cellular cytoskeleton might impair protein secretion and contribute to cell swelling (vid´e infra). Abnormal transport of protein through the Golgi apparatus may also alter the properties and function of proteins destined for the plasma membrane, leading to abnormalities of receptor function and plasma membrane enzyme activities important in cell-signaling (Smith et al., 1989).
Oxidative Stress There is evidence that acetaldehyde can increase the formation of free radicals, possibly via the enzyme xanthine oxidase, generating the reactive superoxide anion. In addition, acetaldehyde itself can react with other free radicals to form an acetaldehyde free radical which could participate in lipid peroxidation and other free radical-mediated damage (Puntarulo & Cederbaum, 1989). Independent of any contribution to free radical formation, acetaldehyde promotes lipid peroxidation by forming adducts with GSH, thus removing one of the cell’s principal free radical scavengers.
Immunologically Mediated Mechanisms Several factors suggest a role for autoimmunity in alcoholic liver disease; however, considerable confusion exists over its precise role in pathogenesis, with many believing that the various immunological abnormalities observed are simply “epiphenomena” of hepatocyte damage. Consideration of the evidence for immunologically-mediated damage in alcoholic liver disease is, however, important, since part of the rationale for the successful treatment of alcoholic hepatitis patients with corticosteroids is suppression of the immune response. Moreover, immuno-genetic factors have been postulated to explain individual susceptibility to alcohol-induced liver disease. Several antigens have been shown to be targets for immune responses in alcoholic liver disease. These include adducts formed between host proteins and acetaldehyde, and the products of lipid peroxidation (Mottaran et al., 2002; Zetterman & Sorrell, 1981). While antibody responses to a variety of antigens are found in heavy drinkers with liver disease the role of humoral immunity in disease pathogenesis is still unclear. As a result, attention has also been focused on the possible role of cellmediated immunity. Many abnormalities of cell-mediated immunity have been
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observed in patients with alcoholic liver disease including: impaired delayed hypersensitivity in vivo, skin-test responsiveness to alcohol in vivo, decreased numbers of circulating T-cells, increased intrahepatic CD4+ve and CD8+ve T cells, lymphocyte transformation to autologous liver homogenates in patients with alcoholic hepatitis, and cell-mediated cytotoxicity for hepatocytes in vitro. A role for cell-mediated immunity in the pathogenesis of alcohol-related liver damage was further suggested by two studies showing immune events (increased lymphocyte-mediated cytotoxicity in vitro) preceding the development of alcoholic hepatitis in both humans and baboons (Lue et al., 1981). It is, however, still unclear whether these cell-mediated immunological events have a significant role in disease pathogenesis or are simply an epiphenomenon reflecting damaged hepatocytes.
Endotoxin-Mediated Cytokine Release Cytokines are low molecular weight polypeptides with distinct sequences, structures and receptors. They are produced by many nucleated cells in the body including peripheral blood monocytes, Kupffer cells, endothelial cells and hepatocytes. They are produced and released in response to a variety of stimuli including infection, trauma, inflammation and endotoxin (the lipopolysaccharide component of Gram-negative bacterial cell walls). Cytokines considered important in alcoholic liver disease include the proinflammatory cytokines, Interleukin-1 (IL1), lnterleukin-8 (IL-8), Tumour Necrosis Factor (TNF), and lnterleukin-6 (IL-6). Cytokines have a diverse range of biological activities. Strikingly, many of their metabolic effects mimic the metabolic complications of alcoholic hepatitis. In addition, TNF can cause hepatocyte necrosis (Schilling et al., 1992) and IL-8 is a major chemoattractant for neutrophils (Mukaida & Matsushima, 1992), infiltration of which is a classical histological feature of alcoholic hepatitis. In view of their properties, it is perhaps not surprising that cytokines have been postulated to play a role in the pathogenesis of alcohol-related injury (Shedlofsky & McClain, 1991). Evidence in support of this hypothesis has been provided by several studies showing increased bioactivity of IL-1, IL-6 and TNF in the sera, peripheral blood monocytes and livers of patients with alcoholic hepatitis (Hill et al., 1992; McClain et al., 1986). The correlation between levels of TNF and prognosis in alcoholic hepatitis patients (Khoruts et al., 1991) further supports its role as a potential mediator of liver cell injury. While it seems likely that cytokines play a role in both the perpetuation of alcohol-related liver damage and many of the clinical manifestations of alcoholic hepatitis, it is not clear whether they are released as part of the general inflammatory response to hepatocyte injury initiated by other mechanisms or whether cytokine-mediated damage is
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a primary event in alcoholic hepatitis. In this respect, the role of endotoxin as the initial stimulus to cytokine release in alcoholics has received considerable attention. It is well established that endotoxin causes liver injury (Nolan, 1989) and alcoholics frequently have systemic endotoxemia, possibly due to increased intestinal permeability to gut-derived toxins (Bjarnason et al., 1984). Moreover, levels of endotoxin have been correlated with other prognostic indicators in alcoholic liver disease (Bigatello et al., 1987). Chronic endotoxin exposure may therefore stimulate cytokine release in alcoholics and initiate cytokine-mediated hepatocyte necrosis. Furthermore, animal studies have demonstrated that chronic alcohol intake somehow sensitizes cells to secrete more TNF in response to endotoxin than controls (Hansen et al., 1994). Cytokine-mediated liver damage has therapeutic implications. The beneficial effects of corticosteroids in patients with alcoholic hepatitis may be due, in part, to their suppression of cytokine release, and therapies directly aimed at preventing cytokine and endotoxin-mediated hepatotoxicity are currently being developed.
“Other” Potential Mechanisms of Hepatocyte Necrosis Several other mechanisms have been suggested to play a role in irreversible hepatocyte damage in alcoholics but have yet to have any therapeutic implications. Hepatocyte swelling secondary to accumulation of fat and protein has been suggested to lead to hepatocyte damage by impairing intracellular and membrane trafficking, interfering with cell-surface signaling and decreasing hepatocyte oxygen supply due to sinusoidal compression. Adaptive changes in the fluidity of cell surface and organelle membranes have been reported in animal models (Dawidowicz, 1985) and, together with membrane damage secondary to lipid peroxidation, are thought to contribute to abnormalities of mitochondrial function, and may also interfere with receptor function including receptor-mediated endocytosis (Casey et al., 1987).
Alcoholic Fibrosis Fibrosis is a prominent part of alcoholic liver disease and may be seen very early in the natural history. It is a bad prognostic feature, indicating that those patients with fatty liver are likely to go on to serious forms of liver disease if they continue to drink (Nakano et al., 1982). A number of mechanisms of alcohol-related hepatic fibrosis have been elucidated but several controversial issues remain unresolved. The most obvious mechanism for alcohol-related fibrosis is that it represents the
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healing response to the necrosis and inflammation occurring in alcoholic hepatitis and yet, even with continued alcohol intake, progression to cirrhosis is seen in only 35% of patients with histologically documented alcoholic hepatitis (Pares et al., 1986). Furthermore, it is now recognized that alcoholic fibrosis/cirrhosis is not always preceded by hepatitis, and can progress directly from simple fatty liver. This suggests a stimulus to fibrosis other than a response to inflammation/necrosis and candidates for this role have been identified. Mechanisms of hepatic fibrosis have been extensively reviewed (Friedman, 1990; Maher, 1990) and only details pertinent to alcohol-induced fibrosis will be discussed here. In normal liver, the synthesis of collagen is a function of parenchymal cells (hepatocytes) and nonparenchymal cells, including sinusoidal endothelial cells and perisinusoidal stellate cells (lipocytes, fat-storing cells, Ito cells), whereas in fibrotic liver, stellate cells play the predominant role. Stellate cells are located in the perisinusoidal space and their normal function appears to be storage of Vitamin A in the form of retinol esters forming characteristic macrovesicular fat droplets. Following stimulation, they undergo proliferation and morphological transformation to transitional cells and eventually to myofibroblasts which are characterized by loss of vitamin A and enhanced production and secretion of collagen. Proliferation and transformation of stellate cells has been observed in the alcoholic fibrosis model in rats and in the livers of humans with alcoholic fibrosis (French et al., 1988; Horn et al., 1986). The factors initiating the stellate cell activation in alcoholics can be considered under two headings.
Factors Released as Part of the Inflammatory Response to Hepatocyte Injury Stellate cells are regulated by cytokines and growth factors released by Kupffer cells, peripheral blood monocytes, platelets and neutrophils as part of the generalized inflammatory response to hepatocyte injury. Factors considered important in stimulation of stellate cell proliferation include platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and TNF␣. Transforming growth factor  (TGF) released from Kupffer cells inhibits proliferation but markedly enhances collagen synthesis by cultured stellate cells (Matsuoka et al., 1989). TGF has been localized immunohistochemically to sites of fibrosis in human hepatic fibrosis, including patients with an alcohol-related etiology (Nagy et al., 1991), and may further contribute to collagen deposition by increasing the synthesis of Tissue Inhibitor of Metalloproteinases (TIMP) which inhibits the collagenases responsible for collagen breakdown (Edwards et al., 1987).
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Factors Unrelated to the Inflammatory Response Factors stimulating stellate cell activation have been identified which are unrelated to the inflammatory response. They may play a role in the initiation of fibrosis in livers that do not pass through the stage of alcoholic hepatitis and progress directly from fatty liver to cirrhosis. Candidates for this role include acetaldehyde which has been shown to increase collagen synthesis by cultured fibroblasts at the level of gene transcription (Brenner & Chojkier, 1987), lactate, and more recently, products of lipid peroxidation. Early manifestations of stellate cell activation include collagen deposition in the space of Disse, reduction of the fenestrations of sinusoidal endothelial cells and formation of basement membranes adjacent to the endothelial cells (Mak & Lieber, 1984). This so-called capillarization of the sinusoids may isolate the hepatocyte from its blood supply and contribute to hypoxic damage, while an increased resistance to blood flow may contribute to portal hypertension. A further benefit of corticosteroid therapy in alcoholic hepatitis may be a reduction in collagen synthesis at the level of gene transcription. This has so far been demonstrated in hepatocytes but not yet in Ito cells (Guzelian et al., 1984).
WHO GETS ALCOHOLIC LIVER DISEASE? Considering the many potential mechanisms postulated to play a role in the pathogenesis of alcoholic liver disease, it might be expected that everyone who consumes alcohol excessively will develop severe liver damage. This is not the case. Even for fatty liver the reported incidence in alcoholic populations varies from 10 to 91%, while the incidence of more severe lesions is much less (Derr et al., 1990) (see Fig. 4). Importantly, normal light microscopy appearances are found in between 5 and 29% of confirmed alcoholics. Factors considered important in determining this highly individual response to alcohol intake include; the total cumulative dose of alcohol consumed, environmental factors, gender and genetic determinants.
The Dose Response Relationship Between Alcohol Intake and Liver Injury There is some evidence to support a dose-response relationship between the total amount of alcohol consumed and the risk of developing cirrhosis (Lelbach, 1975).
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Fig. 4. The Frequency of Each Histological Lesion in Unselected Heavy Drinkers.
For males the relative risk of cirrhosis has been estimated at six times greater at 4–6 units of alcohol/day than at two units/day, and 14 times greater at 6–8 units/day. On the basis of large epidemiological studies such as this, “safe” limits of alcohol consumption, below which liver disease is unlikely, have been recommended. In the UK these limits are currently 21 units/week for men and 15 units/week for women. However, within drinking categories there is considerable variability in the presence and severity of liver damage. In Lelbach’s study, individuals drinking 21 units/day for 22 years had only a 50% incidence of cirrhosis and recent prospective studies have been unable to confirm any linear relationship between alcohol intake and the development of cirrhosis (Marbet et al., 1987; Sorensen et al., 1984). Even for steatosis, the severity has been found to be unrelated to amount, duration or type of alcohol consumed (Leevy, 1962). Clearly, therefore, factors other than the cumulative amount of alcohol consumed are involved in determining which patients develop liver disease.
Environmental Factors The two environmental factors worthy of consideration as potential determinants of the development of alcohol-related liver disease are hepatitis viruses and nutritional status.
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Hepatitis Viruses Markers of past or current hepatitis B virus (HBV) infection have been found more commonly in patients with alcoholic liver disease than the general population in some studies but not others. In view of these discrepant results, it seems unlikely that coexisting HBV infection plays a significant role in determining the severity of alcohol-related liver damage, although in some cases it may contribute to the development of hepatocellular carcinoma (HCC) (Brechot et al., 1982). The discovery of the hepatitis C virus (HCV) and the development of diagnostic tests to measure anti-HCV antibodies have led to several studies examining the role of this virus in alcoholic liver disease. These have shown a correlation between the presence of anti-HCV and the severity of liver disease in alcoholics, and suggest that HCV and alcohol may act synergistically to produce liver damage (Mendenhall et al., 1991; Pares et al., 1990; Wands & Blum, 1991).
Diet and Nutritional Status The specific contribution of malnutrition to the pathogenesis of alcoholic liver disease has been extensively reviewed and remains highly controversial (Derr et al., 1990; Sherlock, 1984). It has been suggested that all of the liver injury in alcoholic animals and humans is secondary to malnutrition rather than a direct consequence of alcohol and its metabolites. There is no doubt that a proportion of patients with alcoholic liver disease have evidence of malnutrition, both “primary” (deficient intake) and “secondary” (deficient nutrient utilization). In experimental animals malnutrition produces a range of liver damage including both fatty liver and fibrosis. However, malnutrition is not a universal finding in alcoholics (Morgan & Levine, 1988), and fibrosis and cirrhosis have been produced in the baboon model of alcoholic liver disease in the presence of diets purportedly high in nutritional value, although this has been disputed (Ainley et al., 1988). In view of these observations, it seems unlikely that malnutrition per se is a primary cause of alcohol-related liver damage. However, particular nutrient deficiencies may play a contributory role. Secondary vitamin A deficiency, due to increased breakdown by induced microsomal enzymes, may contribute to fibrosis, since preliminary evidence suggests that vitamin A reduces stellate cell collagen synthesis (Shiratori et al., 1987). Inadequate intake of trace elements such as selenium, manganese and zinc, may also contribute to hepatocyte damage, since these elements are essential constituents of several antioxidant protective enzymes, including GSH peroxidase (selenium) and superoxide dismutase (zinc and manganese) (Brunt, 1988).
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Gender There is no doubt that gender differences play a role, with women more susceptible to alcohol-induced liver injury than men. As discussed previously, this is probably due both to the lower volume of distribution, and the lower first-pass gastric metabolism of alcohol in women compared to men. Clearly, however, gender differences do not explain differences in susceptibility to alcohol among members of the same sex.
Genetic Factors The absence of any clear cut environmental influences on the variable hepatic response to alcohol has led diverted attention toward a search for genetic factors that might offer an explanation. Evidence supporting a genetic component to predisposition comes mainly from a large study of 15,924 male twin pairs (Hrubec & Omenn, 1981). The concordance rate for alcoholic cirrhosis was 14.6% in monozygotic twins compared to 5.4% in dizygotic twins. Importantly, the difference in concordance rates could not be accounted for by the different concordance rates for alcoholism alone. This remarkable study has provided the stimulus for a number of investigations examining the role of various “candidate genes” suspected of involvement in predisposition to alcohol-induced liver injury. Two main problems have confronted investigators using this approach. First, there is convincing evidence that predisposition to alcoholism itself has a significant genetic component (Devor et al., 1988) and, therefore, to examine the role of any genetic factors in predisposition to end-organ damage distinct from predisposition to alcoholism, any study should ideally include two groups of alcoholics, well-matched for alcohol intake, with and without end-organ damage. In the case of alcoholic liver disease, this is logistically difficult. Secondly, since it is likely that “predisposition” involves several genes, rather than variation at a single locus, investigators must therefore also cope with problems inherent in investigating a “polygenic disease.” This requires examining “candidate genes” in very large populations of alcoholics with and without liver disease ideally originating from the same “genetic pool.” Considering these potential problems, it is not surprising that studies examining “candidate genes” are rather sparse and the strength of any conclusions limited.
Genes Encoding Enzymes Involved in Alcohol Metabolism Since many of the pathogenic mechanisms considered important in the pathogenesis of alcoholic liver disease are related to the metabolism of alcohol,
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the recent identification of polymorphisms at the gene loci encoding alcohol dehydrogenase and aldehyde deydrogenase has led to speculation concerning their potential role in predisposition to alcoholic liver disease. The inactive form of ALDH2, present in about 50% of Orientals is associated with high acetaldehyde concentrations, an exaggerated flush response to alcohol, and appears to protect against alcoholism (Harada et al., 1982; Mizoi et al., 1983). It is now evident that possession of the most active forms of ADH2 and ADH3 (ADH2∗2 and ADH3∗1) is also protective against alcoholism presumably due to faster rates of acetaldehyde accumulation and lower alcohol tolerance (Thomasson et al., 1991). Perhaps not surprisingly, in view of its negative association with alcoholism, patients with alcoholic liver disease have a much lower incidence of the deficient ALDH2∗2 allele compared to controls, with no ALDH2∗2 homozygotes and only occasional heterozygotes in the disease populations studied (Enomoto et al., 1991; Shibuya & Yoshida, 1988). However, in the study of Enomoto et al. (1991), habitual drinkers who were heterozygotes for the normal and mutant ALDH2 genes developed alcoholic liver disease at lower levels of alcohol intake than alcoholics who were homozygotes for the normal gene. This is consistent with the finding of an increased frequency of the more active ADH3 allele, ADH3∗1, in patients with fibrotic alcoholic liver disease compared to locally matched healthy controls (Day et al., 1991). Together, these studies suggest that although individuals with the less active form of ALDH and more active forms of ADH are less likely to become alcoholic, they have a greater risk of developing alcoholic liver disease if they do drink than individuals with the normal form of ALDH and less active forms of ADH. These results strongly implicate acetaldehyde as one of the important pathogenetic factors in the development of alcoholic liver disease. The role of genes encoding other alcohol metabolizing enzymes, including the CYP2E1 gene, in genetic predisposition to alcoholic liver damage has yet to be explored.
Genes Involved in Fibrogenesis Since the final common pathway in the pathogenesis of cirrhosis must be either excess production or deficient degradation of collagen by the liver, genes involved in these mechanisms are a second obvious group of potential candidates for consideration as the basis for predisposition to alcoholic fibrosis. Type I collagen is the predominant collagen in cirrhotic livers, and polymorphisms have been observed at both of the loci encoding its two different constituent polypeptide chains; COL1A1 on chromosome 17, encoding the ␣1(1) chain and COL1A2 on chromosome 7, and encoding the ␣2(1) chain (Sykes et al., 1986). However, contrary to the findings of an initial small study, an investigation with a large number of patients and controls, drawn from an identical “genetic pool,” showed
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no particular haplotype of either type I collagen gene to be associated with cirrhosis (Bashir et al., 1992).
Genes Encoding Histocompatibility Antigens In view of the evidence to support an immunological component in the pathogenesis of alcoholic liver disease, numerous groups have investigated the role of the highly polymorphic HLA antigen system (which partly determines immune responsiveness) in susceptibility to alcohol-related liver damage. Several studies in diverse populations, have reported isolated and conflicting associations of the class I HLA-A and B antigens and the class II DR antigens with alcoholic liver disease, while other studies have failed to show any relationship between HLA antigens and susceptibility. Many of these reports need to be interpreted with caution in the light of several factors that can account for misleading results. Only one investigation took into account the patient’s alcohol history; interestingly, this showed a shorter duration of intake in cirrhotic patients with HLA-B8/DR3 compared to those without this haplotype (Saunders et al., 1982). Many of the studies screened large numbers of HLA antigens in large population samples, which can lead to the occurrence of significant associations by chance. If the results are corrected for the number of alleles screened, the significance of the reported associations disappears (Gilligan & Cloninger, 1987).
Other “Candidate Genes” Considering the numerous pathogenic mechanisms currently thought to play a role in the hepatotoxicity of alcohol, the number and range of potential candidate genes explaining susceptibility to alcoholic liver disease is considerable. Each of these mechanisms may involve proteins encoded by polymorphic genes which would therefore be potential “candidate genes” explaining individual susceptibility to alcoholic liver injury. Further genetic studies must await more precise information on the biochemical pathways underlying these different mechanisms.
CLINICAL AND PATHOLOGICAL MANIFESTATIONS OF ALCOHOLIC LIVER DISEASE Chronic alcohol abuse produces a wide range of morphological changes in the liver, the most frequent being fatty liver, alcoholic hepatitis and cirrhosis (Baptista, 1981;
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MacSween & Burt, 1986). For ease of presentation the three principal lesions will be discussed separately in terms of their pathology, clinical features, prognosis and treatment, but it is important to appreciate that alcohol-related liver damage is a spectrum, with the various lesions occurring more commonly in combination than in isolation. Significantly, the clinical manifestations of each of these histological lesions are extremely variable; ranging from completely asymptomatic forms to a first presentation with severe hepatic failure. Patients with none or minimal symptoms are, however, more likely to have the earlier, more reversible, forms of liver disease and therefore the early recognition of these patients is critical to allow intervention at a stage when it is likely to be of most benefit.
Early Recogniton and Diagnosis of Alcohol-Related Liver Disease “Mr. Smith is a 45 year old man, currently unemployed but previously working in the catering trade. He has been a frequent patient to his family doctor over the past 10 years with complaints including anorexia, morning nausea, diarrhea, anxiety, depression, insomnia and lethargy. He lost his job in a hotel due to frequent absenteeism, particularly on Mondays, and has recently experienced marital difficulties. Clinical examination has always been largely unremarkable other than his increasingly unkempt appearance and recently diagnosed hypertension. He now presents with vague right upper quadrant tenderness and on examination has hepatomegaly. On direct questioning he admits to long-standing excessive alcohol intake and blood tests reveal an abnormal liver biochemical profile. Following referral to the local hospital, liver biopsy reveals the presence of established cirrhosis with superimposed alcoholic hepatitis.” This hypothetical patient demonstrates many of the pitfalls in the early recognition of a patient with alcohol-related liver disease. Patients most commonly present with symptoms unrelated to the liver, typically non-specific digestive symptoms or vague psychiatric complaints. The patient may seek advice concerning the social effects of alcohol abuse on family life or work performance. Often, physical examination will be normal, other than occasional plethora, suffused conjunctivae, tremulousness and aggressive behavior. Up to 30% of patients with alcoholic liver disease have no symptoms related to excessive alcohol intake and may “present” following the chance finding of hepatomegaly or abnormal blood tests at routine medical examination. The key to the early recognition of patients with alcohol-related disease is a high index of suspicion. Once the diagnosis is suspected it is usually easy to confirm by direct questioning for alcohol history and alcohol-related symptoms, careful clinical examination and supportive laboratory investigations.
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History Features in the history important for both the confirmation of alcohol abuse and to aid in its subsequent management, include the amount and duration of alcohol intake, the pattern of intake, precipitating factors of drinking bouts and evidence of physical dependence such as early morning tremor, blackouts and morning drinking. Confirmation of the history should be sought from a family member or close associate. Specific liver-related symptoms, such as jaundice and hematemesis should be sought but are uncommon even in patients with established disease. In addition, since all alcoholics with liver disease do not necessarily have disease of alcoholic etiology (Levin et al., 1979), enquiries should be made concerning other risk factors for liver disease, including a history of foreign travel, blood transfusions or intravenous drug use.
Clinical Examination Important features to note on examination are the signs of chronic liver disease such as hepatomegaly and signs indicative of alcohol-related pathology in other organs such as hypertension, atrial fibrillation and a cushinoid appearance. It is important to understand that many of the classical signs of chronic liver disease, including, spider nevi, Dupuytren’s contracture, palmar erythema and parotid swelling, can occur in alcoholics in the absence of cirrhosis. Clinical signs and history cannot be relied upon to distinguish the various histological sub-types of alcoholic liver disease, since patients with cirrhosis can be asymptomatic while patients with hepatocellular failure may have only severe fatty change (Morgan et al., 1978).
Laboratory Investigations Biochemical and hematological tests can confirm the presence of alcohol abuse and indicate the presence of liver damage, but are not useful in determining the severity of the histological lesion. Blood alcohol estimations are an oftenunderused method of confirming a suspicion of excess drinking, with levels greater than 100 mg/100 ml at a morning clinic, or levels greater than 150 mg/100 ml without obvious intoxication, strongly suggestive of alcohol abuse. Elevation of ␥-glutamyl transferase (␥GT) has been reported in up to 90% of patients abusing alcohol (Wu et al., 1976). The rise is mainly due to hepatic microsomal induction, and is independent of the presence of liver disease, although hepatocellular
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necrosis and cholestasis may be contributing factors. It is not specific for alcohol abuse and is raised in other forms of liver injury, and in patients taking other enzyme-inducing drugs (Rosalki et al., 1971). Its main clinical use is probably in monitoring a period of supposed abstinence, since it falls within a week of cessation of drinking. Other biochemical markers of alcohol abuse rather than liver disease include, elevated levels of serum uric acid (Drum et al., 1981) hypertriglyceridemia, and desialyated transferrin (Storey et al., 1987). The classical hematological marker of alcohol abuse is a raised mean corpuscular volume (MCV), which has been reported to occur in between 80 and 100% of alcoholics with and without liver disease (Buffet et al., 1975) and may be more common in alcoholic women. It is due to a direct toxic effect of alcohol on the marrow, although nutritional folate and B12 deficiencies may contribute in some patients. In regard to biochemical markers of alcohol-related liver damage, a rise in serum aspartate transaminase activity (AST) of up to five times normal is common in patients abusing alcohol. It reflects the presence, but not the severity, of liver damage (Skude & Wadstein, 1977). However, unlike non-alcoholic liver disease, alanine transaminase (ALT) activity is raised less often than AST, and the AST/ALT ratio has been suggested as a means of distinguishing liver disease of alcoholic and non-alcoholic etiology (Clermont & Chalmers, 1967). Biochemical markers of the stage of liver disease have so far proved elusive. Possible exceptions include plasma IgA, which is twice normal in less than 30% of alcoholics with early disease and greater than three times normal in 60% of patients with cirrhosis (Bailey et al., 1976), and more recently, the procollagen peptides. Levels of procollagen III in particular, have been shown to distinguish advanced from early alcoholic liver disease (Bell et al., 1989).
Liver Biopsy Liver biopsy is a mandatory investigation in all patients chronically abusing alcohol who have hepatomegaly and/or abnormal liver blood tests. Firstly, it is used to establish the diagnosis of alcohol-related liver disease. This is important since it has been shown that up to 20% of liver disease in alcoholics with abnormal liver function is due to an alternative etiology (Levin et al., 1979). Secondly, it is the only way of accurately staging the disease, which cannot be achieved by any combination of clinical, or laboratory data (Bruguera et al., 1977). Without knowledge of the histological severity, no prognostic information can be given to the patient and no rational treatment plan can be devised.
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Fig. 5. Alcoholic Fatty Liver Showing Fat with No Hepatocyte Injury and Normal Liver Architecture.
Fatty Liver Pathology Fatty liver is the earliest lesion seen in alcoholic liver disease (Fig. 5). The classical appearance is of a single large fat droplet displacing the nucleus occurring predominantly in perivenular hepatocytes (macrovesicular steatosis). Very rarely, the steatosis is panacinar and may be associated with severe cholestasis, cholangiolitis, and clinical presentation with hepatic failure (Morgan et al., 1978). Inflammation is rare in simple fatty liver, although occasional lipogranulomas can be seen as a response to the extrusion of cellular lipid. Mild fibrosis may occur in response to lipogranulomas and is usually considered reversible. However, the presence of marked perivenular fibrosis in an otherwise uncomplicated fatty liver may be a marker of high risk of progression to cirrhosis (Van Waes & Lieber, 1977). Microvesicular steatosis, in the form of finely dispersed lipid droplets may also occur in some patients (alcoholic foamy degeneration) and is associated with bilirubinostasis, and focal liver necrosis (Uchida et al., 1983). This lesion resolves with abstention. Alcoholic fatty liver is histologically
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indistinguishable from other non-alcoholic fatty liver (NAFLD) associated with the metabolic syndrome (hyperlipidemia, hypertension, type II diabetes and obesity). Clinical Features Patients with fatty liver are usually asymptomatic or present with non-specific digestive symptoms. Rarely fatty liver may be associated with hyperlipidemia, hemolytic anemia and jaundice (Zieve’s syndrome) or hepatic failure. Smooth nontender hepatomegaly is usually the only clinical finding, although signs of portal hypertension may be observed if perivenular fibrosis (central hyaline sclerosis) is present. All, or none, of the laboratory investigations discussed may be abnormal, most commonly, the ␥GT, AST and MCV are mildly raised. Prognosis and Treatment It is widely considered that fatty liver is an entirely benign lesion reversible with abstention from alcohol. Certainly, in the majority of patients with fatty liver who stop drinking the laboratory abnormalities quickly return to normal (Devenyi et al., 1970), and the histological abnormality rapidly regresses (Leevy, 1962). Accordingly, no treatment options have been evaluated in patients with fatty liver other than abstention and a well-balanced diet. However, there are reports that alcoholic fatty liver per se is not always benign, with occasional mortality due to hepatic failure, fat emboli and hypoglycemia. Furthermore, fatty liver may be a precursor of alcoholic cirrhosis. In a study by Sorenson et al. (1984), it was found that the extent of fatty liver on initial liver biopsy was a better predictor of subsequent progression to cirrhosis 10 years later than alcohol history. This suggests that fatty liver may be causative in the development of cirrhosis rather than simply an epiphenomenon of alcohol abuse.
Alcoholic Hepatitis Pathology Alcoholic hepatitis consists of a constellation of histological abnormalities (see Fig. 6), and the features obligatory for diagnosis are the following (Baptista, 1981): (i) Liver cell damage typified by ballooning degeneration progressing to necrosis ± Mallory bodies. Ballooning degeneration is characterized by hepatocyte swelling, a pale granular cytoplasm and a small hyperchromatic nucleus. Mallory bodies are intracytoplasmic inclusions staining purplish-red with hematoxylin and eosin and consisting of aggregates of intermediate filament proteins, reflecting impaired function of the microtubular system. (ii) Inflammatory
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Fig. 6. Alcoholic Hepatitis Showing the Typical Features of Hepatocyte Ballooning, Leukocyte Infiltration and Mallory’s Hyaline.
cell infiltrate, predominantly neutrophils. These are typically arranged round necrotic hepatocytes that contain Mallory bodies (satellitosis). (iii) Pericellular fibrosis producing a chicken-wire appearance. In addition, there is often fibrous thickening around the hepatic vein radicals and eventual obliteration of the veins, a process referred to as central hyaline sclerosis. (iv) Perivenular distribution – unless cirrhosis is present, when lesions occur at the periphery of nodules. As the severity increases, the damage extends to involve the whole lobule. Other features, which are often present but are not obligatory for diagnosis, include fatty change, bridging necrosis, bile-duct proliferation, cholestasis and giant mitochondria. Histological features considered to indicate a high risk of progression to cirrhosis are: the extent and degree of fibrosis (central hyaline sclerosis is the worst sign), a panlobular distribution, and widespread Mallory body formation. In contrast, megamitochondria may indicate a favorable prognosis. It is important that the pattern of lesions described can also occur in other conditions including diabetes mellitus, obesity, jejunal-ileal bypass, total parenteral nutrition and following treatment with various drugs, when it is referred to as non-alcoholic steatohepatitis (NASH).
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Clinical Features There is no good correlation between the severity of the histological lesion and the clinical presentation, which can range from asymptomatic to life-threatening hepatic decompensation (Hislop et al., 1983). However, patients with the milder histology are more likely to present with non-specific symptoms, incidental hepatomegaly or raised transaminases, while patients with severe histology usually present with symptoms specifically related to hepatocellular failure such as jaundice, ascites and encephalopathy, or variceal bleeding. The episode of decompensation leading to clinical presentation may be precipitated by vomiting, diarrhea, anorexia, increased alcohol intake or intercurrent infection. The majority of patients have tender, smooth, hepatomegaly with an arterial bruit in severe cases. Signs of chronic liver disease may be present, even without co-existing cirrhosis, and the more advanced cases may also have signs of portal hypertension and encephalopathy. Non-liver signs commonly present include pyrexia, signs of associated vitamin deficiency and malnutrition, a hyperdynamic circulation and cyanosis due to intrapulmonary arteriovenous shunting. Abnormalities of liverrelated blood tests are always present and include decreased albumin and increased ␥GT, AST, bilirubin, alkaline phosphatase and prothrombin time (PT). In addition, blood urea and serum sodium and potassium are low, unless hepato-renal syndrome supervenes, and hypoglycemia may be present. Macrocytic anemia, neutrophil leukocytosis and thrombocytopenia are present in all but the mildest cases. A peculiar clinical feature of patients with severe alcoholic hepatitis is that they often rapidly deteriorate in the days immediately following hospital admission (Hardison & Lee, 1966). This has been observed in up to 40% of patients and varies from deteriorating blood tests to increasing encephalopathy or variceal bleeding. The pathophysiological basis of this is not clear but suggestions have included the nutritional implications of withdrawing an alcoholic from their principal source of calorific intake and a reduction in hepatic blood flow consequent upon a reduction in levels of acetaldehyde, which, via conversion to adenosine, has vasodilatory actions. Prognosis The short-term outcome in patients with alcoholic hepatitis depends largely on the severity of the initial histological lesion. Thus, in the Veterans Administration (VA) Cooperative study, 30 day mortality was 1% in those with a mild alcoholic hepatitis, 12% in those with moderately severe histology and 34% in patients with severe disease (Mendenhall, 1985). The principal management problem is that many patients will have prolongation of the PT that precludes transabdominal liver biopsy, and yet are the very patients in whom severe disease is likely and where aggressive and experimental therapy might be justified. In view of this difficulty,
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many clinical and laboratory variables have been suggested as indicators of histological severity and therefore of potential use in predicting short-term mortality in alcoholic hepatitis. Based on these variables there have been three main attempts at creating prognostic indices. First, is the modified Child’s criteria which combines the presence of encephalopathy and ascites, with serum albumin, bilirubin and PT (Gluud et al., 1988). Second, is a more complex system combining 12 different variables to derive a combined clinical and laboratory index (CCLI) (Orrego et al., 1983), and third is the discriminant function of Maddrey and colleagues, which is based on PTT and bilirubin only (Carithers et al., 1989). This has been confirmed prospectively and, in view of its simplicity, is probably the most clinically useful index at present. Perhaps surprisingly, none of these indices includes the presence of renal failure, which is not uncommon in the most severely ill patients. This presumably reflects that the occurrence and outcome of the hepatorenal syndrome is entirely dependent on the severity of the hepatocellular dysfunction which is better indicated by other clinical and laboratory variables. If the patient survives to hospital discharge, then the long-term prognosis is determined by the initial histology, the progression to cirrhosis and the subsequent drinking behavior. Thus, the five year survival falls from 70 to 50% in patients with severe compared to mild alcoholic hepatitis (Alexander et al., 1971), and in the VA study, patients with mild hepatitis who developed cirrhosis had a 71% 2 year survival compared to 81% in those who did not (Goldberg et al., 1986). In addition, the seven year survival has been reported to fall from 80 to 50% in patients who continue to drink compared with abstainers (Alexander et al., 1971), which is presumably due, at least in part, to the influence of intake on the risk of progression to cirrhosis. In men with mild histology, drinking behavior is the major factor determining progression to cirrhosis, while in women and men with severe histology, progression can occur independently of drinking behavior (Pares et al., 1986). Treatment There is no doubt that abstinence from alcohol in combination with supportive, nutritional and symptomatic care remain the mainstay of therapy for patients with alcoholic hepatitis. However, in view of the high mortality associated with severe disease, there have been attempts to develop and evaluate new forms of treatment. These attempts have faced two main problems. First, the controversy surrounding the precise pathogenesis of alcohol-induced liver necrosis has complicated the rational design of new treatment options, and, secondly, the decision to treat a patient with experimental therapy is often extremely difficult without a reliable method of determining which patients will do badly with supportive therapy only. Despite these difficulties, a variety of treatment modalities have been evaluated in formal clinical trials, and the rationale for the majority of them has been
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discussed earlier. As yet the only treatment that has reached widespread clinical use is corticosteroid therapy in acute alcoholic hepatitis. Several independent trials and a recent, large meta-analysis have shown improved survival with treatment in patients with severe disease identified by the presence of encephalopathy or Maddrey’s discriminant function of >32 (Carithers et al., 1989; Mathurin et al., 2002; Ramond et al., 1992). These trials have stressed the importance of excluding patients with active infection or gastrointestinal hemorrhage. Another drug that has been shown to be of benefit in acute severe alcoholic hepatitis in one trial is the nonselective phosphodiesterase inhibitor pentoxifylline (Akriviadis et al., 2000). This drug has an effect on monocyte TNF␣ secretion in vitro in addition to a direct effect on red cell deformability. Possibly by both these mechanisms, pentoxifylline was shown to improve mortality and the incidence of hepatorenal syndrome in patients with a Maddrey score of >32.
