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1.
J Clin Transl Hepatol ; 7(3): 213-220, 2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31608212

RESUMO

Background and Aims: Ravidasvir (RDV) is a new generation pangenotypic hepatitis C virus (HCV) NS5A inhibitor, with high barrier to baseline resistance-associated species. This is the first phase 2/3 study conducted in Mainland China confirming the efficacy and safety of RDV + ritonavir-boosted danoprevir + ribavirin for 12 weeks in treatment-naïve noncirrhotic patients with genotype 1 infection in a large population. Methods: In this multicenter, randomized, double-blinded, placebo-controlled phase 2/3 trial (NCT03362814), we enrolled 424 treatment-naïve, noncirrhotic adult HCV genotype 1 patients. All patients were randomized at 3:1 ratio to receive a combination of RDV 200mg once daily plus ritonavir-boosted danoprevir 100mg/100mg twice daily and oral ribavirin 1000/1200mg/day (body weight <75/≥75 kg) (n = 318) or placebo (n = 106) for 12 weeks. The primary end-point was the rate of sustained virologic response 12 weeks after the end of treatment, and the safety was evaluated and compared between treatment and placebo groups. Results: The overall rate of sustained virological response at 12 weeks after treatment is 99% (306/309, 95%, CI: 97%-100%) under per protocol set analysis. All patients harboring baseline NS5A resistance-associated species in the treatment group (76/76, per protocol set) achieved sustained virological response at 12 weeks after treatment. No treatment-related serious adverse events were reported. Laboratory abnormalities showed mild or moderate severity (grade 1 and grade 2) in liver function tests. Conclusions: In treatment-naïve, noncirrhotic HCV Chinese patients infected with HCV genotype 1, all-oral regimen of RDV + ritonavir-boosted danoprevir + ribavirin for 12 weeks was highly efficacious, safe, and well tolerated.

2.
J Clin Transl Hepatol ; 7(3): 221-225, 2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31608213

RESUMO

Background and Aims: Genotype (GT) 1 remains the predominant hepatitis c virus (HCV) GT in Chinese patients. Over 80% of those Chinese patients harbor the interferon-sensitive CC allele of IFNL4rs12979860, which is favorable for interferon-based treatment regimens. This phase III clinical trial aimed to evaluate the efficacy and safety of the ritonavir-boosted danoprevir plus pegylated-interferon α-2a and ribavirin regimen for 12 weeks in treatment-naïve mainland Chinese patients infected with HCV GT1 without cirrhosis. Methods: One hundred and forty-one treatment-naïve, non-cirrhotic HCV GT1 Chinese patients (age ≥18 years) were enrolled for this single-arm, multicenter, phase III MANASA study (NCT03020082). Patients received a combination of ritonavir-boosted danoprevir (100 mg/100 mg) twice a day plus subcutaneous injection of weekly pegylated-interferon α-2a (180 µg) and oral ribavirin (1000/1200 mg/day body weight <75/≥75 kg) for 12 weeks. The primary end-point was sustained virologic response rate at 12 weeks after the end of treatment. The secondary end-points were safety outcomes, tolerability, virologic response over time and relapse rate. Results: All enrolled patients were HCV GT1-infected, and most among them (97.9%, 123/141) had the HCV GT1b subtype. Single-nucleotide polymorphism test showed that the majority of patients were of the IFNL4 rs12979860 CC genotype (87.2%, 123/141). Overall, 140 patients completed the 12-week treatment, and 97.1% (136/140) patients achieved sustained virologic response at 12 weeks (per protocol population group, 95% confidence interval: 92.9-99.2%). Only drug-related serious adverse event occurred. Most of the adverse events were grade 1 and grade 2 alanine aminotransferase elevation or liver dysfunction. One patient discontinued treatment because of severe head injury in a car accident. Conclusions: The triple regimen of ritonavir-boosted danoprevir plus pegylated-interferon α-2a and ribavirin produced a sustained virologic response rate of 97.1% after 12 weeks treatment in noncirrhotic HCV GT1-infected Chinese patients, and was safe and well tolerated. Trial Registration Clinical-Trials.gov Identifier: NCT03020082.

