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1.
Liver Transpl ; 30(7): 728-741, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38315053

RESUMO

Chronic HCV infection induces interferon and dysregulates immune responses through inflammation and chronic antigenic stimulation. Antiviral drugs can cure HCV, providing a unique opportunity to examine the immunological restoration that does and does not occur when a chronic viral infection is eradicated. We quantified blood cytokines levels and used mass cytometry to immunophenotype peripheral blood mononuclear cells before and after HCV cure in 2 groups of patients and controls. At baseline, serum interferon α and soluble CD163 (a macrophage product) were elevated in both liver transplant and nonliver transplant patients compared to controls; the frequencies of several peripheral blood mononuclear cell populations differed from controls; and programmed death protein 1-positivity was increased in nearly all T cell subsets. Many abnormalities persisted after HCV cure, including elevated programmed death protein 1 expression on CD4 naïve and central memory T cells, elevated soluble CD163, and expansion of the plasmablast/plasma cell compartment. Several myeloid-lineage subsets, including Ag-presenting dendritic cells, remained dysregulated. In mechanistic studies, interferon α treatment increased programmed death protein 1 on human T cells and increased T cell receptor signaling. The data identify immunological abnormalities that persist after curative HCV treatment. Before cure, high levels of interferon α may stimulate programmed death protein 1 expression on human T cells, causing persistent functional changes.


Assuntos
Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Antivirais , Hepatite C Crônica , Interferon-alfa , Transplante de Fígado , Humanos , Transplante de Fígado/efeitos adversos , Masculino , Antivirais/uso terapêutico , Pessoa de Meia-Idade , Feminino , Antígenos CD/imunologia , Antígenos CD/sangue , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/sangue , Antígenos de Diferenciação Mielomonocítica/imunologia , Hepatite C Crônica/imunologia , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/sangue , Hepatite C Crônica/cirurgia , Interferon-alfa/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Receptores de Superfície Celular/sangue , Receptores de Superfície Celular/imunologia , Adulto , Estudos de Casos e Controles , Idoso , Hepacivirus/imunologia , Hepacivirus/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Citocinas/sangue , Imunofenotipagem , Resultado do Tratamento
2.
Hepatology ; 66(2): 357-370, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-27642141

RESUMO

Hepatitis C virus (HCV) is unique among RNA viruses in its ability to establish chronic infection in the majority of exposed adults. HCV persists in the liver despite interferon (IFN)-stimulated gene (ISG) induction; robust induction actually predicts treatment failure and viral persistence. It is unclear which forms of HCV RNA are associated with ISG induction and IFN resistance during natural infections. To thoroughly delineate HCV RNA populations, we developed conditions that fully separate the strands of long double-stranded RNA (dsRNA) and allow the released RNAs to be quantified in reverse transcription/polymerase chain reaction assays. These methods revealed that dsRNA, a pathogen-associated molecular pattern (PAMP), comprised 52% (standard deviation, 28%) of the HCV RNA in the livers of patients with chronic infection. HCV dsRNA was proportionally higher in patients with the unfavorable IL28B TT (rs12979860) genotype. Higher ratios of HCV double-stranded to single-stranded RNA (ssRNA) correlated positively with ISG induction. In Huh-7.5 cells, IFN treatment increased the total amount of HCV dsRNA through a process that required de novo viral RNA synthesis and shifted the ratio of viral dsRNA/ssRNA in favor of dsRNA. This shift was blocked by ribavirin (RBV), an antiviral drug that reduces relapse in HCV patients. Northern blotting established that HCV dsRNA contained genome-length minus strands. CONCLUSION: HCV dsRNA is the predominant form in the HCV-infected liver and has features of both a PAMP and a genomic reservoir. Interferon treatment increased rather than decreased HCV dsRNA. This unexpected finding suggests that HCV produces dsRNA in response to IFN, potentially to antagonize antiviral defenses. (Hepatology 2017;66:357-370).


Assuntos
Antivirais/farmacologia , Hepacivirus/genética , Hepatite C/patologia , Interferon-alfa/farmacologia , RNA de Cadeia Dupla/genética , Adulto , Biópsia por Agulha , Western Blotting , Células Cultivadas , Feminino , Citometria de Fluxo , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C/genética , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , RNA de Cadeia Dupla/efeitos dos fármacos , RNA Viral/efeitos dos fármacos , RNA Viral/genética , Valores de Referência , Sensibilidade e Especificidade
3.
J Hepatol ; 63(6): 1323-33, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26220749