Cirrhosis Pathology With progressive injury the features of cirrhosis, namely fibrous septa linking hepatic and portal veins, and regenerative nodules eventually appear. The cirrhosis is usually micronodular, possibly reflecting the inhibition of regenerative activity by alcohol, and frequently reverts to a macronodular cirrhosis with abstention (Rubin et al., 1962). The coexistence of steatosis and hepatitis is common and usually indicates continued consumption. In contrast, alcohol withdrawal at the cirrhotic stage can make the histological determination of etiology almost impossible. Clinical Features As with other forms of cirrhosis, the clinical presentation of alcoholic cirrhosis can range from asymptomatic hepatomegaly to hepatic failure and the complications of portal hypertension, such as ascites or variceal bleeding. Presentation with severe hepatic decompensation usually implies the presence of continued drinking and superimposed alcoholic hepatitis, or may occasionally signal the development of HCC. The clinical findings will depend on the presence of portal hypertension or encephalopathy and do not differ significantly from those observed in other forms of cirrhosis. Patients with compensated cirrhosis, particularly if abstinent from alcohol, can have completely normal laboratory investigations, while patients with continued intake will have a similar range of abnormal laboratory investigations to those seen in patients with alcoholic hepatitis. In addition, a raised ␣-fetoprotein suggests the presence of HCC and indicates the need for further investigations including CT scanning ± hepatic angiography.
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Prognosis The survival of patients with alcoholic cirrhosis is determined by the clinical and histological severity of the disease at presentation and their subsequent drinking behavior. It has also been shown in some studies that gender (Gines et al., 1987) and ethnicity (Mendenhall et al., 1989) may influence survival. Several studies have shown that patients who present with decompensated disease do significantly worse than those presenting with compensated disease (D’Amico et al., 1986; Saunders et al., 1981). The influence of drinking behavior on this trend is best illustrated by the seminal study of Klatskin and Powell (Powell & Klatskin, 1968). They showed that in patients with compensated disease, continued drinking reduced the five-year survival from 89 to 68%. Abstaining patients with ascites or jaundice had lower survival rates than compensated patients, but higher survival rates than patients with ascites or jaundice who continued to drink. The lowest survival was seen in patients with variceal bleeding and alcohol habits had no effect on their mortality. The presence of coexisting alcoholic hepatitis on initial biopsy also affects the prognosis adversely (Orrego et al., 1987b). HCC develops in 5–15% of patients with alcoholic cirrhosis (Lee, 1966). It is most common in abstaining men and the majority of patients die within a few months of diagnosis (D’Amico et al., 1986). Treatment There are at present no effective treatments for patients with alcoholic cirrhosis. Treatment is directed at achieving abstention and treating the complications of hepatic failure and portal hypertension, mainly variceal bleeding and ascites. It is important to stress that even cirrhotic patients with jaundice and ascites can dramatically improve their survival by cessation of drinking. Our experience is that this can be achieved in at least 50% of patients with alcoholic liver disease with intensive medical follow-up supplemented, where necessary, by psychiatric counseling. It is clear, however, that in some patients the disease progresses despite abstention, while in patients presenting with variceal bleeding, subsequent drinking habits appear to have little influence on prognosis, although endoscopic sclerotherapy and treatment with -blockers such as propranolol may improve survival as well as decrease the rebleeding rate (Hayes et al., 1990; Pagliaro et al., 1990). In view of these considerations, liver transplantation has been considered for patients with decompensated alcoholic cirrhosis. Several studies of transplantation have shown that the survival rate is as good as for other chronic liver diseases (Kumar et al., 1990). Contraindications to transplantation include severe extrahepatic disease; cerebral disease, pancreatic disease, neuropathy, malnutrition, cardiomyopathy or abnormal cardiac electrocardiography, which may predict a high risk of sudden cardiac death (Day et al., 1993b). It would also seem reasonable
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to require a period of abstinence prior to transplantation, first, to ensure that the stage of disease does not improve markedly following abstention; second, to counter the concern that alcohol-dependent patients are unlikely to withstand the psychological stresses associated with the operation and follow-up and, third, to satisfy public opinion.
SUMMARY Together with hepatitis viruses, alcohol abuse is the most important cause of liver disease in the world. It produces a wide range of clinico-pathological syndromes from asymptomatic fatty infiltration to life-threatening liver necrosis and established cirrhosis. Clues to the pathogenesis of the liver damage, and to the nature of individual predisposition to disease, most probably lie in a detailed understanding of the metabolism of alcohol, as this process, and the resulting metabolites are probably responsible for many of its toxic effects. In addition, the metabolism of alcohol also induces a potentially injurious “hypermetabolic state,” and leads to the generation of toxic free radicals capable of damaging cell membranes, proteins and nucleic acids. Recent advances in our understanding of the pathogenesis of alcoholic liver disease are at last leading to the more rational design of new treatment modalities for a disease in which, up until now, we have had little therapeutic options other than advising abstention or organ replacement for end-stage disease. Furthermore, understanding the precise biochemical nature of predisposition may eventually lead to disease prevention through targeted counseling to individuals identified to be at high risk.
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FULMINANT HEPATIC FAILURE
Watson Ng, Ian D. Norton and D. Brian Jones DEFINITION Acute liver failure may be defined as rapidly developing severe hepatocellular dysfunction, often in a previously normal liver. Occasionally it may be the first manifestation of previously established but compensated chronic liver disease such as Wilson’s disease or autoimmune chronic active hepatitis. Fulminant hepatic failure (FHF) is defined as acute liver failure associated with the development of hepatic encephalopathy within eight weeks of the onset of symptoms attributable to hepatocellular dysfunction. Generally, this definition assumes that there is no pre-existing liver disease and that if survival occurs, hepatic structure and function may return to normal. This return to normality depends on the liver retaining its capacity to regenerate on the removal of the underlying pathogenic insult. The syndrome is further defined by the time course (Bernuau et al., 1986); that is, The development of encephalopathy within eight weeks of the onset of symptoms in a patient with a previously normal healthy liver, or The appearance of encephalopathy within two weeks of developing jaundice, even in a patient with underlying liver dysfunction. The term subfulminant hepatic failure is sometimes used when acute liver failure is complicated by hepatic encephalopathy which develops after eight weeks or more following the onset of symptoms (Bernuau et al., 1986).
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MAGNITUDE OF THE CLINICAL PROBLEM Comprehensive registry or population based surveillance programs for acute liver failure are lacking, although estimates of incidence can be made from liver transplantation programs, population based programs (such as the CDC Viral Hepatitis Surveillance Program) and single hospital reports (Kim et al., 2002). Of the 2800 or so cases of FHF in the USA each year between 250 and 350 patients undergo liver transplantation. Fulminant hepatic failure accounts for about 5% of liver transplantations in the USA. In 1997 the National Institutes for Health funded a collaboration of 14 U.S. academic centers with the purpose of collecting structured data on all cases of acute liver failure. This consortium, known as the Acute Liver Failure Study Group (ALFSG), collected retrospective data and serum samples from patients with acute liver failure from 1994 through 1998, and has thus far prospectively collected data on almost 500 patients (Anon, 2002).
ETIOLOGY Temporal changes in the etiology of FHF are evident. In retrospective series from the 1980s, viral hepatitis (HAV, HBV, non-A non-B hepatitis) was the most common cause of FHF, whereas later reports listed drug-induced (especially, acetaminophen) as the most common cause of FHF in the USA (Rakela et al., 1985; Schiodt et al., 1997). The ALFSG reported that between 1998 and 2001 the causes of FHF among patients referred for transplant assessment were acetaminophen in 38%, other drugs in 14%, hepatitis B in 8%, hepatitis A in 4%, miscellaneous other causes in 19% and indeterminate in 18% (Ostapowicz et al., 2000). It should be noted that databases dependent upon transplant assessment for FHF may not be representative of all cases of FHF. For example, patients with acetaminophen overdose may have psychiatric comorbidity which would preclude transplantation. This is supported by data from a single urban hospital of all cases of FHF which estimated the annual incidence of acetaminophen-induced FHF to be 7.5 cases per 1 million population (Schiodt et al., 1997, 1999). The CDC captures case fatality rates for severe viral-associated FHF. In 1999 the case fatality rate for HAV was 0.14%, HBV was 0.24% and “rare” for HCV (Anon, 2000) An interesting but not entirely explained phenomenon has been that of HAV causing FHF in chronic HCV carriers but not in chronic HBV carriers, leading to the recommendation that chronic HCV infected individuals require vaccination against HAV (Vento et al., 1998). Hepatitis B is the most common viral cause of FHF worldwide, complicating 0.1–0.5% of patients with acute hepatitis B. Hepatic inflammation due to HBV is
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due to an intense immunological response to the virus. Fulminant HBV usually occurs as part of the immune response to the patient’s initial infection. Rarely, it can occur in HBV carriers who pass through a period of immune suppression (allowing increased HBV replication and viral burden) followed by the patient returning to an immune competent state. The classic scenario for this is in patients undergoing cytotoxic chemotherapy. Thus, patients carrying HBV and cytotoxic chemotherapy should be considered for prophylactic treatment with antiviral therapy such as lamivudine. Patients surviving HBV-associated FHF usually clear the infection and develop immunity. Fulminant liver disease due to HBV occurs more often if the patient is co-infected with hepatitis D (delta hepatitis) (Govindarajan et al., 1984). Other viral causes of FHF include hepatitis E (especially in pregnant women and the elderly in endemic areas such as India and Nepal), Epstein-Barr virus, cytomegalovirus, herpes simplex and varicella zoster. A number of drugs and other chemicals have been shown to cause hepatitis and, in some cases, FHF. Some of these reactions are dose dependant (e.g. carbon tetrachloride, toluene, acetaminophen, Amanita phalloides) while many others are idiosyncratic (the majority of drug reactions). Acetaminophen is the most common toxin-related cause of FHF and is usually seen in the setting of deliberate overdose. However, individuals who have underlying liver disease, or those with induction of cytochrome P450–2E1 (usually through alcohol or medications, such as anticonvulsants) are at risk of acetaminophen toxicity at relatively low (therapeutic) doses (Vale & Proudfoot, 1995). Table 1. Etiology of Fulminant Hepatic Failure. Infection Hepatitis A, B, C, D, E EBV, herpes simplex Drugs Hepatocellular necrosis Acetaminophen Halothane Isoniazid Steatosis Tetracycline Valproate Chemicals and poisons Amanita phalloides Chlorinated hydrocarbons Aflatoxin
Circulatory Ischemic injury Hepatic vein thrombosis Veno-occlusive disease Neoplastic infiltration Microvesicular steatosis Acute fatty liver of pregnancy Reye’s syndrome Metabolic Wilson’s disease Galactosemia Miscellaneous Heatstroke Jejuno-ileal bypass
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Other drugs that have been implicated in FHF include halothane, isoniazid, valproic acid, sulfonamides, propylthiouracil, and phenytoin. Recently, the illicit drug ecstasy has also been described to cause FHF (Farrell, 1994). The ingestion of the mushroom Amanita phalloides is a relatively common cause of FHF in continental Europe. Poisoning with industrial solvents such as chlorinated hydrocarbons can also lead to FHF. Rare causes of FHF include derangement of the hepatic circulation such as in ischemic hepatic injury due to hypotension or hepatic vein thrombosis, veno-occlusive disease, and metastatic infiltration of the liver such as with breast cancer. A proportion of FHF cases are of indeterminate etiology. This cryptogenic group may include patients with non A-E viral hepatitis, unrecognized drug toxicity (particularly herbal preparations) and possibly unrecognized metabolic disorders in the pediatric age group. The various etiologies of FHF are summarized in Table 1.
PATHOLOGY Light microscopic examination of liver from patients with FHF usually shows one of two characteristic appearances. The type 1 lesion is characterized by massive hepatocellular necrosis, which may be centrilobular or diffuse. There may be profound loss of hepatocytes with confluent necrosis that involves adjacent lobules. This extensive necrosis may cause disruption of the reticulin framework of the lobule (Portmann et al., 1975). Remaining hepatocytes may be shrunken, or swollen and vacuolated. A variable inflammatory infiltrate may be present. This is illustrated in Fig. 1. The type 2 lesion, microvesicular steatosis, features hepatocytes containing numerous fat-filled inclusions within the cytoplasm. Typically, the nuclei are not
Fig. 1. Fulminant Hepatic Failure. Note: Example of severe zones 2 and 3 necrosis due to acetaminophen overdose. There is extensive “drop out” of hepatocytes in zones 2 and 3 with relative preservation of periportal hepatocytes. Source: Unpublished data of Ng, Norton and Jones.
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Fig. 2. Fulminant Hepatic Failure. Note: Example of type 2 lesion in a patient with acute fatty liver of pregnancy. There is evidence of large fat globules (macrosteatosis) between hepatocytes. There is also a fine reticular pattern of the cytoplasm of the hepatocytes due to fat infiltration (microsteatosis). Source: Unpublished data of Ng, Norton and Jones.
displaced from the central position. The appearance is best demonstrated in sections specially stained for fat. There may be minimal necrosis, and aminotransferases are more modestly elevated than in patients with the type 1 lesion. Acute fatty liver of pregnancy, tetracycline or sodium valproate-related liver injury, and Reye’s syndrome are examples of the type 2 lesion, as shown in Fig. 2. The mechanism by which various agents cause hepatocellular injury and dysfunction remains poorly understood. Understanding these mechanisms may help in the development of specific therapies. The treatment of acetaminophen overdosage illustrates this fact: early replenishment of depleted hepatic glutathione stores with agents such as N-acetylcysteine is protective against continued hepatocellular necrosis. Host susceptibility and the degree of exposure to a hepatotoxic agent may also influence the severity of the lesion.
CLINICAL FEATURES The clinical features of FHF reflect the effects of the failing hepatocyte function and also the sequelae of multi-organ failure. Hepatic Encephalopathy Encephalopathy is invariably present and is a sine qua non for the diagnosis of FHF. Although the encephalopathy of FHF is similar to that seen in chronic liver disease, there are some important differences. Portal-systemic shunting of blood is
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Table 2. Clinical Staging of Acute Hepatic Encephalopathy. Stage I: Prodrome
Euphoria Occasionally depression Fluctuant, mild confusion Slowed mentation and affect Slurred speech Disordered sleep rhythm Slight asterixis
Stage II: Impending coma
Accentuation of Stage I Drowsiness, inappropriate behavior incontinence Marked asterixis
Stage III: Stupor
Asleep but arouseable Incoherent speech and confusion Asterixis present
Stage IV: Deep coma
May be unresponsive to painful stimulus Asterixis usually absent
important in the pathogenesis of encephalopathy seen with chronic liver disease, but not with FHF. Furthermore, cerebral edema with raised intracranial pressure is only rarely seen in patients with encephalopathy from chronic liver disease, but is frequent in FHF. The pathophysiological processes of encephalopathy associated with FHF are not well understood and are probably multifactorial (Ferenci, 1994; Riordan & Williams, 1997) Many features of FHF, including metabolic derangements, cerebral edema, hypoglycemia, sepsis, and hypoxemia, can all contribute to neurological abnormalities (Cordobe & Blei, 1996). Other factors thought to be important include decreased metabolism of ammonia, enhanced inhibitory neurotransmission via ␥-aminobutyric acid (GABA) receptors in the central nervous system and changes in central neurotransmitters and circulating amino acids (Schafer & Jones, 1982). Other contributing factors include the effects of cytokines released from necrotic liver tissue (Saija et al., 1995), impaired cerebral perfusion from dysfunctional autoregulation of cerebral blood flow in liver failure (Larsen et al., 1997; Strauss et al., 1997), use of sedatives, and serum electrolyte imbalance (Gabuzda & Hall, 1996). The clinical staging of acute hepatic encephalopathy is shown in Table 2. Cerebral Edema The major cause of mortality in patients with FHF is cerebral edema, found in over 80% of patients at autopsy and often associated with brain stem coning
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(Ede & Williams, 1986; Lee, 1993; Ware & D’Agostino Awn Conises, 1971). Cerebral edema is seen almost only in patients with the most severe (stage IV) encephalopathy (Ware et al., 1971; Wendu et al., 1992). The pathogenesis of cerebral edema is controversial and multifactorial. It may involve alteration in the permeability of the blood-brain barrier, inhibition of Na+ -K+ -ATPase, and failure of autoregulation of cerebral blood flow (Munoz et al., 1993). Mechanical ventilation may also contribute. Cerebral edema can be detected on clinical examination. One of the earliest signs of raised intracranial pressure (ICP) is an increase in muscle tone in the limbs. This may progress to full decerebrate posturing. Hyperventilation occurs and may be marked. As ICP rises, trismus and opisthotonus occur. The pupillary reflexes become abnormal, reacting sluggishly to light, and the pupils are dilated. The pupillary changes are especially important as the use of paralyzing agents and mechanical ventilation will mask other signs. The changes may be unilateral or bilateral. Bilateral dilated pupils unresponsive to light usually denote irreversible coning of the brain stem. However, pupillary dilatation is also seen with marked sympathetic stimulation; so this sign alone does not establish irreversibility. Infection Infections develop in up to 80% of patients with FHF, and bacteremia is present in 20–25% (Wyke et al., 1982). Uncontrolled sepsis accounts for approximately 25% of patients with FHF being excluded from liver transplantation and for approximately 40% of postoperative deaths (Lidofshy, 1995; Rolando et al., 1990). Sepsis may be categorized by the components of the systemic inflammatory response syndrome (SIRS) and include temperature greater than 38 ◦ C, heart rate greater than 90 per minute, tachypnea greater than 20 breaths per minute, PaCO2 less than 4.3 kPa, and white count greater than 12 × 109 /. There is a significant association between the SIRS score and the development of encephalopathy and subsequent mortality in FHF (Rolando et al., 2000). There are at least three factors that increase the risk of infection in patients with FHF. These are: Gut-derived microorganisms may enter the systemic circulation from portal circulation as a result of damage to hepatic macrophages (Kupffer cells). Impaired neutrophil function may result from reduced hepatocellular synthesis of acute-phase reactants, such as components of the complement cascade. Multiple invasive procedures are often necessary (such as intravascular and urethral catheterization, endotracheal intubation). Thus, physical barriers to
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infection are compromized. Indeed, the major sites of infection are the respiratory and urinary tracts. Therefore, it is not surprising that the most common microorganisms isolated are staphylococcal species, streptococcal species, and gram-negative rods. In addition to bacterial infection, up to 30% of patients with FHF develop fungal infection (Rolando et al., 1991). The majority of these are caused by Candida albicans. Aspergillus infection is probably more prevalent than previously appreciated and may account for up to half of fatal infections in the period immediately after liver transplantation for FHF (Castells et al., 1993). It is likely that these infections are acquired preoperatively. Risk factors for fungal infections include renal failure and prolonged antibiotic therapy for existing bacterial infections. Characteristically, fungal infection is associated with high fever and/or leucocytosis refractory to broad-spectrum antibiotics. It is unknown whether the high prevalence of fungal infections among patients with FHF is a result of disturbances in immune function or prolonged antibiotic administration. Coagulopathy As the liver has a central role in hemostasis, patients with FHF invariably have a hemorrhagic diathesis. Platelet number, function, and structure may all be abnormal (Rubin et al., 1977). Platelet counts of less than 80,000 have been reported in up to 50% of cases (Saunders et al., 1972). Reasons for this include hypersplenism, bone marrow suppression, increased consumption of platelets, and loss of platelets in extracorporeal hemoperfusion. The liver synthesizes fibrinogen, prothrombin, and factors V, VII, IX, and X, as well as fibrinolytic factors and inhibitors of coagulation (primarily antithrombin III). Impaired hepatic protein synthesis results in reduced production of these factors. In contrast, factor VIII levels are elevated. Factor VII, with its short half-life, is the first clotting factor to decrease in concentration and levels rise rapidly if recovery occurs. The prothrombin time may be prolonged early in the course of FHF. It is a useful index of the severity of hepatocellular dysfunction reflecting hepatic protein synthetic capacity (Roberts & Cederbann, 1972). It may be prolonged prior to clinical deterioration. The partial thromboplastin time is also prolonged, reflecting diminished synthesis of clotting factors. There is often a low-grade disseminated intravascular coagulation (DIC) in patients with FHF (Hillenbrand et al., 1974). Fibrin degradation products are elevated, fibrinogen catabolism is increased, and thrombi are found at autopsy. Clinically, overt bleeding occurs most frequently from the upper gastrointestinal tract and is often severe (Gazzard et al., 1975).
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Cardiac and Circulatory Complications Cardiac arrhythmias are frequent, especially in patients in stage IV coma. Sinus tachycardia is very common. Other arrhythmias, that may include heart block, ectopic beats, or bradycardia, usually occur in the setting of hypoxemia, acidosis, electrolyte disturbances, or raised ICP (Trewby & Williams, 1977). Non-specific ST segment and T wave changes are seen on the electrocardiogram (Weston et al., 1976). Hypotension is common in FHF for several reasons. Hypovolemia from inadequate fluid therapy or hemorrhage must always be considered. Sepsis and cardiopulmonary dysfunction can also contribute. Often, however, there is no readily correctable cause (Trewby & Williams, 1977). Systemic vascular resistance is inappropriately low, possibly due to the effects of endotoxins or diminished hepatic clearance of endogenous vasodilators (such as VIP or substance P) (Hillenbrand et al., 1974; Hortnagel et al., 1984; Lidofshy, 1995; Schafer & Jones, 1982; Sullivan et al., 1981). Cardiac output may be increased. If this compensatory mechanism fails, hypotension with a hyperdynamic circulation results. Bradycardia in this setting suggests central vasomotor depression secondary to cerebral edema. These hemodynamic abnormalities in FHF may result in severe tissue hypoxia (Bihari et al., 1985). A defect in autoregulation of the microcirculation may be a final common pathway that leads to widespread tissue damage and necrosis. Abnormal tissue oxygen uptake may compound this. Pulmonary Complications Arterial hypoxemia as a result of inadequate pulmonary function is common and tends to worsen as coma deepens. Arterio-venous intrapulmonary shunting of blood contributes to this. Pulmonary edema is well recognized in FHF and may be due to overvigorous fluid therapy, but may occur in the absence of fluid overload (Trewby et al., 1978). In these cases, it is thought that increased capillary permeability within the lungs may be responsible for fluid accumulation. The development of adult respiratory distress syndrome (ARDS) is frequently seen (Baudocrin et al., 1995). Patients with an impaired level of consciousness are prone to aspiration pneumonia and pulmonary atelectasis. Renal Complications Renal failure commonly occurs during the course of FHF (seen in more than 30% of patients) (Bihari et al., 1986). Although the degree of renal failure does not
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correlate with the severity of the hepatic failure, it has been associated with a poorer prognosis (Bernuau et al., 1986). However, with early aggressive dialysis, this may no longer be true. Acute tubular necrosis may occur in the setting of hypotension, sepsis, or hemorrhage. Most often, functional renal failure occurs (Ring-Larsen & Palazzo, 1981). This “hepatorenal syndrome” is associated with a normal urine sediment, a urinary sodium concentration of less than 20 mmol/ and resolution if liver function improves. The blood urea nitrogen is not a reliable guide in assessing renal failure as urea production in the failing liver is markedly diminished.
Metabolic Complications Hypoglycemia Massive liver necrosis impairs hepatic glucose release as glycogen stores are depleted and gluconeogenesis diminished. This may lead to hypoglycemia that can be severe (Samson et al., 1967). In addition, hepatic catabolism of insulin may be diminished. Profound hypoglycemia may cause coma independently or may accentuate hepatic encephalopathy. Acid-Base Abnormalities Massive hepatic necrosis coupled with hypotension and hypoxemia predisposes to accumulation of lactic acid and metabolic acidosis. Inadequate ventilation from any cause, with carbon dioxide retention, may add a respiratory component to this acidosis. Hypokalemia tends to cause a metabolic alkalosis. Hyperventilation, either mechanical or from abnormal central regulation, may exacerbate alkalosis. The resultant complex acid-base disturbance is a result of the interaction of these factors. Electrolyte Disorders Hypokalemia frequently occurs in FHF and may be severe. It may be due to inadequate replacement, to increased losses, or to secondary hyperaldosteronism. Moreover, it may worsen coma or provoke cardiac arrhythmias. Hyponatremia is common. Total body sodium is elevated because of secondary hyperaldosteronism, but water excretion is diminished proportionally. In addition, inhibition of Na+ K+ -ATPase may contribute to the development of hyponatremia.
PREDICTORS OF OUTCOME There are two populations of patients with FHF: patients in whom intensive supportive medical care enables recovery of hepatic function and patients who require liver transplantation to survive.
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Table 3. King’s College Criteria – Prognostic Indicators Associated with Adverse Outcome in Fulminant Hepatic Failure. Patients Without Acetaminophen Toxicity
With Acetaminophen Toxicity
Disease etiology: Cryptogenic or drug/toxin, non-A, non-B viral hepatitis Age: <10 or >40 years
Arterial pH: <7.3
Duration of jaundice: >1 week before development of encephalopathy Serum bilirubin concentration: >18 mg/dL Prothrombin time: International normalized ratio >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.
REFERENCES Allen, J. W., Hassanein, T., & Bhatia, S. N. (2001). Advances in bioartificial liver devices. Hepatology, 34, 447–455. Anon (2000). Hepatitis surveillance report. Atlanta, GA: Centers for Disease Control and Prevention. Anon (2002). The acute liver study group. Hepatology, 36, 1326. Ash, S. (1994). Hemodiabsorption in treatment of acute hepatic failure and chronic cirrhosis with ascites. Artif. Organs, 18, 355–362.
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Baudocrin, S. U., Howlle, P., & O’Grady, C. (1995). Acute lung injury in fulminant hepatic failure following paracetamol poisoning. Thorax, 50, 399–402. Bernuau, J., Reuff, B., & Benhamou, J. P. (1986). Fulminant and subfulminant liver failure: Definitions and causes. Sem. Liver Dis., 6, 97–106. Bihari, D., Gimson, A., & Waterson, M. (1985). Tissue hypoxia in fulminant hepatic failure. Crit. Care Med., 13, 1034–1039. Bihari, D., Gimson, A., & Williams, R. (1986). Cardiovascular, pulmonary and renal complications of fulminant hepatic failure. Sem. Liver Dis., 6, 119–128. Blei, A., Olafsson, S., Webster, S., & Levy, R. (1993). Complications of intracranial monitoring in fulminant hepatic failure. Lancet, 341, 157–159. Canalese, J., Gimson, A., & Davis, C. (1982). Controlled trial of dexamethasone and mannitol for cerebral oedema of fulminant hepatic failure. Gut, 23, 625–629. Caraceni, P., & Vanthiel, D. (1995). Acute liver failure. Lancet, 345, 163–169. Castells, A., Salmeron, J. M., & Navasa, M. (1993). Liver transplantation for acute liver failure: Analysis of applicability. Gastroenterology, 105, 532–538. Cordobe, J., & Blei, A. T. (1996). Brain edema and hepatic encephalopathy. Sem. Liver Dis., 16, 271–275. Davenport, A., Will, E., & Davidson, A. (1993). Improved cardiovascular stability during continues modes of renal replacement therapy in critically ill patients with acute hepatic and renal failure. Crit. Care Med., 21, 326–338. Detre, K., Belle, S., & Beringor, K. (1997). Liver transplantation for fulminant hepatic failure in United States October 1987 through December 1997. Clin. Transplant, 8, 274–280. Detry, O., Arkadopoulos, N., & Ting, P. (1999). Clinical use of a bioartificial liver in the treatment of acetaminophen-induced fulminant hepatic failure. Am. Surg., 65, 934–936. Ede, R. J., & Williams, R. (1986). Hepatic encephalopathy and cerebral oedema. Sem. Liver Dis., 6, 107–1018. Ellis, A., Hughes, R., & Nicholl, D. (1999). Temporary extracorporeal liver support for severe acute alcoholic hepatitis using the BioLogic-DT. Int. J. Artif. Organs, 22, 27–34. Ellis, A., Hughes, R., & Wendon, J. (1996). Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure. Hepatology, 24, 1446–1449. Farrell, G. C. (1994). Drug-induced liver disease. Edinburgh: Churchill Livingstone. Ferenci, P. (1994). Brain dysfunction in fulminant hepatic failure. J. Hepatol., 21, 487–490. Forbes, A., Alexander, G., & O’Grady, J. (1989). Thiopental infusion in the treatment of intracranial hypertension complicating fulminant hepatic failure. Hepatology, 10, 306–310. Gabuzda, G. J., & Hall, P. W. (1996). Relation of potassium depletion to renal ammonium metabolism and hepatic coma. Medicine, 45, 481–490. Gazzard, B. G., Portmann, B., & Murray-Lyon, I. M. (1975). Causes of death in fulminant hepatic failure and relationship to quantitative histological assessments of parenchymal damage. Q. J. Med., 44, 615–626. Govindarajan, S., Chin, K., & Redeker, A. (1984). Fulminant B viral hepatitis: Role of delta agent. Gastroenterology, 86, 1417–1420. Hanam, C., Munoz, S., & Rubin, R. (1995). Histopathological heterogeneity in fulminant hepatic failure. Hepatology, 21, 345–351. Harrid, M., Davis, M., & Mellen, P. (1980). Clinical monitoring of intracranial pressure in fulminant hepatic failure. Gut, 21, 866–869. Harrison, P., Keays, R., & Bray, G. (1990). Improved outcome of paracetamol-induced fulminant hepatic failure by late administration of acetylcysteine. Lancet, 335, 1572–1573.
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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
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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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Saleh, M. G., Tibbs, C. J., Koskinas, J., Pereira, L. M. M. B., Bomford, A. B., Portmann, B. C., McFarlane, I. G., & Williams, R. (1994). Hepatic and extrahepatic hepatitis C virus replication in relation to response to interferon therapy. Hepatology, 20, 1399–1404. Schalm, S. W., Heathcote, J. E., Cianciara, J., Farrell, G., Sherman, M., Willems, B., Dhillon, A., Moorat, A., Barber, J., & Gray, D. F. (2000). Lamivudine and alpha interferon combination treatment of patients with chronic hepatitis B infection: A randomised trial. Gut, 46, 562–568. Scheuer, P. J. (1995). The nomenclature of chronic hepatitis: Time for a change. Journal of Hepatology, 22, 112–114. Schilsky, M. L., & Sternlieb, I. (1999). Management of Wilson’s disease. In: E. L. Krawitt (Ed.), Medical Management of Liver Disease (pp. 397–406). New York: Marcel Dekker. Schmidt, W., Wu, P., Han, J. Q., Perino, M. J., LaBrecque, D., & Stapleton, J. (1997). Distribution of hepatitis C virus (HCV) in whole blood and blood cell fractions: Plasma HCV RNA analysis underestimates circulating virus load. Journal of Infectious Diseases, 176, 20–26. Schvarz, R., Glaumann, H., & Weiland, O. (1993). Survival and histological resolution of fibrosis in patients with autoimmune chronic active hepatitis. Journal of Hepatology, 18, 15–23. Seef, L. B., & Hoofnagle, J. H. (2002). National Institutes of Health Consensus Development Conference: Management of Hepatitis C. Hepatology, 36(Suppl. 1), 1–252. Shouval, D., & Samuel, D. (2000). Hepatitis B immune globulin to prevent hepatitis B virus graft reinfection following liver transplantation: A concise review. Hepatology, 32, 1189–1195. Simpson, K. J., Lukacs, N. W., Colletti, L., Strieter, R. M., & Kunkel, S. L. (1997). Cytokines and the liver. Journal of Hepatology, 27, 1120–1132. Sveger, T., & Eriksson, S. (1995). The liver in adolescents with alpha-1-antitrypsin deficiency. Hepatology, 22, 514–517. Szostecki, C., Guldner, H. H., & Will, H. (1997). Autoantibodies against “nuclear dots” in primary biliary cirrhosis. Seminars in Liver Disease, 17, 71–78. Terjung, B., & Worman, H. J. (2001). Anti-neutrophil antibodies in primary sclerosing cholangitis. Best Practice Research in Clinical Gastroenterology, 15, 629–642. Treichel, U., & Meyer zum Buschenfelde, K.-H. (1998). The asialoglycoprotein receptor in autoimmune liver disease. In: E. L. Krawitt, R. H. Wiesner & M. Nishioka (Eds), Autoimmune Liver Diseases (2nd ed., pp. 273–286). Amsterdam: Elsevier. Treichel, U., Paietta, E., Poralla, T., Meyer zum Buschenfelde, K.-H., & Stockert, R. J. (1994). Effects of cytokines on synthesis and function of the hepatic asialoglycoprotein receptor. Journal of Cell Physiology, 158, 527–534. Yano, M., Kumada, H., Kage, M., Ideda, K., Shimamatsu, K., Inoue, O., Hashimoto, E., Lefkowitch, J. H., Ludwig, J., & Okuda, K. (1996). The long-term pathological evolution of chronic hepatitis C. Hepatology, 23, 1334–1340. Zaman, S. N., Melia, W. M., Johnson, R. D., Portmann, B. C., Johnson, P. J., & Williams, R. (1985). Risk factors for development of hepatocellular carcinoma in cirrhosis: Prospective study of 613 patients. Lancet, i, 1357–1360. Zimmerman, H. J. (2000). Drug-induced liver disease. Clinics in Liver Disease, 4, 73–96.
