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1.
Hepatol Int ; 13(4): 422-430, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31172416

RESUMO

BACKGROUND: The safety of nucleos(t)ide analogue (NA) treatment cessation remains one of the most controversial topics in the management of chronic hepatitis B (CHB) patients. This study investigated the efficiency of 48-week pegylated-interferon (peg-IFN) alfa-2a consolidation therapy on viral relapse after discontinued NA treatment in CHB patients who achieved hepatitis B e antigen (HBeAg) seroconversion for > 1 year. METHODS: NA-treated HBeAg-positive patients who achieved the standard of discontinued NA treatment (i.e. time of HBeAg seroconversion > 1 year) were randomly assigned to receive peg-IFN consolidation (n = 24) treatment or continue original NA therapy (n = 24) for 48 weeks. The treatments were then discontinued, and the patients were observed up to 144 weeks. The primary endpoint was the proportion of patients with viral relapse at week 144 among those who received at least one dose of study drug or had at least one study visit [modified intention-to-treat population (mITT)]. RESULTS: Of the 24 patients who received peg-IFN treatment, 6 (25%) experienced viral relapse and 8 (36.3%) showed HBsAg loss during 96 weeks of treatment-free follow-up. Of the patients who underwent NA consolidation treatment, only 1 (4.3%) of 23 patients showed HBsAg loss and 14 (58.3%) of 24 patients experienced viral relapse during follow-up. HBsAg level decline < 0.25 log10 IU/mL at week 96 was significantly associated with viral relapse. CONCLUSION: A 48-week peg-IFN alfa-2a consolidation therapy increased the rate of HBsAg loss and sustained viral replication suppression in HBeAg-positive patients who achieved HBeAg seroconversion for > 1 year after NA treatment discontinuation.


Assuntos
Antivirais/uso terapêutico , Guanina/análogos & derivados , Hepatite B Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Polietilenoglicóis/uso terapêutico , Adulto , Feminino , Guanina/uso terapêutico , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Hepatite B Crônica/imunologia , Humanos , Masculino , Projetos Piloto , Proteínas Recombinantes/uso terapêutico , Soroconversão , Resposta Viral Sustentada , Resultado do Tratamento
2.
Med Sci Monit ; 25: 4665-4674, 2019 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-31230063

RESUMO

BACKGROUND High rates of HBeAg and/or HBsAg seroconversion or clearance have been achieved in chronic hepatitis B (CHB) patients receiving pegylated interferon (pegIFN) in addition to ongoing nucleos(t)ide analogue (NUC) treatment. In the present study, we aimed to evaluate HBsAg kinetics to predict HBeAg seroconversion and HBsAg clearance in these patients. MATERIAL AND METHODS A total of 33 HBeAg-positive and 17 HBeAg-negative patients were enrolled between January 2010 and January 2018. At the end of pegIFN treatment, 9 of 50 patients achieved HBsAg clearance, and 9 of 33 HBeAg-positive patients achieved HBeAg seroconversion. RESULTS The cutoff value of 0.41 log10 IU/mL in HBsAg decline at week 12 had a positive predictive value (PPV) of 58.3% and a negative predictive value (NPV) of 94.6% for HBsAg clearance. The cutoff value of 1.94 log10 IU/mL in HBsAg decline at week 24 had a PPV of 80.0% and an NPV of 97.5% for HBsAg clearance. The cutoff value of 0.47 log10 IU/mL in HBsAg decline at week 12 had a PPV of 83.3% and an NPV of 85.2% for HBeAg seroconversion. The cutoff value of 1.29 log10 IU/mL in in HBsAg decline at week 24 had a PPV of 85.7% and an NPV of 88.5% for HBeAg seroconversion. CONCLUSIONS Early HBsAg drop has a high predictive value for HBsAg clearance and HBeAg seroconversion in patients who were treated with combination therapy of pegIFN and NUCs.


Assuntos
Antígenos de Superfície da Hepatite B/análise , Hepatite B Crônica/tratamento farmacológico , Adulto , Antivirais/uso terapêutico , Biomarcadores Farmacológicos/sangue , DNA Viral/sangue , Feminino , Guanina/análogos & derivados , Antígenos de Superfície da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/sangue , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Hepatite B Crônica/sangue , Humanos , Interferon alfa-2/farmacocinética , Interferon alfa-2/farmacologia , Interferon-alfa/farmacologia , Masculino , Pessoa de Meia-Idade , Polietilenoglicóis/farmacologia , Proteínas Recombinantes/farmacologia , Soroconversão , Resultado do Tratamento
3.
PLoS Pathog ; 15(4): e1007690, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30998767