3.
Biochem Biophys Res Commun ; 515(2): 366-371, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31155294

RESUMO

Flaviviruses are emerging arthropod-borne viruses posing a great threat to human beings worldwide. The E dimer configuration of the flavivirus was prominent during viral assembly, maturation and entry. Neutralization antibodies targeting E dimer played the important role in controlling the flavivirus infection. Previously, the ideal drug target of small molecular inhibitors of JEV was viral proteases and polymerases. The crystal structure of JEV E protein showed a conserved pocket in it is important at membrane fusion step. Recently, a set of anti-virus drugs has been found by virtual screening. Here, we show that the fusion-loop pocket of JEV E protein was a conservative region and an ideal drug target. ChemDiv-3 from virtual screening as the lead compound was found to show a relatively modest inhibition effect for JEV in vitro and in vivo test and could interfere with the formation of JEV sE dimer. ChemDiv-3 interacts with the amino acid residues ASN 313, PRO 314, ALA 315, and VAL 323 in E protein via hydrogen bonds for occupation of the fusion-loop pocket. The key binding sites LYS 312, ALA 513 and THR 317 forming the fusion-loop pocket are the same and other auxiliary sites are similar among the flavivirus. Taken together, the fusion-loop pocket of the flavivirus could be one promising target for drug discovery.

4.
Glycobiology ; 29(3): 242-259, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30535277

RESUMO

The hepatitis B virus (HBV)-induced chronic liver diseases are serious health threats worldwide. There is evidence to display the alterations of salivary N-linked glycans related to the development of HBV-infected liver diseases. Here, we further investigated the alterations of fucosylated N/O-glycans recognized by LTL in saliva from 120 subjects (30 healthy volunteers (HV), 30 patients with hepatitis B (HB), 30 patients with hepatic cirrhosis (HC), and 30 patients with hepatocellular carcinoma (HCC)) using salivary microarrys and MALDI-TOF/TOF-MS. The results showed that the expression level of fucosylated glycans recognized by LTL was significantly increased in HCC compared with other subjects (P < 0.0001). Besides, the fucosylated glycoproteins were isolated from pooled saliva of HV, HB, HC, and HCC by LTL-magnetic particle conjugates. Then, N/O- glycans were released from the isolated glycoproteins with PNGase F and NaClO, and were identified by MALDI-TOF-MS, respectively. Totally, there were 21/20, 25/18, 29/19, and 28/24 N/O-glycan peaks that were identified and annotated with proposed structures in saliva of HV, HB, HC, and HCC. Among the total, there were 8 N-glycan peaks (e.g., m/z 1905.634, 2158.777 and 2905.036) and 15 O-glycan peaks (e.g., 1177.407, 1308.444 and 1322.444) that only presented in patients with HBV-induced liver diseases. One N-glycan peak (m/z 2205.766) was unique in HC, and 9 O-glycan peaks (e.g., m/z 1157.420, 1163.417 and 1193.402) were unique in HCC. This study could facilitate the discovery of biomarkers for HC and HCC based on precise alterations of fucosylated N/O-glycans in saliva.


Assuntos
Biomarcadores Tumorais/genética , Vírus da Hepatite B/genética , Polissacarídeos/genética , Análise Serial de Proteínas , Biomarcadores Tumorais/química , Biomarcadores Tumorais/isolamento & purificação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Feminino , Fibrose/genética , Fibrose/virologia , Gangliosídeo G(M1)/análogos & derivados , Gangliosídeo G(M1)/química , Gangliosídeo G(M1)/genética , Vírus da Hepatite B/isolamento & purificação , Vírus da Hepatite B/patogenicidade , Hepatite Crônica/genética , Hepatite Crônica/virologia , Humanos , Lectinas/química , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Masculino , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Saliva/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
Cell Death Dis ; 9(9): 900, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-30185784