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16.
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% <1% Adefovir (48 wk)
DNA loss eAg loss eAb conversion sAg loss
21% (51 in eAg neg) 24% 12% 0%
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Durability of response to interferon in eAg seroconversions over five years is 80–90%, and roughly two-thirds with lamivudine responders (Dienstag et al., 2003). Unfortunately, the duration of therapy with lamivudine is not defined, but is limited largely by the development of YMDD mutation. Many centers are treating well beyond one year now. Long term benefits of therapy are not clear, but lower viral replication and lower ALT levels are likely associated with reduced risk of HCC and decompensated cirrhosis (Fontana et al., 2002). Adefovir dipivoxil is a nucleotide analog and also causes HBV DNA chain termination. Initially evaluated as an HIV medication, it was found to cause renal toxicity at higher doses. Current FDA approved dosing is at 10 mg daily, with reductions for renal insufficiency. Efficacy is similar to lamivudine in both HBeAg positive and negative patients (Hadziyannis et al., 2003) (see Table 3). The advantage of fewer mutations makes this agent a very attractive drug, although with time, resistance may be seen, since two cases at two years of therapy have been reported out of 79 patients. The use of adefovir is particularly advantageous for long term therapy of HBeAg negative patients. Both lamivudine and adefovir are well tolerated with few side effects, but renal function should be closely monitored in those undergoing adefovir therapy, especially in those with underlying renal insufficiency.
OTHER NUCLEOSIDE ANALOGS Early studies using ganciclovir showed that while there was efficacy in the intravenous formulation, the oral drug was not very effective in HBV therapy. Famciclovir, while well tolerated, showed low efficacy and a high mutation rate, and could not be used as a single agent to treat HBV (de Man et al., 2000). Entacavir, a guanosine analog, is very effective in vitro, and shows promise in phase II studies at doses of 0.1 and 0.5 mg daily (Lai et al., 2002). Ongoing studies will determine its place in the treatment algorithm of HBV. Tenofovir, a nucleotide reverse transcriptase inhibitor, is an HIV approved therapy, but has similar efficacy against HBV as adefovir, to which it is quite similar. While no specific HBV treatment trials have evaluated this agent, it has been shown to reduce viral DNA loads by up to 4 log10 copies/mL at 300 mg daily (Benhamou et al., 2003). Given its proven track record in HIV, it is perhaps an ideal drug in the HIV/HBV co-infected population. It should be added in passing that it is a remarkable fact that the research in the immunopathogenesis of human immunodeficiency virus (HIV) has led to several treatment options for HBV that have allowed suppression and in some cases complete clearance of the virus. A great deal of work is required to further understand this virus, and especially in optimizing treatment regimens. Vaccination
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programs are the key to controlling the epidemic proportions of infections seen in some countries. It is very likely that future therapies will center around evaluating combination therapies with various nucleoside/nucleotide analogs together and in combination with pegylated interferons, to determine if efficacy can be improved while resistance reduced. Any of the FDA approved therapies can be used as a first line, depending on certain variables. In the recently infected, HBeAg positive patient with high ALT and low HBV DNA, or the HDV co-infected individual, interferon may be the best choice. In those with peri-natal infection, very high DNA levels, low to moderate ALT, and HBeAg positive or negative, lamivudine or adefovir should be first line therapy. Neither have a distinct advantage over the other in the short term. Therapy beyond one year and especially beyond two to three years, is associated with lamivudine resistance, and in those individuals, adefovir may be a better choice. Length of therapy with these agents is not known yet, and prolonged therapy beyond one year is probably indicated for most. Future studies will provide the answers to these questions.
SUMMARY AND CONCLUSION Hepatitis B continues to be a worldwide health concern, with rising incidence of hepatocellular carcinoma and liver related mortality. While developed countries have a relatively low prevalence of disease, endemic areas in Asia and Africa bear the brunt of the disease burden, and have the highest morbidity and mortality rates. The therapy as it currently stands is not ideal, and true clearance of virus is generally not achieved with nucleoside analog agents. Interferon remains a viable treatment option, but is indicated in a minority of individuals. The future of therapy will likely revolve around multi-drug combinations, and perhaps novel therapies such as therapeutic vaccines. Vaccination programs are ultimately the most important intervention in preventing new disease especially in endemic areas. The cost of treating HBV will continue to rise in the near future with combination therapies, but ultimately will lead to lower late stage liver complications.
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Bodsworth, N., Donovan, B., & Nightingale, B. N. (1989). The effect of concurrent human immunodeficiency virus infection on chronic hepatitis B: A study of 150 homosexual men. J. Infect. Dis., 160, 577–582. Buckwold, V. E., Xu, Z., Chen, M., Yen, T. S., & Ou, J. H. (1996). Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication. J. Virol., 70, 5845–5851. Cooksley, W. G., Piratvisuth, T., Lee, S. D., Mahachai, V., Chao, Y. C., Tanwandee, T., Chutaputti, A. et al. (2003). Peginterferon a-2a (40kDa): An advance in the treatment of hepatitis B e antigen-positive chronic hepatitis B. J. Viral. Hepatitis, 10, 298–305. Davis, G. L., Hoofnagle, J. H., & Waggoner, J. G. (1984). Spontaneous reactivation of chronic hepatitis B virus infection. Gastroenterology, 86, 230–235. De Jongh, F. E., Janssen, H. L. A., De Man, F. A., Hop, W. C. J., Schalm, S. W., & Van Blankenstein, M. V. (1992). Survival and prognostic indicators in hepatitis B surface antigen-positive cirrhosis of the liver. Gastroenterology, 103, 1630–1635. de Man, R. A., Marcellin, P., Habal, F., Desmond, P., Wright, T., Rose, T., Jurewicz, R. et al. (2000). A randomized, placebo-controlled study to evaluate the efficacy of 12-month famciclovir treatment in patients with chronic hepatitis B e antigen-positive hepatitis B. Hepatology, 32, 413–417. Dienstag, J. L., Cianciara, J., Karayalcin, S., Kowdley, K. V., Willems, B., Plisek, S., Woessner, M. et al. (2003). Durability of serologic response after lamivudine treatment of chronic hepatitis B. Hepatology, 37, 748–755. Dienstag, J. L., Schiff, E. R., Wright, T. L., Perrillo, R. P., Hann, H. W., Goodman, Z., Crowther, L. et al. (1999). Lamivudine as initial treatment for chronic Hepatitis B in the United States. N. Engl. J. Med., 341, 1256–1263. Fontana, R. J., Hann, H. W. L., Perrillo, R. P., Vierling, J. M., Wright, T., Rakela, J., Anschuetz, G. et al. (2002). Determinants of early mortality in patients with decompensated chronic hepatitis B treated with antiviral therapy. Gastroenterology, 123, 719–727. Ganem, D., & Schneider, R. (2001). Hepadnaviridae: The viruses and their replication. In: D. M. Knipe & P. M. Howley (Eds), Fields Virology (pp. 2923–2970). Philadelphia: Lippincott-Raven. Hadziyannis, S. J., Tassopoulos, N. C., Heathcote, E. J., Chang, T. T., Kitis, G., Rizzetto, M., Marcellin, P. et al. (2003). Adefovir dipivoxil for the treatment of hepatitis B e antigen-negative chronic hepatitis B. N. Engl. J. Med., 348, 800–807. Hadziyannis, S. J., & Vassilopoulos, D. (2001). Hepatitis B e antigen-negative chronic hepatitis B. Hepatology, 34, 617–624. Kann, M., & Gerlich, W. (1998). Hepadnaviridae: Structure and molecular virology. In: A. Zuckerman & H. Thomas (Eds), Viral Hepatitis (pp. 77–105). London: Chuchill Livingstone. Kao, J. H., Liu, C. H., & Chen, D. S. (2002). Hepatitis B viral genotypes and lamivudine resistance. J. Hepatol., 36, 303–304. Lai, C. L., Rosmawati, M., Lao, J., Anderson, F. H., Thomas, N., & Dehertogh, D. (2002). Entecavir is superior to lamivudine in reducing hepatitis B virus DNA in patients with chronic hepatitis B infection. Gastroenterology, 123, 1831–1838. Lau, D. T., Doo, E., Park, Y., Kleiner, D. E., Schmid, P., Kuhns, M. C., & Hoofnagle, J. H. (1999). Lamivudine for chronic delta hepatitis. Hepatology, 30, 546–549. Leung, N. W. Y., Lai, C. L., Chang, T. T., Guan, R., Lee, C. M., Ng, K. Y., Wu, P. C. et al. (2001). Extended lamivudine treatment in patients with chronic hepatitis B enhances hepatitis B e antigen seroconversion rates: Results after 3 years of therapy. Hepatology, 33, 1527–1532.
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Liaw, Y.-F., Chien, R.-N., Yeh, C.-T., Tsai, S.-L., & Chua, C.-M. (1999). Acute exacerbation and hepatitis B virus clearance after emergence of YMDD motif mutation during lamivudine therapy. Hepatology, 30, 567–572. Liaw, Y. F., Sheen, I. S., Chen, T. J., Chu, C. M., & Pao, C. C. (1991). Incidence, determinants and significance of delayed clearance of serum HBsAG in chronic hepatitis B virus infection: A prospective study. Hepatology, 13, 627–631. Lindh, M., Andersson, A. S., & Gusdal, A. (1997). Genotypes, nt 1858 variants, and geographic origin of hepatitis B virus-larage scale analysis using a new genotyping method. J. Infect. Dis., 175, 1285–1293. Lok, A. S., Heathcote, E. J., & Hoofnagle, J. H. (2001). Management of Hepatitis B 2000, Summary of a Workshop. Gastroenterology, 120, 1828–1853. Lok, A. S., & Lai, C. L. (1988). A longitudinal follow-up of asymptomatic hepatitis B surface antigenpositive Chinese children. Hepatology, 8, 1130–1133. Mast, E. E., Alter, M. J., & Margolis, H. S. (1999). Strategies to prevent and control hepatitis B and C virus infections: A global perspective. Vaccine, 17, 1730–1733. McMahon, B. J. (1997). Hepatocellular carcinoma and viral hepatitis. In: R. A. Wilson (Ed.), Viral Hepatitis (pp. 315–330). New York: Marcel Dekker. McMahon, B. J., Bulkow, L., Harpster, A., Snowball, M., Lanier, A., Sacco, F., Dunaway, E. et al. (2000). Screening for hepatocellular carcinoma in Alaska Natives infected with chronic hepatitis B: A 16-year population-based study. Hepatology, 32, 842–846. McMahon, B. J., Holck, P., Bulkow, L., & Snowball, M. M. (2001). Serologic and clinical outcomes of 1536 Alask Natives chronically infected with hepatitis B virus. Ann. Int. Med., 135, 759–768. Rehermann, B. (2003). Immune responses in hepatitis B virus infection. Semin. Liver Dis., 2321–2338. Ruiz-Moreno, M. R., Otero, M., Millan, A., Castillo, I., Cabrerizo, M., Jimenez, F. J., Oliva, H. et al. (1999). Clinical and histologic outcome after hepatitis B e antigen to antibody seroconversion in children with chronic hepatitis B. Hepatology, 29, 572–575. Sirma, H., Giannini, C., Poussin, K. et al. (1999). Hepatitis B virus X mutants, present in hepatocellular carcinoma tissue abrogate both the antipoliferative and transactivation effects of HBx. Oncogen., 18, 4848–4859. Schalm, S. W., Heathcote, J., Cianciara, J., Farrell, G., Sherman, M., Willems, B., Dhillon, A. et al. (2000). Lamivudine and alpha interferon combination treatment of patients with chronic hepatitis B infection: A randomized trial. Gut, 46, 562–568. Tassopopoulos, N. C., Papaevangelou, G. J., Sjogren, M. H. et al. (1987). Natural history of acute hepatitis B surface antigen-positive hepatitis in Greek adults. Gastroenterology, 92, 1844–1850.
17.
CURRENT ISSUES IN HEPATITIS B VACCINES
Jane N. Zuckerman and Arie J. Zuckerman INTRODUCTION The availability of hepatitis B vaccines is a major development in preventive medicine. Currently available vaccines are produced either from the plasma of asymptomatic carriers of hepatitis B or by recombinant DNA technology. The vaccines are safe, immunogenic and highly efficacious. Nevertheless, there are three major current issues: (1) A comprehensive strategy for the use of the vaccine. (2) The emergence of hepatitis B surface antigen escape mutants. (3) Non-responders to the current vaccine in 5–15% of healthy individuals. These issues are considered in this chapter. Hepatitis B, which is a public health problem throughout the world, is preventable, but a consensus on immunization strategies is notable by its absence. Current policies in some countries have had little influence on the epidemiology of this important infection and the strategy of selective immunization of groups at “high risk” of infection has had little impact on hepatitis B outside, for example, health care personnel. The World Health Organization has set a target of global control of hepatitis B, and recommended that all countries integrate hepatitis B vaccine into their national immunization programs by 1997. Some 152 countries have introduced such programs.
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A number of strategic options are outlined below including a proposal for immediate implementation of universal antenatal screening and immunization of infants born to carrier mothers as a minimum policy initiative. There is strong support for the introduction of universal antenatal screening to identify hepatitis B carrier mothers and the vaccination of their babies. It is important that any other strategies do not interfere with the delivery of vaccine to this group. Immunization of this group will have the greatest impact in reducing the number of new hepatitis B carriers. For children outside this group, it is difficult to estimate the life time risk of acquiring a hepatitis infection, and four main approaches should be considered (Banatvala et al., 1991):
To continue vaccination of “high risk” babies as defined above; Universal infant immunization; Universal adolescent immunization; Vaccinate everybody.
Vaccination of Adolescents This approach delivers immunization at a time close to the time when “risk behaviour” would expose adolescents to infection. Vaccination could be delivered as part of a wider package on health education in general, to include sex education, risk of AIDS, dangers of drug abuse, smoking, benefits of a healthy diet and life style. The problems with this approach are as follows: Persuading parents to accept vaccination of the children against a sexually transmitted disease, a problem they may not wish to address at that time. Problem of ensuring a full course of three doses is given. There would be difficulty evaluating and monitoring vaccine cover. The systems for monitoring uptake of vaccine in this age group may not operate efficiently.
Vaccination of Infants The advantages of this approach are: It is known that vaccination can be delivered to babies. Parents would accept vaccination against hepatitis B along with other childhood vaccinations without reference to sexual behaviour.
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The disadvantages of this approach are: Whether immunity would remain until exposure occurred in later life. This was thought to become less of a problem as more people were vaccinated as the chance of exposure to infection was reduced. That the introduction of further childhood vaccination would reduce the uptake of other childhood vaccinations. This problem would be avoided if hepatitis B vaccine could be delivered in a combined vaccine containing DPT and such preparations are under evaluation. Vaccination of infants is preferable to vaccination of adolescents as there are sufficient mechanisms to ensure, monitor and evaluate cover. A booster dose could be given in early adolescence combined with a health education package. A rolling program could be introduced, giving priority to urban areas.
HEPATITIS B SURFACE ANTIBODY MUTANTS Production of antibodies to the group antigenic determinant a mediates crossprotection against all sub-types, as has been demonstrated by challenge with a second subtype of the virus following recovery from an initial experimental infection. The epitope a is located in the region of amino acids 124–148 of the major surface protein, and appears to have a double-loop conformation. A monoclonal antibody which recognizes a region within this a epitope is capable of neutralizing the infectivity of hepatitis B virus for chimpanzees, and competitive inhibition assays using the same monoclonal antibody demonstrate that equivalent antibodies are present in the sera of subjects immunized with either plasmaderived or recombinant hepatitis B vaccine. During a study of the immunogenicity and efficacy of hepatitis B vaccines in Italy, a number of individuals who had apparently mounted a successful immune response and become anti-surface antibody (anti-HBs)-positive, later became infected with HBV. These cases were characterized by the co-existence of non-complexed anti-HBs and HBsAg, and in 32 of 44 vaccinated subjects there were other markers of hepatitis B infection (Zanetti, Tanzi, Manzillo et al., 1988). Furthermore, analysis of the antigen using monoclonal antibodies suggested that the a epitope was either absent or masked by antibody. Subsequent sequence analysis of the virus from one of these cases revealed a mutation in the nucleotide sequence encoding the a epitope, the consequence of which was a substitution of arginine for glycine at amino acid position 145 (Carman, Zanetti, Karayiannis et al., 1990).
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There is now considerable evidence for a wide geographical distribution of the point mutation in hepatitis B virus from guanosine to adenosine at position 587, resulting in an amino acid substitution at position 145 from glycine to arginine in the highly antigenic group determinant a of the surface antigen. This stable mutation has been found in viral isolates from children several years later, and it has been described in many countries and in liver transplant recipients with hepatitis B in the USA, Germany and the U.K. who had been treated with specific hepatitis B immunoglobulin or humanized hepatitis B monoclonal antibody (Zuckerman, 2000; Zuckerman et al., 1994). The region in which this mutation occurs is an important virus epitope to which vaccine-induced neutralizing antibody binds as discussed above, and the mutant virus is not neutralized by antibody to this specificity. It can replicate as a competent virus, implying that the amino acid substitution does not alter the attachment of the virus to the liver cell. This has been confirmed in experimental transmission studies at the National Institutes of Health, USA (Ogata, Miller, Ishak et al., 1994). During a study in progress in Singapore three groups of babies were immunized against hepatitis B: 50 babies born to mothers without hepatitis B surface antigen (HBsAg) and 600 born to mothers with HBsAg but without e antigen were immunized successfully. However, among the 600 babies born to mothers with HBsAg and e antigen there were 40 vaccine failures, and all had HBsAg and core antibody. Direct sequencing has been completed for 26 isolates from these 40 infants. Fifteen had wild-type sequences, and serological profiles usually indicated inutero infection. However, the other 11 had variant sequences, namely the 145 glycine-to-arginine variant alone (4 cases) or with other changes (2), alanine at position 144 (1 twin), or other changes yet to be evaluated (Oon, Lim, Ye et al., 1994). In a more recent study, Nainen et al. (2002) compared direct sequencing of amplified or cloned PCR products, solid phase detection of sequence-specific PCR products (SP-PCR), and limiting dilution cloning PCR (LDC-PCR), in order to determine their sensitivity in detecting differing concentration of HBsAg variants in the same population of the infants studied in the 1981–1993 post-exposure prophylaxis of hepatitis B in infants born to carrier mothers. LDC-PCR had the greatest sensitivity and could detect HBsAg variants at a concentration of 0.1% of the total viral population. HBsAg variants were detected in 47 of 93 (51%) of infants with chronic HBV infection acquired after post-exposure prophylaxis, and more than half of the variants were detected only by the most sensitive methods. The G145R variant (glycine to arginine at aa145) was identified most frequently. A report from Taiwan noted the increase in immunized children in the prevalence of mutants of the a determinant of HBV over a period of 10 years, from eight
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of 103 (7.8%) in 1984 to 10 of 51 (19.6%) in 1989, and 9 of 32 (28.1%) in 1994, is of particular concern. The prevalence of HBsAg mutants among those fully immunized was higher than among those not vaccinated (12/33 vs. 15/153, P = 0.0003). In all 27 children with detectable mutants, the mean age of those vaccinated was lower than those not vaccinated, and mutation occurred in a region with greatest hydrophilicity of the surface antigen (amino acids 140–149), and more frequently among those vaccinated than among those not vaccinated. More mutations to the neutralizing epitopes were found in the 1994 survey in Taiwan (Hsu et al., 1999). Another important aspect in the identification of HBsAg variants is the evidence that these mutants may not be detected by all of the blood donor screening tests and by existing diagnostic reagents (reviewed by Francois et al., 2001). This is emphasised by the finding in Singapore, between 1990 and 1992, of 0.8% of carriers of HBV variants in a random population survey of 2001 people (Oon et al., 1995, 1996). These findings add to the concern expressed in a study of mathematical models of HBV vaccination, which predict, on the assumption of no cross-immunity against the variant by current vaccines, that the variant will not become dominant over the wild-type virus for at least 50 years, but the G145R mutant may emerge as the common HBV in 100 (or more) years’ time (Wilson et al., 1999). In summary: Variants of hepatitis B virus surface antigen proteins were identified over a decade ago and may have a potential impact on immunization against this important infection and on public health (Zuckerman, 2000). The G145R mutant is replication competent and is stable. It appears to be the most common variant and may persist in the host for at least 14 years. There is evidence that sera of 10% (up to 40% in high-risk groups) of individuals with antibodies to hepatitis B core antigen (anti-HBc) as the only marker of HBV infection may contain HBV DNA. At least some of the chronic low level carriers of HBV, where surface antigen is not detected and anti-HBc is the only serological marker of HBV infection, are infected with surface mutants. Further studies are required. Epidemiological monitoring of HBV surface mutants is essential employing test reagents which have been validated for detection of the predominant mutations. Urgent consideration should be given to the introduction of routine screening for hepatitis B by nucleic acid based technology of blood donors and tissue and organ donors for transplantation. Consideration should be given to incorporating into the current hepatitis B vaccines of additional antigenic components which will confer protection
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against infection by the predominant a determinant mutation(s), if dictated by epidemiological findings.
OVERCOMING NON-RESPONSIVENESS TO HEPATITIS B IMMUNIZATION All studies of the antibody response to currently licensed plasma-derived hepatitis B vaccines and hepatitis B vaccines prepared by recombinant DNA technology have shown that between 5% and 15% of healthy immunocompetent subjects do not mount an antibody response (anti-HBs) to the surface antigen component (HBsAg) present in these preparations (non-responders) or that they respond poorly (hypo-responders) (Craven et al., 1986; Dienstag et al., 1984; Westmoreland et al., 1990; Wood et al., 1993). The exact proportion depends partly on the definition of non-responsiveness or hypo-responsiveness, generally less than 10 IU/l or 100 IU/l respectively against an international antibody standard. It is considered that non-responders remain susceptible to infection with hepatitis B virus. While several factors are known to affect adversely the antibody response to HBsAg including the site and route of injection, gender, advancing age, body mass (overweight), immunosuppression and immunodeficiency, the mechanisms underlying non-responsiveness to the S component of hepatitis B surface antigen in humans remain largely unexplained although evidence is accumulating that there is an association between different HLA-DR alleles and specific low responsiveness in different ethnic populations. Considerable experimental evidence is available that the ability to produce antibody in response to specific protein antigens is controlled by dominant autosomal Class II genes of the major histocompatibility complex (MHC) in the murine model (reviewed in Alper et al., 1989; Kruskall et al., 1992; Milich, 1991). Much effort has been devoted to overcoming Class II-linked non-responsiveness to current hepatitis B vaccine (for example Arif et al., 1988; McDermott et al., 1999; Milich et al., 1985a, 1986). There is evidence that the pre-S1 and pre-S2 domains have an important immunogenic role in augmenting anti-HBs responses, preventing the attachment of the virus to hepatocytes and eliciting antibodies which are effective in viral clearance, stimulating cellular immune responses, and circumventing genetic nonresponsiveness to the S antigen (Alberti et al., 1988; Gerlich et al., 1990; Klinkert et al., 1986; Milich et al., 1985a). Thus a number of studies indicated that the inclusion of pre-S components in recombinant or future synthetic vaccines should be developed. For example, the pre-S2 region is more immunogenic at the T and B cell levels than the S regions in the mouse model (Milich et al., 1985a, b), as
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is the case with pre-S1 in the mouse (Milich et al., 1986) and in man (Ferrari et al., 1992) and circumvent S region non-responsiveness at the level of antibody production. Indeed, Milich et al. (1986) demonstrated in the murine model that the independence of MHC-linked gene regulation of immune responses to pre-S1, pre-S2 and S regions of hepatitis B surface antigen would assure fewer genetic nonresponders to a vaccine containing all three antigenic regions. Studies conducted in humans with experimental recombinant hepatitis B vaccines containing all three S components of the viral envelope polypeptides demonstrated the enhanced immunogenicity of such preparations when compared with conventional yeastderived vaccines (Yap et al., 1995 and others) although several earlier studies with vaccines containing the S, pre-S1 and pre-S2 components revealed significant differences from preparations containing only the S antigen (Clements et al., 1994; Ferrari et al., 1992; Marescot et al., 1989; Suzuki et al., 1994). These observations led to the development of a new triple antigen hepatitis B vaccine (Hepacare), a third generation recombinant DNA vaccine containing pre-S1, pre-S2 and S antigenic components of hepatitis B virus surface antigen of both subtypes adw and ayw. All three antigenic components are glycosylated, closely mimicking the surface protein of the virus itself, produced in a continuous mammalian cell line, the mouse c127 clonal cell line, after transfection of the cells with recombinant HBsAg DNA. The vaccine is presented as an aluminium hydroxide adjuvant preparation of purified antigenic protein. Animal studies showed that the vaccine was well tolerated and a viral challenge study in chimpanzees demonstrated protective efficacy. This vaccine was evaluated for reactogenicity and immunogenicity in a number of clinical trials (reviewed by Zuckerman & Zuckerman, 2002). The major conclusions from these studies were that the vaccine was safe and immunogenic and overcame the non-responsiveness to the single S antigen vaccines used widely in some 70% of non-responders, and that even a single dose of 20 mcg of the triple antigen provided significant seroprotection levels of antibody. However, the anticipated high costs of the triple antigen vaccine will limit the use of the triple antigen vaccine initially to the following groups: Vaccination of non-responders to the current single antigen(s) vaccines, who are at risk of exposure to HBV infection. Subjects with inadequate humoral immune response to single antigen hepatitis B vaccines, e.g. those over the age of 40 years, males, obese, smokers and other hyporesponders, and Persons who require protection rapidly, e.g. healthcare employment involving potential exposure to parenteral procedures involving blood-to-blood contact
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(current schedules of immunization with single antigen hepatitis B vaccines involve three doses at 0, 1 and 6 months). Studies are required to determine the efficacy of the triple antigen in patients who are immunocompromised and also to determine whether the inclusion of preS1 and pre-S2 antigenic components in this new vaccine will protect against the emergence of HBV surface antigen mutants (see above).
Attempts to Overcome Non-responsiveness by the Use of Immunomodulators Attempts have been made to enhance the anti-HBs response following immunization, particularly in patients treated by maintenance hemodialysis, but often with conflicting results or in limited studies, which have not been confirmed: Alpha-interferon (Goldwater, 1994; Grob et al., 1984); Interleukin-2 (Jungers et al., 1994; Meuer et al., 1989); Thymopentin (Melappioni et al., 1992; Zaruba et al., 1983). and other substances such as experimental oral adjuvants in mice and estrogen. These are referred to for the sake of completion.
The Kinetics of Antibody Response to Hepatitis B Immunization No empirical data are available for the anti-HBs titer required for protection against particular routes of infection or the size of the infectious inoculum. The minimum protective level following immunization has been set in earlier protective efficacy studies at 10 IU/l or more of anti-HBs (Francis et al., 1982; Szmuness et al., 1981). In both studies most cases of HBV infection occurred in subjects who mounted little or no anti-HBs response. Specifically, a protective level of anti-HBs was defined as 10 IU/l against an international standard (Centers for Disease Control, 1987; Stevens et al., 1984). Various studies have also demonstrated that the risk of HBV infection increases as anti-HBs levels decline to 10 IU/l (Coursaget et al., 1986; Hadler et al., 1986; Stevens et al., 1984; Taylor & Stevens, 1988). For example, Hadler et al. (1986) reported in a follow-up study of vaccinated homosexual men an overall incidence of HBV infection of 2.9 per 100 person years with nearly 75% occurring in subjects with anti-HBs titres < 10 IU/l at the time of infection and only a few with anti-HBs titres > 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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Merola, M., Brazzoli, M., Cocchiarella, F., Heile, J. M., Helenius, A., Weiner, A. J., Houghton, M., & Abrignani, S. (2001). Folding of hepatitis C virus E1 glycoprotein in a cell-free system. J. Virol., 75, 11205–11217. Meyer, K., Basu, A., & Ray, R. (2000). Functional features of hepatitis C virus glycoproteins for pseudotype virus entry into mammalian cells. Virology, 276, 214–226. Michalak, J. P., Wychowski, C., Choukhi, A., Meunier, J. C., Ung, S., Rice, C. M., & Dubuisson, J. (1997). Characterization of truncated forms of the hepatitis C virus glycoproteins. J. Gen. Virol., 78, 2299–2306. Miyamoto, H., Okamoto, H., Sato, K., Tanaka, T., & Mishiro, S. (1992). Extraordinarily low density of hepatitis C virus estimated by sucrose density centrifugation and polymerase chain reaction. J. Gen. Virol., 73, 715–718. Moriya, K., Fujie, H., Shintani, Y., Yotsuyanagi, H., Tsutsumi, T., Ishibashi, K., Matsuura, Y., Kimura, S., Miyamura, T., & Koike, K. (1998). The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat. Med., 4, 1065–1067. Moriya, K., Yotsuyanagi, Y., Shintani, Y., Fujie, H., Ishibashi, K., Matsuura, Y., Miyamura, T., & Koike, K. (1997). Hepatitis C virus core protein induces hepatic steatosis in transgenic mice. J. Gen. Virol., 78, 1527–1531. Neddermann, P., Clementi, A., & De Francesco, R. (1999). Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B, and NS5A encoded on the same polyprotein. J. Virol., 73, 9984–9991. Neddermann, P., Tomei, L., Steinkuhler, C., Gallinari, P., Tramontano, A., & De Francesco, R. (1997). The nonstructural proteins of the hepatitis C virus: Structure and functions. Biol Chem., 378, 469–476. Nolandt, O., Kern, V., Muller, H., Pfaff, E., Theilmann, L., Welker, R., & Krausslich, H. G. (1997). Analysis of hepatitis C virus core protein interaction domains. J. Gen. Virol., 78, 1331–1340. O’Farrell, D., Trowbridge, R., Rowlands, D., & Jager, J. (2003). Substrate complexes of hepatitis C virus RNA polymerase (HC-J4): Structural evidence for nucleotide import and de-novo initiation. J. Mol. Biol., 326, 1025–1035. Op De Beeck, A., Cocquerel, L., & Dubuisson, J. (2001). Biogenesis of hepatitis C virus envelope glycoproteins. J. Gen. Virol., 82, 2589–2595. Pang, P. S., Jankowsky, E., Planet, P. J., & Pyle, A. M. (2002). The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding. EMBO J., 21, 1168–1176. Park, J. S., Yang, J. M., & Min, M. K. (2000). Hepatitis C virus nonstructural protein NS4B transforms NIH3T3 cells in cooperation with the Ha-ras oncogene, Biochemical. Biochemical & Biophysical Research Communications, 267, 581–587. Patel, J., Patel, A. H., & McLauchlan, J. (2001). The transmembrane domain of the hepatitis C virus E2 glycoprotein is required for correct folding of the E1 glycoprotein and native complex formation. Virology, 279, 58–68. Pavio, N., Taylor, D. R., & Lai, M. M. (2002). Detection of a novel unglycosylated form of hepatitis C virus E2 envelope protein that is located in the cytosol and interacts with PKR. J. Virol., 76(3), 1265–1272. Pavlovic, D., Neville, D. C., Argaud, O., Blumberg, B., Dwek, R. A., Fischer, W. B., & Zitzmann, N. (2003). The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives. Proc. Natl. Acad. Sci. USA, 100, 6104–6108. Pawlotsky, J. M. (1999). Hepatitis C virus (HCV) NS5A protein: Role in HCV replication and resistance to interferon-alpha. Journal of Viral Hepatitis, 6(Suppl. 1), 47–48.
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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 (0.1m 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 NFB 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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Morris, S. M. (2002). A role for p53 in the frequency and mechanism of mutation. Mutat. Res., 511, 45–62. Murphy, F. R., Issa, R., Zhou, X., Ratnarajah, S., Nagase, H., Arthur, M. J., Benyon, C., & Iredale, J. P. (2002). Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition: Implications for reversibility of liver fibrosis. J. Biol. Chem., 277, 11069–11076. Murphy, G., Stanton, H., Cowell, S., Butler, G., Knauper, V., Atkinson, S., & Gavrilovic, J. (1999). Mechanisms for pro matrix metalloproteinase activation. APMIS., 107, 38–44. Nakatani, K., Seki, S., Kawada, N., Kobayashi, K., & Kaneda, K. (1996). Expression of neural cell adhesion molecule (N-CAM) in perisinusoidal stellate cells of the human liver. Cell Tissue Res., 283, 159–165. Neubauer, K., Knittel, T., Aurisch, S., Fellmer, P., & Ramadori, G. (1996). Glial fibrillary acidic protein – A cell type specific marker for cells in vivo and in vitro. J. Hepatol., 24, 719–730. Newbold, R. F. (2002). The significance of telomerase activation and cellular immortalization in human cancer. Mutagenesis, 17, 539–550. Odenthal, M., Neubauer, K., Meyer zum Buschenfelde, K. H., & Ramadori, G. (1993). Localization and mRNA steady-state level of cellular fibronectin in rat liver undergoing a CCl4-induced acute damage or fibrosis. Biochim. Biophys. Acta., 1181, 266–272. Ohishi, T., Saito, H., Tsusaka, K., Toda, K., Inagaki, H., Hamada, Y., Kumagai, N., Atsukawa, K., & Ishii, H. (2001). Anti-fibrogenic effect of an angiotensin converting enzyme inhibitor on chronic carbon tetrachloride-induced hepatic fibrosis in rats. Hepatol. Res., 21, 147–158. Ohuchi, E., Imai, K., Fujii, Y., Sato, H., Seiki, M., & Okada, Y. (1997). Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J. Biol. Chem., 272, 2446–2451. Okada, Y., & Nakanishi, I. (1989). Activation of matrix metalloproteinase 3 (stromelysin) and matrix metalloproteinase 2 (‘gelatinase’) by human neutrophil elastase and cathepsin G. FEBS Lett., 249, 353–356. Okuno, M., Akita, K., Moriwaki, H., Kawada, N., Ikeda, K., Kaneda, K., Suzuki, Y., & Kojima, S. (2001). Prevention of rat hepatic fibrosis by the protease inhibitor, camostat mesilate, via reduced generation of active TGF-beta. Gastroenterology, 120, 1784–1800. Okuno, M., Moriwaki, H., Imai, S., Muto, Y., Kawada, N., & Kojima, S. (1997). Retinoids exacerbate rat liver fibrosis by inducing the activation of latent TGF-beta in liver stellate cells. Hepatology, 26, 913–921. Okuno, M., Sato, T., Kitamoto, T., Imai, S., Kawada, N., Suzuki, Y., Yoshimura, H., Moriwaki, H., Onuki, K., Masushige, S., Muto, Y., Friedman, S. L., Kato, S., & Kojima, S. (1999). Increased 9,13-di-cis-retinoic acid in rat hepatic fibrosis: Implication for a potential link between retinoid loss and TGF-beta mediated fibrogenesis in vivo. J. Hepatol., 30, 1073–1080. Paizis, G., Cooper, M. E., Schembri, J. M., Tikellis, C., Burrell, L. M., & Angus, P. W. (2002). Up-regulation of components of the renin-angiotensin system in the bile duct-ligated liver. Gastroenterology, 123, 1667–1676. Paizis, G., Gilbert, R. E., Cooper, M. E., Murthi, P., Schembri, J. M., Wu, L. L., Rumble, J. R., Kelly, D. J., Tikellis, C., Cox, A., Smallwood, R. A., & Angus, P. W. (2001). Effect of angiotensin II type 1 receptor blockade on experimental hepatic fibrogenesis. J. Hepatol., 35, 376–385. Pinzani, M. (2002). PDGF and signal transduction in hepatic stellate cells. Front Biosci., 7, d1720– d1726. Pinzani, M., Marra, F., & Carloni, V. (1998). Signal transduction in hepatic stellate cells. Liver, 18, 2–13.