RESUMO

Chronic hepatitis B virus (HBV) infection is associated with functionally impaired virus-specific T cell responses. Although the myeloid-derived suppressor cells (MDSCs) are known to play a critical role in impairing antiviral T cell responses, viral factors responsible for the expansion of MDSCs in chronic hepatitis B (CHB) remain obscure. In order to elucidate the mechanism of monocytic MDSCs (mMDSCs) expansion and T cell function suppression during persistent HBV infection, we analyzed the circulation frequency of mMDSCs in 164 CHB patients and 70 healthy donors, and found that the proportion of mMDSCs in HBeAg (+) CHB patients was significantly increased compared to that in HBeAg (-) patients, which positively correlated with the level of HBeAg. Furthermore, exposure of peripheral blood mononuclear cells (PBMCs) isolated from healthy donors to HBeAg led to mMDSCs expansion and significant upregulation of IL-1ß, IL-6 and indoleamine-2, 3-dioxygenase (IDO), and depletion of the cytokines abrogated HBeAg-induced mMDSCs expansion. Moreover, HBeAg-induced mMDSCs suppressed the autologous T-cell proliferation in vitro, and the purified mMDSCs from HBeAg (+) subjects markedly reduced the proliferation of CD4+ and CD8+ T cells and IFN-γ production, which could be efficiently restored by inhibiting IDO. In summary, HBeAg-induced mMDSCs expansion impairs T cell function through IDO pathway and favors the establishment of a persistent HBV infection, suggesting a mechanism behind the development of HBeAg-induced immune tolerance.


Assuntos
Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Tolerância Imunológica/imunologia , Leucócitos Mononucleares/imunologia , Células Supressoras Mieloides/imunologia , Linfócitos T/imunologia , Adulto , Proliferação de Células , Citocinas/metabolismo , Feminino , Antígenos E da Hepatite B/imunologia , Hepatite B Crônica/metabolismo , Hepatite B Crônica/virologia , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Ativação Linfocitária , Masculino , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/virologia , Linfócitos T Reguladores/imunologia
4.
J Chemother ; 31(4): 209-213, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30963812

RESUMO

Hepatitis B virus (HBV) infections and sequelae present significant health problems worldwide. Two groups of medications are available for chronic HBV infection treatment: (1) interferons (IFNs) and (2) nucleos(t)ide analogues. This study aimed to evaluate entecavir (ETV) and tenofovir disoproxil fumarate (TDF) efficacies in chronic HBV patients, who achieved virological response during Peg-IFN treatment but did not sustain this response and relapsed a year after treatment end. In this study, 74 patients with chronic HBV infection who had virological responses to 180 µg/week Peg-IFNα-2a treatment were included; 38 (20 and 18 HBeAg positive and negative, respectively) of these patients were treated with 245 mg/day TDF, and 36 (20 and 16 HBeAg positive and negative, respectively) were treated with 0.5 mg/day ETV upon relapse after initial treatment discontinuation. In HBeAg-positive patients biochemical response rates were higher for TDF at weeks 96 and 144 (p = 0.044 and 0.019, respectively). However, biochemical response rates were similar for TDF and ETV in HBeAg-positive and HBeAg-negative groups at other weeks (p > 0.05). Virological and serological response rates were similar in patients treated with TDF and ETV in HBeAg-positive and HBeAg-negative groups (p > 0.05).


Assuntos
Antivirais/uso terapêutico , Guanina/análogos & derivados , Hepatite B Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Polietilenoglicóis/uso terapêutico , Tenofovir/uso terapêutico , Adulto , DNA Viral/efeitos dos fármacos , Feminino , Guanina/uso terapêutico , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/metabolismo , Hepatite B Crônica/virologia , Humanos , Masculino , Proteínas Recombinantes/uso terapêutico , Estudos Retrospectivos , Resultado do Tratamento , Carga Viral/efeitos dos fármacos
5.
Cell Mol Biol (Noisy-le-grand) ; 65(2): 75-81, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30860474

