RESUMO
BACKGROUND: The low incidence and the variable presentation complicate clinical investigations on biliary tract cancer. The results of Valle et al. in 2009 provided, for the first time, an evidence-based palliative treatment for this rare tumor type. So far no data are available in Germany regarding the current daily care practice. METHODS: We started this national survey in May 2011, including about 3,400 members of the AIO (Working Group Medical Oncology), DGHO (German Society of Hematology and Oncology) and GGHBB (Society of Gastroenterology and Hepatology in Berlin and Brandenburg). The standardized online form contained questions concerning field of action and diagnostic and therapeutic procedures. Evaluation was conducted anonymously. RESULTS: 162 responses could be obtained, corresponding to a response rate of about 5%. 70.4% of the respondents were physicians in hospitals, 23.5% stated to work in private practices. 61.7% of the respondents were medical oncologists and 27.2% gastroenterologists. 52.5% of the participants pointed out to use the standard regimen of gemcitabine and cisplatin. For second-line regimen, the most frequent answer (29%) specified the administration of oxaliplatin in combination with 5-fluorouracil (5-FU) or capecitabine. CONCLUSIONS: This survey may help to clarify the current oncologic daily care procedures for patients with biliary tract cancer in Germany. The results can be helpful for further clinical investigations or the implementation of a tumor-specific register.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Sistema Biliar/diagnóstico , Neoplasias do Sistema Biliar/terapia , Endoscopia/estatística & dados numéricos , Oncologia/estatística & dados numéricos , Cuidados Paliativos/estatística & dados numéricos , Padrões de Prática Médica/estatística & dados numéricos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias do Sistema Biliar/epidemiologia , Cisplatino/uso terapêutico , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Feminino , Fluoruracila/uso terapêutico , Alemanha/epidemiologia , Pesquisas sobre Atenção à Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Compostos Organoplatínicos/uso terapêutico , Oxaliplatina , Resultado do Tratamento , Adulto Jovem , GencitabinaRESUMO
UNLABELLED: The cellular basis of liver regeneration has been intensely investigated for many years. However, the mechanisms initiating hepatocyte "plasticity" and priming for proliferation are not yet fully clear. We investigated alterations in gene expression patterns during the first 72 hours of C57BL/6N mouse hepatocyte culture on collagen monolayers (CM), which display a high basal frequency of proliferation in the absence of cytokines. Although many metabolic genes were down-regulated, genes related to mitogen-activated protein kinase (MAPK) signaling and cell cycle were up-regulated. The latter genes showed an overrepresentation of transcription factor binding sites (TFBS) for ETF (TEA domain family member 2), E2F1 (E2F transcription factor 1), and SP-1 (Sp1 transcription factor) (P < 0.001), all depending on MAPK signaling. Time-dependent increase of ERK1/2 phosphorylation occurred during the first 48 hours (and beyond) in the absence of cytokines, accompanied by an enhanced bromodeoxyuridine labeling index of 20%. The MEK inhibitor PD98059 blunted these effects indicating MAPK signaling as major trigger for this cytokine-independent proliferative response. In line with these in vitro findings, liver tissue of mice challenged with CCl(4) displayed hepatocytes with intense p-ERK1/2 staining and nuclear SP-1 and E2F1 expression. Furthermore, differentially expressed genes in mice after partial hepatectomy contained overrepresented TFBS for ETF, E2F1, and SP-1 and displayed increased expression of E2F1. CONCLUSION: Cultivation of murine hepatocytes on CM primes cells for proliferation through cytokine-independent activation of MAPK signaling. The transcription factors ETF, E2F1, and SP-1 seem to play a pronounced role in mediating proliferation-dependent differential gene expression. Similar events, but on a shorter time-scale, occur very early after liver damage in vivo.