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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 <7.3 Prothrombin time >100 sec Serum creatinine >300 umol/l Encephalopathy grade 3–4
0.81 0.71 0.69 0.83
Nonacetaminophen-induced liver failure Age <10 or >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 <2.5 g/dl or prothrombin time 5 seconds greater than control may indicate an appropriate time for OLT. The onset of life-threatening complications, such as the hepatorenal syndrome, spontaneous bacterial peritonitis, biliary sepsis, and hepatic encephalopathy may warrant immediate attention. More complex tests to determine the mass of functioning hepatocytes have been proposed but none are necessarily superior to the combination of routinely available parameters listed above. In particular, rapidly rising serum bilirubin in the absence of reversible factors in an otherwise stable patient should prompt great concern. With the improvement of results, the question asked was whether OLT should be performed early in the disease process rather than at a later time. It is possible that early OLT might be superior because of fewer complications, shorter hospital stays, better outcomes and lower overall costs. This implies that potential candidates should be referred for evaluation to transplantation centers or expert hepatologists early in their disease course. Recently, additional models have been developed for assessing the severity of liver disease. The Model for End Stage Liver Disease (MELD), which is an index of disease severity, has been applied to adult liver patients (Wiesner et al., 2003). The pediatric version of the model is called PELD. This scoring system is designed to improve organ allocation to assure that available organs are directed to transplant candidates based on the severity of
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liver disease rather than the length of time on waiting lists. The MELD score is calculated by a relatively simple formula using three readily available clinical tests: serum creatinine (Scr; mg/dl) with the maximum acceptable value of 4 mg/dl, total serum bilirubin (Tbil; mg/dl), and an international normalized ratio (INR) of prothrombin time. The MELD score is derived from the following formula: 10{0.957 Ln (Scr) + 0.378 Ln (Tbil) + 1.12 Ln (INR) + 0.643}. The maximum value of the MELD score is 40. Currently, MELD is the universal criterion in the United States for organ allocation for OLT. Patients are placed on the OLT waiting list based on their MELD score.
Contraindications to OLT With increasing experience, contraindications to OLT seem to be declining (see Table 4). Age >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 (<10%) go on to develop chronic rejection. Liver rejection is associated with characteristic histological findings but no specific blood tests are available to monitor the severity or progression of rejection. During acute rejection, portal areas are infiltrated with a mixture of cell-types
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with injury to bile duct cells and central vein endothelial cells. In contrast, chronic rejection is specially characterized by involvement of arterioles and decrease or loss of bile ducts (Klintmalm et al., 1989). There may be bridging fibrosis, accumulation of foamy histiocytes in the endothelium, and focal necrosis of bile ducts. Early rejection usually manifests in the first week or within the first 3 months. The clinical presentations are nonspecific with fever, malaise, confusion, myalgias, tender hepatosplenomegaly or decreased bile flow, and increased serum bilirubin, aminotransferases, GGT and alkaline phosphatase levels. In contrast, chronic rejection may develop at any time and could also evolve from an episode of acute rejection that is poorly responsive to therapy. Clinically, patients may be jaundiced with gradual rise in serum aminotransferases, alkaline phosphatase and GGT. The vanishing bile duct syndrome constitutes another form of chronic rejection, where the clinical course is one of rapid decline. Confirmation of rejection requires liver biopsy. The differential diagnosis of rejection includes causes of cholestatic liver disease, such as biliary obstruction, viral hepatitis, drug toxicity and sepsis. Occasionally, recurrence of PBC in the grafted liver causes significant confusion even at histology because many features of PBC are similar to those seen with rejection. Development of superior immunosuppressive agents has revolutionized organ transplantation. Prior to the cyclosporine era, 1 year-patient survival after OLT was only approximately 30%. The combined use of cyclosporine, corticosteroids and azathioprine with or without antilymphocyte globulin (ATG) has had a tremendous impact upon patient survival (Kahan, 1989). Conventionally, the addition of azathioprine to cyclosporine and corticosteroids appears particularly important. Use of corticosteroids and azathioprine is not devoid of toxicity and sodium retention, hypertension, glucose intolerance, bone demineralization, or pancreatitis may impose limitations. This has led to the replacement of azathioprine by newer drugs such as mycophenolate mofetil and rapamycin (Allison, 2000). More recently, tacrolimus, a macrolide isolated from the fungus Streptomyces tukubaensis, and other immunosuppressive agents have arrived on the scene with further improvements in results. Cyclosporine binds to a low molecular weight cytosolic protein, cyclophilin, and inhibits T lymphocyte activation with decreased cytokine production, such as interleukins-2, 3 and 4, tumor necrosis factor-␣, interferon-␣ and ␥, etc. The structure of tacrolimus is different from cyclosporine and tacrolimus binds to ligands other than cyclophilin but exhibits similar suppression of T lymphocytes and cytokine release (Murase et al., 1990). However, tacrolimus is some 100 times more potent than cyclosporine. A major limitation of current immunosuppressive agents, however, is their nonspecificity. If specific effector limbs of the immune system could be modulated, undesired immunosuppression could be avoided. Investigators are addressing this question
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at present. An additional problem is posed by drug side effects. Nephrotoxicity has been the principal and dose-limiting side effect of cyclosporine. Despite monitoring of serum cyclosporine levels to monitor the dose, decreased glomerular filtration rate and hypertension may be observed. In contrast, tacrolimus is less nephrotoxic but worsens glycemic control, induces alopecia, and shows greater neurotoxicity. New immunosuppresive agents currently under testing include, brequinar sodium, rapamycin, 15-deoxyspergualin, prostaglandin E analogues, monoclonal antibodies targeted toward the T cell receptor, specific cytokines, and T cell costimulatory blockers. Once rejection is confirmed, patients are treated by a bolus of corticosteroids as the first line of treatment. On occasion, more resistant episodes of rejection may call for the monoclonal antibody OKT3 or ATG. The OKT3 monoclonal antibody represents the prototype of several antibodies currently under testing for immunosuppresion and was an important advance (Cosimi et al., 1987). The antibody was designed to deplete specific lymphocyte populations expressing the CD3 antigen and is particularly helpful in controlling acute rejection. Side effects arising from OKT3-mediated T cell activation or cytokine release, infection, or development of anti-OKT3 antibodies may limit repeated use of OKT3 antibody. High dose pulses of corticosteroids are also helpful in controlling acute rejection. It is generally more difficult to salvage patients with chronic rejection and retransplantation may be necessary. It has been recognized that many individuals are capable of progressively decreasing immunosuppression to trivial doses or to discontinue immunosuppression entirely with no rejection. In analyzing the basis of this phenomenon, Starzl and colleagues demonstrated two-way trafficking in multiple organs of antigen-presenting dendritic cells derived from the host and transplanted liver (Starzl et al., 2000). This state of “chimerism” is thought to play significant initiating roles in tolerance. Some support for such a hypothesis is provided by demonstrations of improved allograft tolerance in individuals previously exposed to alloantigens, such as kidney transplantation in polytransfused patients.
OVERALL OUTCOMES With improved surgical techniques, including venovenous bypass, the intraoperative mortality of OLT is similar to routinely performed abdominal surgeries with 30-day perioperative mortality <10% for most patient groups. Retransplantation is necessary in approximately 10% due to primary graft nonfunction or early complications, such as hepatic artery thrombosis.
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The results of OLT in patients with acute liver failure are gratifying, yet many die while waiting for a liver. The 1-year survival rates in OLT recipients with acute liver failure have steadily improved and approach 90% in most transplant centers. The outcomes have been especially good in patients with cholestatic disorders. The results in a variety of metabolic disorders have been excellent. The cumulative results in patients with or without alcoholic liver disease are also quite good with 1-year survival of approximately 85% and 5-year survival of 76% or better. These results have improved recently owing in part to better immunosuppression with tacrolimus. Survival after liver retransplantation due to chronic rejection is also good and approaches greater than 70%. In contrast, outcomes in patients with tumors are not so good. Nonetheless, for any tumor stage, survival in patients with cirrhosis is better after OLT than with resection alone (Yao et al., 2001). As discussed above, all liver recipients with active HBV replication develop reinfection and rapid deterioration. However, graft survival may be improved, particularly in individuals with less significant HBV replication, with either passive immunization through large doses of anti-HBs or repeated doses of hepatitis B immunoglobulin (HBIG) as well as nucleoside analogs (Steinmuller et al., 2002). Approximately one third of all liver transplantations are currently performed for HCV disease and these patients demonstrate excellent survival even though virtually all patients continue to harbor HCV and most develop chronic hepatitis (Brillanti et al., 2002). Use of nucleoside analogs and interferon (during the perioperative period may decrease the prevalence of recurrent viral hepatitis. Development of split liver transplantation raised high hopes, as this was one way to extend the usefulness of the limited supply of donor livers. However, for unexplained reasons, results of split-liver transplantation have been less good, with significant retransplantation rates due to early graft dysfunction or rejection. Whether endothelial damage plays important roles in these processes and whether this could be avoided during split liver transplantation requires further studies. Living-related liver transplantation has been effective in a number of patients and results currently approach those of OLT. However, liver resection in donors is not a trivial procedure, since individuals remain at a significant risk during the resection surgery, and therefore counseling is extremely important. Living-related liver transplantation is proving most popular in communities where OLT is not available, such as in Japan, or when lack of a donor liver is life threatening. With the disproportional increase in the number of patients waiting for an OLT, livingrelated liver transplantation is becoming very frequent in the United States. The overall experience with auxiliary liver transplantation is restricted and requires further analysis.
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SUMMARY AND PROSPECTS Improvements in surgical techniques and immunosuppression have helped liver transplantation evolve into a viable option for increasing numbers of patients. A major impediment is posed by the small supply of donor organs. Efforts are ongoing to define whether reseeding of the liver with hepatocytes could facilitate recovery in patients with acute liver failure and whether metabolic deficiency states could be ameliorated or corrected by transplantation of normal hepatocytes. Bioartificial support systems using various types of liver cells are being tested to demonstrate whether survival in seriously ill patients could be improved to serve as a bridge until a donor organ can be found. Finally, efforts are proceeding to develop xenogeneic sources of livers for transplantation into people.
ACKNOWLEDGMENTS This work was supported in part by NIH grants RO1 DK 46952 and MO1 RR12248.
REFERENCES Akabayashi, A., Nishimori, M., Fujita, M., & Slingsby, B. T. (2003). Living related liver transplantation: A Japanese experience and development of a checklist for donors’ informed consent. Gut, 52, 152. Allison, A. C. (2000). Immunosuppressive drugs: The first 50 years and a glance forward. Immunopharmacology, 47, 63–83. Azoulay, D., Samuel, D., Ichai, P., Castaing, D., Saliba, F., Adam, R., Savier, E., Danaoui, M., Smail, A., Delvart, V., Karam, V., & Bismuth, H. (2001). Auxiliary partial orthotopic vs. standard orthotopic whole liver transplantation for acute liver failure: A reappraisal from a single center by a case-control study. Ann. Surg., 234, 723–731. Bishop, G. A., & McCaughan, G. W. (2001). Immune activation is required for the induction of liver allograft tolerance: Implications for immunosuppressive therapy. Liver Transpl., 7, 161–172. Brillanti, S., Vivarelli, M., De Ruvo, N., Aden, A. A., Camaggi, V., D’Errico, A., Furlini, G., Bellusci, R., Roda, E., & Cavallari, A. (2002). Slowly tapering off steroids protects the graft against hepatitis C recurrence after liver transplantation. Liver Transpl., 8, 884–888. Carceller, A., Blanchard, H., Alvarez, F., St-Vil, D., Bensoussan, A. L., & Di Lorenzo, M. (2000). Past and future of biliary atresia. J. Pediatr. Surg., 35, 717–720. Cosimi, A. B., Cho, S. I., Delmonico, F. L., Kaplan, M. M., Rohrer, R. J., & Jenkins, R. L. (1987). A randomized clinical trial comparing OKT3 and steroids for treatment of allograft rejection. Transplantation, 43, 91–95. Durand, F., Belghiti, J., Handra-Luca, A., Francoz, C., Sauvanet, A., Marcellin, P., Farges, O., Bernuau, J., & Valla, D. (2002). Auxiliary liver transplantation for fulminant hepatitis B: Results from
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a series of six patients with special emphasis on regeneration and recurrence of hepatitis B. Liver Transpl., 8, 701–707. Hassoun, Z., Gores, G. J., & Rosen, C. B. (2002). Preliminary experience with liver transplantation in selected patients with unresectable hilar cholangiocarcinoma. Surg. Oncol. Clin. N. Am., 11, 909–921. Lau, J. Y., Bain, V. G., Davies, S. E., O’Grady, J. G., Alberti, A., Alexander, G. J., & Williams, R. (1992). High-level expression of hepatitis B viral antigens in fibrosing cholestatic hepatitis. Gastroenterology, 102, 956–962. Jabbour, N., Genyk, Y., Mateo, R., Peyre, C., Patel, R. V., Thomas, D., Ralls, P., Palmer, S., Kanel, G., & Selby, R. R. (2001). Live-donor liver transplantation: The USC experience. Acta. Chir. Belg., 101, 220–223. Kahan, B. D. (1989). Cyclosporine. N. Engl. J. Med., 321, 1725. Kalayoglu, M., Sollinger, W. H., Stratta, R. J., D’Alessandro, A. M., Hoffman, R. M., Pirsch, J. D., & Belzer, F. O. (1988). Extended preservation of the liver for clinical transplantation. Lancet, 1, 617–619. Kamath, P. S., Wiesner, R. H., Malinchoc, M., Kremers, W., Therneau, T. M., Kosberg, C. L., D’Amico, G., Dickson, E. R., & Kim, W. R. (2001). A model to predict survival in patients with end-stage liver disease. Hepatology, 33, 464–470. Kim-Schluger, L., Florman, S. S., Schiano, T., O’Rourke, M., Gagliardi, R., Drooker, M., Emre, S., Fishbein, T. M., Sheiner, P. A., Schwartz, M. E., & Miller, C. M. (2002). Quality of life after lobectomy for adult liver transplantation. Transplantation, 73, 1593–1597. Klintmalm, G. B. G., Nery, J. R., & Husberg, B. S. (1989). Rejection in liver transplantation. Hepatology, 10, 978–985. Lopez-Santamaria, M., Migliazza, L., Gamez, M., Murcia, J., Diaz-Gonzalez, M., Camarena, C., Hierro, L., De la Vega, A., Frauca, E., Diaz, M., Jara, P., & Tovar, J. (2000). Liver transplantation in patients with homozygotic familial hypercholesterolemia previously treated by end-to-side portocaval shunt and ileal bypass. J. Pediatr. Surg., 35, 630–633. Lucey, M. R., Merion, R. M., Henley, K. S., Campbell, D. A., Jr., Turcote, J. G., Nostrant, T. T., Blow, F. C., & Beresford, T. P. (1992). Selection for and outcome of liver transplantation in alcoholic liver disease. Gastroenterology, 102, 1736–1741. Lyra, A. C., Fan, X., Lang, D. M., Yusim, K., Ramrakhiani, S., Brunt, E. M., Korber, B., Perelson, A. S., & Di Bisceglie, A. M. (2002). Evolution of hepatitis C viral quasispecies after liver transplantation. Gastroenterology, 123, 1485–1493. Mc Peake, J., & Williams, R. (1995). Liver transplantation for hepatocellular carcinoma. Gut, 36, 644–646. Murase, N., Kim, D. G., Cramer, D. V., Fung, J. J., & Starzl, T. E. (1990). Suppression of allograft rejection with FK 506. I: Prolonged cardiac and liver survival in rats following short course of therapy. Transplantation, 50, 186–189. O’Grady, J. G., Smith, H. M., Davies, S., Daniels, H. M., Donaldson, P. T., Tan, K. C., Portmann, B., Alexander, G. J., & Williams, R. (1992). Hepatitis B virus reinfection after orthotopic liver transplantation: Serological and clinicall implications. J. Hepatol., 14, 104–111. Opelz, G., Wujciak, T., Dohler, B., Scherer, S., & Mytilineos, J. (1999). HLA compatibility and organ transplant survival. Collaborative Transplant Study. Rev. Immunogenet., 1, 334–342. Otte, J. B., Goyet, D. V., Alberti, D., Balladur, P., & Hemptinne, B. D. (1990). The concept and technique of split liver transplantation. Surgery, 107, 605–612. Schilsky, M. L., Sheinberg, I. H., & Sternlieb, I. (1994). Liver transplantation for Wilson’s disease: Indications and outcome. Hepatology, 19, 583–587.
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Starzl, T. E., Marchioro, T. L., von Kaulla, K., Herman, G., Brittain, R. S., & Waddel, W. R. (1963). Homotransplantation of the liver in humans. Surg. Gynecol. Obstet., 117, 659. Starzl, T. E., Murase, N., Demetris, A., Trucco, M., & Fung, J. (2000). The mystique of hepatic tolerogenicity. Semin. Liver. Dis., 20, 497–510. Steinmuller, T., Seehofer, D., Rayes, N., Muller, A. R., Settmacher, U., Jonas, S., Neuhaus, R., Berg, T., Hopf, U., & Neuhaus, P. (2002). Increasing applicability of liver transplantation for patients with hepatitis B-related liver disease. Hepatology, 35, 1528–1535. Terpstra, O. T., Reurers, C. B., & Schalm, S. W. (1988). Auxiliary heterotopic liver transplantation. Transplantation, 45, 1003–1007. Wiesner, R., Edwards, E., Freeman, R., Harper, A., Kim, R., Kamath, P., Kremers, W., Lake, J., Howard, T., Merion, R. M., Wolfe, R. A., & Krom, R. (2003). Model for end-stage liver disease (MELD) and allocation of donor livers. Gastroenterology, 124, 91–96. Weisner, R. H., Porayko, M. K., Dickson, R. E., Gores, G. J., LaRusso, N. F., Hay, J. E., Whalstrom, H. E., & Krom, R. A. F. (1992). Selection and timing of liver transplantation in primary biliary cirrhosis and primary sclerosing cholangitis. Hepatology, 16, 1290–1299. Wilde, J., Teixeira, P., Bramhall, S. R., Gunson, B., Mutimer, D., & Mirza, D. F. (2002). Liver transplantation in haemophilia. Br. J. Haematol., 117, 952–956. Yao, F. Y., Ferrell, L., Bass, N. M., Watson, J. J., Bacchetti, P., Venook, A., Ascher, N. L., & Roberts, J. P. (2001). Liver transplantation for hepatocellular carcinoma: Expansion of the tumor size limits does not adversely impact survival. Hepatology, 33, 1394–1403.
21.
BIOLOGICAL PRINCIPLES AND NOVEL THERAPIES IN LIVER CELL TRANSPLANTATION
Sanjeev Gupta, Mari Inada, Vinay Kumaran and Brigid Joseph INTRODUCTION Advances in molecular and cell biology have made it possible to isolate specific types of cells and manipulate these in culture if necessary for cell therapy. These methods raise multiple issues, including the biological properties of individual cell types and their in vitro and in vivo behavior after isolation from organs. Significant progress has been made concerning the ability of transplanted cells to engraft, proliferate and function in the liver. Progress in stem cell biology has also begun to provide important clues about the role of stem cells in fetal development, during diseases affecting individual organs, as well as for cell therapy. Many issues in stem cell biology, including identification of stem cells in specific organs, expansion of stem cell populations, and mechanisms of stem cell plasticity by which stem cells of one type, e.g. hematopoietic stem cells, differentiate into other types of mature cells, e.g. liver, muscle, bone, neuronal cells, etc., are not yet understood. The liver offers an excellent paradigm for obtaining insights into stem cell biology and cell therapy. The liver is the body’s factory for protein synthesis, a major defense system against dietary toxins, chemicals, toxic metabolites, drugs,
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as well as a system to process antigens and participate in host immune responses. The regenerative capacity of the liver has been known since ancient times, and represents a unique example of the body’s capacity for healing and self-repair. Impaired liver function has major effects on the body, e.g. acute hepatitis may lead to death due to hepatic insufficiency, whereas defective gene expression in the liver may lead to multiple consequences, including permanent brain injury (e.g. due to the accumulation of unconjugated bilirubin), cardiovascular damage (e.g. due to impaired low density lipoprotein receptor activity and hypercholesterolemia), renal failure (e.g. due to oxalosis), or coagulation abnormalities (e.g. hemophilia A with deficiency of coagulation factor VIII). Therefore, liver-directed cell therapy has a direct bearing on multiple genetic, metabolic and deficiency states (Gupta & Roy Chowdhury, 2002).
CELLULAR ORGANIZATION OF THE LIVER The human liver originates from the foregut endoderm after four weeks of gestation and the adult organization of the organ becomes manifest during the subsequent weeks with production of bile by 14 weeks of gestation, indicating establishment of hepatobiliary function. Formation of hepatocytes requires that embryonic cells are first “specified” by transcription factor switches regulating gene expression, e.g. hepatocyte nuclear factor-3, followed by the activation of a cascade of additional genes that serve roles in “differentiation” of specified cells into mature hepatocytes and other liver cell types (Zaret, 2002). In the adult liver, hepatocytes are the most abundant cell type (∼60%), although the fetal liver is a major site for extramedullary hematopoeisis, and hematopoietic cells constitute approximately 50% of the fetal liver. Other liver cell types include bile duct cells, stellate cells located in the space of Disse, which is juxtaposed between hepatocytes and liver sinusoids, endothelial cells lining liver sinusoids, fibroblasts, pit cells, and Kupffer cells. The hepatic stroma originates from the primitive cardiac mesoderm and development of endothelial cells is critical for hepatocytes at this stage. Optimal hepatic function requires complex interactions between various liver cell types, as well as signals arriving in the liver via the systemic circulation or through neural transmission. Each liver cell type may impact liver function in health and disease. The regulation of liver gene expression is an important reflection of how cell function is affected. For instance, hepatocytes during fetal development, express genes that may be replaced after birth by other genes, e.g. ␣-fetoprotein during the fetal stage is replaced by albumin and expression of biliary genes in fetal hepatoblasts is extinguished in mature hepatocytes. Such profile changes in gene expression affords one way in which to determine whether stem/progenitor
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cells are activated in the liver during pathophysiological states. On the other hand, loss of expression of genes found in embryonic cells and gain of expression of genes found in mature liver cells provide ways to demonstrate whether stem cells have begun to differentiate along hepatic lineages. Such findings are used widely to establish the potential of stem cells, both in cell culture and in intact animals.
THE ROLE OF LIVER STEM CELLS The liver can regenerate after surgical resection of up to 70% of the liver with DNA synthesis and hypertrophy of existing hepatocytes (Michalopoulos & DeFrances, 1997). During this process of liver regeneration following partial hepatectomy, individual hepatocytes need not undergo more than 2–3 cycles of DNA synthesis. In general, mature hepatocytes are difficult to propagate in culture conditions, in agreement with the constraints on somatic cells, as enunciated by the Hayflick limit. This limit predicts that the replication potential of cells is progressively attenuated, such that after 40–50 cell doublings, cells begin to enter a stage of replicative senescence. Indeed, mature hepatocytes exhibit similar features, e.g. after partial hepatectomy, remnant hepatocytes show decreased cell proliferation capacity and evidence for greater p21 expression, senescence-associated -galactosidase activity, and accumulation of lipid peroxidation products with cytoplasmic complexity, which are features of replicative senescence (Sigal et al., 1999). However, the potential of mature hepatocytes is different under in vivo conditions. For instance, serial transplantation studies in fumarylacetoacetate hydrolase (FAH) mutant mice, where accumulation of tyrosine leads to progressive hepatic injury and provides a potent stimulus for proliferation of normal hepatocytes, showed that transplanted cells can proliferate indefinitely, repopulating the liver of recipient animals down over seven generations (Overturf et al., 1997). These studies established that hepatocytes can undergo more than 80 cell divisions without the loss of replication potential, which is a “stem cell-like” property. The liver harbors stem/progenitor cells, especially during the fetal stage (Shafritz & Dabeva, 2002). Cell lines have occasionally been derived from the fetal liver, e.g. HBC-3 cells from the 9.5 d embryonic mouse liver with differentiation along biliary and hepatocyte lineages suggesting progenitor cell-properties (Ott et al., 1999). Similarly, progenitor cell populations have been isolated from the fetal rat liver (Dabeva et al., 2000) whose cells differentiate into hepatocytes after transplantation into tissue-type matched recipients. Fetal liver cells complete differentiation programs after transplantation, and acquire cytochrome P450 activity, which reflects advanced hepatic differentiation. Moreover, fetal rat hepatoblasts showed
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greater replication potential in the intact liver compared with mature hepatocytes (Sandhu et al., 2001). Similarly, epithelial cells can be isolated from the fetal human liver (Malhi et al., 2002) which exhibit a variety of markers observed in liver progenitor cells, including ␣-fetoprotein, ␥-glutamyltranspeptidase, plasminogen activator inhibitor type-1, and various cytokeratins, etc., as well as the capacity to proliferate extensively in cell culture, and to produce mature hepatocytes following transplantation in immunodeficient animals. However, fetal human liver cells are unable to proliferate indefinitely in cell culture and begin to show shortening of telomere length, a feature of aging. On the other hand, when telomerase activity was reconstituted in fetal human liver cells, populations of cells arose with indefinite replication potential and cells became “immortal.” These cells continued to exhibit the stem/progenitor properties of the original cells, with multilineage gene expression patterns. Moreover, in response to the introduction and expression of the Pdx-1 gene, which is a master switch regulating insulin expression in pancreatic  cells, immortalized human fetal liver cells began to express insulin and corrected diabetes mellitus in mice (Zalzman et al., 2003). These findings are in line with the presence of stem/progenitor cells in the fetal liver. The adult liver contains different types of progenitor cells, including cells that have been designated “oval cells,” which originally referred to poorly differentiated cholangiolar cells with an oval shaped nucleus that arose in response to carcinogenic treatments. Oval cells become apparent during liver regeneration induced by chemicals, e.g. carbon tetrachloride, D-galactosamine (GalN), etc., and during acute liver injury or chronic viral hepatitis in humans (Sell, 2001). Oval cells display a variety of hepatocytic and biliary markers, including albumin, ␣-fetoprotein, glycogen, glucose-6-phosphatase, and hybrid isoenzymes. Primary oval cells isolated from the normal rat liver, as well as from the diseased liver of LEC rats with copper toxicosis can generate mature hepatocytes (Malouf et al., 2001; Yasui et al., 1997). Similarly, cell lines isolated from the rat liver, with oval cell properties, generate hepatocytes following transplantation in animals, although cell differentiation is dependent on microenvironmental cues. The liver microenvironment promoted differentiation of transplanted cells into hepatocytes, whereas extrahepatic sites, such as spleen and peritoneal cavity, were less effective in inducing hepatic differentiation. Interestingly, oval cell-like ductular cells from the rodent pancreas can produce mature hepatocytes (Wang et al., 2001), which is again in agreement with the notion of lineage relationships between endodermallyderived liver and pancreatic cells. Whether stem cells isolated from other organs, notably hematopoeitic stem cells (HSC) from the bone marrow, peripheral blood or umbilical cord blood, and mesenchymal stem cells, can differentiate into hepatocytes has aroused considerable interest. For instance, Petersen and colleagues initially demonstrated
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that bone marrow-derived hematopoiatic stem cells (HSC) differentiated into hepatocytes (Petersen et al., 1999). This study was subsequently extended in the mouse, as well as humans, where several groups established the presence of liver cells originating from donor-derived HSC (Korbling et al., 2002; Lagasse et al., 2000; Theise et al., 2000). Transplantation studies of HSC in the FAH mouse were particularly convincing in respect of the potential of these cells for therapeutic liver repopulation (Lagasse et al., 2000). However, subsequent studies revealed additional aspects of the plasticity of HSC; in particular, the efficiency with which HSC produce hepatocytes or other non-hematopoietic cell types is quite limited, certainly in normal organs, thus suggesting that circulating stem/progenitor cells are not likely to be a major source of stem cell replenishment under physiological conditions. On the other hand, while diseased organs do seem to incorporate HSC, at least in the FAH mouse, this appears to be largely due to fusion of HSC with native hepatocytes (Wang et al., 2003). During cell fusion, aneuploidy was also observed. Once again, this raises questions about enhanced potential for oncogenesis in fused cells. Therefore, while HSC can be readily obtained from adults, the question that remains to be answered is whether these cells will be helpful for clinical applications in liver-directed cell therapy.
REPLACEMENT OF THE LIVER WITH TRANSPLANTED CELLS AND STUDY OF BIOLOGICAL MECHANISMS In order to demonstrate whether transplanted cells can repopulate the liver for therapeutic purposes, it is necessary to establish the fate of cells in intact animals. Although stem/progenitor cells express major histocompatibility complex (MHC) antigens less efficiently, it is unknown whether these cells will be tolerated better in comparison with mature hepatocytes, which are rejected in allogeneic recipients with first order kinetics. Therefore, analysis of transplanted cell engraftment and survival is best studied in syngenic or congenic animals that share MHC with no rejection of cells. However, to identify transplanted cells in the liver, unique genetic markers are required. This has been accomplished in many ways, including the use of donor cells expressing unique transgenes, mutant recipients with specific deficiencies in gene expressiion, or use of unique chromosomal or protein markers in the case of human cells. The list of animal models for studying transplanted cell survival in the liver is now fairly long (Gupta & Roy Chowdhury, 2002). In early studies, cells were transplanted into ectopic sites, notably the spleen, which shares a sinusoidal structure with the liver, and the peritoneal cavity, where cells required additional anchorage to extracellular matrix components. The studies showed that transplanted hepatocytes could survive and function in these
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extrahepatic sites in the short-term, along with secretion of proteins or transgene products into the bloodstream. However, analysis of gene expression profiles in transplanted cells showed that hepatic genes were regulated most efficiently in the liver itself, and the peritoneal cavity was less effective (Gupta et al., 1994). Several possible reasons for this site-specificity in gene regulation include the absence of cell-cell interactions and portal blood components available in the liver. Another is differences in the composition of soluble cytokines in the peritoneal cavity. It should be added that transplanted cells in the liver failed to evoke immune responses against soluble transgene products, which was in contrast with cell transplantation in the peritoneal cavity. Importantly, transplanted cells were found to survive lifelong in syngeneic mouse and rat recipients, indicating that, in principle, liverdirected cell therapy could be effective for an entire life-time.
THE STUDY OF BIOLOGICAL MECHANISMS Many investigators have used the dipeptidyl peptidase 4 deficient (dpp4-) rat for studying transplanted cell biology (Gupta et al., 1995). Dpp4 is an ectopeptidase of uncertain significance but with roles extending to lymphocyte function and peptide processing. Since the protein is expressed abundantly in the bile canalicular domains of hepatocytes, it has provided ways in which to particularly test whether transplanted cells are integrated in liver parenchyma with the reconstitution of plasma membrane structures. It has become clear that transplanted hepatocytes engraft in the liver permanently and do not proliferate in normal liver. Transplantation is most effective when these cells are deposited in liver sinusoids. Again, due to differences in the size of hepatocytes (20–40 m) and liver sinusoids (3–6 m), transplanted cells are entrapped within hepatic sinusoids, without escaping into the subsequent vascular bed represented by the pulmonary capillaries. Thus, deleterious complications arising from pulmonary embolization are avoided. However, the presence of cells in hepatic sinusoids leads to an increase in portal pressure due to greater vascular impedance. This is associated with early onset of ischemic events in the liver lobule and microcirculatory cessation in some parts of the liver lobule, although portal pressures return to normal within 1–3 hours and the integrity of the hepatic microcirculation is restored thereafter. Nonetheless, early ischemia in the liver does lead to disturbances in Kupffer cell function, as well as other liver cell types, such as endothelial and stellate cells. Kupffer cell activation is associated with a deleterious effect on engraftment of transplanted cells. However, if Kupffer cell activation is blocked, cell engraftment improves but only 20–30% of the transplanted hepatocytes are eventually engrafted in the liver (Joseph et al., 2002).