RESUMO

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) was positively correlated with serological hepatitis B surface antigen (HBsAg) levels in hepatitis B e antigen (HBeAg) positive chronic hepatitis B (CHB) patients. We evaluated whether Thymopentin (TP5) and interferon (IFN-a) had a synergic effect on HBV cccDNA and the effect of TP5 addition therapy on HBsAg clearance in CHB patients. Real-time PCR experiments were performed to test cccDNA in HepG2.2.15 cells. 45 HBeAg-positive CHB patients had been distributed into two groups randomly. Treatment group: 23 patients were treated with a 24-week TP5 on the basis of the treatment entecavir (ETV) and peginterferon alfa-2a (PegIFN alpha-2a). Control group: 22 patients were treated with ETV and PegIFNa-2a. The study period was 72 weeks. In HepG2.2.15 cells, TP5 5ug/ml and 10ug/ml respectively combined with IFN-a 2ku/ml could potently inhibit cccDNA level at 72 hours (P<0.05). In clinical study, mean HBsAg levels in two groups are not significantly different at different time points (p=0.112). However, changes of mean HBsAg levels in TP5 add-on group at different time points are significantly different (p<0.05). Patients with HBsAg levels <1500IU/ml in control group had higher HBsAg levels compared with patients with HBsAg levels <1500IU/ml in TP5 add-on group (P=0.019). The latter had the most pronounced HBsAg reduction. TP5 and IFN had a synergic effect on inhibiting cccDNA levels in HepG2.2.15 cells; Patients in treatment group showed no extra side effects compared with the control group. 24 weeks TP5 add-on treatment was safe and had a tendency to accelerate the decline of HBsAg when HBV-DNA was undetectable.


Assuntos
Guanina/análogos & derivados , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Polietilenoglicóis/uso terapêutico , Timopentina/uso terapêutico , Adulto , DNA Viral/genética , Quimioterapia Combinada , Feminino , Guanina/farmacologia , Guanina/uso terapêutico , Células Hep G2 , Antígenos E da Hepatite B/sangue , Hepatite B Crônica/sangue , Humanos , Masculino , Proteínas Recombinantes/uso terapêutico , Timopentina/farmacologia , Resultado do Tratamento
6.
Life Sci ; 219: 199-208, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30615846

RESUMO

AIM: Many studies have shown that some microRNAs (miRNAs) play an important role in the pathogenesis of chronic hepatitis B (CHB) infection. In this study, we aimed to explore the role and molecular mechanism of miRNA-548ah in the replication and expression of the hepatitis B virus (HBV). MAIN METHODS: Overexpression and knockdown of miRNA-548ah were performed in three hepatoma cell lines with HBV replication and in a murine HBV model injected with adenovirus HBV vector. The effect of miRNA-548ah on its target gene, histone deacetylase (HDAC) 4, were confirmed in in vitro studies and further investigated in liver tissues from CHB patients. KEY FINDINGS: miRNA-548ah significantly increased the expression of HBV in hepatoma cell lines and in a HBV mouse model. The expression level of covalently closed circular DNA (cccDNA) in the miRNA-548ah mimics group was significantly higher than the negative control group and significantly lower in the miRNA-548ah inhibitor group. The HBV core antigen promotes the expression of miRNA-548ah in hepatocytes. Finally, we observed a negative correlation between the expression of miRNA-548ah and HDAC4 in the liver tissue of patients with CHB. SIGNIFICANCE: miRNA-548ah promoted the replication and expression of HBV through the regulation of the target gene, HDAC4. Inhibition of HDAC4 by miRNA-548ah might influence the deacetylation state of histones binding to cccDNA, thereby enhancing the replication of cccDNA. The HBV core antigen might increase the expression of miRNA-548ah. These results may provide new potential molecular targets for the prevention and treatment of CHB.


Assuntos
Vírus da Hepatite B/fisiologia , Histona Desacetilases/metabolismo , MicroRNAs/metabolismo , Proteínas Repressoras/metabolismo , Animais , Western Blotting , Linhagem Celular , Imunoprecipitação da Cromatina , DNA Viral/metabolismo , Células Hep G2 , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Replicação Viral
7.
Clin Microbiol Infect ; 25(4): 514.e1-514.e8, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29906588

RESUMO

OBJECTIVES: To evaluate the performance of a new mathematical model γ-glutamyl transpeptidase to cholinesterase and platelet ratio (GCPR) versus γ-glutamyl transpeptidase to platelet ratio (GPR) in predicting significant fibrosis and cirrhosis of chronic hepatitis B. METHODS: A complete cohort of 2343 patients was divided into early and late cohort depending on the time of liver biopsy. With reference to the Scheuer standard, liver pathologic stage 2 or higher and stage 4 or higher were defined as significant fibrosis and cirrhosis, respectively. Receiver operating characteristic (ROC) curve was used to evaluate the performance of investigated models. RESULTS: In the early cohort, the areas under ROC curves (AUROCs) of GCPR in predicting significant fibrosis of hepatitis B e antigen (HBeAg)-positive and HBeAg-negative patients (0.782 and 0.775) were both significantly greater than those of GPR (0.748 and 0.747) (Z = 8.198 and Z = 6.023, both p <0.0001); the AUROCs of GCPR in predicting cirrhosis of HBeAg-positive and HBeAg-negative patients (0.842 and 0.861) were both significantly greater than those of GPR (0.802 and 0.823) (Z = 6.686 and Z = 6.116, both p <0.0001). In early, late and complete cohorts, using a single cutoff of GCPR > 0.080, the specificities of GCPR in predicting significant fibrosis of HBeAg-positive patients were 83.3%, 88.2% and 85.0% and of HBeAg-negative patients were 87.6%, 87.4% and 87.6%, respectively; and the sensitivities of GCPR in predicting cirrhosis of HBeAg-positive patients were 81.9%, 88.7% and 84.2% and of HBeAg-negative patients were 83.1%, 82.1% and 82.7%, respectively. CONCLUSIONS: GCPR has higher performance than GPR in predicting significant fibrosis and cirrhosis of chronic hepatitis B.


