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Patients with chronic hepatitis C who have not had a sustained hepatitis C virus (HCV)-RNA response or serum alanine transaminase (ALT) response to a 6-month course of interferon (IFN) may respond to higher dose retreatment with consensus interferon (CIFN). Some nonresponders to initial IFN treatment have a transient response defined as undetectable HCV RNA or normalization of ALT during treatment, but subsequently have a "breakthrough" while still on treatment. The aim of this study was to determine if nonresponders who had breakthroughs responded differently to CIFN retreatment than nonresponders without breakthroughs using data from a large, multicenter trial. ALT and HCV RNA were monitored frequently during initial IFN therapy (either 9 mcg CIFN or 3 MU IFN-alpha2b 3 times per week). HCV-RNA breakthroughs were observed in 86 of 467 (18%) of all treated patients, and ALT breakthroughs were observed in 90 of 467 (19%) of all treated patients. There was no association between breakthroughs and the presence of either binding or neutralizing anti-IFN antibodies. When the patients who were nonresponders to initial IFN treatment were retreated with CIFN (15 mcg) for 12 months, 27% of those with viral breakthroughs had a sustained viral response compared with 8% in prior nonresponders without breakthroughs (P =.102). Sustained ALT responses were observed in 39% with breakthroughs compared with 10% in those without breakthroughs (P =.014). The data suggest that prior nonresponders with breakthroughs have a greater chance of responding to retreatment than do nonresponders without breakthroughs. However, most breakthrough patients would be missed unless repeated HCV-RNA testing were conducted during therapy.

The Phyllanthus amarus plant shows potential for treating hepatitis B virus. To define the mechanism of action of P. amarus, we used HepG2 2.2.15 cells, which support hepatitis B virus replication. P. amarus inhibited hepatitis B virus polymerase activity, decreased episomal hepatitis B virus DNA content and suppressed virus release into culture medium. To examine transcriptional control mechanisms, we used G26 hepatitis B virus transgenic mice, which produce serum HBsAg but neither HBcAg nor virion particles. When P. amarus was administered to transgenic mice, hepatic HBsAg mRNA levels decreased, indicating transcriptional or post-transcriptional down-regulation of the transgene. Increase in hepatitis B virus mRNA expression after stimulation of the glucocorticoid responsive element was also suppressed by P. amarus, suggesting involvement of the hepatitis B virus enhancer in this response. Disruption by P. amarus of hepatitis B virus polymerase activity, mRNA transcription and replication supports its role as an antiviral agent.

A plasmid (palb3) was constructed containing the structural gene for human serum albumin driven by mouse albumin enhancer-rat albumin promoter elements. Using an asialoglycoprotein-polycation conjugate consisting of asialoorosomucoid coupled to poly-L-lysine, a soluble DNA complex was formed that was capable of targeting specifically to hepatocytes via asialoglycoprotein receptors present on these cells. Groups of Nagase analbuminemic rats were injected with complexed DNA or controls, followed by two-thirds partial hepatectomy to stimulate hepatocyte replication. Using a cDNA probe for the human albumin structural gene, hybridizable sequences were detected in analbuminemic rats treated with complex as determined by Southern blot analysis. Two weeks post-injection, the targeted DNA was found to exist primarily in plasmid form with an average copy number of 1000/diploid cell. Human albumin mRNA was detected by dot-blot hybridization with a specific oligonucleotide cDNA probe and confirmed by RNase protection assay using a vector-specific probe. Circulating human albumin was detected in the serum of palb3-treated Nagase analbuminemic rats by Western blots using an antibody specific for human serum albumin. A time course demonstrated that circulating human albumin was not detectable 24 h after injection, but became measurable at a level of 0.05 micrograms/ml within 48 h and increased in concentration to a maximum of 34 micrograms/ml by 2 weeks post-injection. This level of expression remained stable through 4 weeks after injection and partial hepatectomy.

Despite its being considered a primary mitogen for hepatocytes, triiodothyronine (T3) has no effect on the proliferation of hepatocytes in vitro, and in our studies, induces significant in vivo hepatocyte proliferation only during liver injury. We hypothesized that T3 may affect hepatocytes proliferation indirectly, by inducing other cells in the liver to secrete hepatic mitogens.In vivo studies: Lipopolysaccharide, T3 and a combination of the two were injected into rats, and hepatocyte proliferation was determined by PCNA staining and mitotic index.a rat hepatic stellate cell line (HSC-6T) was cultured with T3, IL-6 and a combination of the two, and we assessed the effect of these cytokine/hormone combinations on the cell proliferation and on secretion of IL-6 and HGF, measured by ELISA. Expression of thyroid hormone receptors was assessed by RT-PCR.In vivo: T3, together with lipopolysaccharide, enhances PCNA staining and the mitotic index of hepatocytes in the treated rats. In vitro: the hepatic stellate cell line expresses thyroid hormone receptor alpha 1, but not beta 1. Proliferation of stellate cells is not affected by T3, with or without IL-6. T3 has no effect on secreted levels of IL-6 in the stellate cell line. Hepatic stellate cells cultured with T3 and IL-6 show significantly increased amounts of secreted HGF after 48 h in culture.T3 may induce hepatocyte proliferation in vivo during injury by turning on expression of HGF in stellate cells and acting together with IL-6.