Assuntos
Proliferação de Células , Proteínas de Ligação a DNA/fisiologia , Fator de Transcrição E2F1/fisiologia , Hepatócitos/citologia , Fator de Transcrição Sp1/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sítios de Ligação/fisiologia , Intoxicação por Tetracloreto de Carbono/fisiopatologia , Expressão Gênica , Hepatectomia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição de Domínio TEA , Regulação para CimaRESUMO
UNLABELLED: Fas/CD95-induced apoptosis of hepatocytes in vivo proceeds through the so-called type II pathway, requiring the proapoptotic BH3-only Bcl-2 family member Bid for mitochondrial death signaling. Consequently, Bid-deficient mice are protected from anti-Fas antibody injection induced fatal hepatitis. We report the unexpected finding that freshly isolated mouse hepatocytes, cultured on collagen or Matrigel, become independent of Bid for Fas-induced apoptosis, thereby switching death signaling from type II to type I. In such in vitro cultures, Fas ligand (FasL) activates caspase-3 without Bid cleavage, Bax/Bak activation or cytochrome c release, and neither Bid ablation nor Bcl-2 overexpression is protective. The type II to type I switch depends on extracellular matrix adhesion, as primary hepatocytes in suspension die in a Bid-dependent manner. Moreover, the switch is specific for FasL-induced apoptosis as collagen-plated Bid-deficient hepatocytes are protected from tumor necrosis factor alpha/actinomycin D (TNFalpha/ActD)-induced apoptosis. CONCLUSION: Our data suggest a selective crosstalk between extracellular matrix and Fas-mediated signaling that favors mitochondria-independent type I apoptosis induction.
Assuntos
Apoptose/fisiologia , Hepatócitos/metabolismo , Transdução de Sinais/fisiologia , Receptor fas/metabolismo , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 3/metabolismo , Adesão Celular/fisiologia , Células Cultivadas , Matriz Extracelular/metabolismo , Proteína Ligante Fas/metabolismo , Hepatócitos/citologia , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/metabolismo , Modelos Animais , Fator de Necrose Tumoral alfa/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismoRESUMO
Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide, but the study of HCV infection has been hampered by the lack of an in vitro or in vivo small animal model. The tree shrew Tupaia belangeri is susceptible to infection with a variety of human viruses in vivo, including hepatitis viruses. We show that primary Tupaia hepatocytes can be infected with serum- or plasma-derived HCV from infected humans, as measured by de novo synthesis of HCV RNA, analysis of viral quasispecies evolution, and detection of viral proteins. Production of infectious virus could be demonstrated by passage to naive hepatocytes. To assess whether viral entry in Tupaia hepatocytes was dependent on the recently isolated HCV E2 binding protein CD81, we identified and characterized Tupaia CD81. Sequence analysis of cloned Tupaia cDNA revealed a high degree of homology between Tupaia and human CD81 large extracellular loops (LEL). Cellular binding of E2 and HCV infection could not be inhibited by anti-CD81 antibodies or soluble CD81-LEL, suggesting that viral entry can occur through receptors other than CD81. Thus, primary Tupaia hepatocytes provide a potential model for the study of HCV infection of hepatocytes.
Assuntos
Hepacivirus/fisiologia , Hepatócitos/virologia , Proteínas de Membrana , Tupaia , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos CD/metabolismo , Sequência de Bases , Células Cultivadas , DNA Complementar/análise , Hepacivirus/classificação , Hepacivirus/genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Modelos Biológicos , Dados de Sequência Molecular , RNA Viral/análise , Receptores Virais/biossíntese , Receptores Virais/genética , Receptores Virais/metabolismo , Homologia de Sequência do Ácido Nucleico , Tetraspanina 28 , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/biossínteseRESUMO
Infection with hepatitis B virus (HBV) leads to a wide spectrum of clinical presentations ranging from an asymptomatic carrier state to self-limited acute or fulminant hepatitis to chronic hepatitis with progression to cirrhosis and hepatocellular carcinoma. Infection with HBV is one of the most common viral diseases affecting man. Both viral factors as well as the host immune response have been implicated in the pathogenesis and clinical outcome of HBV infection. In this review, we will discuss the impact of virus-host interactions for the pathogenesis of HBV infection and liver disease. These interactions include the relevance of naturally occurring viral variants for clinical disease, the role of virus-induced apoptosis for HBV-induced liver cell injury and the impact of antiviral immune responses for outcome of infection.