Assuntos
Colinesterases/sangue , Antígenos E da Hepatite B/metabolismo , Hepatite B Crônica/diagnóstico , Cirrose Hepática/diagnóstico , gama-Glutamiltransferase/sangue , Adulto , Biópsia , Feminino , Hepatite B Crônica/patologia , Hepatite B Crônica/virologia , Humanos , Fígado/patologia , Fígado/cirurgia , Cirrose Hepática/patologia , Cirrose Hepática/virologia , Masculino , Pessoa de Meia-Idade , Modelos Teóricos , Contagem de Plaquetas , Curva ROC , Estudos Retrospectivos
8.
J Virol ; 93(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30404796

RESUMO

Viruses have adopted diverse strategies to suppress antiviral responses. Hepatitis B virus (HBV), a virus that is prevalent worldwide, manipulates the host's innate immune system to evade scavenging. It is reported that the hepatitis B e antigen (HBeAg) can interfere with NF-κB activity, which then leads to high viral loads, while HBV with the G1896A mutation remains infectious without the production of HBeAg but can induce more severe proinflammatory response and liver damage. The aim of current work was to study the molecular mechanism by which HBeAg suppresses interleukin-1ß (IL-1ß)-stimulated NF-κB activity, which leads to the suppression of the innate immune responses to HBV infection. Our study revealed that HBeAg could interact with NEMO, a regulatory subunit associated with IκB kinase, which regulates the activation of NF-κB. HBeAg suppressed the IL-1ß-induced tumor necrosis factor (TNF)-associated factor 6 (TRAF6)-dependent K63-linked ubiquitination of NEMO, thereby downregulating NF-κB activity and promoting virus replication. We further demonstrated the inhibitory effect of HBeAg on the NF-κB signaling pathway using primary human hepatocytes, HBV-infected HepG2-NTCP cells, and clinical liver samples. Our study reveals a molecular mechanism whereby HBeAg suppresses IL-1ß-induced NF-κB activation by decreasing the TRAF6-dependent K63-linked ubiquitination of NEMO, which may thereby enhance HBV replication and promote a persistent infection.IMPORTANCE The role of HBeAg in inflammatory responses during the infection of hepatitis B virus (HBV) is not fully understood, and several previous reports with regard to the NF-κB pathway are controversial. In this study, we showed that HBeAg could suppress both Toll-like receptor 2 (TLR2)- and IL-1ß-induced activation of NF-κB in cells and clinical samples, and we further revealed novel molecular mechanisms. We found that HBeAg can associate with NEMO, the regulatory subunit for IκB kinase (IKK) that controls the NF-κB signaling pathway, and thereby inhibits TRAF6-mediated K63-linked ubiquitination of NEMO, resulting in downregulation of NF-κB activity and promotion of virus replication. In contrast, the HBeAg-negative HBV mutant can induce higher levels of NF-κB activity. These results are important for understanding the HBV-induced pathogenesis of chronic hepatitis and indicate that different clinical measures should be considered to treat HBeAg-positive and HBeAg-negative infections. Our findings represent a conceptual advance in HBV-related suppression of NF-κB signaling.


