Biomedical subjects

F V Chisari

Publications and source records attributed to F V Chisari.

At least 91 records · Page 5Linked to original sources

PubMed1996

The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice.

In this study, we examined the ability of the hepatitis B virus (HBV) precore, envelope, and X gene products to modulate HBV replication in the livers of transgenic mice that replicate the virus. Hepatic HBV replication was not affected by overexpression of the envelope or X gene products when these animals were crossed with transgenic mice that express the corresponding viral genes in the hepatocyte. Overexpression of the precore protein, however, eliminated nucleocapsid particles from the cytoplasm of the hepatocytes and abolished HBV replication without affecting the hepatic steady-state content of pregenomic HBV RNA. These observations suggest that the precore protein can exert a dominant negative effect on HBV replication, presumably at the level of nucleocapsid particle maturation or stability, suggesting an important role for this enigmatic viral protein in the HBV life cycle.

Animals↗

PubMed1996

Differential cytotoxic T-lymphocyte responsiveness to the hepatitis B and C viruses in chronically infected patients.

Cytotoxic T lymphocytes (CTL) are thought to control hepatitis B virus (HBV) infection, since they are readily detectable in patients who clear the virus whereas they are hard to detect during chronic HBV infection. In chronic hepatitis C virus (HCV) infection, however, the virus persists in the face of a CTL response. Indeed, most infected patients respond to one or more HCV-1 (genotype 1a)-derived CTL epitopes in the core, NS3, and NS4 proteins, and the CTL response is equally strong in patients infected by different HCV genotypes, suggesting broad cross-reactivity. To examine the effect of the HCV-specific CTL response in patients with chronic hepatitis C on viral load and disease activity, we quantitated the strength of the multispecific CTL response against 10 independent epitopes within the HCV polyprotein. We could not detect a linear correlation between the CTL response and viral load or disease activity in these patients. However, the CTL response was stronger in the subgroup of patients whose HCV RNA was below the detection threshold of the HCV branched- chain DNA assay than in branched-chain-DNA-positive patients. These results suggest that the HCV-specific CTL response may be able to control viral load to some extent in chronically infected patients, and they indicate that prospective studies in acutely infected patients who successfully clear HCV should be performed to more precisely define the relationship between CTL responsiveness, viral clearance, and disease severity in this infection.

Adult↗

PubMed1995

Posttranscriptional clearance of hepatitis B virus RNA by cytotoxic T lymphocyte-activated hepatocytes.

Using transgenic mice that replicate the hepatitis B virus (HBV) genome, we recently demonstrated that class I-restricted, hepatitis B surface antigen-specific cytotoxic T lymphocytes (CTLs) can noncytolytically eliminate HBV pregenomic and envelope RNA transcripts from the hepatocyte. We now demonstrate that the steady-state content of these viral transcripts is profoundly reduced in the nucleus and cytoplasm of CTL-activated hepatocytes, but their transcription rates are only slightly reduced. Additionally, we demonstrate that transcripts covering the HBV X coding region are resistant to downregulation by the CTL. These results imply the existence of CTL-inducible hepatocellular factors that interact with a discrete element(s) between nucleotides 3157 and 1239 within the viral pregenomic and envelope transcripts and mediate their degradation, thus converting the hepatocyte from a passive victim to an active participant in the host response to HBV infection.

Animals↗

PubMed1995

The cytotoxic T lymphocyte response to multiple hepatitis B virus polymerase epitopes during and after acute viral hepatitis.

