RESUMO
BACKGROUND & AIMS: Hepatitis C virus (HCV)-RNA detection in peripheral blood mononuclear cells (PBMCs) after recovery from HCV infection, is a type of occult HCV infection although is unclear how the viral persistence in PBMCs affects HCV-specific T-cell responses. The aim of this study was to investigate if cellular immune responses are modified by HCV persistence in PBMCs. METHODS: HCV-specific CD4(+) and CD8(+) T-cell responses against six HCV peptides, situated within the non-structural (NS) proteins NS3, NS4b and NS5b, were measured by flow cytometry-through intracellular detection of gamma interferon (IFN-γ) or interleukin 4 (IL-4) and CD69 expression- in 27 sustained virological responders (SVR): 13 with and 14 without occult HCV infection in PBMCs, detected by strand-specific real-time PCR. Ten healthy individuals and 14 chronically infected patients with viraemia, were included as controls. RESULTS: SVR without occult infection showed a higher percentage of activated CD4(+) cells against peptides belonging to NS3 (p124, p153) and NS5b (p257, p294), activated CD8(+) cells against NS3 (p124, p153, p158) and NS5b-p294, as well as an elevated percentage of CD4(+) cells releasing IFN-γ + IL-4 against NS3-p153, and by CD8(+) cells against NS3 (p124, p153). SVR without occult infection showed a higher percentage of activation and release of IFN-γ + IL-4 by both cell subpopulations than the two group of controls, in contrast to SVR with occult infection. CONCLUSION: The lower HCV-specific T-cell response found in SVR with occult infection indicates that the immune response may be impaired when the virus persists in PBMCs.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Hepacivirus/imunologia , Hepatite C/imunologia , Ativação Linfocitária , Adulto , Idoso , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antivirais/uso terapêutico , Biomarcadores/sangue , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C/diagnóstico , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Interferon gama/metabolismo , Interleucina-4/metabolismo , Lectinas Tipo C/metabolismo , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/imunologia , RNA Viral/sangue , Fatores de Tempo , Resultado do Tratamento , Carga Viral , Proteínas não Estruturais Virais/imunologiaRESUMO
The aim of this study was to characterize timing, kinetic, and magnitude of CMV-specific immune response after hematopoietic stem cell transplantation (HSCT) and its ability to predict CMV replication and clinical outcomes. Using cell surface and intracellular cytokine staining by flow cytometry, CMV-specific T-cell response was measured in blood, while CMV viral load and chimerism were determined by real-time PCR. Patients that reconstituted CMV-specific T-cell response within 6 weeks after Allo-SCT showed a more robust immune response (CD8(+) : 0.7 cells/µl vs. 0.3/µl; P-value = 0.01), less incidence of CMV replication (33% vs. 89.5%; P-value = 0.007), reduced viral loads (1.81 log copies/ml vs. 0 copies/ml; P-value = 0.04), and better overall survival (72%; CI: 0.53-0.96 vs. 42% CI: 0.24-0.71; P-value = 0.07) than patients with a delayed immune reconstitution. Viremic patients had significantly higher transplant-related mortality than nonviremic patients after 1 year (33% CI: 0.15-0.52 vs. 0% CI: 0.05-0.34; P-value = 0.01). Risk factors independently associated with viral replication were receptor pretransplant CMV-positive serostatus (P-value = 0.02) and acquiring CMV-specific T-cell response after 6 weeks post-transplantation (P-value = 0.009). In conclusion, timing of acquiring a positive CMV-specific T-cell immune response after transplantation may identify patients with different risk for viral replication and different clinical outcomes, including survival.
Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Transplante de Células-Tronco Hematopoéticas , Memória Imunológica , Complicações Pós-Operatórias/imunologia , Subpopulações de Linfócitos T/imunologia , Viremia/imunologia , Adolescente , Adulto , Aloenxertos , Antivirais/uso terapêutico , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/etiologia , Infecções por Citomegalovirus/mortalidade , Feminino , Neoplasias Hematológicas/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/tratamento farmacológico , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/mortalidade , Estudos Prospectivos , Especificidade do Receptor de Antígeno de Linfócitos T , Fatores de Tempo , Carga Viral , Viremia/etiologia , Ativação Viral , Replicação Viral , Adulto JovemRESUMO
BACKGROUND: Hepatitis C virus (HCV) is associated with a higher prevalence of steatosis compared to the general population. AIM: Our aim was to assess the impact of PNPLA3 rs738409 G-allele on steatosis in HCV patients. MATERIAL AND METHODS: We included 474 HCV patients treated with peginterferon plus ribavirin. PNPLA3 rs738409 was genotyped and patients were classified according to alleles and genotypes. Steatosis was detected in 46.4% (220/474). Fibrosis was assessed by Scheuer score. Gene expression was analyzed in Huh7.5 and Huh7 cells using Real Time-PCR. RESULTS: PNPLA3 allele-G was associated with steatosis [54.1% (126/233) vs. 39% (94/241)] (p = 0.0001). In HCV-1, allele-G was related to steatosis [50.6% (82/162) vs. 32.3% (53/164)] (p = 0.001), but did not in HCV-3 [61.9% (26/42) vs. 62% (31/50)] (p = 0.993). PNPLA3 allele-G was associated with steatosis in patients with IL28B-CT/TT [57.7% (82/142) vs. 37.1% (56/151)] (p = 0.0001), but did not in IL28B-CC [47.8% (43/90) vs. 42% (37/88)] (p = 0.442). Independent variables associated with steatosis were: PNPLA3 G-allele [O.R. 1.84 (CI95%: 1.06-3.21); p = 0.007], age [O.R. 1.04 (CI95%: 1.01-1.07); p = 0.017], HCV-genotype 3 [O.R. 2.46 (CI95%: 1.30-4.65); p = 0.006], HOMA > 4 [O.R. 2.72 (CI95%: 1.27-5.82); p = 0.010]. Since PNPLA3 RNA could not be detected on PBMC from HCV patients, an in vitro analysis was performed. Huh7.5 cells infected with JFH1 had a decreased PNPLA3 gene expression (fold inhibition = 3.2 ± 0.2), while Huh7 cells presented increased PNPLA3 gene expression (fold induction = 1.5 ± 0.2). CONCLUSION: PNPLA3 allele-G modulated the development of steatosis, particularly in patients with HCV-1 and IL28B-CT/TT genotype, but was not associated with SVR. Metabolic but not viral steatosis seems to be PNPLA3 regulated. Gene interaction may result in differential PNPLA3 gene expression levels in HCV infection.
Assuntos
Fígado Gorduroso/genética , Hepacivirus/genética , Hepatite C Crônica/genética , Interleucinas/genética , Lipase/genética , Fígado/patologia , Proteínas de Membrana/genética , RNA Viral/genética , Adulto , Antivirais/uso terapêutico , Células Cultivadas , Estudos de Coortes , Estudos Transversais , Quimioterapia Combinada , Fígado Gorduroso/virologia , Feminino , Perfilação da Expressão Gênica , Genótipo , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Humanos , Interferon alfa-2 , Interferon-alfa/uso terapêutico , Interferons , Masculino , Pessoa de Meia-Idade , Polietilenoglicóis/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Ribavirina/uso terapêutico , Fatores de Risco , Carga ViralRESUMO
PURPOSE: The biologically active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (vit D), has immunoregulatory properties via binding vitamin D receptor (VDR). In a prospective trial, we previously reported a reduction in the incidence of chronic GvHD (cGvHD) among patients who received vit D after allogeneic stem cell transplantation (allo-HSCT; Clinical Trials.gov: NCT02600988). Here we analyze the role of patients and donors' VDR SNPs on the immunomodulatory effect of vit D. PATIENTS AND METHODS: Patients undergoing allo-HSCT were included in a prospective phase I/II clinical trial (Alovita) in three consecutive cohorts: control (without vit D), low-dose (1,000 IU/day), and high-dose (5,000 IU/day) groups. Vit D was given from day -5 until +100 after transplant. Genotyping of four SNPs of the VDR gene, FokI, BsmI, ApaI, and TaqI, were performed using TaqMan SNP genotyping assays. RESULTS: We observed a decrease in the incidence of overall cGvHD at 1 year after allo-HSCT depending on the use or not of vit D among patients with FokI CT genotype (22.5% vs 80%, P = 0.0004) and among those patients without BsmI/ApaI/TaqI ATC haplotype (22.2% vs 68.8%, P = 0.0005). In a multivariate analysis, FokI CT genotype significantly influenced the risk of cGvHD in patients treated with vit D as compared with the control group (HR 0.143, P interaction < 0.001). CONCLUSIONS: Our results show that the immunomodulatory effect of vit D depends on the VDR SNPs, and patients carrying the FokI CT genotype display the highest benefit from receiving vit D after allo-HSCT.