Assuntos
Apoptose/fisiologia , Vírus da Hepatite B/patogenicidade , Hepatite B/fisiopatologia , Anticorpos Antivirais/imunologia , Antivirais/uso terapêutico , DNA Viral/genética , Progressão da Doença , Hepatite B/tratamento farmacológico , Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Humanos , Mutação/genética , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Glycogen synthase kinase-3 (GSK-3) is known to modulate cell survival and apoptosis through multiple intracellular signaling pathways. However, its hepatoprotective function and its role in activation of NF-kappaB and anti-apoptotic factors are poorly understood and remain controversial. Here we investigated whether inhibition of GSK-3 could induce apoptosis in the presence of TNF-alpha in primary mouse hepatocytes. We show that pharmacological inhibition of GSK-3 in primary mouse hepatocytes does not lead to TNF-alpha-induced apoptosis despite reduced NF-kappaB activity. Enhanced stability of IkappaB-alpha appears to be responsible for lower levels of nuclear NF-kappaB and hence reduced transactivation. Additionally, inhibition of GSK-3 was accompanied by marked upregulation of beta-catenin, AP-1, and CREB transcription factors. Stimulation of canonical Wnt signaling and CREB activity led to elevated levels of anti-apoptotic factors. Hence, survival of primary mouse hepatocytes may be caused by the activation and/or upregulation of other key regulators of liver homeostasis and regeneration. These signaling molecules may compensate for the compromised anti-apoptotic function of NF-kappaB and allow survival of hepatocytes in the presence of TNF-alpha and GSK-3 inhibition.
Assuntos
Apoptose , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Hepatócitos/enzimologia , NF-kappa B/metabolismo , Fator de Transcrição AP-1/metabolismo , beta Catenina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/biossíntese , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , DNA/metabolismo , Indução Enzimática/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Indóis/farmacologia , Interleucina-6/genética , Maleimidas/farmacologia , Camundongos , Inibidor de NF-kappaB alfa , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/genética , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacos , Proteínas Wnt/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
360 million people are chronically infected with the human hepatitis B virus (HBV) and are consequently prone to develop liver cirrhosis and hepatocellular carcinoma. As approved therapeutic regimens-which modulate patients' antiviral defenses or inhibit the viral reverse transcriptase-are generally noncurative, strategies interfering with other HBV replication steps are required. Expanding on our demonstration that acylated peptides derived from the large HBV envelope protein block virus entry in vitro, we show their applicability to prevent HBV or woolly monkey hepatitis B virus infection in vivo, using immunodeficient urokinase-type plasminogen activator (uPA) mice repopulated with primary human or Tupaia belangeri hepatocytes. Accumulation of the peptides in the liver, their extraordinary inhibitory potency and specific mode of action permit subcutaneous delivery at very low doses. Inhibition of hepadnavirus entry thus constitutes a therapeutic approach to prevent primary HBV infection, such as after liver transplantation, and might also restrain virus spread in chronically infected patients.
Assuntos
Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/fisiologia , Hepatite B/prevenção & controle , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/farmacologia , Acilação , Sequência de Aminoácidos , Animais , Atelinae/virologia , Proteínas de Ligação a DNA/genética , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/química , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Fígado/virologia , Masculino , Camundongos , Distribuição Tecidual , Transplante Heterólogo , Tupaia , Ativador de Plasminogênio Tipo Uroquinase/genética , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/farmacocinéticaRESUMO
UNLABELLED: APOBEC3G (A3G) and APOBEC3C (A3C), 2 members of the APOBEC family, are cellular cytidine deaminases displaying broad antiretroviral activity. A3G inhibits hepatitis B virus (HBV) production by interfering with HBV replication without hypermutating the majority of HBV genomes. In contrast, A3C has little effect on HBV DNA synthesis. The aim of this study was to further dissect the mechanisms by which A3G and A3C interfere with the HBV life cycle. Immunoprecipitation experiments demonstrated that both A3G and A3C bind to the HBV core protein. A ribonuclease (RNase) treatment resulted in the nearly complete dissociation of the HBV core protein from A3G, whereas the HBV core-A3C complex was more stable. Interestingly, the majority of the newly synthesized HBV DNA genomes displayed extensive G-to-A mutations in the presence of A3C, whereas no A3C-induced HBV RNA mutations were detected. These findings support a model in which the RNA-dependent entrapment of A3G into the preassembly complex hampers subsequent steps in capsid formation. On the other hand, A3C is readily packaged into replication-competent capsids and efficiently deaminates newly synthesized HBV DNA. CONCLUSION: These findings demonstrate that HBV is highly vulnerable to the editing activity of an endogenous human deaminase and suggest that A3C could contribute to innate anti-HBV host responses.
