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F V Chisari

Publications and source records attributed to F V Chisari.

At least 127 records · Page 7Linked to original sources
PubMed1993

HLA A2 restricted cytotoxic T lymphocyte responses to multiple hepatitis B surface antigen epitopes during hepatitis B virus infection.

Inasmuch as the hepatitis B virus (HBV) is not directly cytopathic for the infected hepatocyte, it is generally presumed that viral clearance and liver cell injury during viral hepatitis are due to a CTL response to HBV encoded Ag presented by HLA class I molecules. We have previously examined the peripheral blood CTL response to two HBV nucleocapsid epitopes in patients with acute and chronic viral hepatitis, one of which is restricted by HLA-A2, whereas the other is dually restricted by HLA-A31 and Aw68. In this study, we defined the HLA-A2-restricted CTL response to the hepatitis B surface Ag (HBsAg) by using a panel of HBsAg-derived synthetic peptides containing the ideal HLA-A2.1 binding motif (-L------V). Several novel aspects of HBV immunobiology and pathogenesis are evident from this study. First, the peripheral blood CTL response to HBV-encoded Ag is remarkably polyclonal and multispecific in most patients with acute hepatitis. Indeed, HLA-A2-restricted CTL specific for as many as four envelope epitopes and one nucleocapsid epitope were found to be present simultaneously in individual patients with acute viral hepatitis. Second, HBV-specific CTL are not detectable in the peripheral blood in a minority of patients with acute hepatitis, nor have we detected a CTL response in any of the patients with chronic hepatitis that we have studied thus far. Although the cellular and molecular basis for CTL nonresponse remains to be determined, the data suggest that it may contribute to viral persistence. Third, the diversity and the specificity of the CTL response is determined in part by the coding sequence of the viral genome present in each infected patient. Indeed, the apparent nonresponse of some acutely infected patients to at least one HBsAg-specific CTL epitope actually reflects infection by a viral variant that contains a critical substitution in one of the anchor residues within the epitope. Finally, at a fundamental level, the data suggest that the presence of the HLA-A2.1-binding motif in a peptide may not be sufficient for binding; and the capacity of a peptide to bind the class I molecule does not guarantee that it will be immunogenic.

Amino Acid Sequence↗
PubMed1993

HLA-A31- and HLA-Aw68-restricted cytotoxic T cell responses to a single hepatitis B virus nucleocapsid epitope during acute viral hepatitis.

We have recently developed the technology to identify and characterize the human histocompatibility leukocyte antigen (HLA) class I-restricted, CD8+ cytotoxic T lymphocyte (CTL) response to hepatitis B virus (HBV)-encoded antigens in patients with acute viral hepatitis. CTL are expanded in vitro by stimulation with HBV-derived synthetic peptides and selected by restimulation with a panel of HLA-matched stable transfectants that express the corresponding HBV protein. We have recently reported the existence of an HLA-A2-restricted, CD8+ CTL response to an epitope located between residues 18 and 27 of the HBV nucleocapsid core antigen (HBcAg). We now report the discovery of a CTL epitope located between HBcAg residues 141 and 151 that completely overlaps a critical domain in the viral nucleocapsid protein that is essential for its nuclear localization and genome packaging functions as well as processing of the precore protein. The CTL response to this epitope is dually restricted by the HLA-A31 and HLA-Aw68 alleles, which, unexpectedly, appear to use a common binding motif based on the results of alanine substitution and competition analysis, and the binding properties of these two alleles predicted from their known primary sequence, and from the three-dimensional structure of HLA-Aw68. We have also demonstrated that the HBV-specific CTL response to this epitope is polyclonal during acute viral hepatitis, since these two restriction elements can present the HBcAg 141-151 epitope to independent CTL clones derived from a single patient; and that the CTL response is multispecific, since HLA-A2-restricted and HLA-Aw68-restricted CTL responses to HBcAg 18-27 and HBcAg 141-151, respectively, have been identified to coexist in another patient. The foregoing argue against the emergence of CTL escape mutants as a significant problem during HBV infection, especially at this locus, where mutations might be incompatible with viral replication. Finally, our data suggest an association between the HBV-specific CTL response and viral clearance, and they have implications for the design of immunotherapeutic strategies to terminate HBV infection in chronically infected patients.