Assuntos
Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B/imunologia , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Adulto , Feminino , Células HEK293 , Células HeLa , Células Hep G2 , Hepatite B/virologia , Vírus da Hepatite B/imunologia , Interações Hospedeiro-Patógeno , Humanos , Quinase I-kappa B/química , Imunidade Inata , Interleucina-1beta/metabolismo , Lisina/metabolismo , Masculino , Pessoa de Meia-Idade , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinação
10.
Life Sci ; 213: 9-17, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30308183

RESUMO

AIMS: To investigate the role and underlying mechanism of miR-185-5p in hepatitis B virus (HBV) expression and replication. MAIN METHODS: The relative levels of hepatitis B surface antigen and hepatitis B e antigen were detected by enzyme-linked immunosorbent assay (ELISA). The HBV DNA copies in the cultures medium were measured by RT-qPCR. The HBV large surface antigen promoter (S1p) activity was analyzed by luciferase reporter assay. The target relationship between miR-185-5p and ELK1 was identified by bioinformatics analysis and EGFP fluorescent reporter assay. The ELK1 expression was determined by RT-qPCR and Western blot. KEY FINDINGS: miR-185-5p significantly decreased HBV large surface antigen promoter activity and subsequently the production of HBV proteins and HBV DNA copies in vitro. Further, we identified the ETS transcription factor ELK1 is a target of miR-185-5p. Overexpression and knockdown experiments showed overexpression of ELK1 stimulated HBV large surface antigen promoter activity and promoted the production of HBV proteins and HBV DNA copies, whereas knockdown of ELK1 has the opposite effects. Moreover, the rescue of ELK1 expression reversed the suppression of miR-185-5p on HBV replication and gene expression. Further mechanistic study showed that the ETS binding sites within the HBV large surface antigen promoter are required for the repression effect of miR-185-5p on HBV. SIGNIFICANCE: There are few reports about the interaction between miRNAs and the transcription from HBV S1p, we found that miR-185-5p decreases HBV S1p activity by targeting ELK1, which may provide a promising therapeutic strategy for HBV infection.


Assuntos
Carcinoma Hepatocelular/virologia , Regulação Viral da Expressão Gênica , Vírus da Hepatite B/genética , Hepatite B/genética , Neoplasias Hepáticas/virologia , MicroRNAs/genética , Proteínas Elk-1 do Domínio ets/genética , Sítios de Ligação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Replicação do DNA , DNA Viral/genética , DNA Viral/metabolismo , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , Replicação Viral/genética , Proteínas Elk-1 do Domínio ets/metabolismo
11.
Viral Immunol ; 31(9): 639-645, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30222506

RESUMO

Nod-like receptor protein 3 (NLRP3), absent in melanoma 2 (AIM2), and interferon gamma inducible protein 16 (IFI16) are innate immune sensors for intracellular microbes, which can be activated by various dangerous signals and subsequently lead to caspase-1 (CASP1) activation and the maturation cleavage of effector molecules pro-IL-1ß and pro-IL-18. Their roles in immunopathology of acute and chronic hepatitis B virus (HBV) infection are still unclear. In this study, we first investigated the activation of NLRP3, AIM2, and IFI16 inflammasomes in peripheral blood mononuclear cells (PBMCs) from patients infected with acute hepatitis B (AHB) and chronic hepatitis B (CHB) by quantitative real-time PCR and enzyme-linked immunosorbent assay. We next analyzed the impact of hepatitis B e antigen (HBeAg) on activation of AIM2 and IFI16 inflammasomes in PBMCs of CHB patients stimulated in vitro with AIM2 and IFI16 agonist ligands, poly (dA:dT) and VACA-70mer, respectively. The results showed that the mRNA expression levels of AIM2, IFI16, and CASP1 in PBMCs from AHB and CHB patients were both upregulated. Furthermore, the mRNA levels of AIM2 and IFI16 in CHB patients were significantly positively correlated with serum HBV loads. However, only in patients with AHB there was elevation of serum IL-1ß and IL-18. There was no activation of NLRP3, AIM2, and IFI16 inflammasomes in CHB patients. Stimulation of PBMCs of CHB patients in vitro with poly (dA:dT) and VACA-70mer induced the activation of AIM2 and IFI16 inflammasomes, respectively. This ligand-induced activation was suppressed by HBeAg. Our results suggest that there exists activation of the AIM2 and IFI16 inflammasomes, but not the NLRP3 inflammasome, in AHB, and the activation of the AIM2 and IFI16 inflammasomes can be inhibited by HBeAg in CHB, which may contribute to HBV-induced immunotolerance.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Hepatite B Crônica/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Doença Aguda , Adulto , DNA Viral/sangue , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/genética , Feminino , Antígenos E da Hepatite B/metabolismo , Hepatite B Crônica/sangue , Hepatite B Crônica/imunologia , Humanos , Imunidade Inata/imunologia , Interleucina-18/sangue , Interleucina-1beta/sangue , Leucócitos Mononucleares/imunologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas Nucleares/agonistas , Proteínas Nucleares/genética , Fosfoproteínas/agonistas , Fosfoproteínas/genética , Polidesoxirribonucleotídeos/antagonistas & inibidores , Polidesoxirribonucleotídeos/farmacologia
12.
Artigo em Inglês | MEDLINE | ID: mdl-30224536