Assuntos
Citidina Desaminase/metabolismo , DNA Viral/química , Vírus da Hepatite B/genética , Desaminase APOBEC-3G , Sequência de Bases , Replicação do DNA , DNA Viral/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Mutação , Nucleosídeo Desaminases/metabolismo , RNA Viral/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleases/química , Replicação ViralRESUMO
The differentiation and functional status of virus-specific CD8+ T cells is significantly influenced by specific and ongoing antigen recognition. Importantly, the expression profiles of the interleukin-7 receptor alpha chain (CD127) and the killer cell lectin-like receptor G1 (KLRG1) have been shown to be differentially influenced by repetitive T-cell receptor interactions. Indeed, antigen-specific CD8+ T cells targeting persistent viruses (e.g., human immunodeficiency virus and Epstein-Barr virus) have been shown to have low CD127 and high KLRG1 expressions, while CD8+ T cells targeting resolved viral antigens (e.g., FLU) typically display high CD127 and low KLRG1 expressions. Here, we analyzed the surface phenotype and function of hepatitis C virus (HCV)-specific CD8+ T cells. Surprisingly, despite viral persistence, we found that a large fraction of peripheral HCV-specific CD8+ T cells were CD127+ and KLRG1- and had good proliferative capacities, thus resembling memory cells that usually develop following acute resolving infection. Intrahepatic virus-specific CD8+ T cells displayed significantly reduced levels of CD127 expression but similar levels of KLRG1 expression compared to the peripheral blood. These results extend previous studies that demonstrated central memory (CCR7+) and early-differentiated phenotypes of HCV-specific CD8+ T cells and suggest that insufficient stimulation of virus-specific CD8+ T cells by viral antigen may be responsible for this alteration in HCV-specific CD8+ T-cell differentiation during chronic HCV infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Hepacivirus/imunologia , Hepatite C/imunologia , Memória Imunológica , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Lectinas Tipo C/metabolismo , Transativadores/metabolismo , Doença Aguda , Adulto , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/metabolismo , Feminino , Hepatite C/virologia , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Humanos , Fígado/citologia , Fígado/imunologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Receptores ImunológicosRESUMO
Peptide aptamers (PAs) can be employed to block the intracellular function of target proteins. Little is known about the mechanism of PA-mediated protein inhibition. Here, we generated PAs that specifically bound to the duck hepatitis B virus (HBV) core protein. Among them, PA34 strongly blocked duck HBV replication by inhibiting viral capsid formation. We found that PA34 led to a dramatic intracellular redistribution of its target protein into perinuclear inclusion bodies, which exhibit the typical characteristics of aggresomes. As a result, the core protein is efficiently removed from the viral life cycle. Corresponding findings were obtained for bioactive PAs that bind to the HBV core protein or to the human papillomavirus-16 (HPV16) E6 protein, respectively. The observation that PAs induce the specific sequestration of bound proteins into aggresomes defines a novel mechanism as to how this new class of intracellular inhibitors blocks the function of their target proteins.