Cytotoxic T lymphocytes (CTL) are thought to contribute to viral clearance and liver cell injury during hepatitis B virus (HBV) infection. Using a strategy involving the in vitro stimulation of peripheral blood mononuclear cells (PBMC) with HBV-derived synthetic peptides containing HLA-A2.1, -A31, and -Aw68 binding motifs, we have previously described CTL responses to several epitopes within the HBV nucleocapsid and envelope antigens in patients with acute hepatitis. In this study we define six HLA-A2-restricted CTL epitopes located in the highly conserved reverse transcriptase and RNase H domains of the viral polymerase protein, and we show that the CTL response to polymerase is polyclonal, multispecific, and mediated by CD8+ T cells in patients with acute viral hepatitis, but that it is not detectable in patients with chronic HBV infection or uninfected healthy blood donors. Importantly, the peptide-activated CTL recognize target cells that express endogenously synthesized polymerase protein, suggesting that these peptides represent naturally processed viral epitopes. DNA sequence analysis of the viruses in patients who did not respond to peptide stimulation indicated that CTL nonresponsiveness was not due to infection by viral variants that differed in sequences from the synthetic peptides. CTL specific for one of the epitopes were unable to recognize several naturally occurring viral variants, except at high peptide concentration, underlining the HBV subtype specificity of this response. Furthermore, CTL responses against polymerase, core, and envelope epitopes were detectable for more than a year after complete clinical recovery and seroconversion, reflecting either the persistence of trace amounts of virus or the presence of long lived memory CTL in the absence of viral antigen. Finally, we demonstrated that wild type viral DNA and RNA can persist indefinitely, in trace quantities, in the serum and PBMC after complete clinical and serological recovery, despite a concomitant, vigorous, and sustained polyclonal CTL response. Since viral persistence is not due to escape from CTL recognition under these conditions, the data suggest that HBV may retreat into immunologically privileged sites from which it can seed the circulation and reach CTL-inaccessible tissues, thereby maintaining the CTL response in apparently cured individuals and, perhaps, prolonging the liver disease in patients with chronic hepatitis.

Acute Disease↗

PubMed1995

Breaking tolerance leads to autoantibody production but not autoimmune liver disease in hepatitis B virus envelope transgenic mice.

Hepatitis B virus (HBV) transgenic mice containing the HBV envelope open reading frame under the transcriptional control of the mouse albumin promoter express hepatitis B surface Ag (HBsAg) in all of their hepatocytes and secrete HBsAg (10 to 40 ng/ml) into the circulation. Because these transgenic mice show no signs of spontaneous liver cell injury or autoimmunity toward the viral (self-) Ag, we asked whether the state of self-tolerance could be reversed by the induction of an acute necroinflammatory liver disease or by immunization with HBV envelope proteins, with the aim of creating a transgenic model for chronic, immune-mediated hepatitis. Our studies indicate that repetitive administration of bacterial LPS, IFN-gamma, or HBsAg-specific CTL, all of which were previously shown to cause liver cell injury and inflammation, does not break tolerance at the T or B cell level, suggesting that the intrahepatic lymphomononuclear cell infiltrate induced by these agents consists of HBsAg-nonspecific cells. The adoptive transfer of HBsAg-primed nontransgenic CD4+ T cells into transgenic mice did not induce anti-HBs autoantibody production by transgenic B cells, even though transgenic B cells were fully responsive to immunization with HBsAg when appropriate T cell help was provided in a nontransgenic environment. Immunization of transgenic mice with purified HBsAg in CFA and repetitive infection with rHBV envelope vaccinia virus led to production of T cell-dependent anti-HBs autoantibodies that cleared HBsAg from the serum, but not to activation of HBsAg-specific CTL. We conclude that HBV envelope transgenic mice are largely tolerant to the transgene product at the T cell but not at the B cell level, and that the activation of an anti-HBs response was not sufficient to induce an autoimmune liver disease in this HBV envelope transgenic mouse model.

Animals↗

PubMed1995

Induction in vitro of a primary human antiviral cytotoxic T cell response.

We report herein the successful priming of human anti-viral cytotoxic T cells (CTL) in vitro using two induction strategies based on the stimulation of peripheral blood mononuclear cells isolated from uninfected donors with synthetic viral peptides. The peptides used contain HLA-A2 binding motifs and have been identified as HLA-A2-restricted CTL epitopes in patients infected by the hepatitis B and C viruses. One approach uses repetitive long-term stimulation and the other uses bulk cultures containing large numbers of naive peripheral blood mononuclear cells. Both approaches successfully induce HLA-A2-restricted CTL specific for several viral epitopes. Some CTL recognize endogenously synthesized antigen on target cells infected with recombinant vaccinia virus expressing the corresponding viral proteins. This simple technique permits easy analysis of the primary human CTL repertoire, and may be exploitable for production of specific CTL effector cells for adoptive immunotherapy and dissection of the cellular and molecular requirements for priming of naive human CTL.

Amino Acid Sequence↗

PubMed1995

Hepatitis B virus immunopathology.