Acute Disease↗
PubMed1993

Interleukin-2 downregulates hepatitis B virus gene expression in transgenic mice by a posttranscriptional mechanism.

We have recently demonstrated that tumor necrosis factor alpha (TNF-alpha) and interleukin-2 (IL-2) downregulate the hepatic steady-state content of hepatitis B virus (HBV) mRNA in vivo in HBV-transgenic mice and that the IL-2 effect is mediated by TNF-alpha. In the current study, we demonstrate that IL-2-induced downregulation of hepatic HBV 2.1-kb mRNA is not due to changes in the transcription rate or the intranuclear maturation or export of this transcript but that it is selectively and profoundly depleted from the cytoplasm of the liver cells in vivo following IL-2 administration. Collectively, these results suggest that IL-2 alters the steady-state content of hepatic HBV mRNA by a posttranscriptional mechanism in vivo, that this effect is mediated by TNF-alpha, and that it probably reflects increased cytoplasmic degradation of the viral transcript.

Animals↗
PubMed1993

Definition of a minimal optimal cytotoxic T-cell epitope within the hepatitis B virus nucleocapsid protein.

Residues 11 to 27 of the hepatitis B virus nucleocapsid antigen contain a cytotoxic T-cell epitope that is recognized by cytotoxic T cells from virtually all HLA-A2-positive patients with acute hepatitis B virus infection. Using panels of truncated and overlapping peptides, we now show that the optimal amino acid sequence recognized by cytotoxic T cells is a 10-mer (residues 18 to 27) containing the predicted peptide-binding motif for HLA-A2 and that this peptide can stimulate cytotoxic T cells able to recognize endogenously synthesized hepatitis B core antigen. Since patients with chronic hepatitis B virus infection fail to mount an efficient cytotoxic T-cell response to it, this epitope might serve as the starting point for the design of synthetic peptide-based immunotherapeutic strategies to terminate persistent viral infection.

Acute Disease↗
PubMed1993

Human leukocyte antigen class I-independent pathways may contribute to hepatitis B virus-induced liver disease after liver transplantation.

The proliferative response of peripheral blood lymphocytes to the HBcAg was compared with serological, molecular and immunohistochemical parameters of hepatitis B virus infection and with biochemical and histological parameters of liver disease in a patient who received a completely human leukocyte antigen class I-mismatched liver allograft for fulminant hepatitis. The proliferative response increased progressively after transplantation, as hepatitis B virus infection became reestablished in the hepatic allograft. Strikingly, the HBcAg-specific T cells suddenly disappeared from the peripheral blood immediately before the acute onset of a severe necroinflammatory liver disease in which more than 80% of the hepatocytes expressed HBcAg. These observations are compatible with the hypothesis that human leukocyte antigen class I-independent hepatitis B virus-specific T cells might play a previously unsuspected role in the pathogenesis of hepatitis B virus-induced liver disease.

Adult↗
PubMed1992

Multiple oncogenes and tumor suppressor genes are structurally and functionally intact during hepatocarcinogenesis in hepatitis B virus transgenic mice.