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A central mechanism directing engraftment of liver cells in the parenchyma involves liver endothelial cells, which must be disrupted during translocation of hepatocytes from the sinusoids to the liver plate (Malhi et al., 2002). When endothelial cells are injured, transplanted cells engraft in a superior fashion. Subsequently, the transplanted hepatocytes develop conjoint bile canaliculi and gap junctions with adjacent native hepatocytes. It is this ability of transplanted cells to incoporate in to the liver parenchyma that has been exploited to understand the basis of position-specific differences in liver gene regulation. Whereas certain genes are expressed more abundantly in the periportal areas, other genes are well expressed in the perivenous areas of the liver lobule. One hypothesis put forward to account for these differences was that younger hepatocytes in periportal areas expressed fewer genes, and as hepatocytes became more mature during transition toward perivenous areas, more genes were expressed. However, transplantation studies showed that hepatocytes did not migrate from the periportal area toward the perivenous area. Gene expression in hepatocytes was found to be a function of microenvironmental differences, and not due to differences in the age of cells. Transplanted cells engraft well in animals with acute liver injury, as well as in animals with cirrhosis and significant hepatic fibrosis. In the normal liver, transplanted cells respond appropriately to proliferative stimuli with transient DNA synthesis following acute liver injury. There also is more persistent cell proliferation in response to chronic injury affecting native hepatocytes but sparing transplanted hepatocytes. In animals with acute liver injury or cirrhosis, transplanted cells show some proliferation, depending mostly on the balance between injury and proliferation in native hepatocytes. On the whole, in the normal liver, transplantation of cells in one session permits repopulation of approximately 0.5–2% of the liver, which can be increased to approximately 5–7% of the liver by transplanting cells repeatedly (Rajvanshi et al., 1996). On the other hand, correction of specific disorders may require a greater magnitude of liver repopulation, which has led to efforts at understanding the regulation of more extensive liver repopulation with transplanted hepatocytes. The most significant concept in inducing transplanted cell proliferation involves selective ablation of native hepatocytes with chemicals, hepatotoxic trangenes, or other types of injury. For instance, extensive liver repopulation occurs in animals with hepatic expression of urokinase-type plasminogen activator (albuPA mouse) (Rhim et al., 1994), the FAH mouse discussed briefly above, which models hereditary tyrosinemia type 1, Fas ligand-induced apoptosis, prodrug activation via herpes simplex virus thymidine kinase (HSV-TK), expression of the cell cycle regulator Mad1, and use of toxic bile salts in mice deficient in mdr 2 gene, which impairs biliary phospholipid excretion with hepatobiliary injury
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(Gupta & Roy Chowdhury, 2002). Genotoxic hepatic damage has been helpful for liver repopulation. For instance, the pyrollizidine alkaloid, retrorsine, inhibits hepatocellular proliferation, and has been highly effective for liver repopulation in rodents (Laconi et al., 1998). More recently, another pyrollizidine alkaloid, monocrotaline, which has effects on hepatocytes, as well as hepatic endothelial cells, was effective for inducing transplanted cell proliferation. These toxic chemicals are unsuitable for clinical use. However, genotoxic liver injury with radiation and partial hepatectomy or ischemia-reperfusion injury has been effective for liver repopulation in rats and offers more suitable clinical strategies for cell therapy (Malhi et al., 2002). Overall, if transplanted cells become integrated in the liver parenchyma in juxtaposition to other liver cell types, i.e. Kupffer cells, stellate cells, and endothelial cells, it should be possible to address mechanisms in cell-cell interactions directing the fate of transplanted hepatocytes. Such studies are at an early stage but should advance our insights into the biology of these various cell types. The knowledge of the fate of rodent cells in the liver provides a basis for demonstrating whether specific populations of human hepatocytes can be similarly shown to engraft and proliferate in the liver of immunodeficient animals capable of tolerating xenografts before embarking on their clinical use.
THERAPEUTIC POTENTIAL OF LIVER CELL TRANSPLANTATION In principle, hepatocyte transplantation is considered suitable for treating liver failure, chronic liver disease, various metabolic deficiency states, coagulation disorders, and other conditions (see Table 1). Take, for example, metabolic deficiency states. These are an important target especially since the liver itself may not be affected at all. The Crigler-Najjar syndrome type 1 was among the first metabolic diseases to be treated with hepatocyte transplantation (Fox et al., 1998). The disorder arises from deficiency in bilirubin-UGT1A1 enzyme activity, which results in the accumulation of unconjugated bilirubin to neurotoxic levels. Page In one young girl with the CriglerNajjar syndrome, type I, transplantation of allogenic human hepotocytes resulted in a substantial decline in bilirubin levels and the requirement for phototherapy also decreased. Such results are analogous to those seen in Gunn rats afflicted with a similar genetic lesion. Patients with ornithine transcarbamylase deficiency, a glycogen storage disorder, and patients with ␣-1 antitrypsin deficiency have shown improvement following hepatocyte transplantation (Muraca et al., 2002; Strom et al., 1997a, b). In several
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Table 1. Candidate Conditions Suitable for Liver Cell Therapy. Genetic Disorders • Congenital hyperbilirubinemia, e.g. Crigler-Najjar syndrome • Defects of carbohydrate metabolism • ␣–1 antitrypsin deficiency • Erythropoietic protoporphyria • Familial hypercholesterolemia • Hyperammoniamia syndromes • Lipidoses, e.g. Niemann-Pick disease • Oxalosis • Tyrosinemia, type 1 • Wilson’s disease • Hemophilia A • Factor IX deficiency • Hereditary angioedema • Thrombotic thrombocytopenic purpura Acquired Disorders • Acute liver failure • Chronic viral hepatitis • Cirrhosis and liver failure
patients known to have familial hypercholesterolemia, the transplantation of genetically modified autologous hepatocytes led to a decline in blood cholesterol levels. This was not unexpected in the light of preclinical studies using the Watanabe heritable hyperlipidemia rabbit model with mutated low density lipoprotein receptor gene, similar to patients with familial hypercholesterolemia (Grossman et al., 1995). As specific strategies become available to increase the mass of transplanted liver cells in people, future studies could potentially be more successful. There are a number of other animal models of metabolic disorders, including the Long-Evans Cinnamon (LEC) rat, which is used as a model for Wilson’s disease, the FAH mouse, used as a model for hereditary tyrosinemia, type-1, and the mdr-2 knockout mice, used as a model for progressive familial intrahepatic cholestasis. In all of them, hepatocyte transplantation has been curative of the liver disease (Gupta & Roy Chowdhury, 2002). In the case of patients with acute liver failure, the unique challenge is to overcome the complications which they often have, namely acute illness, multi-organ failure or raised intracranial pressure. Animal models for acute liver failure have been difficult to assess on account of the variability in the mortality rate in these animals. Altogether, improved mortality in acute liver failure cannot be attributed to cell transplantation because the data are either limited or are not unequivocal. Thus,
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no firm conclusions can be drawn about the efficacy of hepatocyte transplantation in acute liver failure. On the other hand, it is already evident that the treatment of hepatic encephalopathy in rats employing cell transplantation leads to an improvement in mental status and a greater capacity to handle ammonia. And there are indications that the deposition of hepatocytes in the spleen of terminally ill cirrhotic rats is accompanied by improvements in liver chemistry tests, coagulation abnormalities, and outcomes (Nagata et al., 2003). It is scarsely necessary to say that more studies of this type are essential before cell transplantation in patients with chronic liver disease is attempted. Many benefits might reasonably be expected since a large number of patients are unable to undergo orthotopic liver transplantation (OLT), require repeated hospitalization and suffer from poor quality of life.
ACKNOWLEDGMENTS This work was supported in part by NIH grants R01DK46952, P33DK41296, and MO1 RR12248.
REFERENCES Dabeva, M. D., Petkov, P. M., Sandhu, J., Oren, R., Laconi, E., Hurston, E., & Shafritz, D. A. (2000). Proliferation and differentiation of fetal liver epithelial progenitor cells after transplantation into adult rat liver. Am. J. Pathol., 156, 2017–2031. Fox, I. J., Roy Chowdhury, J., Kaufman, S. S., Goertzen, T. C., Chowdhury, N. R., Warkentin, P. I., Dorko, K., Sauter, B. V., & Strom, S. C. (1998). Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation. N. Engl. J. Med., 338, 1422–1426. Grossman, M., Rader, D. J., Muller, D. W. M., Kolansky, D. M., Kozarsky, K., Clark, B. J., III, Stein, E. A., Lupien, P. J., Bryan Brewer, H., Jr, Raper, S. E., & Wilson, J. M. (1995). A pilot study of ex vivo gene therapy for homozygous familiar hypercholesterolaemia. Nature Med., 1, 1148–1154. Gupta, S., Rajvanshi, P., & Lee, C.-D. (1995). Integration of transplanted hepatocytes in host liver plates demonstrated with dipeptidyl peptidase IV deficient rats. Proc. Natl. Acad. Sci. USA, 92, 5860–5864. Gupta, S., & Roy Chowdhury, J. (2002). Therapeutic potential of hepatocyte transplantation. Semin. Cell. Dev. Biol, 13, 439–446. Gupta, S., Vemuru, R. P., Lee, C-D., Yerneni, P., Aragona, E., & Burk, R. D. (1994). Hepatocytes exhibit superior transgene expression after transplantation into liver and spleen compared with peritoneal cavity or dorsal fat pad: Implications for hepatic gene therapy. Human Gene Ther., 5, 959–967. Joseph, B., Malhi, H., Bhargava, K. K., Palestro, C. J., McCuskey, R. S., & Gupta, S. (2002). Kupffer cells participate in early clearance of syngeneic hepatocytes transplanted in the rat liver. Gastroenterology, 123, 1677–1685.
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Korbling, M., Katz, R. L., Khanna, A., Ruifrok, A. C., Rondon, G., Albitar, M., Champlin, R. E., & Estrov, Z. (2002). Hepatocytes and epithelial cells of donor origin in recipients of peripheralblood stem cells. N. Engl. J. Med., 346, 738–746. Laconi, E., Oren, R., Mukhopadhyay, D. K., Hurston, H., Laconi, S., Pani, P., Dabeva, M., & Shafritz, D. A. (1998). Long-term, near-total liver replacement by transplantation of isolated hepatocytes in rats treated with retrorsine. Am. J. Pathol., 153, 319–329. Lagasse, E., Connors, H., Al Dhalimy, M., Reitsma, M., Dohse, M., Osborne, L., Wang, X., Finegold, M., Weissman, I. L., & Grompe, M. (2000). Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat. Med., 6, 1229–1234. Malhi, H., Annamaneni, P., Slehria, S., Joseph, B., Bhargava, K. K., Palestro, C. J., Novikoff, P. M., & Gupta, S. (2002). Cyclophosphamide disrupts hepatic sinusoidal endothelium and improves transplanted cell engraftment in rat liver. Hepatology, 36, 112–121. Malhi, H., Gorla, G. R., Irani, A. N., Annamaneni, P., & Gupta, S. (2002). Cell transplantation after oxidative hepatic preconditioning with radiation and ischemia-reperfusion leads to extensive liver repopulation. Proc. Natl. Acad. Sci. USA, 99, 13114–13119. Malhi, H., Irani, A. N., Gagandeep, S., & Gupta, S. (2002). Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes. J. Cell. Sci, 115, 2679–2688. Malouf, N. N., Coleman, W. B., Grisham, J. W., Lininger, R. A., Madden, V. J., Sproul, M., & Anderson, P. A. (2001). Adult-derived stem cells from the liver become myocytes in the heart in vivo. Am. J. Pathol., 158, 1929–1935. Michalopoulos, G., & DeFrances, M. C. (1997). Liver regeneration. Science, 276, 60–66. Muraca, M., Gerunda, G., Neri, D., Vilei, M. T., Granato, A., Feltracco, P., Meroni, M., Giron, G., & Burlina, A. B. (2002). Hepatocyte transplantation as a treatment for glycogen storage disease type 1a. Lancet, 359, 317–318. Nagata, H., Ito, M., Cai, J., Edge, A. S., Platt, J. L., & Fox, I. J. (2003). Treatment of cirrhosis and liver failure in rats by hepatocyte xenotransplantation. Gastroenterology, 124, 422–431. Ott, M., Ma, Q., Li, B., Gagandeep, S., Rogler, L. E., & Gupta, S. (1999). Regulation of hepatitis B virus expression in progenitor and differentiated cell-types: Evidence for negative transcriptional control in nonpermissive cells. Gene Expression, 8, 175–186. Overturf, K., Al-Dhalimy, M., Ou, C-N., Finegold, M., & Grompe, M. (1997). Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes. Am. J. Pathol., 151, 1273–1280. Petersen, B. E., Bowen, W. C., Patrene, K. D., Mars, W. M., Sullivan, A. K., Murase, N., Boggs, S. S., Greenberger, J. S., & Goff, JP. (1999). Bone marrow as a potential source of hepatic oval cells. Science, 284, 1168–1170. Rajvanshi, P., Kerr, A., Bhargava, K. K., Burk, R. D., & Gupta, S. (1996). Efficacy and safety of repeated hepatocyte transplantation for significant liver repopulation in rodents. Gastroenterology, 111, 1092–1102. Rhim, J. A., Sandgren, E. P., Degen, J. L., Palmiter, R. D., & Brinster, R. L. (1994). Replacement of diseased mouse liver by hepatic cell transplantation. Science, 263, 1149–1152. Sandhu, J. S., Petkov, P. M., Dabeva, M. D., & Shafritz, D. A. (2001). Stem cell properties and repopulation of the rat liver by fetal liver epithelial progenitor cells. Am. J. Pathol., 159, 1323–1334. Sell, S. (2001). Heterogeneity and plasticity of hepatocyte lineage cells. Hepatology, 33, 738–750. Shafritz, D. A., & Dabeva, M. D. (2002). Liver stem cells and model systems for liver repopulation. J. Hepatol., 36, 552–564.
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Sigal, S. H., Rajvanshi, P., Gorla, G. R., Saxena, R., Sokhi, R. P., Gebhardt, D. F., Jr., Reid, L. M., & Gupta, S. (1999). Partial hepatectomy-induced polyploidy attenuates hepatocyte replication and activates cell aging events. Am. J. Physiol., 276, G1260–G1272. Strom, S. C., Fisher, R. A., Rubinstein, W. S., Barranger, J. A., Towbin, R. B., Charron, M., Mieles, L., Pisarov, L. A., Dorko, K., Thompson, M. T., & Reyes, J. (1997). Transplantation of human hepatocytes. Transplant. Proc., 29, 2103–2106. Strom, S. C., Fisher, R. A., Thompson, M. T., Sanyal, A. J., Cole, P. E., Ham, J. M., & Posner, M. P. (1997). Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure. Transplantation, 63, 559–569. Theise, N. D., Badve, S., Saxena, R., Henegariu, O., Sell, S., Crawford, J. M., & Krause, D. S. (2000). Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology, 31, 235–240. Wang, X., Al-Dhalimy, M., Lagasse, E., Finegold, M., & Grompe, M. (2001). Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells. Am. J. Pathol., 158, 571–579. Wang, X., Willenbring, H., Akkari, Y., Torimaru, Y., Foster, M., Al-Dhalimy, M., Lagasse, E., Finegold, M., Olson, S., & Grompe, M. (2003). Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature, 422, 897–901. Yasui, O., Miura, N., Terada, K., Kawarada, Y., Koyama, K., & Sugiyama, T. (1997). Isolation of oval cells from Long-Evans Cinnamon rats and their transformation into hepatocytes in vivo in the rat liver. Hepatology, 25, 329–334. Zalzman, M., Gupta, S., Giri, R. K., Berkovich, I., Sappal, B. S., Karnieli, O., Zern, M. A., Fleischer, N., & Efrat, S. (2003). Reversal of hyperglycemia in mice using human expandable insulinproducing cells differentiated from fetal liver progenitor cells. Proc. Natl. Acad. Sci. USA, 100, 7253–7258. Zaret, K. S. (2002). Regulatory phases of early liver development: Paradigms of organogenesis. Nat. Rev. Genet., 3, 499–512.
22.
FLUID TRANSPORT IN THE GALLBLADDER
Joar Svanvik and Bengt Nilsson INTRODUCTION Reviews concerning the subject of water and electrolyte movements in the gallbladder are available (Diamond, 1968; Reuss, 1991; Reuss et al., 2000; Rose, 1987; Svanvik, 1993). Although the subject falls within the purview of epithelial cell transport, it is important in relation to pathobiology, such as the study of ion transport during gallstone formation (Moser et al., 2000) and cystic fibrosis (Lazarowski et al., 2001) and hepatobiliary tract disorders.
FLUID ABSORPTION BY THE GALLBLADDER MUCOSA Transport of Na+ and Cl− Sodium enters the epithelial cells of the gallbladder passively across the luminal membrane, that is, down the electrochemical gradient which is maintained by the active extrusion of Na+ across the basolateral membrane by an Na+ -K+ -ATPase pump. The rate of transcellular sodium transport depends on the rate of sodium entry across the luminal cell membrane. Influx studies in rabbit gallbladder (Duffey et al., 1978) have demonstrated non-diffusional Na+ entry involving a one to one electrically neutral coupled entry for Na+ and Cl− . The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 555–575 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15022-8
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Fig. 1. The Mechanism of Na+ and Cl− Transport by the Epithelial Cells of the Gallbladder. Note: At the apical side of the cell membrane there is double ion exchange mediating + − counter-transport of Na+ /H+ and Cl− /HCO− 3 . Cremashi et al. (1992) have shown a Na Cl + + − symport and Na -K -2Cl cotransport occur. At the basolateral cell membrane there is active extrusion of Na+ by a Na+ -K+ ATPase pump. Sodium entry across the luminal cell membrane is regarded as the rate-limiting step in transcellular Na+ transport.
NaCl and fluid absorption is enhanced by bicarbonate (Heintze et al., 1979) which is required for the maintenance of intracellular [H+ ] activity, and, in turn, maintaining an Na+ /H+ exchange. This indicates the presence of a double ion exchange mediating parallel counter-transport of Na+ /H+ and Cl− /HCO3 − in the apical membrane (Heintze et al., 1981), as illustrated in Fig. 1. The Na+ /H+ exchange is brought about by the NHE-3 exchanger isoform in the rabbit (Silviano et al., 1996) and human gallbladder (Silviano et al., 1997). In calf gallbladder (Bazzini et al., 2001), the NHE1 and NHE3 Na+ /H+ exchangers are important, while NHE2 and NHE3 exchangers are present in the prairie dog gallbladder (Abedin et al., 2001). The Na+ and Cl− fluxes across the apical membrane may be dissociated which means that the membrane transporters are not obligatorily linked (Reuss, 1984) Further analysis of the apical influx shows two additional forms of neutral Na+ : Cl− : coupled transport: hydrochlorothiazide-sensitive Na+ , Cl− symport and
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bumetanide-sensitive Na+ : K+ : 2Cl− cotransport (Cremashi et al., 1992). When cAMP levels are high, entry occurs by Na+ :Cl− -coupled transport and, when cAMP levels are low, Na+ /H+ , Cl− /HCO3− double exchange predominates. Na+ exit from the cell against its electrochemical gradients is mediated by an Na+ -K+ -ATPase system whose activity correlates directly with the rate of fluid transport (Van Os & Slegers, 1972). Its activity is dependent on intracellular Na+ activity which in turn depends on Na+ entry across the luminal membrane. This represents the rate limiting step of transepithelial Na+ transport (Graf & Giebisch, 1979). Ions are also able to move across epithelia by means of the paracellular pathway. Fromter and Diamond (1972) showed that the this route in Necturus gallbladder accounted for some 97% of passive ion permeation. Their survey of different epithelia revealed that there were marked differences in junctional tightness on the basis on which they classified epithelia as “tight” or “leaky.” Leakiness in gallbladder tight junctions has been demonstrated by also using electron microscopy (Machen et al., 1972). Shachar and Hill (2002) have suggested that a major fraction of water traverses the paracellular route during isotonic fluid transfer. Based on the theory and experimental results derived from extracellular probe studies, they concluded that paracellular probe flows are not due to simple convection generated by osmotic flow through the junctions but are generated by active fluid transport within the junction; that is, a mechano-osmotic process. The geometry of the pathway involved would indicate that some salt accompanies the paracellular fluid, representing a hypoosmotic flow. Transport of salt by the cell route, which may be accompanied by some water, represents hypertonic flow. The problem then is one of balancing the two to produce an isotonic fluid. Using recent data from knockout mice, they suggest that aquaporins could be functioning in different epithelial tissues as osmo-comparators within a feedback loop that regulates the paracellular fluid flow rate. Transport of H+ and HCO3− It has been known since long that bile pH declines over time in the gallbladder lumen. The fasting pH of common duct bile is around 7.3 and that of gallbladder bile 6.9 (Gleeson et al., 1992). It was initially suggested that this could be due to either absorption of bicarbonate ions or secretion of hydrogen ions. Using the isolated rabbit gallbladder, Sullivan and Berndt (1973) demonstrated that the PCO2 rose on the luminal side, and this rise could not be explained by tissue metabolism alone. The authors drew the conclusion that the gallbladder secretes hydrogen
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ions rather than absorbs bicarbonate since PCO2 would then be expected to fall. These findings were later verified by Rege and Moore (1987), who demonstrated secretion of H+ in the gallbladder of the anesthetized dog. The mechanism for this H+ secretion was analyzed by Petersen and co-workers (1985) who demonstrated an active Na+ /H+ exchange at the apical membrane of guinea-pig gallbladder epithelium that was reduced by amiloride and cAMP. More recently, an NHE-3 isoform of the Na+ /H+ exchanger was identified in human gallbladder (Silviano et al., 1997). Carbonic anhydrase II (CA II), which is inhibited by acetazolamide, is known to play a role in the regulation of acid-base balance in many tissues. The presence of CA II in the gallbladder has also been demonstrated by immunoblotting. Biliary CA activity has an important function in the regulation of VIP- and secretinstimulated bicarbonate secretion across the gallbladder mucosa (Nilsson et al., 2002). Sutor and Wilkie (1976), showed that there are variations in pH of gallbladder bile in patients pending surgery. As expected, the bile became more acidic with time during fasting but the acidification process was slowed by sleep and inactivity. A study demonstrated an enhanced secretion of H+ in response to stimulation of the splanchnic nerves in the cat (Nilsson et al., 1993). This could explain the findings by Sutor and Wilkie of lower adrenergic activity during sleep and inactivity. A role of pH in gallbladder bile has been implicated in the solubility of CaCO3 (Shiffman et al., 1990). Due to the sharp decline in [CO− 3 ] upon acidification, [CaCO3 ] is strongly reduced in gallbladder bile. This is suggested as being an important mechanism which plays a role in preventing the formation of calcium-containing stones in the gallbladder.
Solute-Linked Water Absorption Movement of water is secondary to active, solute movement, and the result of osmotic equilibration. The gallbladder mucosa, like that of several other epithelia, is able to perform “isotonic” fluid transport. The “standing gradient osmotic flow” theory was first proposed by Diamond and Bossert (1967) to explain intraepithelial osmotic coupling. According to this theory, solute is pumped into the long narrow spaces between adjacent epithelial cells. This creates a hypertonic compartment into which water is drawn, resulting in flow toward the serosal end of the lateral intercellular space. Hence there is a standing osmotic gradient under steady state conditions. Such a mechanism is supported by the finding that the lateral intercellular spaces between epithelial cells are distended during fluid transport, and collapsed by procedures which inhibit transport, such as metabolic inhibitors,
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and ion replacement (Kaye et al., 1966). More recent studies have led to evidence of the presence of aquaporin CHIP (channel-forming integral membrane protein of 28 kD) in the human gallbladder mucosa where it is mainly located on the basolateral membrane of the epithelial cells (Nielsen et al., 1993). How aquaporins are involved in the regulation of water is not currently understood but it was recently suggested that there is cotransport of water along with Na+ , and other small ions (Loo et al., 2002).
FLUID SECRETION BY GALLBLADDER MUCOSA Prostaglandins (Saverymuttu et al., 1979) and gastrointestinal regulatory peptides (Morton et al., 1977; Wood et al., 1982) may induce net fluid secretion by the guinea pig gallbladder in vitro. Furthermore, secretion also has been reported in vivo in the anesthetized cat after infusion of gastrointestinal regulatory peptides (Jansson et al., 1979), local intraarterial infusion of prostaglandin E2 (Thornell et al., 1981) and bradykinin (German et al., 1989). Bradykinin stimulated electrogenic bicarbonate secretion by guinea pig gallbladder (Baird & Margolius, 1989). The possibility of mucosal net secretion of fluid as a normal physiological event is supported by the observation of gallbladder secretion after feeding in the primate (Svanvik et al., 1984). Studies in patients treated with transhepatic gallbladder drainage showed that two hours after a meal, gallbladder contents were opalescent and white in color, and had the composition of an extracellular fluid, indicating net secretion of fluid from the gallbladder mucosa (Igimi et al., 1992). Further, studies using simultaneous measurements with ultrasound and dynamic cholescintigraphy in healthy volunteers support the idea of postprandial fluid secretion in the gallbladder that may help evacuate its contents (R˚adberg et al., 1993). Gallbladder mucosal inflammation is associated with a net secretion of fluid into the lumen in the cat (Svanvik et al., 1981), dog (Svanvik et al., 1986) and man (Dumont et al., 1982). The ability of the gallbladder to secrete fluid has been the subject of discussion in Gastroenterology (Sweeting, 1993). Net secretion of water and electrolytes by the gallbladder mucosa into the lumen is an active process which may take place against hydrostatic and osmotic gradients (Heintze et al., 1976). The ionic events occurring during secretion have been studied in the guinea pig gallbladder. In this species, under control conditions, bicarbonate is secreted into the gallbladder lumen in exchange for chloride (Heintze et al., 1981). This transport continues and is enhanced during prostaglandininduced secretion, where it is associated with a considerable reduction in chloride absorption, and a reversal of sodium and potassium absorption to net secretion.
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VESICULAR SECRETION OF GLYCOPROTEINS The gallbladder epithelial cells contain glycoproteins in their apical portions. Ultrastructural techniques have localized glycoproteins to secretory granules, 0.2–0.3 m in diameter (see Fig. 2). These granules fuse with the apical cell membrane and are discharged by exocytosis (Wahlin et al., 1976). In situ
Fig. 2. Electron Micrograph Demonstrating a Principal Cell of the Mouse Gallbladder (PA-CrA-silver stain). Note: Numerous secretory granules (S) containing glycoproteins are seen in the apical portion of the cell.
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hybridization has demonstrated expression mainly of the genes MUC3, MUC5B and MUC6 in the adult gallbladder (Buisine et al., 2002). There is a low basal level of secretion of glycoproteins in the fasting state, and, rapid secretion observed after injection of cholecystokinin (CCK) or cholinergic agents, and also in response to intragastric olive oil (Axelsson et al., 1978). In animals fed lithogenic diets, hypersecretion of gallbladder glycoproteins occurs (Lee, 1981; Wahlin et al., 1976). Studies of prairie dogs given a cholesterol rich diet have shown that mucin secretion is inhibited by aspirin (Lee et al., 1981), suggesting that prostaglandins are involved in gallbladder epithelial glycoprotein secretion. Arachidonic acid stimulates the glycoprotein secretion of gallbladder explants from prairie dogs in vitro (LaMont et al., 1983). Oxygen radicals stimulate glycoprotein secretion from the guinea pig gallbladder in vitro (Hale et al., 1987). An electron-microscopic and morphometric study demonstrated that intraluminal PGE2 strongly reduced in vivo mucus granules in the mouse gallbladder epithelial cells (Wahlin et al., 1988). There is a basal level of release of PGE2 into the lumen of the feline gallbladder that is enhanced by gallbladder distension and experimentally induced cholecystitis (Thornell et al., 1986). Studies carried out in cholesterol-fed prairie dogs have shown an increased synthesis of gallbladder prostaglandins which was assumed to be mediated by increased phospholipase A2 activity (LaMont et al., 1983). Irritating stimuli in the gallbladder lumen, such as the presence of supersaturated bile, lysolecithin and gallstones, may increase prostaglandin formation which releases mucus from the epithelial cells. There is recent evidence that cyclooxygenase-2 mediates mucin secretion from epithelial cells of the lipopolysaccharide-treated canine gallbladder (Kim et al., 2003). Glycoprotein granules are also present in normal human gallbladder epithelium, and the volume density and size of these granules in the fasted subject correspond closely to those in the mouse (Sahlin et al., 1990). Human cultured gallbladder epithelial cells also secrete mucus (Yoshitomi et al., 1987). In isolated human gallbladder epithelial cells, calcium and protein kinase C are important for stimulation of mucin secretion, while agents known to stimulate cAMP production are less effective in this setting (Dray-Charier et al., 1997). Recent studies have revealed the importance of expression of the biliary mucin gene Glycam1 in the formation of gallstones in inbred mice (Lammert et al., 2002).
REGULATION OF GALLBLADDER FLUID TRANSPORT Several local mediators affect the rate of gallbladder NaCl and water transport both in vivo and in vitro. Some of these mediators may regulate gallbladder water
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and electrolyte transfer under physiological conditions by modifying NaCl influx, active Na+ extrusion, and/or modifying junctional permeability. Cyclic AMP has been proposed as a second messenger for the effects of several such mediators, and has been found to inhibit NaCl coupled influx, the rate limiting step for transepithelial Na+ transport (Diez de los Rios et al., 1981; O’Grady et al., 1989).
Gastrointestinal Regulatory Peptides Among gastrointestinal regulatory peptides, secretin, GIP, glucagon and VIP may inhibit fluid absorption by the gallbladder mucosa (Jansson et al., 1978; Morton et al., 1977; Wood et al., 1982a, b) while CCK (Jansson & Svanvik, 1977) and GIP (Jansson et al., 1978) are without effect. Secretin stimulates electrogenic bicarbonate secretion in the guinea pig gallbladder in vitro while somatostatin antagonizes this effect (Sprakties et al., 1992) The effects of VIP and secretin on cAMP production has been studied in isolated epithelial cells of human and guinea pig gallbladder (Dupont et al., 1981). VIP was found to be a potent stimulant of cAMP production. Studies in porcine gallbladders in vitro, have shown that VIP causes net chloride secretion into the lumen (O’Grady et al., 1989). Such findings suggest that the VIP-ergic nerves demonstrated by immuno-histochemistry in the gallbladder wall of several species (Sundler et al., 1977) may release VIP to act on receptors on the serosal surface of gallbladder epithelial cells, thereby modifying the direction and rate of transmucosal fluid transport. A variety of other gastrointestinal regulatory peptides have also been studied in the isolated guinea pig gallbladder. Neurotensin, bombesin, motilin, cholecystokinin, the CCK-analog caerulein, and somatostatin were all without effect on absorption (Wood et al., 1982). Of these regulatory peptides, only VIP and secretin were able to modify gallbladder fluid transport at presumably physiological concentrations. However, peptides without effect on transport in vitro, if administered alone, may act to potentiate or inhibit the effects of other regulatory factors. In regard to endothelin-1, it exerts an inhibiting effect via a Gi protein-coupled receptor, cAMP-dependent anion secretion in human gallbladder epithelium, indicating a role in the control of bile secretion by an autocrine/paracrine mechanism (Fouassier et al., 1998).
Prostaglandins Prostaglandins E1 and F2 have the ability to totally inhibit fluid absorption in the gallbladder and reverse it to net secretion (Jiveg˚ard et al., 1988; Leyssac et al.,
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1974). This effect appears to be mainly mediated by intramural, probably VIP-ergic neurons rather than a direct action on the epithelial cells (Jiveg˚ard et al., 1988). Prostaglandin E, I and F-like substances have been isolated from the mucosa and smooth muscle of pathological gallbladders (Kaminski et al., 1985; Myers et al., 1993; Wood & Stamford, 1977). They are released from guinea pig gallbladder in vitro (Kaminski, 1985) and into the lumen of the cat gallbladder in vivo (Thornell et al., 1986). Whether or not prostanoid formation results in modifying transport under physiological conditions is not yet known, but there is convincing evidence of a role of prostanoids in pathological changes in gallbladder transport observed in cholecystitis. It was shown that selective blockade of cyclooxygenase-2 reduced PGE2 release, as well as fluid secretion to the lumen of the inflamed gallbladder (Nilsson et al., 1998).
Nitric Oxide Nitric oxide is synthesized from L-arginine and metabolized to nitrate and nitrite. Fluid secretion in inflamed gallbladders was reversed to net absorption in response to nitric oxide synthase blocker NW -nitro-L-arginine. Formation of nitrate was reduced. These effects were reversed by L-Arginine, the substrate for nitric oxide synthesis (Nilsson et al., 1996). The nitric oxide synthase blocker had no effect on gallbladder fluid transport in normal gallbladders (Thune et al., 1995).
Hormones Ingestion of contraceptive steroids in the cat did not affect basal gallbladder fluid absorption (R˚adberg & Svanvik, 1986). Several hormones and biologically active substances including prolactin (Diamond, 1965; Mainoya et al., 1974), oxytocin and ADH (Cremaschi et al., 1968; Cremashi & Galante, 1969), angiotensin II (Leyssac et al., 1974), and serotonin (Donowitz et al., 1980) inhibit absorption or are without effect. There is no evidence at present favoring a role for any of these substances in the physiological regulation of gallbladder water and electrolyte transport.
Role of the Autonomic Nervous System Adrenergic, cholinergic and peptidergic nerve fibers reach the gallbladder wall (Baumgarten & Lange, 1969; Bj¨orck et al., 1983; Ky¨osola & Penttil¨a, 1977),
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where a rich network of nerve fibers close to the epithelium is present (Gilloteaux et al., 1989). Vasoactive intestinal peptide (VIP), enkephalin (ENK), and substance P (SP) have been found in nerves in the gallbladder wall. The adrenergic nerve supply travels in the splanchnic nerves, and also to a minor degree, in the vagus nerves. Cholinergic and peptidergic fibers have been found in the vagus nerves and electrical stimulation of these nerves releases vasointestinal peptide (VIP) into the gallbladder lumen (Bj¨orck et al., 1986). Norepinephrine enhances net water absorption by the human gallbladder in vitro (Onstad et al., 1967) and in situ in the anesthetized cat (Bj¨orck et al., 1982). In the porcine gallbladder in vitro, norepinephrine reduces the short circuit current attributable to net Cl− secretion, and reduces intracellular cAMP, while VIP has the opposite effect (O’Grady et al., 1989). Acetylcholine is without effect on water transport by the gallbladder in vitro (Brennan et al., 1981), or in vivo (Bj¨orck et al., 1984) at doses which cause gallbladder contraction. Electrical stimulation of the splanchnic nerves increases the rate of water absorption by the gallbladder of the anesthetized cat, an effect which can be abolished by ␣-adrenergic receptor blockade (Bj¨orck et al., 1982). The site of action of the sympathetic nerves on net water transport by the gallbladder remains undefined. Besides a possible direct effect on mucosal cells, sympathetic nerves may act on local ganglia to inhibit the release of neurotransmitter which inhibits water absorption. A plausible transmitter released by local reflexes is VIP. Electrical stimulation of the cervical vagus nerves in the cat reduced the gallbladder net water absorption in atropinized animals(Bj¨orck et al., 1982), indicating the presence of a non-cholinergic mechanism capable of reducing net water absorption by the gallbladder. In view of the increased release of VIP to the gallbladder lumen (Bj¨orck et al., 1986), it seems most likely that vagal stimulation activates local VIP fibers. Truncal vagotomy enhances net water absorption by the feline gallbladder mucosa (Bj¨orck et al., 1984). When applied to patients, this procedure is accompanied by an increase in the size of the gallbladder, and a raised incidence of cholesterol gallstone formation.