Approximately 5% of the world population is infected by the hepatitis B virus (HBV) which causes a necroinflammatory liver disease of variable duration and severity. Chronically infected patients with active liver disease carry a high risk of developing cirrhosis and hepatocellular carcinoma. The immune response to HBV-encoded antigens is responsible both for viral clearance and for disease pathogenesis during this infection. While the humoral antibody response to viral envelope antigens contributes to the clearance of circulating virus particles, the cellular immune response to the envelope, nucleocapsid and polymerase antigens eliminates infected cells. The class I- and class II-restricted T cell responses to the virus are vigorous, polyclonal and multispecific in acutely infected patients who successfully clear the virus, and they are relatively weak and more narrowly focussed in chronically infected patients who do not. The pathogenetic and antiviral potential of the cytotoxic T lymphocyte (CTL) response to HBV have been demonstrated by the induction of a severe necroinflammatory liver disease following the adoptive transfer of HBV surface antigen-specific CTL into HBV transgenic mice, and by the noncytolytic suppression of viral gene expression and replication in the same animals by a post-transcriptional mechanism mediated by interferon-gamma, tumor necrosis factor-alpha and interleukin-2. The dominant cause of viral persistence during HBV infection is the development of a weak antiviral immune response to the viral antigens. While neonatal tolerance probably plays an important role in viral persistence in patients infected at birth, the basis for poor responsiveness in adult onset infection is not well understood and requires further analysis. Viral evasion by epitope inactivation and T cell receptor antagonism may contribute to the worsening of viral persistence in the setting of an ineffective immune response, as can the incomplete down-regulation of viral gene expression and the infection of immunologically privileged tissues. Chronic liver cell injury and the attendant inflammatory and regenerative responses create the mutagenic and mitogenic stimuli for the development of DNA damage that can cause hepatocellular carcinoma. Elucidation of the immunological and virological basis for HBV persistence may yield immunotherapeutic and antiviral strategies to terminate chronic HBV infection and reduce the risk of its life-threatening sequellae.

Animals↗

PubMed1995

In vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells stimulated with synthetic peptides.

A protocol for in vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells (PBMCs) was developed. Antigen presenting cells (APCs) consisted of Staphylococcus aureus Cowan-I (SAC-I) activated PBMCs treated with a citrate-phosphate buffer at pH 3 to release endogenous peptides bound to surface MHC. This treatment resulted in transient expression of empty class I molecules which could be subsequently stabilized with peptide and beta 2-microglobulin (beta 2m). SAC-I activated PBMCs from HLA-A2.1 normal donors loaded with HBV core 18-27 peptide following acid treatment were used to stimulate PBMCs depleted of CD4+ T cells, in the presence of recombinant interleukin-7 (rIL-7). After 12 days, cells were restimulated with autologous, peptide-pulsed, adherent cells and tested for CTL activity 7 days later. In 23 independent experiments from 13 different HLA-A2.1 donors, this protocol resulted in induction of primary CTL more than 90% of the time. As indicated by both the frequency and magnitude of the response against peptide-sensitized target cells, SAC-I activated PBMCs treated with acid were the most efficient stimulator APC. Thirteen per cent of the cultures generated were capable of lysing target cells transfected with the HBV core antigen and, in general, these CTL cultures exhibited high avidity for the HBV core peptide. This protocol is generally applicable to different antigens and class I alleles, and thus, may be utilized to screen large numbers of peptides to identify human CTL epitopes.

Antigen-Presenting Cells↗

PubMed1995

Hepatitis B virus transgenic mice: insights into the virus and the disease.