In the current study we sought to elucidate the molecular mechanisms which might contribute to hepatocarcinogenesis in a hepatitis B virus (HBV) envelope transgenic mouse model in which chronic hepatocellular injury and inflammation lead to regenerative hyperplasia and eventually to the development of chromosomal abnormalities and hepatocellular carcinoma (HCC), thereby reiterating many of the pathophysiological events that occur prior to the development of HCC in chronic HBV infection in humans. We have previously demonstrated that HBV envelope gene expression is decreased in regenerating hepatocytes and preneoplastic nodules early in the disease process and that expression of alpha-fetoprotein and the multidrug transporter gene mdr-III is activated in the tumors that develop in this model, but not prior to tumor development. In the current study, we examined the structure and expression of a large panel of dominant acting oncogenes and tumor suppressor genes in the liver at all stages of the disease process in order to determine the extent to which they contribute to hepatocarcinogenesis in these transgenic mice. To our surprise, no changes were observed in the structure or function of any of these genes, many of which are commonly activated in other rodent models of hepatocarcinogenesis but rarely activated in human HCC. These findings suggest that the HBV transgenic mouse model is different from most other rodent models of hepatocarcinogenesis and that it may relate more closely to the events involved in HBV-induced human hepatocarcinogenesis, where generalized chromosomal abnormalities are common, while structural and functional changes in most of the commonly studied positive-acting oncogenes examined herein are not. Since p53 and RB mutations have recently been reported to be late events in human hepatocarcinogenesis, the structural integrity of the RB locus and the absence of p53 mutations in the HBV transgenic mouse model suggest that they may represent a relatively early stage of hepatocellular tumorigenesis and that further manipulation of this model is warranted in order to more fully reproduce the molecular-genetic events that characterize HBV-induced HCC in humans.

Animals↗
PubMed1992

Reactivation of insulin-like growth factor II during hepatocarcinogenesis in transgenic mice suggests a role in malignant growth.

We have studied the expression of insulin-like growth factor II (IGF-II) during hepatocarcinogenesis in four independent transgenic mouse lines. In all four lines liver-directed transgene expression induces a stepwise and relatively synchronized tumorigenesis. IGF-II reexpression occurs in all four lines irrespective of the mechanism of tumor induction. Reexpression is chronologically associated with late progression steps toward hepatocellular carcinoma and correlated with the respective tumor progression rate in each line. IGF-II activation is focal and topographically associated with high replicative activity. IGF-II mRNAs in hepatocellular carcinomas show similarities to the expression pattern in fetal liver, and a M(r) 15,000 IGF-II polypeptide accumulates intracellularly in distinct cytoplasmic preferentially perinuclear compartments. These data indicate that IGF-II reexpression is a marker for progression to hepatocellular carcinoma and may contribute to hepatocarcinogenesis via an autocrine mechanism.

Animals↗
PubMed1992

HBsAg retention sensitizes the hepatocyte to injury by physiological concentrations of interferon-gamma.

The role that inflammatory cytokines may play in the life cycle of the hepatitis B virus and in the pathogenesis of its associated liver disease has not been carefully delineated. In this report, we demonstrate that bacterial lipopolysaccharide, a potent inducer of inflammatory cytokines in vivo, causes a severe acute liver disease in transgenic mice whose hepatocytes produce the hepatitis B virus large envelope polypeptide and retain HBsAg within the endoplasmic reticulum. In contrast, 100-fold higher doses of bacterial lipopolysaccharide do not induce liver cell injury in nontransgenic littermate controls or in transgenic mice whose hepatocytes secrete HBsAg rather than retain it. Coincident with the hepatocellular injury and the influx of inflammatory cells into the liver, a marked reduction occurs in the intrahepatic content of hepatitis B virus steady-state messenger RNA, thereby confirming the selectivity of this process for the HBsAg-positive hepatocyte. Bacterial lipopolysaccharide-induced hepatocellular injury appears to be principally mediated by interferon-gamma because it can be markedly reduced by the prior administration of neutralizing interferon-gamma-specific monoclonal antibodies and because recombinant interferon-gamma is also selectively cytotoxic for the HBsAg-positive transgenic hepatocyte in vivo. Tumor necrosis factor-alpha is also involved in this process because bacterial lipopolysaccharide-induced liver cell injury is significantly reduced by tumor necrosis factor-alpha specific monoclonal antibodies. The role of tumor necrosis factor-alpha in bacterial lipopolysaccharide-induced liver cell injury is less clear than interferon-gamma, however, because unlike interferon-gamma it is also toxic for nontransgenic hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine Transaminase↗
PubMed1992

Fine specificity of the human T cell response to hepatitis B virus core antigen.