Transport Across the Mucosa in the Inflamed Gallbladder In a study of transport by human gallbladder excised at cholecystectomy, absorptive function correlated with histological and clinical findings (Nahrwold et al., 1976). Patients with more marked clinical findings had more extensive histological changes and poor absorptive function in the gallbladder. These observations lend credence to the finding of a reduced gallbladder contents/hepatic bile ratio of
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biliary lipids and bilirubin in patients having gallstones and extensive lesions in the gallbladder wall of a functioning gallbladder (R˚adberg et al., 1988). Fluid transport in situ by the gallbladder, has been studied in experimental cholecystitis induced by implanting human gall stones into cat gallbladder (Svanvik et al., 1981). In animals with gallstones induced by cystic duct obstruction whose gallbladder mucosa was markedly inflamed continuously secreted fluid into the gallbladder lumen, thus forming hydrops (edema). Indomethacin, a prostaglandin synthetase inhibitor, promptly reversed fluid secretion to a role of absorption suggesting that endogenous prostaglandin formation may be responsible for the observed fluid transport change. Subsequent work has shown that inflammatory fluid secretion by feline gallbladder mucosa in experimental cholecystitis can be induced by activation of cyclooxygenase-2, which leads to an increase in prostaglandin formation (Nilsson et al., 1998). In dogs (Svanvik et al., 1986), obstruction of the gallbladder in combination with infection of its contents induced continuous fluid secretion into its lumen. This was sustained for several days. Secretion was markedly reduced by indomethacin, thus further supporting the role of prostaglandins in fluid secretion by inflamed gallbladder. Corradini et al. (1998) demonstrated that the mucosal absorption of fluid and apolar lipids in humans is a physiological process that is impaired by the presence of gallstones. Addition of lysolethicin to an electrolyte solution in the gallbladder lumen at a concentration comparable with that found in bile of patients with acute cholecystitis reversed the direction of transport to a net secretion (Niederhiser, 1983). This fluid secretion was abolished by indomethacin suggesting that the change in fluid transport may result from endogenous prostaglandin formation. In acute cholecystitis the gallbladder neck is usually obstructed by a gallstone. A reversal of the direction of fluid transport across the gallbladder mucosa to a net secretion into the lumen will cause distension of the obstructed gallbladder. Distension, per se has been reported to stimulate prostaglandin synthesis by the gallbladder wall (Thornell, 1981). The clinical course of acute cholecystitis, sometimes with necrosis and gallbladder perforation can be explained by these changes (Thornell, 1982). The intraluminal pressure in the gall bladder in acute cholecystitis is markedly raised, sometimes to values exceeding 90 mm Hg (Thornell et al., 1985). Prostaglandin E2 , formed by the inflamed gallbladder, induces a secretory response by the mucosa, which is inhibited by opiates and tetrodotoxin but not by hexamethonium or atropine (Jiveg˚ard et al., 1988), indicating that PGE2 acts mainly on intrinsic non-cholinergic, non-adrenergic nerves that influence the function of the gallbladder epithelial cells. Morphological evidence indicates gallbaldder wall nerve fibers containing VIP, SP, CCK and ENK. These peptides are transmitter candidates for the nervous response to PGE2 . VIP induces a secretory
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response (Jansson et al., 1979) but neither SP nor CCK affects fluid transport, while ENK reduces fluid secretion in the gallbladder (Bj¨orck et al., 1983). VIP occurs in raised amounts in the gallbladder lumen in experimental cholecystitis (Jiveg˚ard et al., 1989). Anti-antibodies interrupt VIP inflammatory secretion (Nilsson et al., 1994). Taken together, these findings suggest that the non-adrenergic, noncholinergic nerves activated by prostaglandins in the gallbladder wall are VIP-ergic neurons. Mucosal fluid secretion in inflamed gallbladder is abolished by indomethacin and when intramural nerves are blocked by lidocaine or tetrodotoxin but only partially by hexamethonium (Jiveg˚ard et al., 1987). Such findings support the hypothesis that fluid secretion in cholecystitis at least in part is due to activation of nerves by prostaglandins. Opioids like morphine, loperamide and enkephalin abolish or drastically reduce active fluid secretion in experimental cholecystitis (Jiveg˚ard et al., 1985). It is therefore possible that the opioid action on gallbladder fluid secretion in cholecystitis is mediated by inhibition of intramural secretory nerves. We have obtained evidence suggesting that endogenous opioids are released in response to distension of the inflamed gallbladder, thereby abolishing active fluid secretion when the intraluminal pressure increases (Jiveg˚ard & Svanvik, 1988).
PHYSIOLOGICAL REGULATION OF GALLBLADDER FLUID TRANSPORT Fasting and Feeding A number of studies suggest that gallbladder fluid transport across the gallbladder mucosa is subject to physiological regulation. Johnston and coworkers (1932) found that the hourly absorption of water by dog gallbladder was thrice more during the day than that during sleep at night. The influence of fasting and feeding on the concentrating function of the gall bladder in primates has also been investigated (Svanvik et al., 1984). During the day fasting animals had a net hourly absorption rate corresponding to one third of the fasting gallbladder volume. Feeding reverses the direction of transport from net absorption to net secretion into the gallbladder lumen. This particular study also confirmed the findings of Johnston and coworkers that net water absorption from the gallbladder is reduced at night during sleep as compared with that seen during the awake state. The finding of postprandial fluid secretion by the mucosa are in agreement with those of R˚adberg et al. (1993) who studied healthy volunteers and those of Igimi et al. (1992) who studied patients. Infusion of acid into the dudodenum of the anesthetized cat reduces fluid absorption by the gallbladder mucosa (Jansson, 1978), an effect that is blocked
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by intravenous infusion of somatostatin (Bj¨orck & Svanvik, 1984), suggesting mediation by the release of peptide hormones or peptide transmitters. Net water transport across the gallbladder wall may be influenced by both humoral factors and autonomic nerves. The gallbladder secretory response to feeding may result from an increased release of VIP by intramural nerves. As discussed above exogenous VIP can induce a net fluid secretion into the gallbladder lumen. VIP receptors have been demonstrated on the epithelial cells (Dupont et al., 1981) and histochemical studies have visualized VIP-containing nerve fibers in close proximity to the gallbladder epithelium (Sundler et al., 1977). The greater net water absorption during daytime as compared with that during sleep can be explained satisfactorily by a general increase in the activity of the sympathetic nervous system, since adrenergic stimulation increases gallbladder net water absorption (Bj¨orck et al., 1982). In addition to gallbladder contraction, net secretion by the mucosa may facilitate more efficient evacuation of the gallbladder after a meal. As might be expected, increased net fluid absorption under conditions of increased adrenergic activity would have the opposite effect.
Pregnancy and Gallbladder Fluid Absorption Riegel and coworkers (1935), and Potter (1936) observed that the gallbladder in pregnant women undergoing caesarian section was distended, and contained bile similar in composition to hepatic bile. Much later Large and coworkers (1960), reported that the chemical composition of gallbladder bile in pregnant women did not indicate impairment in gallbladder concentrating function. Studies using cats show that the net rate of gallbladder water absorption doubles in pregnancy, Table 1. Endogenous Substances and Procedures Enhancing Glycoprotein Release From Gallbladder Epithelial Cells. Substance
Exptl. Condition
Species
Reference
Prostaglandin E2 Lysolecithin Oxygen radicals Lithogenic diet Arachidonic acid Bile salts Gallstone formation
In vivo In vivo In vitro In vivo In vivo In vitro In vivo
Mouse Cat Pig Mouse Prairie dog Guinea pig Human
Intracellular Ca2+ concentration
In vitro
Human
Wahlin et al. (1988) Thornell et al. (1986) Hale et al. (1987) Wahlin et al. (1976) LaMont et al. (1983) Oleary et al. (1991) Sahlin et al. (1990) Shiffman et al. (1993) Dray-Charier et al. (1997)
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Table 2. Endogenous Substances and Procedures Enhancing Gallbladder Water Absorption. Substance
Exptl. Condition
Species
Reference
Norepinephrin
In vitro In vivo
Human Cat
Onstad et al. (1977) Bj¨orck et al. (1982)
In vivo In vivo In vivo
Cat Cat Cat
Bj¨orck et al. (1982) Bj¨orck et al. (1984) R˚adberg and Svanvik (1986)
In vivo In vivo
Guinea pig Prairie dog
Conter et al. (1986) Saunders et al. (1992)
Stim. of splanch Nic nerves Vagotomy Pregnancy Gallstone Formation
while oophorectomy does not affect this variable (R˚adberg & Svanvik, 1986). One explanation given is that the enlarged gallbladder in pregnancy is probably associated with an increase in transporting epithelial cells. Tables 1–3 list the endogenous and biogenic substances and procedures enhancing glycoprotein release, water absorption and reversing water absorption to water secretion. Table 3. Biogenic Substances and Procedures Reversing Gallbladder Net Water Absorption to Water Secretion. Substance
Exptl. Condition
Species
Reference
Cholera toxin Prostaglandin
In vivo In vitro
Dog Guinea-pig
VIP
In vivo In vitro In vivo In vitro
Cat Guinea-pig Cat Guinea-pig
PHI Feeding
In vivo In vitro In vivo
Cat Guinea-pig Monkey Human
In vitro In vivo In vivo In vivo
Human Cat Dog Cat
Shafer et al. (1969) Heintze et al. (1975) Saverymuttu et al. (1979) Thornell et al. (1981) Morton et al. (1977) Jansson and Svanvik (1977) Morton et al. (1977) Wood et al. (1982) Jansson et al. (1979) Brennan et al. (1982) Svanvik et al. (1984) Igimi et al. (1992) R˚adberg et al. (1993) Dumont et al. (1982) Svanvik et al. (1981) Svanvik et al. (1986) Niederhiser et al. (1983)
Secretin
Cholecystitis
Lysolecithin
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INTEGRATED FUNCTION OF THE GALLBLADDER Pressures within the biliary tract remain low and fairly constant despite large variations in bile flow (Hallenbeck, 1967). The pressure itself is determined by the rate of hepatic bile secretion, the rate of reabsorption of fluid in the bile ducts, and in the gallbladder, the resistance to flow exerted by the sphincter of Oddi, and compliance of the system. If compliance in the system is reduced by removal of the gallbladder, then contraction of the sphincter of Oddi would be expected to cause a prompt rise in the distending pressure in the bile duct system (Tanaka et al., 1984). The physiological events causing movement of fluid into and out of the gallbladder are first the transport of fluid across the gallbladder mucosa, and second, contraction and relaxation of the gallbladder wall. Rapid fluid absorption by the gallbladder epithelium increases the capacity of the gallbladder to retain the organic contents of bile in its lumen by 5–8-fold, while contractions themselves of the gallbladder wall may rapidly deliver this enriched bile to the duodenum. In-between meals, most of the hepatic bile is diverted to the gallbladder which undergoes rhythmic contractions with intermittent delivery of bile to the duodenum. Bile entering the gallbladder is concentrated in terms of its organic contents by mucosal water absorption, and is also acidified by H+ secretion by the gallbladder mucosa. In this balance between gallbladder emptying and concentrating activity, the latter dominates, as a result of which bile acids and other organic constituents accumulate in the gallbladder. In response to a meal, secreted CCK causes the gallbladder to contract. Fluid absorption by the gallbladder mucosa is both reduced and replaced by net secretion of fluid which helps evacuation of the gallbladder of its contents.
ACKNOWLEDGMENT Author’s studies carried out in the authors laboratory are supported by grants from The Swedish Medical Research Council (17X-04984).
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Baird, A. W., & Margolius, H. S. (1989). Bradykinin stimulates electrogenic bicarbonate secretion by the guinea pig gallbladder. J. Pharmacol. Exp. Ther., 248, 268–272. Baumgarten, H. G., & Lange, W. (1969). Extrinsic adrenergic innervation of the extrahepatic biliary duct system in guinea pig, cats and rhesus monkeys. Z. Zellforssch. Mikrosk. Anat., 100, 606–615. Bazzini, C., Botta, G., Meyer, G. et al. (2001). The presence of NHE1 and NHE3 Na+ /H+ exchangers and apical cAMP-independent Cl− channel indicate that both absorptive and secretory functions are present in calf gallbladder epithelium. Exp. Physiol., 86, 571–583. Bj¨orck, S., Fahrenkrug, J., Jiveg˚ard, L., & Svanvik, J. (1986). Release of immunoreactive vasoactive intestinal peptide (VIP) from the gallbladder in response to vagus stimulation. Acta Physiol. Scand., 128, 639–642. Bj¨orck, S., Jansson, R., & Svanvik, J. (1982). The adrenergic influence on concentrating function in the feline gallbladder. Gut, 23, 1019–1023. Bj¨orck, S., Jansson, R., & Svanvik, J. (1983). Influence of electrical vagal stimulation and acetylcholine on the function of the feline gallbladder. Scand. J. Gastroenterol., 18, 129–135. Bj¨orck, S., Jansson, R., & Svanvik, J. (1984). The concentrating function of the gallbladder after truncal vagotomy. Acta Chir. Scand., 150, 393–397. Bj¨orck, S., Lundberg, J. M., & Svanvik, J. (1983). Substance P, enkephaline and VIP in the feline gallbladder: Distribution of radioimmunoactivity and functional effects by exogenous administration. In: S. Bj¨orck (Ed.), Autonomic Nerves and Neuropeptides in Concentrating Function and Motility in the Gallbladder. Thesis, University of G¨oteborg, G¨oteborg, Sweden. Bj¨orck, S., & Svanvik, J. (1984). The influence of somatostatin on gallbladder response to intraduodenal acid and autonomic nerve stimulation in the cat. Scand. J. Gastroenterol., 19, 173–177. Buisine, M. P., Devisme, L., Degand, P. et al. (2002). Developmental mucin gene expression in the gastroduodenal tract and accessory digestive glandsII. Duodenum and liver, gallbladder and pancreas. J. Histochem. & Cytochem., 48, 1667–1676. Conter, R. L., Roslyn, J. J., Porter-Fink, V., & DenBesten, L. (1986). Gallbladder absorption increases during early cholesterol gallstone formation. Am. J. Surg., 151, 184–191. Corradini, S. G., Yamashita, G., Nuutinen, H. et al. (1998). Human gallbladder mucosal function. Effects on intraluminal fluid and lipid composition in health and disease. Dig. Dis. Sci., 43, 335–343. Cremaschi, D., Giordana, B., Lippe, C., & Capraro, V. (1968). Effect of neurohypophysial hormones and their mechanism of action on gallbladder transport. Arch. Int. Physiol. Biochim., 76, 813–822. Cremashi, D., & Galante, M. (1969). Action of posthypophyseal hormones on the “in vivo” isosmotic net water transport and on adeny cyclase in rabbit gallbladder. Arch. Physiol. Biochim., 77, 819–828. Cremashi, D., Porta, C., Bott´a, G., & Meyer, G. (1992). Nature of the neutral Na -Cl coupled entry at the apical membrane of rabbit gallbladder epithelium IV, Na+ /H+ , Cl− /HCO3 double exchange, hydrochlorothiazide-sensitive Na+ , Cl− symport and Na+ , K+ , 2Cl− cotransport are all involved. J. Membrane Biol., 129, 221–235. Diamond, J. M. (1965). The concentrating activity of the gallbladder. In: W. Taylor (Ed.), The Biliary System (pp. 495–514). Oxford: Blackwell. Diamond, J. M. (1968). Transport mechanisms in the gallbladder. In: C. F. Code & W. Herder (Eds), Handbook of Physiology (pp. 2451–2482, The Alimentary Canal, Vol. V). Baltimore: Williams and Wilkins.
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AUTHOR INDEX Abedin, M. Z., 556 Abid, K., 468 Adams, D. H., 394 Afford, S. C., 191 Agarwal, D. P., 324 Aggarwal, B. B., 174 Ago, H., 478 Ahmad, F., 175 Aimes, R. T., 506 Ainley, C. C., 337 Akabayashi, A., 530 Akgun, A., 409 Akriviadis, E., 349 Al, R. H., 478 Alberti, A., 444 Alcolado, R., 497, 505 Alexander, C. A., 233, 235 Alexander, J. F., 348 Ali, N., 480 Alison, M. R., 34, 151 Allen, J. W., 378 Allison, A. C., 526, 537 Alper, C. A., 444 Alpert, S., 281 Alpini, G., 24, 28, 30–32 Alter, H. J., 455 Alter, M. J., 455 Altomare, E., 329 Alvarez, F., 406, 407 Alvaro, D., 23–25, 27, 39, 110 Amakawa, M., 510 Ambrosch, F., 447 Amenta, P. S., 500 Ananthanarayanan, M., 101 Anantharaju, A., 13 Anderson, B. R., 183 Anderson, R. M., 151 Ando, Y., 277 Andre, P., 469
Andreassen, O. A., 132 Andus, T., 507 Anon Anon, 362 Anonymous, 455 Anwer, M. S., 82, 84–86, 89, 97, 99, 102–106, 108, 109 Aono, S., 277, 278 Arai, M., 327 Argiles, J. M., 195 Arias, I. M., 278, 281, 499, 512 Arif, M., 444 Arima, N., 476 Arner, E. S., 265 Arrese, M., 101, 102 Arthur, M. J. P., 502, 506 Asabe, S.-I., 476 Ash, S., 377 Atkinson, J. M., 105 Atkinson, L. R., 275 Axelsson, H., 561 Azoulay, D., 530 Azzaroli, F., 128 Badur, S., 409 Bahr, M. J., 408 Bailey, R. J., 343 Baird, A. W., 559 Bajt, M. L., 183 Ballardini, G., 393, 418 Bamberger, M. J., 235 Bamburg, J., 55 Banatvala, J. E., 440 Bancroft, J. D., 277 Banerjee, R., 474 Bar On, H., 242 Bara˜nano, D. E., 261, 262, 264, 267 Barasch, J., 509 Barrett, S., 412 Bartenschlager, R., 461, 463, 474 577
578 Barth, H., 467 Bartosch, B., 467 Bashir, R., 340 Bass, C. L., 262 Bassendine, M. F., 318 Bassilian, S., 243 Bataller, R., 504, 505 Baudocrin, S. U., 369 Baumert, T. F., 470 Baumgarten, H. G., 563 Bautista, A. P., 176, 192 Bazan, J. F., 172 Bazzini, C., 556 Bazzone, T. J., 321 Beard, M. R., 456 Beckett, G. J., 298 Bedossa, P., 505 Behrens, S. E., 478 Bell, H., 343 Ben-Ari, Z., 417 Benedetti, A., 24, 26, 33, 34, 39, 102 Benhamou, Y., 435 Benyon, R. C., 194, 502, 506, 508 Benzeroual, K., 106 Bergasa, N. V., 388 Berger, G. M., 244 Berggren, S., 319 Berk, P. D., 273, 277 Berndt, W. O., 557 Bernier, R. H., 431 Bernstein, J., 324, 388 Bernuau, J., 361, 370 Berthelot, P., 282 Bertini, G., 272 Bertoletti, A., 189 Beuers, U., 38, 106, 107, 110, 130 Beylot, M., 247 Bigatello, L. M., 333 Bihari, D., 369, Bird, T. A., 175 Bishop, G. A., 527 Bissell, D. M., 158 Bissig, K. D., 157 Bj¨orck, S., 235, 245, 246, 249, 563, 564, 566–568 Bjarnason, I., 333 Bjorkegren, J., 235
AUTHOR INDEX Bjornsson, O. G., 245, 246 Blackberg, J., 410 Blakolmer, K., 5 Blaner, W. S., 501 Blaschke, T. F., 277–279 Blei, A. T., 366 Blei, A., 374 Blight, K. J., 461, 462, 468, 470, 476, 477, 479, 480 Block, T. M., 456 Blomhoff, R., 501, 502 Blomstrand, R., 327 Blum, H. E., 337 Blumberg, W., 281 Boberg, K. M., 417 Bodsworth, N., 431 Bohan, A., 89, 99, 208, 224 Bohlinger, I., 177 Bone-Larson, C. L., 185–187 Boogaerts, J. R., 242 Borchardt, R. A., 235 Borowski, P., 475 Bosma, P. J., 276–278 Bosron, W. F., 322, 324 Bossert, W. H., 558 Boulton, R., 158 Bourgeois, C. S., 249 Bouscarel, B., 103–105, 107 Boveris, A., 323 Boyer, J. L., 22, 23, 27, 28, 30, 89, 91, 95, 98, 108, 208, 224, Bradley, D. W., 469 Brass, V., 476 Br´echot, C., 337, 410 Bree, R. T., 504 Brennan, L. J., 564 Brenner, D. A., 335 Bressanelli, S., 478, 479 Briggs, D. C., 393 Bright, N., 70 Brilla, C. G., 504 Brillanti, S., 539 Brodie, B. B., 326 Bruccoleri, A., 187 Bruck, R., 106, 192 Bruguera, M., 343 Bruguerolle, B., 213
Author Index Brunt, P., 337 Bruschi, S. A., 224 Bucher, N. L. R., 147, 149 Buchler, M., 273 Buckwold, V. E., 432 Budd, G., 147 Buffet, C., 343 Buisine, M. P., 561 Bukara, M., 176 Bukh, J., 456, 461 Bull, L. N., 51 Bulow, J., 245, 247 Burgart, L. J., 403 Burnham, F. J., 240 Burt, A. D., 341 Buschenfelde, K. H., 419 Buss, F., 70 Butler, P., 396 Caballeria, J., 321 Caligiuri, A., 30, 31, 39 Calmus, Y., 130 Campos, S. P., 175 Canalese, J., 373 Cantley, L. C., 107, 108 Cao, T. T., 65, 71 Caraceni, P., 374 Carithers, R. L., 348, 349 Carloni, V., 511 Carman, W. F., 441 Carrick, R. J., 469 Carruthers, J. S., 9 Casagrande, S., 266 Cases, S., 241 Casey, C. A., 333 Castells, A., 368 Castet, V., 467 Cauch-Dudek, K., 404 Caulin, C., 159 Cavanaugh, V. J., 188 Cederbann, A. L., 368 Chalasani, N., 279, 287 Chalmers, T. C., 343 Chander, G., 456 Chang, S. C., 464 Charlotte, F., 35 Chawla, A., 124
579 Chen, S. C., 193 Chi, X., 503 Chiang, J. Y., 99–101 Chin, H., 185 Chisari, F. V., 188, 418, 419 Cho, H. S., 471, 472 Cho, W. K., 22, 23, 27, 28, 30 Choi, A. M., 259 Choi, J., 465 Chojkier, M., 335 Choo, Q. L., 456, 477 Choukhi, A., 466 Chuang, L. M., 175 Chuck, S. L., 233 Chvatchko, Y., 178 Cianflone, K. M., 236 Ciotti, M., 276 Clayton, L. M., 104 Clement, B., 445, 500 Clermont, R. J., 343 Cloninger, C. R., 340 Cocquerel, L., 466 Cohen, D. E., 85 Cohen, J. C., 244 Cohen, L. E., 281 Cohen, P., 246 Cohn, S. M., 23 Cole, T. G., 232 Colletti, L. M., 178, 179, 187 Collier, A. J., 462 Columbano, A., 151, 159 Conter, R. L., 568 Cooksley, W. G., 403, 434 Cookson, S., 419 Coppack, S. W., 249 Coppel, R. L., 390 Corasanti, J. G., 106 Cordobe, J., 366 Cornelius, C. E., 91 Corradini, S. G., 565 Corsi, A. K., 232 Cosimi, A. B., 526, 538 Costelli, P., 195 Courouce, A. M., 446, 448 Coursaget, P., 446, 448 Courvalin, J.-C., 416 Crabb, D. W., 324
580 Crapper, R. M., 414 Craven, D. E., 444 Crawford, A. R., 6, 8, 9, 11, 17, 86, 87, 90 Crawford, J. M., 1, 2, 4, 5, 9, 17, 87, 90 Cremaschi, D., 563 Cressman, D. E., 153, 187 Cross, C. E., 329 Cruveilhier, J., 147 Cui, Y., 271 Cunningham, C. C., 57 Czaja, A. J., 403, 405, 407, 417 Czaja, M. J., 194 Dabeva, M. D., 545 Daemen, M. A., 181 Dambach, D. M., 186 Dani, C., 258 Darnay, B. G., 174 Darnell, J. E., 174 Das, K. C., 266 Dasgupta, A., 474 Dashti, N., 237 Davenport, A., 376 Davidson, A. R., 277 Davidson, H. W., 103 Davidson, L. S., 371 Davidson, N. O., 238 Davies, S. E., 532 Davis, G. L., 432 Davis, R. A., 235, 238 Dawidowicz, E. A., 333 Day, C. P., 318, 328, 339, 350 De Bleser, P. J., 512 De Francesco, R., 471, 472, 474, 478 De Groote, J., 401 De Jongh, F. E., 432 de Man, R. A., 435 De Vos, R., 469 De Vree, J. M., 98 Defrances, M. C., 154, 545 del Giudice, E. M., 277 DeLeve, L. D., 207, 226 DeMeo, A. N., 183 Demmer, L. A., 238 Demori, I., 154 Denson, L. A., 273 Derr, R. F., 335, 337
AUTHOR INDEX Desmet, V. J., 3, 4, 403 Detre, K., 376 Detry, O., 378 Deutschman, C. S., 178 Devenyi, P., 345 Devor, E. J., 338 Dhumeaux, D., 282 Di Marco, S., 472 Diakonova, M., 35 Diamond, J. M., 555, 557, 558, 563 Diehl, A. M., 174, 187 Dienes, H. P., 403 Dienstag, J. L., 402, 434, 435, 444 Diez de los Rios, A., 562 Dimitrova, M., 479 Diraison, F., 247 Doctor, R., 65, 72 Donaldson, P. T., 405, 407 Donner, M. G., 98, 103 Dranoff, J. A., 102, 103, 108 Dray-Charier, N., 561, 567 Drum, D. E., 343 Drummond, G. S., 275 Du, W. D., 502, 505, 506 Duan, W.-M., 137, 138 Dubuisson, J., 466, 479 Duerden, J. M., 240 Duffey, M. E., 555 Dufour, J. F., 408 Dumont, A. E., 559, 568 Dupont, C., 562, 567 Durand, F., 530 Eckfeldt, J., 272 Edakuni, G., 39 Ede, R. J., 367 Edidin, M., 67 Edwards, D. R., 334 Efsen, E., 185 Egger, D., 464, 475 Eisenberg, C., 232 Ekataksin, W., 10, 16 Elferink, R. O., 87, 88, 98 Elias, H., 9 Elias, I., 24 Ellis, A., 377, 378 Elsasser, H. P., 34
Author Index Elsing, C., 31 Engelking, L. R., 84, 91 Enomoto, N., 339, 477 Ezelle, H. J., 470 Faa, G., 5 Faber, K. N., 271, 272 Fabrikant, J. I., 151 Factor, V. M., 39 Failla, C., 471 Failli, P., 503 Falcon, V., 469, 470 Falkow, S., 76 Fang, J. W. S., 189 Faouzi, S., 182 Farber, J. M., 189 Farrell, G. C., 224, 364 Fattovich, G., 411 Faubion, W. A., 124 Fausto, N., 153, 159 Fazio, S., 237 Felsher, B. F., 277 Feng, S. L., 156 Feranchak, A. P., 108 Ferenci, P., 366 Fernandezbotran, R., 180 Ferrari, C., 188, 418, 419, 445 Ferrari, E., 478 Fevery, J., 278 Fieldsend, J. K., 236 Figueroa, R. B., 324 Fiorucci, S., 128 Fisher, N. C., 192 Fishman, J. A., 456 Fitz, J. G., 23, 27, 65, 107 Flint, M., 467, 485 Folli, F., 107 Fontana, R. J., 435 Forbes, A., 374 Foresti, R., 271 Forsberg, E., 500 Forton, D. M., 387 Foster, D. W., 239 Fouassier, L., 58, 68, 72, 74, 562 Fox, I. J., 279, 550 Foxwell, B. M. J., 174 Francavilla, A., 156, 159
581 Francis, D. P., 446 Francois, G., 443 Francoual, J., 279 Frayn, K. N., 229, 247, 249 French, S. W., 325, 327, 334 Frezza, M., 321 Friebe, P., 462, 463 Friedman, S. L., 334, 497, 502, 505 Fromter, E., 557 Fukuda, R., 189 Fulop, A. K., 153 Gabuzda, G. J., 366 Gaca, M. D., 502, 503, 510, 513 Gaeta, G. B., 409 Galante, M., 563 Gale, M. J., 477 Gale, M., 477 Galle, P. R., 180 Gallinari, P., 474 Gallucci, R. M., 154 Galun, E., 160 Ganem, D., 428 Gangopadhyay, A., 195 Ganguly, T. C., 102 Ganter, F., 177 Gantla, S., 278 Gardner, C. R., 224 Gardner, J. P., 467 Gatmaitan, Z. C., 102, 108 Gaudio, E., 26, 28, 34 Gazzard, B. G., 368 George, J., 512 Gerber, M. A., 403, 413 Gerlich, W. H., 428, 444 German, D., 559 Gershwin, M. E., 390 Gewirtz, D. A., 104, 105, 107 Gibas, A., 447 Gibbons, G. F., 230, 235, 238–246, 248, 249 Gilligan, S. B., 340 Gilloteaux, J., 564 Gimson, A. E., 372 Gines, P., 350 Ginsberg, G., 212, 213 Glaser, S. S., 13, 23, 24, 27, 30–35, 39
582 Glavy, J. S., 110 Gleeson, D., 557 Gluud, C., 348 Goddard, S., 191 Godlewski, G., 3 Goedde, H. W., 324 Goh, P. Y., 476 Goldberg, L., 319 Goldberg, S., 348 Goldstein, B. J., 175 Goldstein, J. L., 249 Goldstein, R. S., 33 Goldwater, P. N., 446 Gomez, D. E., 507 Gordon, D. A., 235 Gordon, G. G., 323 Gordon, S. C., 404, 406 Gores, G. J., 34 Gosert, R., 475, 479 Gottlieb, T. A., 69 Gough, A., 416 Govindarajan, S., 363 Grakoui, A., 457, 460, 466, 470, 471 Granner, D. K., 248 Grant, A. J., 193 Green, D. R., 123 Green, R. P., 238 Greenfield, M. S., 249 Gregorio, G. V., 406, 407, 416, 417 Greisler, H. P., 149 Gressner, A. M., 195 Griffin, S. D., 468 Grob, P. J., 446 Groen, A. K., 87–89 Groothuis, G. M., 88, 89 Grossman, M., 551 Gruber, B. L., 507 Gu, B., 474 Guicciardi, M. E., 34 Guidotti, L. G., 188 Guo, J. T., 461 Gupta, S., 149, 544, 547, 548, 550, 551 Gurantz, D., 31 Gutmann, D., 73, 74 Gutstein, S., 281 Guzelian, P. S., 335
AUTHOR INDEX Hadler, S. C., 446, 448, 449 Hadziyannis, S. J., 429, 435 Hagedorn, C. H., 478 Hagenbuch, B., 85 Hale, W. B., 561, 567 Hall, P. W., 366 Hall, R., 58 Hallenbeck, G., 569 Halliwell, B., 270 Hamilton, R. L., 230, 235 Hamm-Alvarez, S. F., 110 Hammerton, R. W., 68 Han, H. J., 34 Hanam, C., 371 Handler, J. A., 33 Hannon, G. J., 262, 263 Hansen, J., 333 Hara, H., 139 Harada, K., 193 Harada, S., 339 Harada, T., 468 Harbrecht, B. G., 175 Harder, D. R., 150 Hardison, W. G., 347 Hardy, R. W., 480 Harrid, M., 374 Harris, W. S., 243 Harrison, P., 372, 375 Hartsfield, C. L., 260 Hashimoto, K., 280 Hassoun, Z., 535 Havel, R. J., 240 Hay, R., 235 Hayakawa, T., 102 Hayes, J. D., 298 Hayes, P. C., 350 Heathcote, E. J., 456, 486 Hebbachi, A. M., 235 Hegedus, T., 58 Heiland, M., 248 Heintze, K., 556, 559, 568 Hellerstein, M. K., 240 Hellerstein, N. S., 240 Hemmann, U., 154 Hempel, S. L., 269 Hemptinne, B. D., 4 Henderson, A. R., 295, 299, 303