Assuntos
Aptâmeros de Peptídeos/química , Vírus da Hepatite B/genética , Proteínas/antagonistas & inibidores , Sequência de Aminoácidos , Antivirais/farmacologia , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/metabolismo , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo , Vimentina/química , Replicação ViralRESUMO
Initiation of hepatitis C virus (HCV) infection is mediated by docking of the viral envelope to the hepatocyte cell surface membrane followed by entry of the virus into the host cell. Aiming to elucidate the impact of this interaction on host cell biology, we performed a genomic analysis of the host cell response following binding of HCV to cell surface proteins. As ligands for HCV-host cell surface interaction, we used recombinant envelope glycoproteins and HCV-like particles (HCV-LPs) recently shown to bind or enter hepatocytes and human hepatoma cells. Gene expression profiling of HepG2 hepatoma cells following binding of E1/E2, HCV-LPs, and liver tissue samples from HCV-infected individuals was performed using a 7.5-kd human cDNA microarray. Cellular binding of HCV-LPs to hepatoma cells resulted in differential expression of 565 out of 7,419 host cell genes. Examination of transcriptional changes revealed a broad and complex transcriptional program induced by ligand binding to target cells. Expression of several genes important for innate immune responses and lipid metabolism was significantly modulated by ligand-cell surface interaction. To assess the functional relevance and biological significance of these findings for viral infection in vivo, transcriptional changes were compared with gene expression profiles in liver tissue samples from HCV-infected patients or controls. Side-by-side analysis revealed that the expression of 27 genes was similarly altered following HCV-LP binding in hepatoma cells and viral infection in vivo. In conclusion, HCV binding results in a cascade of intracellular signals modulating target gene expression and contributing to host cell responses in vivo. Reprogramming of cellular gene expression induced by HCV-cell surface interaction may be part of the viral strategy to condition viral entry and replication and escape from innate host cell responses.
Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Fator C1 de Célula Hospedeira/imunologia , Proteínas do Envelope Viral/metabolismo , Antígenos Virais/metabolismo , Sequência de Bases , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Regulação da Expressão Gênica , Hepacivirus/genética , Hepatite C/genética , Fator C1 de Célula Hospedeira/metabolismo , Humanos , Dados de Sequência Molecular , Análise Serial de Proteínas , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Técnicas de Cultura de Tecidos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
Virus-specific CD8+ T cell responses play an important role in the natural course of infection; however, the impact of certain CD8+ T cell responses in determining clinical outcome has not been fully defined. A well-defined cohort of women inoculated with HCV from a single source showed that HLA-B27 has a strong association with spontaneous clearance. The immunological basis for this association is unknown. However, the finding is especially significant because HLA-B27 has also been shown to have a protective role in HIV infection. We report the identification of an HLA-B27 restricted hepatitis C virus (HCV)-specific CD8+ T cell epitope that is recognized in the majority of recovered HLA-B27 positive women. In chronically HCV-infected individuals, analysis of the corresponding viral sequence showed a strong association between sequence variations within this epitope and expression of HLA-B27, indicating allele-specific selection pressure at the population level. Functional analysis in 3 chronically HCV-infected patients showed that the emerging variant viral epitopes represent escape mutations. In conclusion, our results suggest a dominant role of HLA-B27 in mediating spontaneous viral clearance as well as viral evolution in HCV infection and mechanistically link both associations to a dominant novel CD8+ T cell epitope. These results support the central role of virus-specific CD8+ T cells and the genetically determined restriction of the virus-specific T cell repertoire in HCV infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/genética , Antígeno HLA-B27/imunologia , Hepacivirus/imunologia , Hepatite C/imunologia , Adulto , Idoso , Epitopos de Linfócito T/imunologia , Feminino , Predisposição Genética para Doença , Antígeno HLA-B27/genética , Hepacivirus/genética , Hepatite C/genética , Hepatite C/virologia , Humanos , Imunidade Celular , Masculino , Pessoa de Meia-IdadeRESUMO
APOBEC3G is a cellular cytidine deaminase displaying broad antiretroviral activity. Recently, it was shown that APOBEC3G can also suppress hepatitis B virus (HBV) production in human hepatoma cells. In the present study, we characterized the mechanisms of APOBEC-mediated antiviral activity against HBV and related hepadnaviruses. We show that human APOBEC3G blocks HBV production in mammalian and nonmammalian cells and is active against duck HBV as well. Early steps of viral morphogenesis, including RNA and protein synthesis, binding of pregenomic RNA to core protein, and self-assembly of viral core protein, were unaffected. However, APOBEC3G rendered HBV core protein-associated full-length pregenomic RNA nuclease-sensitive. Ongoing reverse-transcription in capsids that had escaped the block in morphogenesis was not significantly inhibited. The antiviral effect was not modulated by abrogating or enhancing expression of the accessory HBV X protein, suggesting that HBV X protein does not represent a functional homologue to the HIV vif protein. Furthermore, human APOBEC3F but not rat APOBEC1 inhibited HBV DNA production. Viral RNA and low-level DNA produced in the presence of APOBEC3F or rat APOBEC1 occasionally displayed mutations, but the majority of clones were wild-type. In conclusion, APOBEC3G and APOBEC3F but not rat APOBEC1 can downregulate the production of replication-competent hepadnaviral nucleocapsids. In contrast to HIV and other retroviruses, however, APOBEC3G/3F-mediated editing of nucleic acids does not seem to represent an effective innate defense mechanism for hepadnaviruses.