The fine specificity of the human T cell response to the hepatitis B virus core antigen (HBcAg) was investigated in 23 patients with acute hepatitis B virus (HBV) infection using a panel of short synthetic peptides covering the entire core region. An immunodominant T cell epitope which was recognized by all except one patient, was identified within the core sequence 50-69. Two further important T cell recognition sites were represented by the amino acid sequences 1-20 and 117-131, which were stimulatory for the T cells of 69% and 73% of the patients, respectively. T cell recognition of the synthetic peptides was HLA class II restricted because the peptide-induced T cell proliferation was inhibited by anti-HLA class II but not by anti-HLA class I monoclonal antibodies. These findings may be relevant to the development of future preventive and therapeutic strategies against HBV infection.

Acute Disease↗
PubMed1992

Cellular and molecular mechanisms of hepatocarcinogenesis.

For colorectal carcinomas, the rate of tumor development is proportional to the fourth to sixth power of elapsed time, suggesting that four to six independent events are necessary. Although similar calculations have not been made for HBV-associated HCCs, it is likely that this is also the case for HCCs, since individuals with persistent HBV infection do not usually develop HCC until they are 45 or greater years old. As evidence for specific genetic and epigenetic changes in HCCs accumulate, the important players in multistep hepatocarcinogenesis are becoming clearer. However, even though Myc family oncogenes are clearly implicated in woodchuck HCC, similar integrations have not been found in human HCCs. Therefore, although rodent and human systems have many similarities, we must realize that important differences may also exist. Regarding tumor suppressor genes, the evidence for p53 alterations in HCC is strong. A growing body of evidence suggests further that alterations in the retinoblastoma gene and one or more tumor suppressor genes on chromosome 11 are also involved in HCC. HBV integrations may certainly play a role in the generation of chromosome aberrations leading to loss of tumor suppressor alleles, since chromosomes 11 and 17 are the most common integration sites. Finally, the role of X proteins as participants in malignant transformation has been demonstrated for certain immortalized, nontransformed hepatocytes. Altered autocrine mechanisms of cell growth control, possibly involving IGF-II, are clearly implicated in HCC. Paracrine mechanisms for the control of hepatocyte growth and differentiated functions may also be altered as a result of the synthesis and secretion of a complex array of interleukins, HGF, and basic and acidic FGFs by cells in the inflammatory and cirrhotic lesions of precancerous livers. Whether the order of molecular changes in the hepatocyte is important for malignant progression is presently not clear. What is clear, however, is that hepatocarcinogenesis involves alterations in the concerted action of protooncogenes, growth factor, and tumor suppressor genes. How these factors interact will provide a more complete understanding of the mechanism or mechanisms of hepatic oncogenesis.

Animals↗
PubMed1992

Hepatitis B virus (HBV)-specific cytotoxic T-cell (CTL) response in humans: characterization of HLA class II-restricted CTLs that recognize endogenously synthesized HBV envelope antigens.

In this study, we show that CD4+, hepatitis B virus (HBV) envelope-specific T-cell clones produced by stimulation with a particulate antigen preparation are able to recognize and kill not only autologous antigen-presenting cells incubated with exogenous HBV envelope antigens but also autologous HLA class II-positive cells expressing endogenously synthesized HBV envelope antigens following infection with recombinant vaccinia viruses or transfection with recombinant Epstein-Barr virus expression vectors. Experiments with lysosomotropic agents and brefeldin A suggest that the endosomal compartment is likely involved in the processing of endogenously synthesized viral proteins for recognition by CD4+ T cells. Our study indicates that HBV envelope-specific, HLA class II-restricted CD4+ cytotoxic T lymphocytes can potentially participate in the immune clearance of HBV-infected cells and the pathogenesis of hepatocellular injury in hepatitis B.