Recently, we described a new model for hepatocyte transplantation with nearly total replacement of the liver by exogenous hepatocytes (E. Laconi et al., Am. J. Pathol., 153: 319-329, 1998). The model is based on the mitoinhibitory effect of the pyrrolizidine alkaloid retrorsine on hepatocytes in the resident liver while transplanted hepatocytes proliferate. In this study, we exploit this novel approach to address the important and controversial issue of whether hepatocytes, when proliferating extensively, undergo dedifferentiation and give rise to foci of undifferentiated hepatocytes. Genetically marked hepatocytes (isolated from normal Dipeptidyl peptidase IV+ Fischer 344 rats) were delivered intraportally (2 x 10(6) cells) into the liver of retrorsine-treated Dipeptidyl peptidase IV- mutant Fischer 344 rats in conjunction with partial hepatectomy. Transplanted hepatocytes were detected histochemically or immunohistochemically, and cell proliferation was studied by in situ hybridization for histone-3 mRNA. Expression of alpha-fetoprotein (AFP) mRNA, a marker of hepatocyte dedifferentiation, was also revealed by in situ hybridization. One day after partial hepatectomy and hepatocyte transplantation, endogenous hepatocytes and oval cells expanding in the liver expressed histone-3 mRNA (cells had entered S phase); 2 days later, transplanted hepatocytes and nonparenchymal cells also expressed histone-3 mRNA. Although the majority of endogenous hepatocytes did not divide and became arrested as quiescent megalocytes, the exogenous hepatocytes, as well as newly formed small hepatocytes, most probably derived from liver progenitor cells, underwent extensive proliferation. After 7-14 days, the nonparenchymal cells stopped proliferating, but transplanted hepatocytes and small endogenous hepatocytes continued to proliferate for 1 month, forming foci of dividing parenchymal cells. Although many of the hepatocytes in clusters were in S phase (histone-3 mRNA positive), none expressed AFP mRNA. In contrast, high expression of AFP mRNA was observed in proliferating oval and transitional cells, forming duct-like structures of cytokeratin-19-positive cells. From these studies, we conclude that hepatocyte proliferation in the adult liver is not associated with dedifferentiation.

The pattern of mRNA expression for liver-specific proteins and liver-enriched transcription factors was studied in two models of facultative gut epithelial progenitor cells activation: D-galactosamine (GalN)-induced liver injury and dietary copper depletion leading to pancreatic acinar atrophy. After 5 weeks of copper deficiency (CuD), pancreatic acini of Fischer 344 rats underwent atrophy, associated with intense proliferation of small duct-like cells with oval-shaped nuclei. These cells resemble morphologically epithelial progenitor cells of the liver that proliferate after GalN administration. Activated pancreatic epithelial cells express mRNAs for liver-specific genes normally expressed in fetal liver, including alpha-fetoprotein, albumin, alpha-1 antitrypsin, glucose-6-phosphatase, and others, but not genes that are turned on after birth such as serine dehydratase, tyrosine aminotransferase, and multidrug resistance gene-1b. They express mRNAs for liver-enriched transcription factors including HNF-1 alpha, HNF-3 beta and gamma, HNF-4, and members of the CCAAT-enhancer binding protein (C/EBP) family. The only mRNA for a liver-enriched transcription factor not detected in the pancreas of CuD animals was HNF-3 alpha. Expression of HNF-3 alpha, beta, and gamma, and C/EBP-beta mRNA was highly activated in proliferating liver epithelial cells on days 2 and 3 after GalN injury. Increased expression of C/EBP-delta was observed first in the liver on day 1 after GalN administration and in the pancreas at 4 weeks after initiating CuD. We suggest that C/EBP-delta could be involved in the initial activation of epithelial progenitor cells and that HNF-3 alpha, beta, and gamma, and C/EBP-beta might participate in their maturation. We conclude further that pancreatic epithelial progenitor cells undertake differentiation through the hepatocyte lineage but cannot complete the differentiation program within the pancreatic milieu.