RESUMO

BACKGROUND & AIMS: Although patients infected by genotype 1b hepatitis C virus (HCV) with Q(70) and/or M(91)core gene mutations have an almost five-fold increased risk of developing hepatocellular carcinoma (HCC) and increased insulin resistance, the absence of a suitable experimental system has precluded direct experimentation on the effects of these mutations on cellular gene expression. METHODS: HuH7 cells were treated long-term with human serum to induce differentiation and to produce a model system for testing high-risk and control HCV. For clinical validation, profiles of infected cells were compared to each other and to those of liver biopsies of patients with early-stage HCV-related cirrhosis followed prospectively for up to 23 years (n=216). RESULTS: Long-term culture in human serum produced growth-arrested, hepatocyte-like cells whose gene profile overlapped significantly with that of primary human hepatocytes. High-risk (Q(70)/M(91)) and control (R(70)/L(91)) viruses had dramatically different effects on gene expression of these cells. The high-risk virus enhanced expression of pathways associated with cancer and type II diabetes, while the control virus enhanced pathways associated with oxidative phosphorylation. Of special clinical relevance, the transcriptome of cells replicating the high-risk virus correlated significantly with an HCC high-risk profile in patients (Bonferroni-corrected p=0.03), whereas no such association was observed for non-HCC-related clinical outcomes. CONCLUSIONS: The cell-based system allowed direct head-to-head comparison of HCV variants, and provided experimental support for previous clinical data indicating an oncogenic effect of core gene mutations. This simple experimental system distinguished HCV variants and will enable future mechanistic analysis and exploration of interventional approaches.


Assuntos
Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/virologia , Genes Virais , Hepacivirus/genética , Hepacivirus/patogenicidade , Antígenos do Núcleo do Vírus da Hepatite B/genética , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/virologia , Mutação , Proteínas do Core Viral/genética , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Variação Genética , Genótipo , Hepacivirus/fisiologia , Hepatite C Crônica/complicações , Hepatite C Crônica/virologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Cirrose Hepática/complicações , Cirrose Hepática/virologia , Oncogenes , Estudos Prospectivos , Fatores de Risco , Transcriptoma , Virulência/genética , Replicação Viral
4.
J Infect Dis ; 208(5): 830-8, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23757341

RESUMO

BACKGROUND: Clinical studies have shown faster disease progression and stronger immune activation in human immunodeficiency virus (HIV)-1-infected females when compared with males for the same level of HIV-1 replication. Here we determine whether the elevated levels of HIV-1-induced interferon-alpha (IFN-α) production observed in females are associated with higher interferon-stimulated gene (ISG) expression levels in T cells, hence suggesting type-I IFN as a mechanism for the higher HIV-1-associated immune activation observed. METHODS: T-cell and dendritic cell populations were isolated from treatment-naive chronically HIV-1-infected individuals enrolled in the Adult Clinical Trials Group 384 by fluorescence-activated cell sorting. The expression of 98 genes involved in Toll-like receptor and type I IFN signaling pathways were quantified using Nanostring technology. RESULTS: Several ISGs were significantly correlated with HIV-1 viral load and/or CD4(+) T-cell count. Higher expression levels of a subset of these ISGs were observed in cells derived from females as compared to males after adjusting for viral load and were correlated to higher levels of T-cell activation. CONCLUSION: These data show that higher IFN-α production is associated with higher ex vivo expression of several ISGs in females. This might contribute to higher levels of immune activation and the observed faster HIV-1 disease progression in females for a given level of viral replication.


Assuntos
Regulação da Expressão Gênica , Infecções por HIV/imunologia , HIV-1/imunologia , Interferon-alfa/imunologia , Linfócitos T/imunologia , Adulto , Células Cultivadas , Células Dendríticas/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade
5.
Cancer Med ; 12(9): 10175-10186, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37078924