Ammonium Chloride↗
PubMed1992

Hepatitis B virus (HBV)-specific cytotoxic T-cell response in humans: production of target cells by stable expression of HBV-encoded proteins in immortalized human B-cell lines.

To analyze the hepatitis B virus (HBV)-specific cytotoxic T-cell (CTL) response during acute and chronic viral hepatitis, target cells that express HBV-encoded antigens in the context of the appropriate HLA restriction element must be available for each subject studied. Since HBV is not infectious for human cells in vitro, such target cells must be produced by DNA-mediated gene transfer into cultured human primary cells or cell lines. For this purpose, we have developed a panel of Epstein-Barr virus-based episomal expression vectors containing each of the HBV open reading frames under the transcriptional control of the simian virus 40 early promoter. Transfection of Epstein-Barr virus-immortalized B-cell lines with this panel of recombinants consistently leads to stable expression of the HBV envelope, nucleocapsid, and polymerase proteins. The HBV X gene product is transiently expressed following transfection, but stable expression of this protein cannot be maintained on a long-term basis. To assess the suitability of this system for the identification of HBV-specific CTL in humans, a panel of EBO-HBV transfectants of defined HLA haplotype was used to monitor the HBV-specific CTL response in a patient with acute viral hepatitis type B. Transfectants that stably express the HBV nucleocapsid (core) antigen were found to serve as excellent targets for the detection of HLA class I-restricted CTL that recognize endogenously synthesized HBV core antigen in this patient; they were also successfully used to stimulate the specific expansion of these CTL in vitro.

B-Lymphocytes↗
PubMed1992

Tumor necrosis factor alpha negatively regulates hepatitis B virus gene expression in transgenic mice.

It is well known that several inflammatory cytokines can modulate hepatocellular gene expression in a complex physiological process known as the hepatic acute-phase response. Since hepatitis B virus (HBV) characteristically induces a vigorous lymphomononuclear inflammatory response in the liver during acute and chronic hepatitis, it is possible that hepatocellular HBV gene expression may also be modulated by one or more of the cytokines produced by these cells. Using bacterial lipopolysaccharide (LPS) as a surrogate inducer of inflammatory cytokines in vivo, we have tested this hypothesis in a transgenic mouse model system. In experiments with two independent transgenic mouse lineages that express the HBV envelope region under the control of either HBV or cellular promoters, we observed a 50 to 80% reduction in the hepatic steady-state content of a 2.1-kb HBV mRNA following administration of a single intraperitoneal dose of LPS. The regulatory influence of several inflammatory cytokines known to be induced by LPS was also examined in this system. The negative regulatory effect of LPS was consistently reproduced by the administration of a single nontoxic dose of tumor necrosis factor alpha, and it was occasionally observed following the administration of high doses of alpha interferon and interleukin-6, while no effect was detectable in response to high-dose interleukin-1 alpha or to gamma interferon. These observations suggest that tumor necrosis factor alpha and perhaps other cytokines may activate a heretofore unsuspected intracellular pathway that negatively regulates HBV gene expression. The intracellular mechanism(s) responsible for this effect and its pathophysiologic relevance remain to be elucidated.

Acute-Phase Reaction↗
PubMed1992

Activation of multidrug resistance (P-glycoprotein) mdr3/mdr1a gene during the development of hepatocellular carcinoma in hepatitis B virus transgenic mice.