RESUMO

Currently available therapies for chronic hepatitis B virus (HBV) infection can efficiently reduce viremia but induce hepatitis B surface antigen (HBsAg) loss in very few patients; also, these therapies do not greatly affect the viral covalently closed circular DNA (cccDNA). To discover new agents with complementary anti-HBV effects, we performed a drug repurposing screen of 1,018 Food and Drug Administration (FDA)-approved compounds using HBV-infected primary human hepatocytes (PHH). Several compounds belonging to the family of retinoic acid receptor (RAR) agonists were identified that reduced HBsAg levels in a dose-dependent manner without significant cytotoxicity. Among them, tazarotene exhibited the most potent anti-HBV effect, with a half-maximal inhibitory concentration (IC50) for HBsAg of less than 30 nM in PHH. The inhibitory effect was also observed in HBV-infected differentiated HepaRG (dHepaRG) models, but not in HepG2.215 cells, and HBV genotypes A to D were similarly inhibited. Tazarotene was further demonstrated to repress HBV cccDNA transcription, as determined by the levels of HBV cccDNA and RNAs and the activation of HBV promoters. Moreover, RNA sequence analysis showed that tazarotene did not induce an interferon response but altered the expression of a number of genes associated with RAR and metabolic pathways. Inhibition of RARß, but not RARα, by a specific antagonist significantly attenuated the anti-HBV activity of tazarotene, suggesting that tazarotene inhibits HBV in part through RARß. Finally, a synergistic effect of tazarotene and entecavir on HBV DNA levels was observed. Therefore, RAR agonists as represented by tazarotene were identified as potential novel anti-HBV agents.


Assuntos
Antivirais/farmacologia , Guanina/análogos & derivados , Vírus da Hepatite B/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Ácidos Nicotínicos/farmacologia , Receptores do Ácido Retinoico/genética , Acitretina/farmacologia , Adapaleno/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fármacos Dermatológicos/farmacologia , Reposicionamento de Medicamentos , Sinergismo Farmacológico , Expressão Gênica , Guanina/farmacologia , Células Hep G2 , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/genética , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/crescimento & desenvolvimento , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/virologia , Ensaios de Triagem em Larga Escala , Interações Hospedeiro-Patógeno/genética , Humanos , Ceratolíticos/farmacologia , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/metabolismo , Transcrição Genética/efeitos dos fármacos , Tretinoína/farmacologia , Replicação Viral/efeitos dos fármacos
13.
Structure ; 26(10): 1314-1326.e4, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30100358

RESUMO

Hepatitis B virus (HBV) is the leading cause of liver disease worldwide. While an adequate vaccine is available, current treatment options are limited, not highly effective, and associated with adverse effects, encouraging the development of alternative therapeutics. The HBV core gene encodes two different proteins: core, which forms the viral nucleocapsid, and pre-core, which serves as an immune modulator with multiple points of action. The two proteins mostly have the same sequence, although they differ at their N and C termini and in their dimeric arrangements. Previously, we engineered two human-framework antibody fragments (Fab/scFv) with nano- to picomolar affinities for both proteins. Here, by means of X-ray crystallography, analytical ultracentrifugation, and electron microscopy, we demonstrate that the antibodies have non-overlapping epitopes and effectively block biologically important assemblies of both proteins. These properties, together with the anticipated high tolerability and long half-lives of the antibodies, make them promising therapeutics.


Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos E da Hepatite B/química , Vírus da Hepatite B/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Antivirais , Sítios de Ligação , Cristalografia por Raios X , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/química , Humanos , Microscopia Eletrônica , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Ultracentrifugação
15.
Stem Cell Res Ther ; 9(1): 177, 2018 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-29973264