RESUMO

BACKGROUND: Successful treatment of hepatitis C reduces liver inflammation and fibrosis; however, patients remain at risk of developing hepatocellular carcinoma (HCC). AIMS: To identify risk factors for new-onset HCC in patients cured of hepatitis C. METHODS: Imaging, histological, and clinical data on patients whose first HCC was diagnosed >12 months of post-SVR were analyzed. Histology of 20 nontumor tissues was analyzed in a blinded manner using the Knodel/Ishak/HAI system for necroinflammation and fibrosis/cirrhosis stage and the Brunt system for steatosis/steatohepatitis. Factors associated with post-SVR HCC were identified by comparison with HALT-C participants who did not develop post-SVR HCC. RESULTS: Hepatocellular carcinoma was diagnosed in 54 patients (45 M/9F), a median of 6 years of post-SVR [interquartile range (IQR) =1.4-10y] at a median age of 61 years (IQR, 59-67). Approximately one-third lacked cirrhosis, and only 11% had steatosis on imaging. The majority (60%) had no steatosis/steatohepatitis in histopathology. The median HAI score was 3 (1.25-4), indicating mild necroinflammation. In a multivariable logistic regression model, post-SVR HCC was positively associated with non-Caucasian race (p = 0.03), smoking (p = 0.03), age > 60 years at HCC diagnosis (p = 0.03), albumin<3.5 g/dL (p = 0.02), AST/ALT>1 (p = 0.05), and platelets <100 × 103 cells/µL (p < 0.001). Alpha fetoprotein ≥4.75 ng/mL had 90% specificity and 71% sensitivity for HCC occurrence. Noncirrhotic patients had larger tumors (p = 0.002) and a higher prevalence of vascular invasion (p = 0.016) than cirrhotic patients. CONCLUSIONS: One-third of patients with post-SVR HCC did not have liver cirrhosis; most had no steatosis/steatohepatitis. Hepatocellular carcinomas were more advanced in noncirrhotic patients. Results support AFP as a promising marker of post-SVR HCC risk.


Assuntos
Carcinoma Hepatocelular , Fígado Gorduroso , Hepatite C Crônica , Hepatite C , Neoplasias Hepáticas , Humanos , Pessoa de Meia-Idade , Idoso , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/etiologia , Hepatite C Crônica/complicações , Hepatite C Crônica/tratamento farmacológico , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/etiologia , Antivirais/uso terapêutico , Resposta Viral Sustentada , Fatores de Risco , Hepatite C/complicações , Cirrose Hepática/complicações , Fígado Gorduroso/complicações , Fígado Gorduroso/tratamento farmacológico , Hepacivirus
6.
Sci Rep ; 11(1): 1551, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452360

RESUMO

Viral hepatitis leads to immune-mediated liver injury. The rate of disease progression varies between individuals. We aimed to phenotype immune cells associated with preservation of normal liver function during hepatitis C virus (HCV) infection. Clinical data and specimens were obtained from 19 HCV-infected patients undergoing liver transplantation. Liver and peripheral blood mononuclear cells were isolated and eight subsets of innate immune cells were delineated by multiparameter flow cytometry. Cytokine assays and microarrays were performed. Intrahepatic CD56Bright/CD16- natural killer (NK) cells comprised the only subset correlating with better liver function, i.e., lower bilirubin (p = 0.0002) and lower model for end stage of liver disease scores (p = 0.03). The signature of liver NK cells from HCV-infected patients included genes expressed by NK cells in normal liver and by decidual NK cells. Portal vein blood had a higher concentration of interleukin (IL)-10 than peripheral blood (p = 0.03). LMCs were less responsive to toll-like receptor (TLR) stimulation than PBMCs, with fewer pro-inflammatory gene-expression pathways up-regulated after in vitro exposure to lipopolysaccharide and a TLR-7/8 agonist. Hepatic CD56Bright/CD16- NK cells may be critical for maintaining liver homeostasis. Portal vein IL-10 may prime inhibitory pathways, attenuating TLR signaling and reducing responsiveness to pro-inflammatory stimuli.


Assuntos
Hepatite C/imunologia , Células Matadoras Naturais/metabolismo , Fígado/patologia , Idoso , Progressão da Doença , Feminino , Citometria de Fluxo/métodos , Hepacivirus/patogenicidade , Hepatite C/metabolismo , Hepatite C/fisiopatologia , Humanos , Imunidade Inata/imunologia , Imunidade Inata/fisiologia , Imunofenotipagem , Células Matadoras Naturais/patologia , Leucócitos Mononucleares/metabolismo , Fígado/imunologia , Testes de Função Hepática/métodos , Masculino , Pessoa de Meia-Idade
7.
Hepatol Commun ; 2(1): 21-28, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29404509

RESUMO

Hepatitis C virus (HCV) is one of the most prevalent causes of chronic blood-borne infections worldwide. Despite developments of highly effective treatments, most infected individuals are unaware of their infection. Approximately 75% of infections are in low- and middle-income countries; therefore, continuing research in HCV molecular virology and the development of vaccines and affordable diagnostics is required to reduce the global burden. Various intracellular forms of the HCV nucleocapsid (core) protein are produced in cell culture; these comprise the conventional p21 core and the newly discovered shorter isoforms (minicores). Minicores lack the N-terminus of p21 core. This study was conducted to determine if minicores are secreted in cell culture and more importantly if they circulate in the blood of individuals infected with HCV. We also developed a new monoclonal antibody that detects minicores targeting a C-terminal region common to p21 core and minicores. Direct evidence of minicores requires western blot analysis to distinguish the detection of p21 core from minicores. However, the sensitivity for western blot detection of HCV proteins from blood is nil without their prior purification/enrichment from blood. Therefore, we developed a purification method based on a heparin/Mn+2 precipitation of apolipoprotein B-containing lipoproteins because HCV is thought to circulate as a hybrid lipoviral particle. Minicores are secreted in culture when cells are grown in the presence of human serum. The heparin/Mn+2 precipitate from HCV-infected cell culture supernatants and from the blood of 4 patients with high-titer genotype-1 HCV contained minicores. Conclusion: Minicores are major newly discovered HCV proteins that are secreted and circulate in blood during natural infections. Minicore proteins have translational potential as targets in diagnostic assays and in vaccine development. (Hepatology Communications 2018;2:21-28).