It should be apparent from the foregoing that the transgenic mouse model system has contributed substantially to our understanding of many aspects of HBV biology, immunobiology, and pathogenesis in the past several years. We have learned that HBV can replicate within the mouse hepatocyte, as well as other mouse cell types, suggesting that there are probably no strong tissue or species-specific constraints to viral replication once the viral genome enters the cell. However, the failure thus far to detect viral cccDNA in the hepatocyte nucleus in several independently derived transgenic lineages suggests that other, currently undefined, constraints on host range and tissue specificity may also be operative. Thanks to the transgenic mouse model, we now understand the pathophysiological basis for HBsAg filament formation and "ground glass" cell production, and we have learned that at least this viral gene product can be toxic for the hepatocyte, first by compromising its ability to survive the hepatocytopathic effects of lipopolysaccharide and gamma interferon, and eventually by causing it to die in the absence of any obvious exogenous stimulus. In recent studies, it has been shown that preformed nucleocapsid particles do not cross the nuclear membrane, in either direction, at least in the mouse hepatocyte. If this is confirmed, it will have two important implications: first, that nucleocapsid disassembly must occur in the cytoplasm before the nascent viral genome can enter the nucleus; second, that the intranuclear nucleocapsid particles are empty, and therefore serve no currently defined purpose in the viral life cycle. This should stimulate new interest in the analysis of the function of these particles that are a prominent feature of mammalian hepadnavirus infection. The transgenic mouse model has also established definitively that HBV-induced liver disease has an immunologic basis, and that the class I restricted CTL response plays a central role in this process. Additionally, the mouse studies have taught us that when the CTL recognize their target antigen on the hepatocyte they cause them to undergo apoptosis, forming the acidophilic Councilman bodies that are characteristic of viral hepatitis. Further, we have learned that although the CTL initiate the liver disease, they actually contribute more to disease severity indirectly by recruiting antigen nonspecific effector cells into the liver than by directly killing the hepatocytes themselves; and that by releasing gamma interferon when they recognize antigen, the CTL can destroy enough of the liver to cause fulminant hepatitis in mice whose hepatocytes overproduce the large envelope protein and are hypersensitive to the cytopathic effects of this cytokine.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Viral mutations, TCR antagonism and escape from the immune response.

Persistent viruses use several mechanisms to evade the immune response, including the generation of mutations that affect TCR recognition. It has recently been reported that spontaneous mutations at TCR contact sites within individual viral epitopes in certain persistent human viruses can abrogate or antagonize the recognition of the corresponding wild-type epitope, and it has been suggested that such mutations may contribute to viral persistence.

Humans↗

PubMed1995

Nucleic acid vaccination primes hepatitis B virus surface antigen-specific cytotoxic T lymphocytes in nonresponder mice.

The efficiency of different vaccination techniques to prime in vivo major histocompatibility complex class I-restricted murine cytotoxic T-lymphocyte (CTL) precursors to hepatitis B virus small surface antigen (HBsAg) was investigated. Mice were immunized either by injection of a low dose of recombinant HBsAg protein preparations (native HBsAg particles or denatured HBsAg monomers) without adjuvants, by infection with recombinant vaccinia virus carrying an HBsAg-encoding gene, or by intramuscular transfer of plasmid DNA encoding HBsAg under appropriate promoter control. In H-2d mice, an Ld-restricted, S28-39-specific CTL response was efficiently primed by all alternative vaccination techniques tested, but the most potent priming of class I-restricted CTL to HBsAg in vivo was observed with DNA immunization. Priming of anti-HBsAg CTL in H-2b mice was not detectable after infection with a recombinant vaccinia virus or after injection with exogenous recombinant HBsAg preparations. After DNA immunization, however, both Kb- and Db-restricted CTL reactivity to HBsAg emerged in H-2b mice. Hence, nucleic acid immunization revealed class I-restricted CTL responsiveness to HBsAg in a mouse strain previously considered to be a nonresponder at the CTL level. These results demonstrate that the simple technique of nucleic acid immunization not only is extremely efficient but also reveals an extended spectrum of potentially immunogenic epitopes of protein antigens.

Animals↗

PubMed1995

High-level hepatitis B virus replication in transgenic mice.

Hepatitis B virus (HBV) transgenic mice whose hepatocytes replicate the virus at levels comparable to that in the infected livers of patients with chronic hepatitis have been produced, without any evidence of cytopathology. High-level viral gene expression was obtained in the liver and kidney tissues in three independent lineages. These animals were produced with a terminally redundant viral DNA construct (HBV 1.3) that starts just upstream of HBV enhancer I, extends completely around the circular viral genome, and ends just downstream of the unique polyadenylation site in HBV. In these animals, the viral mRNA is more abundant in centrilobular hepatocytes than elsewhere in the hepatic lobule. High-level viral DNA replication occurs inside viral nucleocapsid particles that preferentially form in the cytoplasm of these centrilobular hepatocytes, suggesting that an expression threshold must be reached for nucleocapsid assembly and viral replication to occur. Despite the restricted distribution of the viral replication machinery in centrilobular cytoplasmic nucleocapsids, nucleocapsid particles are detectable in the vast majority of hepatocyte nuclei throughout the hepatic lobule. The intranuclear nucleocapsid particles are empty, however, suggesting that viral nucleocapsid particle assembly occurs independently in the nucleus and the cytoplasm of the hepatocyte and implying that cytoplasmic nucleocapsid particles do not transport the viral genome across the nuclear membrane into the nucleus during the viral life cycle. This model creates the opportunity to examine the influence of viral and host factors on HBV pathogenesis and replication and to assess the antiviral potential of pharmacological agents and physiological processes, including the immune response.