RESUMO

BACKGROUND: In cell-based therapy, the transmission of communicable diseases imposes a substantial threat to recipients. In this study, we investigated whether cell-based screening could detect hepatitis B virus (HBV) in human umbilical cord-derived mesenchymal stem cells (HUMSCs) isolated from HBV-infected donors to understand the susceptibility of HUMSCs to HBV infection. METHODS: HBV assay was performed in HUMSCs derived from healthy and HBV-infected donors with enzyme-linked immunosorbent assay (ELISA), fluorescence quantitative PCR (FQ-PCR) assay, and droplet digital PCR (ddPCR) assay. Further, HBV DNA was assayed in HUMSCs derived from healthy donors after incubation with human sera containing a high titer of HBV using FQ-PCR. RESULTS: HBV antigen/antibody and DNA failed to be detected using ELISA, FQ-PCR, and ddPCR. After incubation with HBV infection sera, HBV DNA could be detected, but below the valid titer of the assay kit. The HBV DNA levels in HBV-incubated HUMSCs gradually decreased with medium change every 2 days and then significantly decreased, not even detected after passage. CONCLUSIONS: The current cell-based screening methods could not detect HBV in HUMSCs derived from HBV-infected donors, indicating the importance of more stringent donor eligibility to reduce the risk of transmission of communicable diseases in cell-based therapy. To solve the problem of an occult HBV window period in donor eligibility determination, we recommend that the donors undergo another HBV serological test 3 months after the first serological communicable disease screening.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Vírus da Hepatite B/isolamento & purificação , Células-Tronco Mesenquimais/virologia , Adipogenia/fisiologia , Diferenciação Celular/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/normas , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Hepatite B/diagnóstico , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/patogenicidade , Humanos , Mães , Osteogênese/fisiologia , Gravidez , Doadores de Tecidos
16.
Arch Virol ; 163(10): 2829-2833, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29948380

RESUMO

The high prevalence (14.3%) of HIV/HBV co-infections reported in west China makes it necessary to screen concurrent HBV infection in HIV carriers. HBV B genotype was shown to be dominant in 54 cases of HIV/HBV co-infection, accounting for 81.48% of the total. The total drug resistance rate observed was 3.70%. A1762T, G1764A and G1896A mutations were common mutations identified in the BCP/PC region. However, the prevalence of the G1896A mutation was significantly high among the HBeAg negative HIV/HBV co-infected patients, and may be associated with high HBV replication. Mutations in the PC region are related to the loss in synthesis of HBeAg and may accelerate HBV replication in HIV positive patients.


Assuntos
Coinfecção/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Vírus da Hepatite B/genética , Hepatite B/virologia , Regiões Promotoras Genéticas , Replicação Viral , Adulto , Feminino , HIV-1/genética , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos E da Hepatite B/genética , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação
17.
Aliment Pharmacol Ther ; 48(5): 547-555, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29956827

RESUMO

BACKGROUND: Peginterferon induces off-treatment responses in approximately one-third of patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B. AIM: To develop an easy-to-use baseline prediction score to identify hepatitis B virus (HBV) genotype B-/C-infected HBeAg-positive Asian patients likely to respond to peginterferon alfa-2a. METHODS: Generalised additive models, multiple logistic regression (MLR) analysis and internal validation methods were applied to data from 647 HBeAg-positive patients from China, Hong Kong and Taiwan to develop a scoring system to predict response 24 weeks after completing a 48-week course of peginterferon alfa-2a. RESULTS: Five baseline factors (age, sex, alanine aminotransferase ratio, hepatitis B surface antigen (HBsAg) level and HBV DNA level) were retained in the final MLR for HBeAg seroconversion and used to develop a scoring system from 0 to 7. Among patients with scores of 0-1, 2-3, 4 or ≥5, HBeAg seroconversion was achieved in 6.4% (6/94), 23.0% (61/265), 36.4% (67/184) and 54.8% (57/104), respectively, and a combined response (HBeAg seroconversion plus HBV DNA <2000 IU/mL) in 5.3% (5/94), 12.8% (34/265), 25.0% (46/184) and 36.5% (38/104), respectively. Among patients with scores of 0-1, 2-3, 4 or ≥5, 57.0% (53/93), 12.3% (31/253), 3.4% (6/178) and 1.0% (1/100) had HBsAg ≥20 000 IU/mL at treatment Week 12; only 3/91 (3.3%) with HBsAg ≥20 000 IU/mL experienced a combined response at 24 weeks post-treatment (negative predictive value = 97% [88/91]). CONCLUSION: A pre-treatment scoring system using readily available baseline characteristics identifies HBeAg-positive Asian patients likely to experience sustained HBeAg seroconversion after treatment with peginterferon alfa-2a.