Rat liver regeneration was studied from 24 hours to 8 days after a single intraperitoneal injection of D-galactosamine (GalN). Morphological changes in the liver were analyzed in parallel with sequential changes in expression of histone-3 mRNA (a marker of cell proliferation), fetal alpha-fetoprotein (AFP) mRNA and gamma-glutamyl transpeptidase (GGT) (markers of fetal hepatocytes), and albumin mRNA and glucose-6-phosphatase (G6Pase) (markers of adult hepatocytes). Proliferation of nonparenchymal epithelial cells (NPC), detected in situ by [3H]thymidine labeling or histone-3 mRNA expression, began after 24 hours primarily in the portal area around the bile ducts. After 2 days, histone-3 labelling intensity increased in rows and clusters of NPC which expanded from the portal zone and invaded into the parenchyma. On days 3 and 5, NPC expressing his-3 mRNA expanded further, forming pseudo-ducts and islet-like structures (NPC structures). Proliferating NPC were positive for GGT. Some GGT positive cells were also positive for the fetal form of AFP mRNA, which lagged behind GGT by 24 hours and peaked on day 5. On day 3, some cells with the appearance of NPC expressed albumin mRNA. Double label in situ hybridization for fetal AFP and albumin mRNAs and dual histochemistry for GGT and G6Pase showed simultaneous expression of these markers in NPC on day 5. Other cells expressing fetal AFP mRNA or GGT on day 5 had a morphological appearance between NPC and hepatocytes (transitional cells). Proliferation of hepatocytes began on day 2, reached maximum on day 5 and then declined. Proliferating hepatocytes did not express fetal AFP mRNA or GGT. These findings indicate that after GalN injury, the liver responds by activation of progenitor cells that proliferate and then differentiate into mature hepatocytes. Adult hepatocytes can also proliferate after GAlN injury, but these hepatocytes do not undergo dedifferentiation/redifferentiation during regeneration of the hepatic lobule.

Although mankind has suffered the ravages of hepatitis from biblical times, it is only in the past three decades that we have begun to understand the nature of the viruses which infect the liver. We can now confidently talk of at least five viruses designated as hepatitis viruses (А, B, С or post-transfusion non-A non-B, D or delta, and E or water-borne epidemic non-A non-B). All cause acute hepatitis. Clinical manifestations alone are insufficient to allow distinction of one virus from another. However, an understanding of the biology and epidemiology of hepatitis viruses has greatly facilitated diagnosis. Hepatitis D virus (HDV) is unique because it is defective and requires hepatitis B virus (HBV) to survive. Both HBV and HDV (as well as HCV) can persist long-term and cause chronic hepatitis, cirrhosis, and/or death. Therefore, recognition of the onset and resolution of HBV or HDV infection are equally important. Moreover, highly effective vaccines against HBV have been developed and means are at hand to prevent HBV altogether and consequently, HDV.

Hepatocytes from donor transgenic mice that produce an easily assayable circulating marker have been used to develop a novel hepatocyte transplantation system. Isolated G7 HBV transgenic donor hepatocytes secreting HBsAg were transplanted into congeneic or allogeneic mouse recipients. Serum HBsAg was present three days after hepatocyte transplantation in congeneic animals and persisted indefinitely when hepatocytes were transplanted into the spleen. Transplanted hepatocytes within the splenic pulp were identified by morphologic and histochemical analysis. Migration of hepatocytes injected into the spleen to the liver was demonstrated by in situ hybridization using an RNA probe for HBsAg. Transplantation into nonimmunosuppressed allogeneic recipients resulted in disappearance of detectable hepatocytes in the spleen within two weeks. This novel transplantation system should facilitate studies of hepatocyte engraftment and survival, modulation of allograft rejection, and development of hepatocyte-directed gene therapy.

Hepatitis B virus (originally termed serum hepatitis) has been known to exist for more than four decades and the virus particle was physically demonstrated by electron microscopy more than 15 years ago; yet until recently, progress has been very slow in understanding the nature and biology of this virus. The major limitation has been the lack of cell culture systems to effectively propagate hepatitis B virus (HBV) and study its replication cycle. However, with the advent of recombinant DNA methods in the 1970’s, the cloning of the complete HBV genome, the information derived from sequencing this genome, and use of cloned HBV DNA as a molecular probe, significant advances have been made during the past six years. This review will deal with our current understanding of the HBV genome, the nature of its gene products and the virus replication mechanism, all which illustrate unique features. Finally, information will be presented concerning our present understanding of the possible role of HBV in hepatic oncogenesis for which a clear association has been shown by epidemiologic studies.