Animals↗

PubMed1995

Lack of detection of negative-strand hepatitis C virus RNA in peripheral blood mononuclear cells and other extrahepatic tissues by the highly strand-specific rTth reverse transcriptase PCR.

To further explore the controversial potential for extrahepatic replication of hepatitis C virus (HCV), the highly strand-specific rTth method of reverse transcriptase PCR was used to examine sera, liver, peripheral blood mononuclear cells, and other extrahepatic tissues from HCV-infected chimpanzees and humans. Positive-strand HCV RNA was present in the liver at approximately 10-fold-higher levels than negative-strand HCV RNA. No negative-strand RNA was detected in peripheral blood mononuclear cells or other extrahepatic tissues despite the presence of abundant positive-strand RNA. These data demonstrate that within the limits of sensitivity of this highly strand-specific reverse transcriptase PCR method, no extrahepatic replication of HCV was detected.

Animals↗

PubMed1995

Hepatitis B virus immunopathogenesis.

Approximately 5% of the world population is infected by the hepatitis B virus (HBV) that causes a necroinflammatory liver disease of variable duration and severity. Chronically infected patients with active liver disease carry a high risk of developing cirrhosis and hepatocellular carcinoma. The immune response to HBV-encoded antigens is responsible both for viral clearance and for disease pathogenesis during this infection. While the humoral antibody response to viral envelope antigens contributes to the clearance of circulating virus particles, the cellular immune response to the envelope, nucleocapsid, and polymerase antigens eliminates infected cells. The class I- and class II-restricted T cell responses to the virus are vigorous, polyclonal, and multispecific in acutely infected patients who successfully clear the virus, and the responses are relatively weak and more narrowly focused in chronically infected patients who do not. The pathogenetic and antiviral potential of the cytotoxic T lymphocyte (CTL) response to HBV has been demonstrated by the induction of a severe necroinflammatory liver disease following the adoptive transfer of HBsAg-specific CTL into HBV transgenic mice, and by the noncytolytic suppression of viral gene expression and replication in the same animals by a posttranscriptional mechanism mediated by interferon gamma, tumor necrosis factor alpha, and interleukin 2. The dominant cause of viral persistence during HBV infection is the development of a weak antiviral immune response to the viral antigens. While neonatal tolerance probably plays an important role in viral persistence in patients infected at birth, the basis for poor responsiveness in adult-onset infection is not well understood and requires further analysis. Viral evasion by epitope inactivation and T cell receptor antagonism may contribute to the worsening of viral persistence in the setting of an ineffective immune response, as can the incomplete downregulation of viral gene expression and the infection of immunologically privileged tissues. Chronic liver cell injury and the attendant inflammatory and regenerative responses create the mutagenic and mitogenic stimuli for the development of DNA damage that can cause hepatocellular carcinoma. Elucidation of the immunological and virological basis for HBV persistence may yield immunotherapeutic and antiviral strategies to terminate chronic HBV infection and reduce the risk of its life-threatening sequellae.

Adult↗

PubMed1995

Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection. I. Induction of a primary cytotoxic T lymphocyte response in humans.

Our goal is to use peptide epitopes that are recognized by cytotoxic T lymphocytes (CTL) as immunogens for the development of prophylactic and therapeutic vaccines with chronic hepatitis B virus (HBV) infection being our first therapeutic target. Because most CTL peptide epitopes are poor immunogens, we specifically modified them by covalently attaching two additional components: a T helper peptide epitope and two lipid molecules. Using the murine influenza virus CTL epitope NP 147-155 as a model system, we found this construct to be highly immunogenic, and a single injection resulted in memory CTL induction that persisted for > 1 yr. Based on the animal studies, a vaccine was designed and tested for both safety and its ability to induce a primary CTL response in normal subjects. The three vaccine components included HBV core antigen peptide 18-27 as the CTL epitope, tetanus toxoid peptide 830-843 as the T helper peptide, and two palmitic acid molecules as the lipids. A dose escalation trial (5, 50, and 500 micrograms) carried out in 26 normal subjects showed that the vaccine was safe and able to induce a primary HBV-specific CTL response. A dose-response curve was observed and five out of five subjects responded to the 500-micrograms dose.