Assuntos
Antígenos E da Hepatite B/metabolismo , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Polietilenoglicóis/uso terapêutico , Adolescente , Adulto , Idoso , Antivirais/uso terapêutico , Grupo com Ancestrais do Continente Asiático/estatística & dados numéricos , Biomarcadores Farmacológicos/análise , China/epidemiologia , Feminino , Hepatite B Crônica/epidemiologia , Hepatite B Crônica/metabolismo , Hong Kong/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Proteínas Recombinantes/uso terapêutico , Projetos de Pesquisa , Estudos Retrospectivos , Taiwan/epidemiologia , Resultado do Tratamento , Adulto Jovem
18.
Biomed Pharmacother ; 99: 774-780, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29710475

RESUMO

BACKGROUND: Hepatitis B virus (HBV) chronic infection is a health problem in the worldwide, with a underlying higher risk of liver cirrhosis and hepaticocellular carcinoma. A number of studies indicate that microRNAs (miRNAs) play vital roles in HBV replication. This study was designed to explore the potential molecular mechanism of miR-200c in HBV replication. METHODS: The expression of miR-200c, nuclear factor IA (NFIA) mRNA, HBV DNA, and HBV RNA (pregenomic RNA (pgRNA), and total RNA) were measured by qRCR. The levels of HBsAg and HBeAg were detected by ELISA. NFIA expression at protein level was measured by western blot. The direct interaction between miR-200c and NFIA were identified by Targetscan software and Dual-Luciferase reporter analysis. Enhance I activity were detected by Dual-Luciferase reporter assay. RESULTS: miR-200c expression was prominently reduced in pHBV1.3-tranfected Huh7 and in stable HBV-producing cell line (HepG2.2.15). The enforced expression of miR-200c significantly suppressed HBV replication, as demonstrated by the reduced levels of HBV protein (HBsAg and HBeAg) and, DNA and RNA (pgRNA and total RNA) levels. NFIA was proved to be a target of miR-200c and NFIA overexpression notably stimulated HBV replication. In addition, the inhibitory effect of miR-200c on HBV Enhance I activity was abolished following restoration of NFIA. CONCLUSIONS: miR-200c repressed HBV replication by directly targeting NFIA, which might provide a novel therapeutic target for HBV infection.


Assuntos
Vírus da Hepatite B/fisiologia , Hepatite B Crônica/genética , MicroRNAs/genética , Replicação Viral/fisiologia , Western Blotting , Linhagem Celular Tumoral , DNA Viral/metabolismo , Ensaio de Imunoadsorção Enzimática , Regulação Viral da Expressão Gênica , Genes Reporter , Células Hep G2 , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Hepatite B Crônica/virologia , Humanos , Luciferases/genética , Fatores de Transcrição NFI/genética , RNA Viral/metabolismo
19.
Virology ; 519: 190-196, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29734042

RESUMO

Hepatitis B virus (HBV) exists as 9 major genotypes and multiple subtypes, many of which exhibit differences in pathogenicity and treatment response. Genotype H identified in Central America is associated with low incidence of liver disease and HCC, but higher incidence of occult HBV (low level HBV DNA positivity, HBsAg negative). The replication phenotype of genotype H associated with less severe forms of liver disease is unknown. We hypothesized that the reduced pathogenesis associated with this genotype may be due to by lower rates of viral replication and/or secretion compared to other characterised strains. We used transient transfection and infection cell culture models to characterise the replication phenotype, compared to our D3 reference strain. Genotype H exhibited reduced viral replication and altered envelope protein expression compared to genotype D, with functional studies showing that low replication was in part likely due to sequence differences in the major transcriptional regulatory region.


Assuntos
Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Replicação Viral , Adulto , Replicação do DNA , DNA Viral/genética , Genótipo , Células Hep G2 , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica , Humanos , Fígado/patologia , Fígado/virologia , Masculino , Fenótipo , Carga Viral
20.
Molecules ; 23(3)2018 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-29534537

RESUMO

A series of oxime ethers with C6-C4 fragment was designed and virtually bioactively screened by docking with a target, then provided by a Friedel-Crafts reaction, esterification (or amidation), and oximation from p-substituted phenyl derivatives (Methylbenzene, Methoxybenzene, Chlorobenzene). Anti-hepatitis B virus (HBV) activities of all synthesized compounds were evaluated with HepG2.2.15 cells in vitro. Results showed that most of compounds exhibited low cytotoxicity on HepG2.2.15 cells and significant inhibition on the secretion of HBsAg and HBeAg. Among them, compound 5c-1 showed the most potent activity on inhibiting HBsAg secretion (IC50 = 39.93 µM, SI = 28.51). Results of the bioactive screening showed that stronger the compounds bound to target human leukocyte antigen A protein in docking, the more active they were in anti-HBV activities in vitro.


Assuntos
Antivirais/farmacologia , Éteres/farmacologia , Vírus da Hepatite B/metabolismo , Oximas/farmacologia , Antivirais/química , Avaliação Pré-Clínica de Medicamentos , Éteres/química , Antígenos HLA-A/química , Antígenos HLA-A/metabolismo , Células Hep G2 , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/crescimento & desenvolvimento , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Oximas/química
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