The expression of multidrug resistance (mdr) genes was investigated in the livers of transgenic mice that express the human hepatitis B virus large envelope polypeptide under the transcriptional control of a liver-specific promoter. These mice develop a storage disease due to the accumulation of a nonsecretable form of hepatitis B surface antigen in the hepatocyte. Liver cell injury is followed by a hepatocellular proliferative response, dysplasia, microscopic nodular hyperplasia, and finally hepatocellular carcinoma. The expression of mdr1, mdr2, and mdr3 genes was analyzed in livers at different stages of the disease by RNase protection assay, Western blot, and immunohistochemistry. RNase protection assay revealed that mdr3 mRNA expression was moderately increased in tissue with microscopic nodular hyperplasia and significantly overexpressed in hepatocellular carcinoma but undetectable in earlier stages of the disease. Western blot using isoform-specific anti-mdr3 antibody demonstrated that the expression of mdr3 protein reflected the steady-state level of mdr3 mRNA. Immunohistochemical analyses using anti-mdr3 isoform-specific antibody and monoclonal antibody C219, which recognizes all the three mdr isoforms, demonstrated selective overexpression in preneoplastic foci during the stage of microscopic nodular hyperplasia as well as in neoplastic hepatocytes in hepatocellular carcinoma. No consistent activation of mdr1 and mdr2 (but occasional coactivation with mdr1) genes during hepatocarcinogenesis was observed. Our results suggest that the hepatocellular mdr3-specific activation mechanism is associated with the late events of hepatocarcinogenesis in this model. The predictable kinetics of mdr gene expression in this transgenic tumor model suggest that it is suitable for future studies of the mechanism of mdr gene activation and the possible pharmacological consequences for mdr3 gene expression of hepatocellular carcinoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

HLA class I-restricted human cytotoxic T cells recognize endogenously synthesized hepatitis B virus nucleocapsid antigen.

Knowledge of the immune effector mechanisms responsible for clearance of hepatitis B virus (HBV)-infected cells has been severely limited by the absence of reproducible systems to selectively expand and to characterize HBV-specific cytotoxic T lymphocytes (CTLs) in the peripheral blood of patients with viral hepatitis. By using a strategy involving sequential stimulation with HBV nucleocapsid synthetic peptides followed by autologous, or HLA class I-matched, HBV nucleocapsid transfectants, we now report the existence of CTLs able to lyse target cells that express endogenously synthesized HBV nucleocapsid antigen in the peripheral blood of patients with acute viral hepatitis B. The CTL response is HLA-A2 restricted, mediated by CD8-positive T cells, and specific for a single epitope, located between amino acid residues 11 and 27 of HBV core protein; these residues are shared with the secretable precore-derived hepatitis B e antigen. Equivalent lysis of target cells that express each of these proteins suggests that their intracellular trafficking pathways may intersect. The current report provides definitive evidence that HLA class I-restricted, CD8-positive CTLs that recognize endogenously synthesized HBV nucleocapsid antigen are induced during acute HBV infection in humans and establishes a strategy that should permit a detailed analysis of the role played by HBV-specific CTLs in the immunopathogenesis of viral hepatitis.

B-Lymphocytes↗
PubMed1991

Cytotoxic T lymphocytes recognize an HLA-A2-restricted epitope within the hepatitis B virus nucleocapsid antigen.

The absence of readily manipulable experimental systems to study the cytotoxic T lymphocyte (CTL) response against hepatitis B virus (HBV) antigens has thus far precluded a definitive demonstration of the role played by this response in the pathogenesis of liver cell injury and viral clearance during HBV infection. To circumvent the problem that HBV infection of human cells in vitro for production of stimulator/target systems for CTL analysis is not feasible, a panel of 22 overlapping synthetic peptides covering the entire amino acid sequence of the HBV core (HBcAg) and e (HBeAg) antigens were used to induce and to analyze the HBV nucleocapsid-specific CTL response in nine patients with acute hepatitis B, six patients with chronic active hepatitis B, and eight normal controls. By using this approach, we have identified an HLA-A2-restricted CTL epitope, located within the NH2-terminal region of the HBV core molecule, which is shared with the e antigen and is readily recognized by peripheral blood mononuclear cells from patients with self-limited acute hepatitis B but less efficiently in chronic HBV infection. Our study provides the first direct evidence of HLA class I-restricted T cell cytotoxicity against HBV in humans. Furthermore, the different response in HBV-infected subjects who successfully clear the virus (acute patients) in comparison with patients who do not succeed (chronic patients) suggests a pathogenetic role for this CTL activity in the clearance of HBV infection.

Alanine Transaminase↗