Assuntos
Antivirais/fisiologia , Citosina Desaminase/fisiologia , Vírus da Hepatite B do Pato/fisiologia , Vírus da Hepatite B/fisiologia , Proteínas/fisiologia , Desaminase APOBEC-1 , Desaminase APOBEC-3G , Animais , Galinhas , Citidina Desaminase/fisiologia , DNA Viral/biossíntese , Vírus da Hepatite B/genética , Nucleosídeo Desaminases , DNA Polimerase Dirigida por RNA/metabolismo , Ratos , Proteínas Repressoras , Proteínas do Core Viral/biossíntese , Montagem de VírusRESUMO
Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8(+) T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4(+)CD25(+) regulatory phenotype in suppressing virus-specific CD8(+) T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCV-specific CD8(+) T cells were inhibited by CD4(+)CD25(+) T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8(+) T cells but also to influenza virus-specific CD8(+) T cells. Importantly, CD4(+)CD25(+) T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8(+) T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4(+)CD25(+) cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4(+)CD25(+) T cells that are able to suppress CD8(+) T-cell responses to different viral antigens. Our results further suggest that CD4(+)CD25(+) T cells may contribute to viral persistence in chronically HCV-infected patients and may be a target for immunotherapy of chronic hepatitis C.
Assuntos
Antígenos CD4/metabolismo , Linfócitos T CD8-Positivos/imunologia , Hepatite C Crônica/imunologia , Ativação Linfocitária/imunologia , Receptores de Interleucina-2/metabolismo , Linfócitos T Reguladores/imunologia , Adulto , Sequência de Aminoácidos , Comunicação Celular , Feminino , Hepacivirus , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , FenótipoRESUMO
Hepatitis B virus (HBV) core promoter mutations have been implicated in the pathogenesis of fulminant hepatitis B. Due to the limited availability of primary human hepatocytes, the functional characterization of HBV mutants has been performed predominantly in transformed cells, which may not represent ideal model systems for studying virus-cell interactions. We and others have shown that primary hepatocytes of the tree shrew Tupaia belangeri support HBV infection and replication. In this study, we used primary Tupaia hepatocytes to analyze the phenotype of two HBV core promoter mutations that have been associated with a clinical outbreak of fatal fulminant hepatitis. Similar to previous findings in human hepatoma cells, the HBV core promoter mutations resulted in enhanced viral replication and core expression. Surprisingly, however, the presence of the mutations had a marked effect on hepatocyte viability not previously observed in hepatoma cells. Reduced cell viability was found to be due to the induction of apoptosis, as evidenced by caspase-3 activation and nuclear fragmentation. In conclusion, HBV mutants exhibit a novel phenotype in primary hepatocytes distinctly different from previous findings in hepatoma cell lines. This phenotype may have important implications for the understanding of the fulminant clinical course associated with HBV mutations.