The synthesis of protein requires a complex interaction of cellular constituents. Transfer of genetic information from DNA into protein can be divided into two main processes: 1) transcription of DNA into “messenger” RNA, and 2) translation of “messenger” RNA into specific polypeptide chains composed of the appropriate mixture of amino acids. In differentiated cells this process yields proteins for functions common to all eukaryotic cells (“housekeeping” functions), as well as certain specific proteins with unique properties for the given cell type. For example, the liver is the only organ known to synthesize certain serum proteins, such as albumin, fibrinogen, etc. For the synthesis of these specialized proteins, it is not unreasonable to anticipate that a significant portion of the cellular biosynthetic capacity might be devoted toward synthesis of these specialized proteins. Cellular control processes to regulate synthesis of these proteins at either the transcriptional or post-transciptional level could represent an important mechanism for controlling specialized functions in normal as well as physiologically or pathologically altered cells. In order to assess transcriptional and/or translational events controlling synthesis of unique proteins such as albumin, a simple quantitative assay for messenger RNA was needed.

Although serum albumin was originally recognized and described by Ancell as early as 1837 (1 ), it has only been possible to obtain insight into the mechanism for albumin synthesis in the past 10–15 years. The purpose of this chapter is to describe methods developed recently to study albumin synthesis at the molecular level. However, since classical methods still have more clinical relevance, we would like to mention briefly some of these approaches. Initial studies were devoted to the simple measurement of serum albumin levels in normal individuals and patients with various disorders and Dr. Majoor made a number of significant contributions in this area (2). Subsequently, a variety of radioactive labeling procedures were developed which permitted determination of the serum half-life (T1/2) for this protein (3). This involved intravenous injection of tracer doses of radioactive labeled albumin into patients with hypoalbuminenia and determination of the radioactive decay curve for injected material. Assuming that synthesis of albumin is equivalent to its rate of loss or degradation and that the serum pool is at steady state, a rate for albumin synthesis can be calculated indirectly from the serum decay curve (4). The next major advance in studying albumin synthesis was the development by McFarlane and colleagues of a method to measure albumin synthesis rate directly in whole animals. As these investigators recognized, there was a direct correlation between incorporation of arginine into albumin and urea synthesis, and measurement of labelled arginine in albumin after injection of 14C carbonate versus urea production could be used to calculate albumin synthesis (5). Techniques were then advanced to permit studies of albumin synthesis in the isolated—perfused liver (6), and this model has provided us with much useful information on factors involved in the regulation of albumin synthesis. These topics will be explored in depth in subsequent chapters.

A major avenue of our research has been to develop molecular probes (cDNAs) for studying the pathogenesis of liver disease (i.e., applied molecular pathophysiology). During the last 2-3 yr, we have developed and used molecular hybridization to study regulation of albumin synthesis in normal, protein-calorie deprived, uremic, and cirrhotic rat liver. Our current work is directed toward cloning the albumin gene to permit further analysis of albumin transcriptional and posttranscriptional control. Molecular hybridization, DNA cloning, related techniques can be utilized to study the function of virtually any gene. Incorporation of such advances in basic research into meaningful studies of liver disease is an exciting challenge to modern academic hepatology.

The human hepatocellular carcinoma cell line PLC/PRF/5, which synthesizes and secretes hepatitis B surface antigen, was grown under optimal conditions in tissue culture, using Eagle's minimal essential medium supplemented with 10% fetal bovine serum and 10(-11) M triiodothyronine on collagen rafts. Injection s.c. of the PLC/PRF/5 cell line into athymic BALB/c nude mice resulted in the growth of a well-circumscribed, moderately differentiated hepatocellular carcinoma. The intervals until tumor appearance and tumor "take" rates were dependent on inoculum dose. Four to 5 x 10(6) cells induced tumor growth in 29% of 14 injected mice within 29 to 40 days, while 7 to 13 X 10(6) cells induced tumors in all 15 mice within 10 to 12 days after inoculation. Hepatitis B surface antigen was detected in the nude mouse serum and tumor tissue, and its concentration roughly correlated with tumor weight. A low level of antibody against hepatitis B surface antigen was detected in five tumor-bearing animals, as well as in one mouse which did not produce a tumor. Hepatitis B core antigen and its antibody and hepatitis B e antigen and its antibody were not detected in 26 mice, using immunohistochemical and radioimmunoassay methods. alpha-Fetoprotein, carcinoembryonic antigen, and alpha-antitrypsin were detected in nude mice tumors, using the peroxidase-antiperoxidase technique. Finally, hepatitis B virus DNA, identified in the nude mouse tumor by molecular hybridization techniques, was compared to PLC/PRF/5 cell line hepatitis B virus DNA.