Author Index Heneghan, M. A., 405, 408 Herbst, H., 502, 509 Hermann, R., 151 Hernandez, R., 108 Higgin, J. A., 235, 236 Higgins, G. M., 151 Hijikata, M., 469, 471 Hill, D. B., 183, 232 Hilleman, M. R., 447 Hislop, W. S., 347 Hodgson, H. J., 159 Hoffman, A. L., 187 Hofmann, A. F., 27, 31, 309, 315 Hogaboam, C. M., 186 Holmengren, A., 265 Honda, M., 462 Hong, Z., 478 Hoofnagle, J. H., 411, 412, 414 Hoofnagle, J., 376 Hopf, U., 396 Hopkins, P. M., 258 Horn, T., 334 Hortnagel, H., 369 Horuk, R., 173 Hou, Z., 475 Houck, K. A., 158 Howard, B. V., 248 Howe, A. Y., 474 Huang, C. S., 274, 275 Huang, J., 67 Huang, Y. S., 183 H¨ugle, T., 475 Hutchinson, D. R., 307 Hutson, J. L., 235 Hwang, S. B., 463, 469 Ichikawa, T., 159 Igimi, H., 559, 566, 568 Iimuro, Y., 514 Ikeda, K., 461 Ikeda, M., 461 Ikeda, S., 461 Imai, K., 502, 571 Imai, S., 502, 571 Inagi, R., 180 International Group of Pathologists, 401 Inui, T., 157
583 Inui, Y., 237 Invernizzi, P., 389 Ioannides, C., 214 Iredale, J. P., 506, 508, 509, 513–515 Ishak, K. G., 415, 442 Ishido, S., 475, 479 Israel, Y., 329 Issa, R., 511, 513 Ivashkina, N., 477, 478 Iwai, M., 153 Jabbour, N., 530 Jackman, M. R., 69 Jaeschke, H., 224 Jakowlew, S. B., 158 Janero, D. R., 233, 236 Jansen, P. L. M., 85, 86, 87, 98, 224, 271, 272, 278, 279 Jansson, R., 559, 562, 566, 568 Jennett, R. B., 330 Johnson, A. E., 232 Johnson, P. J., 403, 408, 417, 418 Johnston, C. G., 566 Jones, B. A., 106, 107 Jones, D. E. J., 36, 193 Jones, E. A., 361, 364, 365, 388, 404 Jones, J. E., 493 Jones, P. J., 240 Jonsson, J. R., 505 Joseph, B., 543, 548 Jubin, R., 462 Jung, D., 101 Jungermann, K., 23, 24, 418 Jungers, P., 446 Kahan, B. D., 526, 537 Kakimi, K., 183, 190 Kakumu, S., 188 Kalayoglu, M., 528 Kamath, P. S., 532 Kaminski, D. L., 563 Kanai, M., 274 Kaneko, S., 476 Kaneko, T., 476 Kann, M., 428 Kanno, N., 22, 26, 28, 36, 37, 83, 94, 96, 102, 103
584 Kano, K., 30 Kanta, J., 505 Kanto, T., 469 Kanzler, S., 408 Kao, J. H., 428 Kaplan, M., 274, 275, 277 Kaplowitz, N., 207, 219, 220, 223, 226 Kappas, A., 275 Karakash, C., 237 Karayiannis, P., 441 Karpen, S. J., 99–101 Kartenbeck, J., 73, 280 Katayanagi, K., 39 Kato, A., 23, 27, 28 Katz, N., 23, 24 Katze, M. G., 477 Kawarade, Y., 5 Kaye, G. I., 559 Keene, C. D., 132, 133–135 Keiding, S., 314 Keitel, V., 280 Kendrick-Jones, J., 70 Kennedy, J., 172 Keokosky, W. Z., 322 Keppler, D., 73, 98, 103 Keski-Oja, J., 511 Khoruts, A., 332 Kiassov, A. P., 501 Kiba, T., 159 Kidd-Ljunggren, K., 410 Kieft, J. S., 462 Kietzmann, T., 418 Kikuchi, S., 74 Kilic, G., 65, 108 Kim, H. J., 243, 561 Kim, J. L., 472, 474, 476 Kim, T. H., 500 Kim, W. R., 362, 455, 456 Kim-Schluger, L., 530 Kinnman, N., 503, 511 Kipp, H., 102 Kirillova, I., 153 Kishimoto, T., 153 Kitamura, T., 281 Kitamura, Y., 260 Klatskin, G., 350
AUTHOR INDEX Klatz, A. P., 324 Klein, N., 372 Klinkert, M., 444 Klintmalm, G. B. G., 537 Klotz, U., 214 Knittel, T., 501, 502, 512 Knolle, P. A., 180 Koch, J. O., 474 Koepsell, H., 88 Koga, A., 5 Koga, H., 124 Kogure, K., 158 Koiwai, O., 277, 279 Kolykhalov, A. A., 456, 462, 463, 477 Kondrup, J., 241 Koniaris, L. G., 183, 187 Konig, J., 280 Korbling, M., 547 Kordula, T., 175 K¨orner, F., 278 Korsten, M. A., 324 Korth, M. J., 477 Koskinas, J., 413 Kossakowska, A. E., 509 Kossor, D. C., 33 Kranc, K. R., 267 Krause, U., 108 Kreamer, B., 277 Krebs, E. G., 109 Krieger, N., 461 Kruskall, M. S., 444 Kubitz, R., 107, 108 Kuhn, R. J., 470 Kuhn, W. F., 104, 105, 107 Kuipers, F., 98 Kullak-Ublick, G. A., 85, 87, 101 Kumar, P. K. R., 474 Kumar, S., 350 Kummerer, B. M., 471 Kunkel, M., 469 Kuo, G., 67 Kurikawa, N., 505 Kurosawa, H., 35, 124 Kurose, I., 176 Kurz, A. K., 108, 109 Kusano, F., 189 Kwong, A. D., 472
Author Index La Monica, N., 464 Laconi, E., 550 Lagasse, E., 547 Lai, C. L., 431 Lakehal, F., 34 Lammert, F., 561 LaMont, J. T., 561, 567 Lamri, Y., 31 Landing, B. H., 8, 10 Landmann, L., 73 Lane, M. D., 233, 235, 236 Lanford, R. E., 460, 461 Lange, W., 563 Larapezzi, E., 180 Lara-Pezzi, E., 76 Large, A. M., 567 Larrey, D., 220 Larsen, F. S., 366 Larusso, E., 73 LaRusso, N. F., 26 Laskin, D. L., 184, 224 Latasa, M. U., 154 Lau, A. L., 158 Lau, D. T., 434 Lau, D. T.-Y., 411 Lau, J. Y., 532 Lau, L. F., 150 Lawrenson, R. A., 416 Lazaridis, K. N., 27, 31, 110, 126 Lazebnik, Y., 123 Le Couteur, D. G., 212 Lechmann, M., 456 Ledda-Columbano, G. M., 159 Lee, F. I., 347, 350 Lee, J. M., 98, 103 Lee, S. P., 561 Lee, V. M., 34 Lee, W. M., 367, 371, 411 Leevy, C. M., 336, 345 Leiper, J. M., 233 Leist, M., 192 Leitch, C. A., 240 Lelbach, W. K., 317, 335 Lemoine, O., 191 Lemon, S. M., 462 Lentsch, A. B., 179 LeSage, E. G., 24, 39
585 LeSage, G. D., 24, 25, 27, 28, 30, 31, 33–35, 39 Lesburg, C. A., 473, 478 Lester, D., 322 Leu, J. I., 154 Leung, N. W., 327 Leng, N. W. I., 434 Leung, P. S. C., 416 Leuschner, U., 395 Levin, D. M., 342, 343 Levin, M. K., 474 Levine, D., 511 Levine, J. A., 337 Levitt, M. D., 272, 321 Levy, E., 235 Lewis, C., 281 Lewis, G. F., 241, 249 Leyland, H., 508 Leyssac, P., 562, 563 Li, G., 509 Li, T. K., 322, 324 Li, Y., 156 Liang, T. J., 456 Liaw, Y.-F., 4, 11, 29, 32 Liberman, E., 468 Lidofshy, S. D., 367, 369 Lieber, C. S., 34, 323, 326, 329, 330, 335, 344 Lieberman, F. L., 324 Lieser, M. J., 126 Lim, G.-K., 442 Lim, M. S., 509 Lin, R. C., 330 Lindenbach, B. D., 456, 471 Lindh, M., 432 Lindros, K. O., 324 Lingappa, V. R., 233 Lipkin, E. W., 240 Lisowsky, T., 156 Liu, Q., 464 Liu, W. B., 508, 509 Lo, S.-Y., 464, 469 Lohmann, V., 461, 468, 471, 476–480 L¨ohr, H. F., 189, 193 Lok, A. S., 431, 433 Loo, D. D., 559 Lopez Soriano, F. J., 195
586 Lopez-Santamaria, M., 533 Louis, H., 176 Love, R. A., 472, 473 Lu, W., 464 Lucey, M. R., 534 Ludwig, J., 8, 22, 26, 28, 403, 405, 410, 418 Lue, S. L., 332 Lukacs, N. W., 176 Lukas, T. J., 105 Lukavsky, P. J., 462 Lundberg, A. S., 504 Luo, G., 478 Luzio, J., 70 Lyra, A. C., 533 Machen, T. E., 557 Mackay, I. R., 399, 400 MacSween, R. N., 341, 502 Maderazo, E. C., 183 Maher, J. J., 188, 334 Mahler, S. M., 150 Maines, M. D., 258 Mainoya, J. R., 563 Mak, K. M., 335 Makin, A. J., 371 Malaguarnera, M., 188 Malhi, H., 546, 549, 550 Malik, A. H., 411 Malik, R., 159, 161 Malmstrom, R., 246, 249 Malouf, N. N., 546 Maltby, J., 191 Mann, D. A., 503 Manns, M. P., 224, 408 Manzillo, G., 441 Marbet, U. A., 336 Marescot, M. R., 445 Margeli, A. P., 156 Maric, M., 387 Marinelli, R. A., 39 Marra, F., 194 Marshall, A. W., 320 Martin, L., 373 Martinez, O. M., 190, 193 Martinez-Anso, E., 28 Martinez-Hernandez, A., 500
AUTHOR INDEX Martins da Costa, C., 417 Marucci, L., 13, 25, 35 Maruo, Y., 277 Marzioni, M., 22, 26, 36, 37 Mason, A., 411 Massague, J., 158 Masse, L., 317 Mast, E. E., 431 Masyuk, T. V., 22 Mathis, D. M., 34 Mathurin, P., 349, 502 Mato, J. M., 330 Matsuda, Y., 195 Matsukawa, A., 177, 178 Matsumoto, M., 464 Matsumura, S., 391 Matsuoka, M., 334 Matsushima, K., 332 Matsushita, M., 139 Matsuzaki, Y., 103 Matusan, A. E., 474 Mawet, E., 180 Mayer, M., 258 Mayne, M., 139 McCaughan, G. W., 527 McClain, C. J., 332 McCrudden, R., 507 McDermott, A. B., 444 McFarlane, B. M., 419 McFarlane, I. G., 399, 403–406, 407, 408, 410, 415, 419 McGarry, J. D., 239, 248 McGough, A., 56 McGowan, J. A., 147, 157 McGuire, R. F., 500 McIntyre, N., 314 McIntyre, P. J., 447 McLauchlan, J., 463–465 McLean, A. J., 212 McMahon, B. J., 431–433 McNair, A. N. B., 417 McNiven, M.73 Mead, J. E., 147 Means, R. T., 191 Medina, J. F., 103 Meier, P. J., 73, 85, 87, 89 Meijer, D. K., 88, 89
Author Index Melappioni, M., 446 Melero, S., 31 Mendelson, K. G., 175 Mendenhall, C. L., 337, 347, 350 Mendoza, E. C., 188 Meng, L. J., 85 Mennone, A., 27 Mercer, D. F., 461 Merrifield, C., 69 Meuer, S. C., 446 Meyer zum Buschenfelde, K. H., 419 Meyer, K., 467 Michalak, J. P., 466 Michalak, T. I., 420 Michalopoulos, G. K., 154, 157, 158, 545 Migliaccio, G., 464 Milani, S., 509 Milich, D. R., 189, 444, 445 Millar, J. B., 109 Miller, R. G., 442 Misra, S., 108, 378 Miyake, M., 5 Miyamoto, H., 469 Miyoshi, H., 238 Mizoi, Y.,339 Mizuhara, H., 192 Mohammed, H. H., 183 Moir, A. M., 244 Monaghan, G., 275–277 Monica, N., 464 Moore, E. W., 558 Mor-Cohen, R., 280 Moreno, A., 322 Morgan, E. T., 214 Morgan, M. Y., 327, 337, 342, 344 Morita, M., 188 Moriya, K., 464 Morris, S. M., 503 Morton, I. K. M., 559, 562, 568 Moscoso, G. J., 3 Moseley, R. H., 89 Moss, D. W., 295, 299, 303, 309 Mottaran, E., 331 Moyer, B. D., 74 Muallem, S., 67 Mufson, R. A., 174 Mukaida, N., 332
587 Mukhopadhayay, S., 102, 106, 110 M¨uller, M., 86, 224, 271, 272 Mulligan, R., 314 Munoz, S., 367, 373 Muraca, M., 550 Murase, N., 537 Murphy, F. R., 510 Murphy, G., 507 Murray, S., 195 Murray-Lyon, I., 377 Myant, N. B., 250 Myers, N. C., 102 Myers, S., 563 Nagata, H., 552 Nagy, P., 158, 334 Nahrwold, D. L., 564 Nainen, O. V., 442 Nakamura, K., 193 Nakamura, T., 158 Nakanishi, I., 507 Nakano, M., 333 Nakanuma, Y., 2, 12 Nakatani, K., 501 Nanji, A. A., 191, 192 Napoli, J., 189 Narumi, S., 190 Nathans, D., 150 Nathanson, M. H., 22, 23, 91, 95, 106 Nawa, T., 156 Neddermann, P., 471, 474 Negro, F., 408–410 Nelson, D. R., 209, 216 Nelson, S. D., 224 Nemezansky, E., 303 Neubauer, K., 501 Newbold, R. F., 504 Newton, A. C., 106 Ngala Kenda, J. F., 147 Niederhiser, D., 565, 568 Nielsen, S., 23, 24, 559 Niemela, O., 330 Nikias, G. A., 403 Nilsson, B., 558, 563, 565, 566 Noe, B., 109 Noe, J., 107 Nolan, J. P., 333
588 Nolandt, O., 464 Nommensen, F. E., 447 Nordberg, J., 265 Norman, J. E., 409 Nouri-Aria, K. T., 418 Novikoff, P. M., 39 Nowicki, M. J., 102 Ntambi, J. M., 243 Nuutinen, H., 325 Nyberg, S., 378 Obermayer-Straub, P., 224 O’Brien, R. M., 248 Odenthal, M., 505 O’Farrell, D., 478 Ogasawara, J., 181 Ogata, N., 442 Ogawa, K., 273 O’Grady, J. G., 371, 532 O’Grady, S. M., 562, 564 Ohishi, T., 505 Ohtani, O., 26, 27 Ohuchi, E., 506 Okada, Y., 35, 507 Okamoto, T., 188 Okuno, M., 512 Oldstone, M. B. A., 449 O’Leary, D. P., 567 Olofsson, S. O., 233, 245, 246 Olson, J., 501 Omenn, G. S., 338 Ona, V. O., 132 Onstad, G. R., 564, 568 Ontko, J. A., 327 Oon, C.-J., 442, 443 Op de Beeck, A., 466 Opelz, G., 527 Oratz, M., 305 Orrego, H., 329, 348, 350 Oscarson, M., 216 Ostapowicz, G., 362, 371 Ostrow, J. D., 291, 314 Oswald, M., 99 Ott, M., 545 Otte, J. B., 529 Otterbein, L. E., 259 Oude Elferink, R. P., 87, 98
AUTHOR INDEX Overturf, K., 158, 545 Owen, M. R., 241 Pack, R., 39 Packard, C. J., 236, 241 Packer, L., 180 Pagliaro, L., 350 Paizis, G., 504, 505 Palazzo, U., 370 Palmer, K. R., 31 Pang, P. S., 471 Panwels, A., 376 Pares, A., 322, 334, 337, 348 Park, J. S., 475 Patel, J., 466 Paulusma, C. C., 98 Paumgartner, G., 38, 130 Pavio, N., 467 Pavlovic, D., 468 Pawlotsky, J. M., 456, 477 Pease, R. J. 233 Penttil¨a, O., 563 Pereira, L. M. M. B., 413 Perlemuter, G., 464 Perrone, R. D., 38 Persico, M., 277 Pessayre, D., 224 Peters, M., 154 Peters, T. J., 327 Peters, W. H., 272, 278 Petersen, B. E., 547 Petersen, K. U., 558 Petropoulos, C. J., 39 Pfeifer, U., 479 Pflugheber, J., 477 Phillips, M. J., 26 Phinizy, J. L., 24, 32, 39 Piccininni, S., 474 Pieper-Bigelow, C., 272 Pieroni, L., 471 Pietschmann, T., 460, 461 Pinzani, M., 39, 187, 502, 503 Ploix, C., 193 Plumpe, J., 159 Pohlmann, S., 467 Polimeno, L., 156 Pollard, T., 53
Author Index Popper, H., 22, 26, 400–403 Portmann, B., 364 Potter, M. J., 567 Potts, J. L., 245 Powell, L. W., 350 Preaux, A. M., 508, 514 Primhak, R. A., 415 Prince, A. M., 469 Proudfoot, A. T., 363 Pullinger, C. R., 237, 250 Puntarulo, S., 331 Puoti, C., 413 Qi, Z., 512 Qiao, L., 129 Qin, W., 237, 479 Que, F. G., 35 Quigley, J. P., 506 R˚adberg, G., 559, 563, 565, 566, 568 Rai, R. M., 174, 187 Raines, E. W., 511 Rajvanshi, P., 549 Rakela, J., 362 Ramadori, G., 505 Ramalho, F. S., 156 Rameh, L. E., 107, 108 Ramond, M. J., 349 Randall, G., 461 Raposo, G., 59, 71 Ravaggi, A., 465 Reaven, G. M., 249 Redeker, A. G., 277 Reed, K. E., 476 Reesink, H. W., 402, 412, 413 Reeves, H. L., 497, 502 Rege, R. V., 558 Rehermann, B., 431 Reichen, J., 104 Renton, K. W., 215 Reuss, L., 555 Reyes, G. R., 477 Rhim, J. A., 158, 549 Rho, J., 475 Rice, C. M., 456, 461, 462, 471, 476, 480 Rickard, D., 277 Riegel, C., 567
589 Rijnbrand, R. C. A., 462 Rinck, G., 471 Ring-Larsen, H., 370 Riordan, S. M., 366 Ritter, J. K., 278 Rizzetto, M., 408–410 Roberts, H. R., 368 Roberts, S. K., 21, 27, 35–38, 408 Robertson, W., 24, 27, 32, 39 Rodgers, R. E., 24, 27 32, 39 Rodrigues, C. M. P., 123, 124, 126–128, 132, 133, 139–141 Roelofs, H. M., 278 Roelofsen, H., 102 Rolando, N., 367, 368, 375 Roman, R. M., 27, 31, 38 Rosalki, S. B., 291, 294, 295, 299, 303, 306, 309, 343 Rose, R. C., 555 Roskams, T., 4 Rost, D., 73 Rothschild, M. A., 305 Roulot, D., 512 Rowell, D. L., 189 Roy Chowdhury, J., 544, 547, 550, 551 Roy-Chowdhury, N., 275 Rubaltelli, F. F., 272 Rubin, E., 275 Rubin, M. H., 368 Rudolph, K. L., 504 Ruiz-Moreno, M. R., 432 Rusinol, A., 235 Russell, D. W., 120 Russell, W. E., 158 Rust, C., 107, 129 Ryder, S. D., 408, 413 Sabile, A., 464 Sahlin, S., 561, 567 Saija, A., 366 Saile, B., 157 Saint, E. G., 399 Sakamoto, K., 195 Sakamuro, D., 475 Sala, C., 68 Salaspuro, M. P., 325 Saleh, J., 247
590 Saleh, M. G., 413, 414 Salim, M., 261 Sampei, K., 261 Samson, R., 370 Samuel, D., 411 Sandhu, J. S., 546 Santolini, E., 463, 464, 471 Santucci, L., 176 Sasaki, H., 22, 26 Sato, H., 507 Sato, M., 501, 502 Sato, T., 179 Satoh, S., 476 Saunders, J. B., 340, 350 Saunders, K. D., 568 Saunders, S. J., 368 Saverymuttu, S. H., 559, 568 Saxena, R., 7, 12 Scarselli, E., 467 Schaefer, B., 512 Schaeffer, H. J., 109, 110 Schafer, D. F., 366, 369 Schaffner, F., 22, 26, 401 Schalm, S. W., 411, 432 Schaper, F., 175 Schaub, F. J., 181 Schekman, R., 232 Scheuer, P. J., 403 Schilling, P. J., 332 Schilsky, M. L., 417, 531 Schindeler, C., 174 Schiodt, F., 362 Schlenker, T., 27, 30, 31, 38 Schliess, F., 109, 130 Schmidt, W., 413 Schmidt-Mende, J., 477 Schmitt, M., 108 Schnabl, B., 504 Schneider, R., 428 Scholmerich, J., 106 Schreiber, S. S., 305 Schuchmann, M., 180 Schuppan, D., 512 Schvarz, R., 408 Schwartz, J. B., 213, 214 Schwarz, J. M., 248 Seeff, L. B., 323, 455
AUTHOR INDEX Segal, E., 76 Seger, R., 109 Selby, M. J., 466 Selden, C., 155, 160 Sell, S., 18, 39, 546 Sellers, J. A., 235 Selzner, N., 153 Sen, C. K., 180 Senoo, H., 502, 511 Seppen, J., 278 Serizawa, A., 178 Shafer, D. E., 568 Shafritz, D. A., 545 Shaw, S., 329, 531 Shedlofsky, S., 332 Sheetz, M. P., 67 Sheilds, P. L., 189, 190 Shelness, G. S., 235, 238 Sheng, M., 68 Shepherd, J., 236, 241 Sherlock, S., 337 Sherman, L., 76 Sheron, N., 183, 191 Shi, S. T., 464 Shibuya, A., 339 Shiffman, M. L., 558, 567 Shimizu, Y., 182 Shimoda, K., 183 Shimoike, T., 463 Shiratori, Y., 153, 180, 337 Shirota, Y., 479 Shito, M., 178 Short, D., 58 Shouval, D., 411 Shurety, W., 69, 70 Sidossis, L. S., 247 Sigal, S. H., 545 Silviano, V., 556, 558 Simonet, W. S., 183, 191 Simpson, K. J., 419 Sinaasappel, M., 278, 279 Sirma, H., 429 Skak, C., 314 Skude, G., 343 Slegers, J. F. G., 557 Smart, D. E., 503 Smith, H. M., 324, 532
Author Index Smith, R. M., 91 Smith, S. L., 331 Snyder, S. H., 267 Sohara, N., 502, 510 Sol´a, S., 127 Somasundaram, R., 512 Sorensen, T. I., 336 Soroka, C. J., 98, 102, 108 Sorrell, M. F., 331 Soto, H., 193 Spahn, C. M. T., 462 Sparks, C. E., 231, 237, 239, 243–245, 248 Sparks, J. D., 231, 237, 239, 243–246, 248 Spengler, U., 188 Sprakties, G., 562 Spring, D. J., 233, 235 Stamford, I. F., 563 Standaert, M. L., 108 Stange, J., 377 Starzl, T. E., 150, 525, 526, 528, 538 Steen, H., 107 Steer, C. J., 123, 124, 126 Steiger, B., 73 Steiner, J. W., 9 Steinkuhler, C., 471, 472 Steinmuller, T., 539 Stemorowicz, R., 396 Sternlieb, I., 417 Stevens, A., 378 Stevens, C. E., 446, 448 Stieger, B., 73, 85, 87, 89 Stockmann, H., 377, 378 Stoll, S., 210. 212, 218 Storey, E. L., 343 Strand, S., 180 Strauss, G., 366 Strautnieks, S. S., 98 Stravitz, R. T., 106, 128 Strazzabosco, M., 23, 31, 84 Strom, S. C., 550 Sullivan, B., 557 Sullivan, S., 369 Sundler, F., 562, 567 Sussman, N., 378 Sutor, D. J., 558 Suzuki, C., 503 Suzuki, H., 445
591 Suzuki, K., 507 Suzuki, R., 464 Suzuki, S., 178 Svanvik, J., 555, 559, 562, 563, 565–568 Sveger, T., 416 Swain, M. G., 387 Sweeting, J. G., 559 Swift, R., 388 Sykes, B., 339 Szmuness, W., 446, 449 Szostecki, C., 416 Taga, T., 153 Tai, C.-L., 474 Taipale, J., 511 Tait, B. D., 399 Takahara, T., 509 Takahashi, K., 469 Takahashi, M., 261 Tan, C. E. L., 3, 5 Tan, S. L., 477 Tanaka, M., 565 Tanaka, T., 266, 273 Tanaka, Y., 266, 273, 463, 469 Taniguchi, E., 157 Tanji, Y., 476 Tanner, M. S., 415, 449 Tanzi, E., 441 Tassopopoulos, N. C., 429 Tate, G., 280 Taub, R., 187 Tauton, J., 71 Taylor, D. R., 467 Taylor, P. E., 446, 448 te Morsche, R. H., 278 Tedder, R. S., 447 Tellinghuisen, T. L., 480 Terada, T., 12, 26, 35 Terjung, B., 406 Terpstra, O. T., 530 Theise, N. D., 9, 39, 547 Theret, N., 508, 511 Thomasson, H. R., 339 Thomssen, R., 469 Thornberry, N. A., 123 Thornell, E., 559, 561, 563, 565, 567, 568 Thornton, A. J., 176
592 Thune, A., 563 Tietz, P. S., 22, 23, 27, 30, 31 Tilg, H., 183 Tilzey, A. J., 447 Timbrell, J., 209 Toh, S., 280 Toker, A., 107, 109 Toledopereyra, L. H., 178 Tomkins, L., 76 Torok, N., 73 Traber, P. G., 95 Trauner, M., 83–85, 87, 89, 98, 103 Tredger, J. M., 210, 212, 218 Treichel, U., 419 Trewby, P. N., 369 Trey, C., 371 Trim, N., 501, 513 Tsujii, H., 280 Tuma, D. J., 330 Tuma, P., 67 Twisk, J., 238 Tygstrup, N., 312, 313 Tzetis, M., 277 Tzung, S. P., 157 Uchida, T., 344 Ueberham, E., 512 Ueno, Y., 32, 34, 35 Umeno, M., 323 Unger, R. H., 245, 247 Urvil, P. T., 474 Valaes, T., 275 Vale, J. A., 363 Vallee, B. L., 321 van den Abeele, P., 173 van der Veere, C. N., 278 Van Dyke, R. W., 89 Van Eyken, P., 505 van Gool, J., 180 van Hattum, J., 447 van Kuijck, M. A., 280 van Montfoort, J. E., 88 Van Os, C. H., 557 Van Thiel, D. H., 533 Van Waes, L., 344 van Waes, M. A., 232
AUTHOR INDEX Vance, D. E., 236 Vance, J. E., 236 Vanthiel, D., 374 Varaklioti, A., 465, 466 Vartiainen, M., 52 Vassilopoulos, D., 429 Vassy, J., 501 Vendemiale, G., 330 Venkatesan, S., 327, 328 Vento, S., 362 Verges, B. L., 248 Vermeulen, K., 503 Vijayan, V., 3 Vlahos, C. J., 108 Vonk, R. J., 98 Vyas, S. K., 508 Wada, M., 280 Wadstein, J., 343 Waggoner, J. G., 277 Wahlin, T., 560, 561, 567 Wake, K., 16, 501 Walker, J. G., 389 Walley, K. R., 177 Walter-Sack, I., 214 Wands, J. R., 337 Wang, L., 99 Wang, Q. M., 479 Wang, X., 546, 547 Ware, A. T., 367 Washington, K., 6 Watanabe, A., 509, 513 Watanabe, T., 192 Watson, J. P., 469 Watson, R., 384, 396 Waxman, D. J., 218 Waxman, L., 471 Weber, K. T., 504 Weber, M. J., 110 Webster, C. R. L, 103, 106, 108–110 Wei, H., 505 Weisner, R. H., 531 Wells, T. R., 8, 10 Wendel, A., 192 Wendu, J., 367, 375 Westmoreland, D., 444, 447 Weston, M. J., 369
Author Index Wetterau, J. R., 235 White, C. W., 266 Whittle, H., 448 Wiesner, R., 534 Wiggins, D., 239, 241, 242, 246 Wilde, J., 533 Wilkie, L. I., 558 Wilkinson, G. R., 215 Wilkinson, M. G., 109 Will, H., 507 Williams, R., 366, 367, 369, 372, 399, 417, 418, 533 Wilson, J. N., 443 Wismans, P., 447 Wolkoff, A. W., 85, 281, 282 Wolpert, E., 282 Wolters, H., 102 Wondergem, R., 150 Wong, L., 503, 511 Wood, J. R., 559, 562, 563, 568 Wood, R. C., 444 Woodside, W. F., 248 Worman, H. J., 406, 416 Wu, A., 342 Wu, G. Y., 91 Wu, J., 188 Wurmser, A. E., 108 Wyke, R. J., 367 Xie, Q., 128 Xie, W., 274 Xie, Z. C., 460 Xu, Z., 465, 466 Yagnik, A. T., 466 Yam, A., 39 Yamada, Y., 153, 154, 187 Yamaguchi, Y., 106 Yamamoto, A., 275 Yamamoto, K., 26 Yamamoto, M., 240 Yamashita, T., 478 Yan, Y., 472, 473 Yanagi, M., 456, 463 Yang, C., 510 Yano, M., 414
593 Yano, S., 106 Yao, F. Y., 533, 539 Yao, N., 472–474 Yao, X., 52 Yao, Z., 237, 238, 245 Yap, I., 445 Yasui, K., 464 Yasui, O., 546 Ye, Z., 442 Yerushalmi, B., 126 Yi, M., 461 Yoneyama, H., 177, 193 Yoo, B. J., 462 Yoong, K. F., 196 Yoshida, A., 339 Yoshida, R., 193 Yoshie, O., 173, 193 Yoshiji, H., 505, 509, 513, 514 Yoshitomi, S., 561 Yuasa, T., 469 Zafarullah, M., 507 Zahler, M. H., 158 Zajicek, G., 501 Zalzman, M., 546 Zaman, S. N., 411 Zammit, V. A., 230, 244 Zamore, P. D., 263 Zanetti, A. R., 441 Zaret, K. S., 544 Zaruba, K., 446 Zeeh, J., 213 Zern, M. A., 188 Zetterman, R. K., 331 Zeuzem, S., 456 Zhang, H., 185 Zhang, Y. J., 463, 505 Zhong, W., 478 Zhu, Q., 461 Zimmerman, H. J., 207, 214, 219, 220, 224–226, 416 Zlotnik, A., 173 Zollner, G., 99, 273 Zsembery, A., 27, 30, 31 Zuckerman, A. J., 442, 443, 445 Zuckerman, J. N., 445
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SUBJECT INDEX A Acetaminophen hepatotoxicity, 186, 221 Acetaminophen liver toxicity and liver transplantation, 530–534 Actin-associated proteins, 49, 51, 53, 57 Actin cytoskeleton, 49, 51, 57–63, 65, 67–74, 76, 77 Actin depolymerizing factor, 55 Actin monomers polymerize, 52 Activator protein-1 (AP-1), 260 Acute liver failure, 181, 183, 219, 361, 362, 530–535, 539, 540, 551, 552 Acute liver failure and liver transplantation, 530–534 Acute liver injury, 167, 508, 546, 549 Adult liver contains different types of progenitor cells, 346 Albumin, 5, 39, 237, 257, 262, 268, 271, 275, 291–293, 305–307, 309, 312, 315, 347, 348, 377, 385, 405, 413, 534, 544, 546 Albumin-bound bilirubin, 268 Alcoholic liver disease (ALD), 167, 180, 317–319, 325, 328–333, 335, 337–344, 350, 351, 402, 414, 416, 531–534, 539 Acetaldehyde, 210, 318, 321, 323–325, 330, 331, 335, 339, 347 Alcohol dehydrogenase, 217, 319, 321, 339 Alcoholic fatty liver; pathogenesis, 325 Alcoholic fibrosis; pathogenesis, 325 Alcoholic hepatitis, 163, 183, 189, 191, 192, 225, 296, 318, 325, 331–335, 340, 341, 345–350, 414 Clinical features, 341, 345, 347, 349, 365, 383, 412
Prognosis, 38, 281, 318, 332, 341, 345, 346–348, 350, 370, 371, 385, 395, 396, 402, 403, 407, 410, 413, 533 Treatment, 23, 38, 67, 70, 73, 77, 119, 128, 131, 132, 134, 137, 142, 167, 177, 179, 184, 186, 189–191, 196, 226, 227, 248, 264, 275, 277–279, 282, 295, 302, 318, 324, 328, 331, 341, 343, 345, 346–351, 363, 365, 372, 377, 378, 387, 388, 394, 395, 397, 400, 403, 405, 407, 408, 410, 411–415, 417, 427, 433, 435, 436, 456, 504, 532, 538, 540, 546, 552 Cytochrome P450 pathway, 120 Hepatocyte necrosis in, 325, 328, 332, 333, 418 Immune mechanisms in, 419 Liver biopsy in, 220, 277, 279, 298, 316, 341, 343, 345, 347, 400–402, 407, 413, 433, 537 Liver transplantation in, 183, 184, 227, 279, 294, 350, 362, 367, 368, 370–372, 375–378, 395, 396, 408, 411, 417, 456, 525–527, 529–531, 533, 534, 539, 540, 548, 549, 552 Metabolism, 81, 82, 86, 121, 180, 184, 207–209, 212–219, 222, 225, 226, 230–232, 236, 239, 242–244, 247–250, 365, 295, 305, 307, 310, 312, 314, 315, 318–321, 338, 351, 366, 418, 464, 533, 551, 557 Susceptibility, 38, 182, 204, 224, 225, 318, 319, 321, 323, 331, 338, 340, 365, 392, 393, 405, 448, 449 Alkaline phosphatases, 299, 300 Alkaline phosphatase isoenzymes, 302 Alkaline phosphatase and liver disease, 301 595
596 Increased serum alkaline phosphatase, 292–302 Allograft rejection and liver transplantation, 181, 190, 191 Alpha Fetoprotein, 308 Alpha-1 Antitrypsin, 308, 533 Alpha-interferon, 446 Aminopyrine tests Aminopyrine breath test, 312, 313 Aminopyrine removal test, 312, 313 Aminotransferases, aspartate and alanine, 391–416 Ammonia, blood ammonia and liver disease, 310 Animal models for studying cell therapy, 543–554 Animal models for studying cell transplantation, 548, 551, 552 Animals with acute liver injury, 549 Antenatal screening, 440 Anti-HBs, 430, 433, 434, 441, 444, 446–449, 539 Anti-mitochondrial antibodies, 383, 397 Antibody response, 331, 444, 446, 449 Antibody titers, 446 Antigenic components, 443, 445, 446 Antioxidant, 126, 222, 258, 259, 260, 262, 263, 265–268, 282, 337 -1 antitrypsin deficiency, 305, 308, 402, 415, 522, 550, 551 Apolipoprotein B and, 250 Apoptosis, 33–35, 40, 107, 119, 122–126, 128, 131, 132, 134, 135, 137–141, 151, 157, 159, 176, 177, 180–182, 194, 223, 269, 282, 391, 419, 465, 499, 503, 504, 507, 510, 512–514, 549 Arginine for glycine at amino acid position, 441 Arias syndrome, 278 Arp2/3 complex, 53, 54, 61, 70, 71, 76 ASBT is expressed in large but not small (less than 15 m) bile ducts, 32 Assembly and Secretion of hepatic very-low density lipoprotein, 229, 236, 247, 464 Autoimmune, 36, 128, 168, 180, 189, 193, 220, 272, 273, 283, 294, 297, 305, 306, 360, 384–386, 391, 396, 397,
SUBJECT INDEX 402, 404–407, 416, 417, 419, 455, 496, 509, 531 Autoimmune cholangitis, 359, 386, 417 autoimmune hepatitis, 168, 193, 220, 273, 306, 386, 402, 404, 405, 531 Autoimmune hepatitis and liver transplantation, 128, 168, 193, 220, 273, 297, 305, 386, 404, 405, 531 Autologous tissues, 527 Autosomal recessive diseases, 279–280 Auxiliary liver transplantation, 525, 530, 539 Auxiliary partial orthotopic liver transplantation, 530 B Bile Bile acids stimulate proliferation and secretion only in large cholangiocytes in vitro, 32 Conjugation with glycine or taurine, 121 Dehydroxylation, 309, 310 Dynamic studies of bile acid metabolism, 315 Entero-hepatic circulation, 310 Bile duct, 1, 2 Bile duct cells, stellate cells, 334 Bile duct obstruction, 97, 273, 283, 294, 301 Bile ductule, 1 Bile formation and cholestasis, 82, 97, 106, 107, 111 Hepatocellular transporters Inorganic ions in, 82 Organic anions in, 84 Lipids in, 81, 119, 565 Organic cations in, 88 Organic neutral compounds in, 89 Mechanism of bile formation, 91 Bile acid dependent bile flow in, 91 Bile acid independent bile flow in, 91, 93 Acinar heterogeneity in, 91, 95 Ductular bile formation in, 96 Paracellular Pathway in, 82, 90, 97, 557
Subject Index Bile secretion problems, 273 Biliary atresia, 38, 509, 531, 532 Biliary atresia and liver transplantation, 38 Biliary epithelium is morphologically and functionally heterogeneous, 21 Bilirubin, 63, 74, 81, 82, 84–86, 94, 95, 130, 208, 212, 257–268, 270–283, 291–295, 302, 304, 306, 314, 315, 347, 348, 371, 377, 385, 395, 400, 405, 406, 413, 429, 532, 534, 535, 537, 544, 550, 565 Bilirubin conjugates, 257, 272, 273 Bilirubin diglucuronide, 86, 257 Bilirubin glucuronidation, 278 Bilirubin glucuronidation is partially deficient, 278 Bilirubin in prematures, 258 Bilirubin level, 258, 261, 272, 273, 275, 278, 279, 282, 292, 294, 302, 550 Bilirubin metabolism, 257, 314 Bilirubin synthesis, 277 Bilirubin UGT1A1 contains a TATAA box, 276 Bilirubin, serum Bili-alb and biliproteins, 293 Conjugated and unconjugated, 268, 272, 281 Dynamic studies of bilirubin metabolism, 314 ‘Normal’ range, 296, 303, 413 Unconjugated hyperbilirubinemia, 294 Bilirubin-1X, 262 Bilirubin-glucuronides are secreted into the bile, 273 Biliverdin, 261, 262, 265, 282, 283 Biliverdin 1X (reductase), 262 Biliverdin reductase (BVR), 258, 261, 282, 283 Biomarkers of lipid peroxidation, 270 Biotripyrrins, 267 Black lysosomal pigment, 279, 281 Bound form, 154, 233, 262 Brain cells, 185, 261, 264 Bridging fibrosis, 537 Bromosulphthalein, hepatic removal, 313, 315 Bundling proteins, 57 Buthionine sulfoxine (BSO), 264