Adolescent↗

PubMed1995

Cytotoxic T lymphocyte response to hepatitis C virus-derived peptides containing the HLA A2.1 binding motif.

The HLA class I-restricted cytotoxic T lymphocyte (CTL) response is a major defense mechanism in viral infections. It has been suggested that the CTL response may contribute to viral clearance and liver cell injury during hepatitis C virus (HCV) infection. To test this hypothesis requires an understanding of the characteristics of HCV-specific cytotoxic effector cells and identification of the target antigens to which they respond. To begin this process we stimulated peripheral blood mononuclear cells (PBMC) from a group of HLA-A2 positive patients with chronic hepatitis C with a panel of 130 HCV-derived peptides containing the HLA-A2 binding motif. Effector cells were tested for their capacity to lyse HLA-A2-matched target cells that were either sensitized with peptide or infected with a vaccinia virus construct containing HCV sequences. Using this approach we have identified nine immunogenic peptides in HCV, three of which are derived from the putative core protein, three from the nonstructural (NS) 3 domain, two from NS4 and one from NS5. Selected responses were shown to be HLA-A2 restricted, mediated by CD8+ T cells and to recognize endogenously synthesized viral antigen. Unexpectedly, peptide-specific CTL responses could also be induced in sero-negative individuals, suggesting in vitro activation of naive CTL precursors. The precursor frequency of peptide-specific CTL was 10 to 100-fold higher in infected patients compared to uninfected controls, and the responses were greatly diminished by removal of CD45 RO+ (memory) T cells. Further quantitative studies are clearly required to establish whether a correlation exists between the HCV-specific CTL response and the clinical course of this disease. Definition of the molecular targets of the human CTL response to HCV creates this opportunity, and may also contribute to the development of a T cell-based HCV vaccine.

Amino Acid Sequence↗

PubMed1995

Hepatitis B virus (HBV) sequence variation of cytotoxic T lymphocyte epitopes is not common in patients with chronic HBV infection.

It has been suggested that immune selection pressure exerted by the cytotoxic T lymphocyte (CTL) response could be responsible for viral persistence during chronic hepatitis B virus infection. To address this question, in the current study we compared the DNA and amino acid sequences of, and the CTL responses to, multiple HLA-A2-restricted CTL epitopes in the hepatitis B virus in several HLA-A2-positive patients with acute and chronic hepatitis. Our results indicate that the CTL response to these epitopes is barely detectable in the majority of patients with chronic hepatitis. Further, we show that the weak CTL response is not secondary in infection by mutant viruses lacking these epitopes, and we show that the CTL response did not select for escape mutants in any of these patients. We conclude that an ineffective hepatitis B virus specific CTL response is the primary determinant of viral persistence in chronic hepatitis and that immune selection of viral variants is not a common event in the majority of patients.

Acute Disease↗

PubMed1995

Strong, sustained hepatocellular proliferation precedes hepatocarcinogenesis in hepatitis B surface antigen transgenic mice.

Hepatocyte turnover rates were studied in two lineages of transgenic mice that overproduce the hepatitis B virus (HBV) large envelope protein and retain filamentous hepatitis B surface antigen (HBsAg) particles in the endoplasmic reticulum, resulting in the formation of ground glass hepatocytes. The high producer lineage (50-4) develops a necroinflammatory liver disease that progresses to hepatocellular carcinoma (HCC), whereas the low producer lineage (107-5) displays no histopathologic changes other than ground glass hepatocytes. Bromodeoxyuridine (BrdU)-labeling studies of S-phase hepatocytes provide quantitative evidence for a strong, sustained proliferative response in the hepatocytes in lineage 50-4 that occurs after the onset of hepatocellular injury but long before the development of liver cell tumors. In contrast, the level of hepatocellular proliferation in lineage 107-5 is the same as nontransgenic controls. The findings support the concept that sustained hepatocellular proliferation plays an important role in the development of hepatocellular carcinoma (HCC).

Animals↗