The hepatitis B virus (HBV) genome contains a specific DNA binding site for the glucocorticoid receptor. Using DNase I footprinting, this binding site was localized at HBV map positions 341-370 clockwise from the EcoRI site. The DNA sequence protected in the footprint contains two tandem copies of the GRE core hexanucleotide 5'-TGTTCCT-3'. Deletion analysis and reconstruction experiments in plasmid expression vectors demonstrated that this glucocorticoid receptor binding sequence serves as a signal for augmenting glucocorticoid-dependent activity of the HBV enhancer, which is located approximately 730 nucleotides downstream in the HBV genome. Even though it does not serve as an independent enhancer element, the HBV glucocorticoid receptor domain can therefore be categorized as a functional GRE.

Nuclear RNAs prepared from rat liver and rat hepatoma cell line H4AZC2 have been fractionated and examined for albumin mRNA sequences by annealing to specific albumin [3H]cDNA. In both instances, sucrose gradient analysis revealed nuclear RNA molecules containing albumin RNA sequences which sedimented at 26 S (26 S albumin RNA). In contrast, cytoplasmic albumin messenger RNA sediments exclusively at 17 S. 26 S albumin RNA is resistant to both heat denaturation (65 degrees C X 5 min) and denaturation in 85% formamide (v/v), and 75% of these molecules are polyadenylated. These results provide evidence for the existence of an intact, high molecular weight, polyadenylated nuclear RNA which contains albumin mRNA sequences.

Translation of capped and uncapped eukaryotic mRNAs is stimulated by addition of eIF-4B to an mRNA-dependent reticulocyte lysate system. m7G5 ppp inhibits translation of capped but not uncapped mRNAs and reduces translation of capped vaccinia mRNA to the level obtained with uncapped vaccinia mRNA. Exogenous eIF-4B but no other initiation factor reverses inhibition of protein synthesis by m7G5'ppp. Both capped and uncapped mRNAs interact directly with eIF-4B to form a stable complex, which can be detected by a simple nitrocellulose filter binding assay. However, addition of a 5'-cap to beta-eliminated globin mRNA or satellite tobacco necrosis virus RNA (normally uncapped) increased binding affinity of these mRNAs for eIF-4B and causes binding of these mRNAs to become sensitive to inhibition by m7G5'ppp. These results indicate that the role of the mRNA 5'-cap in translation is related specifically to the function of eIF-4B in forming a complex with mRNA (prior to association of mRNA with the 40 S ribosomal subunit) and that both cap and non-cap sequences participate in this process.

Recently, using molecular hybridization techniques with albumin [3H]cDNA, we have determined that in normally fed rats 98% of total liver polyribosomal albumin mRNA sequences are found in membrane-bound polyribosomes (Yap, S. H., Strair, R. K., and Shafritz, D. A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5397-5401). We now observe that a 24- to 30-h withdrawal of food leads to major changes in the amount and subcellular distribution of albumin mRNA molecules. The total amount of cytoplasmic albumin mRNA per liver and concentration of albumin mRNA per unit of membrane-bound polyribosomal RNA are decreased. However, the proportion of albumin mRNA present in the postribosomal supernatant fraction increases dramatically in a short term fast, so that it now represent 60% of total cytoplasmic albumin mRNA sequences. Most of the albumin mRNA sequences in the postribosomal supernatant fraction sediment between 30 S and 50 S. These findings suggest that albumin mRNA is probably stored in the messenger ribonucleoprotein fraction during the fasting state.

HBV DNA was assayed by molecular hybridization in the serum of 21 patients with HBsAg + /delta Ag + chronic liver disease (CLD). The results were compared to those in 21 HBsAg + /delta Ag - cases matched for histological diagnosis and HBeAg/anti-HBe status and to HBcAg and delta Ag detection in the liver by direct immunofluorescence. In the delta Ag + cases serum HBV DNA was either negative (17/21 or 81%) or transiently positive (4/21 or 19%) in small or trace amounts (trace to 1 +). By contrast 9 of 21 (43%) of HBsAg + delta Ag - cases were serum HBV DNA positive, 6 of them having values ranging from 2 + to 4 +. HBcAg was detected in the liver of patients with strong serum HBV DNA reactions and was absent from all serum HBV DNA negative ones. These results indicate a significant difference in the HBV replication level between delta Ag positive and negative patients with HBsAg + CLD and with comparable liver histology and HBeAg/anti-HBe status, and support the need for wider application of molecular hybridization techniques in the pathogenetic and epidemiological study of HBV infection.