Assuntos
Apoptose , Vírus da Hepatite B/genética , Hepatite B/fisiopatologia , Hepatite B/virologia , Hepatócitos/virologia , Mutação , Tupaia/virologia , Animais , Células Cultivadas , Antígenos de Superfície da Hepatite B/metabolismo , Hepatócitos/imunologia , Regiões Promotoras Genéticas/genética , Transdução GenéticaRESUMO
The precise mechanisms responsible for the failure of intrahepatic hepatitis C virus (HCV)-specific CD8+ T cells to control the virus during persistent infection have not been fully defined. We therefore studied the CD8+ T-cell response in 27 HLA-A2-positive patients using four previously well-defined HLA-A2-restricted HCV epitopes. The corresponding HCV sequences were determined in several patients and compared with the intrahepatic HCV-specific CD8+ T-cell response. The results of the study indicate: (1) intrahepatic HCV-specific CD8+ T cells are present in the majority of patients with chronic HCV infection and overlap significantly with the response present in the peripheral blood. (2) A large fraction of intrahepatic HCV-specific CD8+ T cells are impaired in their ability to secrete interferon gamma (IFN-gamma). This dysfunction is specific for HCV-specific CD8+ T cells, since intrahepatic Flu-specific CD8+ T cells readily secrete this cytokine. (3) T-cell selection of epitope variants may have occurred in some patients. However, it is not an inevitable consequence of a functional virus-specific CD8+ T-cell response, since several patients with IFN-gamma-producing CD8+ T-cell responses harbored HCV sequences identical or cross-reactive with the prototype sequence. (4) The failure of intrahepatic virus-specific CD8+ T cells to sufficiently control the virus occurs despite the presence of virus-specific CD4+ T cells at the site of disease. In conclusion, different mechanisms contribute to the failure of intrahepatic CD8+ T cells to eliminate HCV infection, despite their persistence and accumulation in the liver.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Fígado/imunologia , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/metabolismo , Epitopos/imunologia , Feminino , Humanos , Interferon gama/metabolismo , Fígado/citologia , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND/AIMS: Transplantation of primary human hepatocytes and establishment of hepatitis B virus (HBV) infection in immunodeficient urokinase plasminogen activator (uPA) transgenic mice was shown. However, the availability of usable primary human hepatocytes is very limited. Therefore, alternative and more accessible sources of hepatocytes permissive for HBV infection are highly desirable. Here we investigated the potential of primary hepatocytes from the tree shrew Tupaia belangeri that were shown to be susceptible to HBV infection. METHODS: Freshly isolated or cryopreserved primary tupaia hepatocytes were transplantated via intrasplenic injection into immunodeficient uPA/RAG-2 mice. Engrafted mice were then infected with HBV and woolly monkey (WM)-HBV positive sera. RESULTS: Extensive proliferation of xenografted cells was demonstrated by the stable production of tupaia alpha1-antitrypsin in serum and liver of transplanted mice. Quantitative PCR assays demonstrated the presence of circulating viral particles as well as intracellular viral DNA, including covalently closed circular (ccc) DNA, in transplanted mice. Viral infection could be serially passaged in mice. Furthermore, viral replication was strongly inhibited by treating mice with adefovir dipivoxil. CONCLUSIONS: uPA mice repopulated with tupaia hepatocytes represent a useful and more accessible model for HBV infection studies, including the evaluation of antiviral therapy and cccDNA.
Assuntos
Adenina/análogos & derivados , Antivirais/uso terapêutico , Modelos Animais de Doenças , Hepatite B Crônica/etiologia , Hepatócitos/transplante , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Adenina/uso terapêutico , Animais , DNA Circular/análise , DNA Viral/análise , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Camundongos , Organofosfonatos/uso terapêutico , Transplante Heterólogo , Tupaia , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The study of early steps during HCV infection has been hampered by the lack of suitable in vitro or in vivo models. Primary Tupaia hepatocytes (PTH) have been shown to be susceptible to HCV infection in vitro and in vivo. Human scavenger receptor class B type I (SR-BI) represents an HCV receptor candidate mediating the cellular binding of E2 glycoprotein to HepG2 hepatoma cells. However, the function of SR-BI for viral infection of hepatocytes is unknown. In this study, we used PTH to assess the functional role of SR-BI as a putative HCV receptor. Sequence analysis of cloned tupaia SR-BI revealed a high homology between tupaia and human SR-BI. Transfection of CHO cells with human or tupaia SR-BI but not mouse SR-BI cDNA resulted in cellular E2 binding, suggesting that E2-binding domains between human and tupaia SR-BI are highly conserved. Preincubation of PTH with anti-SR-BI antibodies resulted in marked inhibition of E2 or HCV-like particle binding. However, anti-SR-BI antibodies were not able to block HCV infection of PTH. In conclusion, our results demonstrate that SR-BI represents an important cell surface molecule for the binding of the HCV envelope to hepatocytes and suggest that other or additional cell surface molecules are required for the initiation of HCV infection. Furthermore, the structural and functional similarities between human and tupaia SR-BI indicate that PTH represent a useful model system to characterize the molecular interaction of the HCV envelope and SR-BI on primary hepatocytes.