8.
Sci Rep ; 6: 27025, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27246310

RESUMO

Mutations at positions 70 and/or 91 in the core protein of genotype-1b, hepatitis C virus (HCV) are associated with hepatocellular carcinoma (HCC) risk in Asian patients. To evaluate this in a US population, the relationship between the percentage of 70 and/or 91 mutant HCV quasispecies in baseline serum samples of chronic HCV patients from the HALT-C trial and the incidence of HCC was determined by deep sequencing. Quasispecies percentage cut-points, ≥42% of non-arginine at 70 (non-R(70)) or ≥98.5% of non-leucine at 91 (non-L(91)) had optimal sensitivity at discerning higher or lower HCC risk. In baseline samples, 88.5% of chronic HCV patients who later developed HCC and 68.8% of matched HCC-free control patients had ≥42% non-R(70) quasispecies (P = 0.06). Furthermore, 30.8% of patients who developed HCC and 54.7% of matched HCC-free patients had quasispecies with ≥98.5% non-L(91) (P = 0.06). By Kaplan-Meier analysis, HCC incidence was higher, but not statistically significant, among patients with quasispecies ≥42% non-R(70) (P = 0.08), while HCC incidence was significantly reduced among patients with quasispecies ≥98.5% non-L(91) (P = 0.01). In a Cox regression model, non-R(70) ≥42% was associated with increased HCC risk. This study of US patients indicates the potential utility of HCV quasispecies analysis as a non-invasive biomarker of HCC risk.


Assuntos
Carcinoma Hepatocelular/diagnóstico , Hepacivirus/genética , Hepatite C Crônica/diagnóstico , Neoplasias Hepáticas/diagnóstico , Quase-Espécies , RNA Viral/genética , Proteínas do Core Viral/genética , Substituição de Aminoácidos , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/virologia , Feminino , Expressão Gênica , Genótipo , Hepacivirus/classificação , Hepacivirus/isolamento & purificação , Hepatite C Crônica/complicações , Hepatite C Crônica/virologia , Humanos , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Estudos Prospectivos , Risco
9.
AIDS ; 26(5): 533-41, 2012 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-22210629

RESUMO

OBJECTIVE AND DESIGN: The objective of this study was to determine changes in toll-like receptor (TLR) responses of monocytes, myeloid dendritic cells and plasmacytoid dendritic cells during primary and chronic HIV-1 infection. TLRs serve as important innate receptors to sense pathogens, and have been implicated in mediating immune activation in HIV-1 infection. Studies assessing the consequences of HIV-1 infection on the ability of innate immune cells to respond to TLR stimulation have come to varying conclusions. METHODS: Using intracellular flow cytometry, cytokine production by cryopreserved peripheral blood mononuclear cells from healthy controls and HIV-1-infected individuals were examined after TLR stimulation. RESULTS: We observed that the effect of HIV-1 infection on TLR responses not only depended on the stage of HIV-1 infection, but was also dependent on the individual receptor and cell type examined. Monocyte and myeloid dendritic cell responses to TLR8 stimulation were associated with HIV-1 viral load and CD4 T-cell count, whereas plasmacytoid dendritic cell responses to TLR7 stimulation were not. Responses to TLR2 stimulation were not affected by HIV-1 infection, whereas responses to TLR9 stimulation were universally decreased in all HIV-1-infected individuals examined regardless of treatment or clinical parameters. CONCLUSION: Responsiveness to TLR7/8 stimulation, which have been shown to recognize HIV-1 ssRNA, did not decrease in chronic infection, and may represent a contributing factor to ongoing T-cell immune activation in the setting of chronic viremic HIV-1 infection.


Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Receptores Toll-Like/imunologia , Doença Aguda , Adulto , Idoso , Contagem de Linfócito CD4 , Estudos de Casos e Controles , Doença Crônica , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Citometria de Fluxo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Receptor 7 Toll-Like/imunologia , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/imunologia , Receptor 8 Toll-Like/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Carga Viral , Adulto Jovem
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