597 C Cadaveric solid organs in the United States, 528 Canal of hering, 9–11, 13 Canalicular protein of the ATP-binding cassette superfamily, 272 Cancer cells, 264, 267 Capacity and activity, 268 CAR (constitutive androstane receptor), 211, 218, 274, 275 Carbon monoxide, CO, 259 Carbon tetrachloride, 24, 48, 95, 184, 186, 363, 505, 509, 511, 513, 514, 546 Carcinoid of the liver and liver transplantation, 531 Carriers, 232, 362, 409, 413, 427, 432, 433, 439, 440, 443, 449, 450, 527, 533 Caspase, 123, 124, 126–128, 132, 134, 137–139, 159, 181, 182, 264, 476 Caused by a reduction of the activity of the bilirubin-conjugating enzyme, 276 Cell culture, 37, 67, 124, 461, 470, 475, 476, 545, 546 Cell cycle, 147, 150–154, 188, 503, 549 cell transplantation, 548, 551, 552 Cell-cell interactions, 170 Cell-matrix interaction, 513, 515 Cellular mechanisms of cholestasis Role of Nuclear receptors in, 99 Role of cyclic AMP in, 102 Role of calcium in, 103 Role of Protein kinase C in, 106 Role of PI3K signalling in, 105, 107, 110 Role of MAPK signalling in, 109 Role of protein phosphatases in, 110 Cholangiocarcinoma has a strong predilection for involving the major bile duct bifurcation, 37 Cholangiocytes, 5, 14–16, 24, 27, 33, 63, 65 Cholangiopathies differentially affect the biliary epithelium leading to selective alteration and destruction of specific sized ducts, 36 Cholestasis, 36, 81, 82, 97, 122, 123, 224, 274, 386, 393
598 Cholestasis, 36, 38, 73, 82, 85, 97–100, 102–104, 106, 107, 110, 111, 122, 124, 126, 142, 220, 224, 226, 272, 304, 343, 344, 387, 389, 394, 531, 532, 551 Cholestasis and liver transplantation, 311 Cholesterol and, 1, 230, 232, 236, 239, 311 Cholesterol, serum cholesterol in liver disease, 311 Cholinesterase, serum, 292, 309 Chronic Active Hepatitis (CAH), 361, 399, 401–403, 509 Alcoholic liver disease and, 167, 180, 181, 183, 191, 192, 296, 299, 317–319, 328, 329, 333, 337, 339, 340, 414, 416, 531, 532, 534, 539 Alpha-1-antitrypsin deficiency and, 308, 402, 415, 533 Autoimmune hepatitis and, 128, 168, 193, 220, 273, 297, 386, 402, 404, 405, 531 Definition of, 9, 444 Diagnosis of, 341, 409, 415 Treatment and prognosis of, 407, 410, 413 Chronic hepatitis B and D and, 408 Chronic hepatitis C and, 304, 402, 412–414, 527 Diagnosis of, 341, 409, 415 Treatment and prognosis of, 407, 410, 413 Cryptogenic liver disease and, 402 Current nomenclature of, 402 Drug-induced liver disease and, 207, 220, 416 Interface hepatitis and, 401, 402, 405, 411–420 Pathogenesis of, 38, 67, 68, 72, 73, 81, 88, 92, 98, 119, 132, 319, 325, 328, 418 Non-A, non-B (NANB) hepatitis and, 362, 371, 402, 456 Wilson’s disease and, 181, 307, 361, 402, 417, 531, 551 Chronic cholestasis, 227, 388, 531 Chronic HBV infection, 362, 430, 432, 434 Chronic hepatitis and liver transplantation, 527
SUBJECT INDEX Chronic hepatitis C, 296, 298, 299, 304, 402, 412–414, 527 Chronic Lobular Hepatitis (CLH), 401 Chronic Persistent Hepatitis (CPH), 400, 403 Bile acid biosynthesis, 120 Bile acids in liver disease, 119 Bile acids in neurodegenerative disorders, 119 Caspases, 123–125, 127, 128, 132, 513 Cholestasis, 97–104, 122–124, 272–274, 386–389, 393, 394, 531, 532, 551 Huntington’s disease, 119, 131 Mitochondrial pathway of apoptosis, 132 Neurodegeneration, 134, 139 Neuroprotection, 137 Nigral transplantation, 136 Parkinson’s disease, 136 Survival signaling pathways, 128 Tauroursodeoxycholic acid, 16, 119, 126 Chronic rejection, 536–539 Chronic renal failure, 535 Chronic viral hepatitis, 191, 296, 400, 402, 414, 419, 531, 532, 546, 551 Circulating forms of bilirubin, 268 Cirrhosis, 193–195, 301–306, 334–351, 402–406, 431–433, 497–499 CNS-1, 278, 279 CNS-2, 278, 279 Cobalt, 262 Conditions suitable for cell transplantation, 551 Confirmation of rejection requires liver biopsy, 537 Congenital hyperbilirubinemia, 74, 551 Congenital metabolic deficiencies, 534 Congenital spherocytosis, 277 Conjugated bilirubin, 272–274, 279–282, 293, 309, 310, 313–315 Conjugated bilirubin cannot be secreted into bile, 280 Conjugation defects, 272, 273 Consequence of a secretion defect, 272 Converted to bilirubin mono- and diglucuronide, 273 Coproporphyrin isomer I excretion is elevated, 280
Subject Index
599
Crigler Najjar syndrome, 278, 279, 293, 550, 551 Crigler Najjar syndromes type I and II, 273, 294 Crigler-Najjar syndrome type, 1, 278, 550 Cross-talk between the redox proteins, 266 Current vaccine in 5–15%, 39 Cyclosporine, 30, 132, 134, 208, 225, 526, 537, 538 Cystic fibrosis, 15, 17, 23, 37, 48, 65, 73–75, 83, 555 Cytochromes, 209, 214, 215 Cytoprotective, 35, 142, 258, 260, 267 Cytosolic xanthine oxidase, 268 D Damage of the basal ganglia, 258, see Kernicterus Deficiency of the transporter protein MRP2, 280 Degradation products, 267, 368 Degree of hyperbilirubinemia, 531 Derived from hemoglobin, 258 Diabetes and, 250, 345 Diagnosis of organ rejection, 536 Diagnostic tests, 220, 337 Diarrhea, 73, 277, 341, 347 Die of kernicterus, 278 Differentiate between conjugated and unconjugated bilirubin, high-pressure liquid chromatography should be used, 272 Differentiation of stem cells, 543 Dipeptidyl peptidase IV deficiency and cell transplantation, 548 Direct bilirubin, 257 Disadvantages of this approach, 441 Dissection phase, anhepatic phase, 529 Disseminated malignancy, 527, 535 Disturbances in Kupffer cell, 548 DNA synthesis, 147, 150, 151, 153, 156–158, 187, 434, 545, 549 Do not proliferate in normal liver, 548 Drug hepatotoxicity and liver transplantation, 207 Drug Metabolism Age and, 212
Circadian rhythms and, 213 Common pathways of, 207 Diet and, 214 Disease and, 215 Environment and, 217 Enzyme induction and, 217 Enzyme inhibition and, 218 Gender and, 213 Genetics and, 215 Hormones and, 214 Drug-induced cholestasis, 97, 273 Dubin-Johnson Syndrome, 74, 75, 87, 98, 279–282 Dubin-Johnson Syndrome Mutations in the Canalicular Transport Protein MRP2, 281 Ductal plate, 2–6, 12 E Efficacy of hepatitis B vaccination, 448 Elevated serum levels of both conjugated and unconjugated bilirubin, 281 Elevated unconjugated, 277 Elevation of conjugated bilirubin, 279 Elevation of unconjugated bilirubin, 278 Encoded by the UGT1 gene locus on chromosome, 2q37, 276 Endothelial, 34, 151, 158, 169, 176, 178, 180, 187, 190, 191, 194, 195, 209, 224, 260, 266, 269, 332, 334, 335, 499, 500–502, 505, 512, 515, 528, 536, 537, 539, 544, 548–550 Endothelial cells, 151, 158, 169, 176, 178, 180, 187, 190, 209, 224, 260, 266, 269, 332, 334, 335, 499, 500–502, 505, 512, 515, 537, 544, 548–550 Enzyme induction, 211, 217, 225, 304 Epithelial cells, 1, 3, 4, 5, 9, 10, 12, 13, 22–24, 54–56, 58, 59, 61, 62, 64, 66–72, 556, 558–563, 565 Epithelioid hemangioendothelioma and liver transplantation, 531 Epitope, 391, 392, 441–443, 450 Exceeding 2.5 mg per deciliter, 257 Exclusionary criteria.systemic disseminated malignancy and irreversible liver damage, 527
600
SUBJECT INDEX
Expression in hepatocytes was found to be a function, 549 Extrahepatic causes, 272 Ezrin-radixin-moesin, 58, 65 F2-isoprostane, 270 F Familial hypercholesterolemia, 239, 533, 551 Fatty acid residues in lipids, 272 Fe2+ complex, 260 Fe3+, 260 Fecal urobilinogen, 257 Fenton reaction, 269 Fetal development, 543, 544 Fetal human liver cells are unable to proliferate indefinitely, 546 Five cysteine residues, 266 Fluid secretion in, 14, 30, 559, 563, 566 Fluid transport in, 555, 563, 565 Foamy histiocytes in the endothelium, 537 Focal necrosis of bile ducts, 537 Following bile duct injury, small ducts proliferate to provide compensatory secretory function, whereas large ducts are repaired, 39 Formation of the intrahepatic biliary tree, 3 Free bilirubin, 268 Free form, 262 Free unbound bilirubin, 271 From damage by peroxyl radicals, 264 Fulminant Hepatic Failure (FHF), 160, 310, 311, 361, 362, 364 Acid base complications in, 378 Cardiac complications in, 369 Cerebral edema in, 373 Coagulopathy in, 372 Definition, 9, 12, 82, 267, 268, 361, 401, 444 Electrolyte disturbances in, 369 Etiology, 36, 37, 219, 220, 227, 277, 334, 342, 349, 362, 364, 371, 372, 394, 400, 405 Experimental therapy, 347, 348, 377 Liver support systems in, 378 Liver transplantation in, 279, 294, 456
Management of, 227, 315, 372, 375, 376, 387, 413, 536 Metabolic complications in, 332 Multiple organ failure in, 375 Pathology, 74, 132, 212, 227, 317, 318, 341, 342, 343, 345, 349, 415, 504 Prognosis in, 332, 371, 396 Pulmonary complications in, 369 Renal complications in, 369 Fusion of hematopoietic stem cells, 546 Future therapy, 436 G G145R variant, 442 Galactose, Elimination Capacity, Gallstones, 215, 277, 561, 565 Gamma-glutamyl transferase (GGT), 303 Serum activity, ‘normal’ range, In liver disease, 298, 303 And alcohol intake, 304 In diabetes mellitus, 305 And drug intake, 214 Gene silencing technique, 262 General protein-disulfide reductases, 265 Genetic defects in, 249 Genetic defects of UGT1A1, 273 Genetic identity, 527 Genome organization, 474, 479 Genome replication, 474, 479 HCV lifecycle, overview, 459, 460 Limitations of therapeutics, 456 Genotoxic liver injury, 550 Gilbert syndrome, 273, 275, 277, 278 Gilbert syndrome mutations, 276 Glucose, blood glucose and liver disease, 311, 372 Glucose-6-phosphate dehydrogenase, 275, 277 Glutathione (GSH), 34, 81, 84, 87, 93–95, 130, 159, 184, 209, 217, 222, 260, 263, 268, 272, 282, 292, 304, 313, 330, 365, 528 Glutathione peroxidase, 264 Glutathione-S-Transferase, 34, 86, 292, 298 Gradual rise in serum aminotransferases, alkaline phosphatase and GGT, 537
Subject Index
601
Growth factors, 34, 99, 107, 109, 150, 151, 152, 153, 157, 158, 160, 170, 214, 334, 513, 530 GSH reduces peroxides, 264 H Halothane hepatotoxicity, 220 HbsAg, 400, 409, 410, 427, 428, 432, 434, 441, 444, 447 HBsAg mutants, 443 HBV DNA, 409, 410, 411, 429, 430, 434, 443 HBV e antigen, 430, 449 HBV mutations, 446 HCV, 188, 189, 337, 362, 412, 413, 420, 427, 455, 456, 458, 460, 471, 476, 480, 527, 539 Heat shock-protein-32, 260 HeLa cells, 264, 266 Hematopoietic cells constitute approximately 50% of the fetal liver, 193, 470, 544 Heme, 257, 258, 260, 261, 262, 265, 271, 275, 291, 294 Heme oxygenase, 258, 260, 261, 265, 271, 275, 283 Heme-Heme Oxygenase System, 258 Hemin, 261, 271 Hepatic function in health and disease, 295 Hepatic insufficiency and liver transplantation, 307, 544 Hepatic oval cells and production of hepatocytes, 34, 546 Hepatic production of circulating proteins, 176 Hepatic stellate cell, 157, 187, 194, 497, 498, 500, 501, 508, 509, 511, 514, 515 Hepatic vein thrombosis and liver transplantation, 363, 364 Hepatitis B carriers, 440 Hepatitis B core antigen, 189, 443 Hepatitis B Immunization, 444 Hepatitis B infection, 71, 89, 436, 441, 447, 448 Hepatitis B surface antibody mutants, 428, 429, 439, 442, 443, 450
Hepatitis B surface antigen, 409, 427, 439, 442, 450 Hepatitis B surface antigen escape mutants, 439 Hepatitis B vaccines, 439, 441, 444–446 Hepatitis B vaccines prepared by recombinant DNA technology, 444 hepatitis B virus (HBV), 76, 337, 419, 427, 527 Hepatitis B virus and liver transplantation, 527 Hepatitis C virus and liver transplantation, 527 Hepatoblastoma and liver transplantation, 531 Hepatocellular carcinoma (HCC), 39, 76, 196, 301, 302, 308, 309, 337, 385, 408, 411, 414, 416, 429, 430, 432, 436, 456, 531, 533 Hepatocellular carcinoma and liver transplantation, 531 Hepatocyte growth factor, 100, 154, 155, 160, 187. See growth factors hepatocyte to bile, 273 Hepatocyte transplantation, 279, 377, 550, 551, 552 Hepatocyte transplantation, 544 Hepatocytes, 1, 5, 9, 11, 12, 16, 17, 18, 22, 23, 24, 26, 31, 34, 39, 49, 50, 85, 101, 105, 107, 110, 111, 122, 129, 142, 147, 149–151, 154, 156, 169, 171, 175, 180, 185, 279, 280, 299, 308, 333, 337, 344, 364, 377, 393, 461, 479, 499, 500, 540 Hepatocytes did not migrate from the periportal area toward the perivenous area, 549 Hepatocytes engraft, 578 Hepatotoxicity, 186, 207, 211, 219, 220, 222, 224, 225, 226, 323, 329, 333, 340 Diagnosis, 279, 282, 296, 297, 315, 341, 345, 384, 385, 395, 405, 407, 409, 412, 415, 416, 512, 537 Incidence of, 71, 195, 219, 277, 335, 349, 362, 378, 389, 396, 409, 430, 436, 446, 536, 564
602
SUBJECT INDEX
Pathogenesis of, 38, 41, 73, 98, 110, 132, 167, 172, 192, 319, 321, 324, 328, 332, 337, 338, 348, 351 Screening for, 310, 378, 443 Susceptibility to, 207, 224, 318, 323, 338, 340, 405, 449 Treatment of, 23, 70, 73, 119, 131, 137, 142, 191, 248, 275, 278, 318, 331, 365, 372 Types of, 51, 89, 109, 215, 220, 242, 282, 297, 301, 305, 310, 315, 342, 412, 540, 546 Hepatotoxins and liver transplantation, 278 Hereditary nonhemolytic unconjugated hyperbilirubinemia, 74, 75, 86, 217, 258, 261, 270, 272, 278, 282, 292, 315, 531, 551 Heritable hyperlipidemia, 24 Heterogeneity of cholangiocyte secretory functions, bile duct reactions to injury, and cholangiocyte proliferation and differentiation, 24 Heterotopic, 12, 376, 525 “High risk” babies, 132, 536 Histological findings, 182, 186, 281, 282, 318, 347, 348, 371, 383, 386, 395 Histology, 407, 537 HLA, 340, 392, 393, 394, 396, 397, 444, 461, 463, 464, 465, 474, 527 HLA-DR alleles, 444 HLA-matching and liver transplantation, 527 Hormones and, 15, 85, 89, 208, 212, 260, 271, 563 Human Leukocyte Antigen (HLA), 407, 527 Hypersplenism, 277, 368 Hypo-responders, 444 Hypoxanthine, 268 I Icterus, 257, 258, 275, 277 Identification of stem cells in specific organs, 543 Immune system, 131, 168, 175, 262, 391, 465, 527, 532 Immunodeficiency viruses, 195, 527
Immunogenic role, 444 Immunoglobulins, 303, 385, 469 Immunological memory, 447 Immunomodulators, 446 Immunosuppression and liver transplantation, 449, 526–540 Immunosuppressive drugs and liver transplantation, 408, 534 Immunosuppressive regimes using cyclosporine, 526 Immunotolerance, 449 In contrast, chronic rejection, 537 In patients with conjugation defects, 272 In the classic bile duct injury model, the bile duct ligated (BDL) rat, only large bile ducts proliferate, 25 Incidence of HBV infection of 2.9 per 100 person years, 44, 61 Increased neonatal bilirubin levels, 275 Increased sensitivity of large bile ducts to injury, 24 Indications for liver transplantation, 530, 531 Indocyanine Green, 313, 314 Induced by chemicals, 546 Inducible isoform, 260 Infection, drug toxicity, organ rejection, delayed bone disease, 536 Infections – bacterial, 368 Injury is restricted to limited ranges of bile duct sizes, 22 Injury to bile duct cells and central vein endothelial cells, 537 Insulin resistance, obesity and, 248 Interleukin-2, 446 Intracellular bilirubin concentration, 261 Intrahepatic biliary tree, 1–3, 5–9 Involvement of arterioles, 537 Ischemia in the liver, 548 Ischemia-reperfusion injury, 137, 550 Isomeric monopyrrole derivatives, 267 J Jaundice, 183, 212, 257, 258, 262, 272–275, 277, 281, 283, 293, 294, 297, 301
Subject Index
603 K
Kernicterus, 258, 275, 277–279 Kupffer cells, 192, 544
Living-related liver transplantation, 527, 529, 539 Loss of bile ducts, 537 Lower risk for coronary artery disease, 258
L
M
Lactate dehydrogenase, 292, 299 LD5 isoenzyme, 299 Large intrahepatic bile ducts secreting fluid in response to secretin, 24 Lecithin cholesterol acyltransferase (LCAT), 311 Serum activity in liver disease Effect on serum lipids and lipoproteins, 298 Leptin and, 245 Leucine aminopeptidase, 72, 292, 303 Linear tetrapyrrole bilirubin IX, 259 Liver biopsy, 220, 277, 279, 298, 316, 341, 343, 345, 347, 400–402, 407, 409, 413, 433, 537 Liver cancer and liver transplantation, 464 Liver cell transplantation, 543, 545, 547, 549, 550 Liver development, 160 Liver disease, 180–183, 189, 191–194, 296–299, 301–313, 315–319, 328–333, 335–342, 408–410, 414–416, 515–527 Liver fibrosis, 192, 195, 325, 497–499, 505–507, 509–515 Liver injury, 177–179, 182–187, 190–192, 337, 338, 340, 343, 365, 418, 497, 502, 504, 505, 508, 509, 511, 512, 546, 549, 550 Liver regeneration, 150, 151, 153, 154, 156, 158, 159, 161, 184, 188, 227, 372, 545, 546 Liver repopulation, 547, 549, 550 Liver tests, 293, 296, 383, 385, 400, 409, 413, 415, 416 Liver transplantation before irreversible organ damage occurs, 534 Liver transplantation surgery, 543–554 Liver tumor and liver transplantation, 543–554
Major histocompatibility complex and liver transplantation, 128, 130, 173, 393, 444, 527, 547 Major histocompatibility complex antigens, 128, 130, 173, 393, 444, 527, 547 Major indication for OLT, 532 Major surface protein, 441 Many of the MRP2 substrates, 280 Marker of HBV infection, 443 Mayo Model, 532 Mechanisms directing liver cell engraftment, 547 Mediated by the ATP-dependent transport protein MRP2 (Multidrug Resistance-associated Protein), 273 MELD, 534, 535 Mental status and a greater capacity to handle ammonia, 552 Metabolic disorders, 364, 533, 539, 551 Metabolic liver disease and liver transplantation, 525–542 Metabolic syndrome and method of RNA interference, 262 Meulengracht syndrome, 276 Microchimerism and liver transplantation, 525–542 Microenvironmental differences, 546 Minimum protective level, 446, 447 Mitochondria, 36, 89, 123, 125, 127, 132, 134, 140, 209, 210, 222, 244, 266, 268, 321, 346, 389, 415 Model for end-stage liver disease (MELD), 534 Molecular virology, 427, 428, 456, 460, 480 Monozygotic twins, 527 More than 80 cell divisions, 545 Most causes of jaundice, 273 Mouse model, 38, 134, 142, 444 MRP2 (ABCC2), 272
604
SUBJECT INDEX
MRP3, 85, 86, 98, 103, 273, 280 Mushroom poisoning and liver transplantation, 525–542 Mutation, 37, 38, 98, 216, 275, 279, 429, 432, 435, 441, 442, 445, 477 Mutations in the UGT1A1 gene, 278 Myosin, 52, 58, 67, 70, 111 N NADPH ® NADP+ reaction, 267 Natural history, 333, 384, 385, 394, 395, 397, 408, 427, 531 Natural history and epidemiology, 431 2nd and 8th day of life, 274 Neonatal jaundice, 212, 274, 277 Neonates with CNS-1, 278 Neuroendocrine tumor and liver transplantation, 531 Non-responders, 439, 444, 445, 447, 449 Non-responders and silent infection, 449 non-responsiveness to the S antigen, 444 Non-structural proteins, 457, 458, 461, 470, 476, 479, 480 NS2 protein, 458, 470 NS4B protein, 475 NS5A protein, 474, 476, 477 NS5B protein, 459, 477 Polyprotein processing, 457, 468 Protein membrane topology, 458, 470, 475, 476, 479, 480 Structural proteins, 457, 458, 461, 464, 468, 470, 475, 476, 479, 480 Virion morphogenesis, 468, 471 Nonacetaminophen-induced acute liver failure, age, duration of jaundice, serum factor V, 531 Normal serum bilirubin levels do not exceed 100 micromol/l, 272, 294 Nucleoside analogs, 434, 435, 539 5 -Nucleotidase, 67, 292, 303, 385 Null phenotype, 263 60% of normal term newborns, 10, 61, 392 O OKT3, 526, 538 OLT in patients with alcoholic liver disease, 534, 539
Organ donation and liver transplantation, 525–542 Organ preservation, 528 Organ rejection and complications, 536 Organ rejection and liver transplantation, 525–542 Organic anion transport protein, 2, 83, 271 Orthotopic liver transplantation (OLT), xvi, 371, 376, 396, 525, 552 Other liver cell types, 544, 556 Outcomes compared favorably with nonalcoholic individuals, 534 Oval cells become apparent during liver regeneration, 546 Overproduction of bilirubin from excessive hemolysis, 272 Oxidative stress, 128, 175, 222, 224, 260, 266, 329, 431 Ozone (O3 ), 270 Ozone stimulates peroxidation, 270 P Partial hepatectomy, 33, 34, 39, 41, 147, 149, 153, 154, 157, 159, 171, 187 Pathogenesis, xiv, 38, 41, 73, 77, 98, 99, 119, 131, 167, 168, 172, 188, 192, 319, 321, 325, 328, 332, 464 Pathophysiology, 23, 49, 134, 316 Patients with CNS-2, 278, 279 Patients with Dubin-Johnson syndrome its expression is increased, 191, 266, 280 Patients with ornithine transcarbamylase deficiency, 550 PBC is characterized by spotty rather than diffuse proliferation/loss of certain sized ducts (i.e., small interlobular bile ducts), 33, 36 PCR (LDC-PCR), 442 PDZ domain proteins, 58, 65, 68 Pediatric model for end-stage liver disease (PELD), 534 Phase I Drug Metabolism, 33 Phase II Drug Metabolism, 34 Phenobarbital, 211, 275, 278, 279, 302 Phosphatidyl inositol (4,5) bis-phosphate (PIP2 ), 59, 100, 108, 506 Phototherapy, 273, 275, 279, 550
Subject Index Physiological jaundice of the newborn, 274 Physiological jaundice of the newborn, 262 Pigment, 257, 279, 281, 293 Plasma Proteins, 90, 305 Plasma-derived hepatitis B vaccines, 444 Plasmids, 263, 508 Plasticity of hematopoietic stem cells, 543 Polycystic disease of the liver and liver transplantation, 531 Portal blood supply, 530 Portoenterostomy (Kasai operation), 532 Post-exposure prophylaxis, 442 Pre-S1, 444, 445, 450 Pre-S2, 444 Predictors of mortality in liver failure, 317–359 Preservation for up to 24 hours, 528 Prevalence, 277, 368, 383, 384, 396, 397, 431, 433, 436, 442, 443, 539 Primary antioxidants, 268 Primary Biliary Cirrhosis 23, 36, 103, 124, 130, 167, 193, 220, 273, 294, 296, 302, 311, 383, 385, 387, 402, 406, 416, 502, 531 Primary biliary cirrhosis and liver transplantation, 383–398 Primary sclerosing cholangitis, 23, 36, 130, 167, 273, 302, 395, 402, 416, 531 Primary sclerosing cholangitis and liver transplantation, 383–398 Procollagen-III-peptide, 307 Produce gene knockouts, 262 Profilin, 54, 55 Progress in stem cell biology, 543 Proliferative stimulus, 147, 150 Prolonged neonatal jaundice, 277 Protoporphyrin IX, 259, 260, 262 Pruritus, 227, 383, 387, 388, 404, 530 PSC has a propensity to primarily affect extrahepatic and large bile ducts in the liver although a small duct variant of PSC has been described where the large ducts are spared, 37 PXR (nuclear pregnane X-receptor), 274 Pyrollizidine alkaloid, retrorsine, 550
605 R Radiation, 109, 313, 550 Rat, 5, 104–108, 137, 139 Real-time RT-PCR (polymerase chain reaction), 263 Recombinant DNA vaccine, 445 Redox cycle model, 267 Regulation of fluid transport in, 555 Regulation of liver development, 160 Regulation of transplanted cell proliferation, 549, 550 Renal bilirubin-glucuronide secretion, 529 Reperfusion phase, 547 Replacement of the liver by a donor organ, 525 Replicative senescence, 504, 545 Rh factor incompatibility, 258 RISC, 262 Risk of HBV infection, 446, 448 Risk of HBV infection increases as anti-HBs levels decline to 10 IU/l in responders, 448 RNA interference, 262, 282 RNA polymerase II, 276, 457 Rotor syndrome 281, 282 S Secondary antioxidants, 268 Selective ablation of native hepatocytes, 549 Selenium, 264, 337 Selenocysteine, 264 Serial monitoring of liver function, 534 Serologic patterns of HBV, 450 Serum bilirubin, 257, 258, 272, 273, 275, 277, 278, 282, 291, 292, 293, 294, 315, 371, 385, 395, 532, 534, 535, 537 Serum bilirubin levels do not respond to phenobarital treatment, 278 Serum unconjugated bilirubin (30-80 micromol/liter), 276 Severe intracranial hypertension, 535 Severity of encephalopathy serves as a positive predictor of mortality, 531 SH reactive agents, 262 Significant concept in inducing transplanted cell proliferation, 549
606
SUBJECT INDEX
Significant hepatic fibrosis, 549 Sinusoidal membrane, 82, 83, 84, 86, 90, 103, 108, 271, 273 SiRNA vectors, 263 Site-specificity in gene regulation, 548 Small ducts function as progenitor cells for biliary cells and hepatocytes, 39 Solute-linked water absorption in, 558 Sources of triacylglycerol for, 240 Split liver transplantation, 527, 529, 539 States for organ allocation for OLT, 535 Stearoyl CoA desaturase and, 242, 243 Stellate cells, 157, 169, 176, 187, 195, 222, 224, 334, 498, 500, 508, 509, 550 Stem cell plasticity, 543 “Stem cell-like” property, 545 Stem/progenitor cells, 544–547 Stercobilin, 257 Stercobilinogen, 257 Stimulate the binding of transcriptional factors to DNA, 266 Stones, 273, 302, 395, 558, see Gallstones Structure of VLDL, 230 Subarachnoid hemorrhage, 267 Substance abuse, inability to comply with immunosuppression, 535 Substrates, 85, 87, 88, 111, 123, 130, 207, 208, 214, 271, 272, 273, 278, 280, 326, 472, 506, 508 Superoxide dismutase (SOD), 268 Supramolecular complexes, 68 Surface mutants, 443 Surgery for OLT, 529 Survival of hepatocytes in ectopic locations, 149 T T and B cell levels than the S regions, 444 Tacrolimus, 395, 526, 537, 538, 539 TATAA box, 275–277 Telomere length, 546 Tertiary antioxidants, 268 Thalassemia, 277 The FAH mouse, 547, 549, 551 The G145R mutant, 443 The Gambia, 448 The MHCis, 527
The Model for End Stage, 534 The Molecular Virology of Hepatitis C Virus (HCV), 455 Therapeutic potential of cell transplantation, 550 There are specific compartments from which cholangiocytes proliferate (i.e., small and large sized ducts) and that differentially respond to injury, hepatic toxins or diets, 33 Thioredoxin (TRX), 264–266, 282 TRX, 266 TRX acts as a regulator of other antioxidants e.g. Mn-dependent superoxide dismutase, 266 TRX peroxidase, 266 TRX-2, 266 Thioredoxin reductase, 264–266 Thioredoxin reductase defense system, 265 Third generation vaccines incorporating pre-S1 and pre-S2 epitopes, 450 Thymopentin, 446 Tin, 262 Tin-mesoporphyrin, 275 Titer of vaccine induced anti-HBs declines, often rapidly, during the months and years following immunization, 447 Topomerase I inhibitor irinotecan, 277 Total antioxidant status, 267 Total urinary coproporphyrin excretion is normal, 282 Toxic metals, 262 Transcription, 99, 111, 121, 124, 129, 140, 152–154, 156, 179, 180, 187, 188, 216, 217, 223, 238, 239, 243, 244, 261, 263, 276 Transcription factors and embryonic liver development, 4 Transcriptional control of HO-1, 260 Transplantation antigens and liver transplantation, 527 Transplanted cells engraft, 549 Transport of H+ and HCO− 3 in, 92 Transport protein, 65, 74, 98, 249, 271, 273 Treating liver failure, chronic liver disease, various metabolic deficiency states, coagulation, 550
Subject Index
607
Treatment, 38, 70, 73, 226, 277, 279, 282, 345, 348, 350, 377, 407, 410, 413, 427, 433 Treatment of genetic disorders with liver transplantation, 533 Treatment of hepatic encephalopathy, 552 Treatment of organ rejection, 536 Triglyceride, serum levels in liver disease, 292 Tripeptide with a single cysteine residue, 266 Triple antigen hepatitis B vaccine, 445 Tumor recurrence could occur after OLT, 533 Tumors, 73, 147, 195, 196, 215, 273, 301, 302, 531, 533, 539 Two general approaches, 525 Type 1, 278, 364, 365, 546, 549, 550, 551 Type 2, 98, 278, 364, 365 U Ubiquinone cytochrome C reductase, 268 UDP-glucuronosyltransferase family, 1, 274 UDPglucuronosyltransferase, 273, 277 UGT1A1, 217, 273–279, 550 UGT1A1 Locus with Crigler Najjar syndrome mutations, 279 Unbound unconjugated bilirubin, 275 Unconjugated, 84–86, 92, 93, 126, 217, 257, 268, 272–279, 281, 292, 293, 294, 313, 314, 544, 550 Unconjugated bilirubin, 268, 272–279, 281, 314, 544, 550 Unconjugated hyperbilirubinemia, 217, 272, 278, 292, 293 Unique exon 1 is spliced to exon, 2, 276 United Network for Organ Sharing (UNOS), 528 Universal adolescent immunization, 440 Universal antenatal screening, 440 Universal criterion in the United, 535 Universal infant immunization, 440
University of Wisconsin (UW) solution, 528 Up-regulated during cholestasis, 273 Uridine-diphosphoglucuronate glucuronosyltransferase, 276 Urobilin, 257 Urobilinogen, urinary, 292 Urokinase-type plasminogen activator (alb-uPA mouse), 154, 549 Ursodeoxycholic acid, 16, 38, 119, 120, 121, 126, 129, 388, 394, 395, 397, 417 V Vaccinated homosexual men, 446 Vaccination of adolescents, 440, 441 Vaccination of infants, 440, 441 Vaccine-associated mutants, 450 Vanishing bile duct syndrome, 537 Vasoactive fragments of bilirubin, 267 Vesicular secretion, in, 560 Vesicular trafficking, 65, 468 Viral hepatitis, 181–184, 295–297, 530–532 W Watanabe rabbit model, 551 Water insoluble bilirubin, 258 When endothelial cells are injured transplanted cells engraft, 549 When larger ducts are involved in biliary atresia the prognosis is poor, 38 Wild-type sequences, 442 Wilson’s disease, 181, 307, 361, 363, 402, 417, 531, 533, 551 with CNS-1, 278, 279 with CNS-2, 278, 279 Within most mammalian cells, the concentration of GSH, 263 World reservoir of 350 million carriers, 450 X Xenobiotics, 207, 208, 323, 397, 418
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