RESUMO

T cells play a crucial role in viral clearance and vaccine responses; however, the mechanisms that regulate their homeostasis during viral infections remain unclear. In this study, we investigated the machineries of T-cell homeostasis and telomeric DNA damage using a human model of hepatitis C virus (HCV) infection. We found that naïve CD4 T cells in chronically HCV-infected patients (HCV T cells) were significantly reduced due to apoptosis compared with age-matched healthy subjects (HSs). These HCV T cells were not only senescent, as demonstrated by overexpression of aging markers and particularly shortened telomeres; but also DNA damaged, as evidenced by increased dysfunctional telomere-induced foci (TIF). Mechanistically, the telomere shelterin protein, in particular telomeric repeat binding factor 2 (TRF2) that functions to protect telomeres from DNA damage, was significantly inhibited posttranscriptionally via the p53-dependent Siah-1a ubiquitination. Importantly, knockdown of TRF2 in healthy T cells resulted in increases in telomeric DNA damage and T-cell apoptosis, whereas overexpression of TRF2 in HCV T cells alleviated telomeric DNA damage and T-cell apoptosis. To the best of our knowledge, this is the first report revealing that inhibition of TRF2 promotes T-cell telomere attrition and telomeric DNA damage that accelerates T-cell senescent and apoptotic programs, which contribute to naïve T-cell loss during viral infection. Thus, restoring the impaired T-cell telomeric shelterin machinery may offer a new strategy to improve immunotherapy and vaccine response against human viral diseases.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Dano ao DNA/fisiologia , Hepatite C/metabolismo , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/antagonistas & inibidores , Apoptose/fisiologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Hepacivirus/patogenicidade , Hepatite C/virologia , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Proteínas Supressoras de Tumor/metabolismo
6.
Cell Discov ; 4: 16, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29644094

RESUMO

T cells have a crucial role in viral clearance and vaccine response; however, the mechanisms regulating their responses to viral infections or vaccinations remain elusive. In this study, we investigated T-cell homeostasis, apoptosis, DNA damage, and repair machineries in a large cohort of subjects with hepatitis C virus (HCV) infection. We found that naive CD4 T cells in chronically HCV-infected individuals (HCV T cells) were significantly reduced compared with age-matched healthy subjects. In addition, HCV T cells were prone to apoptosis and DNA damage, as evidenced by increased 8-oxoguanine expression and γH2AX/53BP1-formed DNA damage foci-hallmarks of DNA damage responses. Mechanistically, the activation of DNA repair enzyme ataxia telangiectasia mutated (ATM) was dampened in HCV T cells. ATM activation was also diminished in healthy T cells exposed to ATM inhibitor or to HCV (core protein) that inhibits the phosphoinositide 3 kinase pathway, mimicking the biological effects in HCV T cells. Importantly, ectopic expression of ATM was sufficient to repair the DNA damage, survival deficit, and cell dysfunctions in HCV T cells. Our results demonstrate that insufficient DNA repair enzyme ATM leads to increased DNA damage and renders HCV T cells prone to apoptotic death, which contribute to the loss of naive T cells in HCV infection. Our study reveals a novel mechanism for T-cell dysregulation and viral persistence, providing a new strategy to improve immunotherapy and vaccine responses against human viral diseases.

7.
Medicine (Baltimore) ; 97(15): e0208, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29642144

RESUMO

We used lectin microarray and mass spectrometric analysis to identify the N-linked glycosylation patterns of hepatitis C virus (HCV) particles. HCV J6/JFH-1 chimeric cell culture (HCVcc) in the culture supernatant was concentrated and purified by ultrafiltration and sucrose gradient ultracentrifugation. Twelve fractions were collected from the top and analyzed for viral infectivity and HCV RNA content after sucrose gradient separation. HCV RNA and proteins were separated by ultracentrifugation in a continuous 10% to 60% sucrose gradient to purify viral particles based on their sedimentation velocities. HCVcc particles were found mainly in fractions 6 to 8, as determined by quantitative polymerase chain reaction (qPCR) analysis for HCV RNA and ELISA of the HCV core protein. The N-glycans on HCV proteins were analyzed by lectin microarray and mass spectrometry. We identified that 32 of 37 lectins displayed the positive binding signals and 16 types of N-glycoforms of which the major HCV glycoforms were high mannose-type N-linked oligosaccharides, hybrid N-glycans, and fucosylated N-glycans. Our study provided new detailed information regarding the majority of the glycan-protein profile, complementing to previous findings of glycan-HCV protein interactions.


Assuntos
Hepacivirus/fisiologia , Hepatite C/virologia , Polissacarídeos/metabolismo , RNA Viral/análise , Proteínas Virais/metabolismo , Técnicas de Cultura de Células , Descoberta de Drogas , Glicoproteínas/metabolismo , Glicosilação , Hepatite C/tratamento farmacológico , Humanos
8.
Front Immunol ; 8: 1435, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29163508

RESUMO

Background: CD100, also known as Sema4D, is an immune semaphorin constitutively expressed on natural killer (NK) cells and T cells. As an immune activation molecule, CD100 has important immunoregulatory effects on NK functions by enhancing the interactions between NK cells and target cells. The aim of this study was to investigate whether hepatitis C virus (HCV) infection affects CD100 expression, and whether interferon-α treatment enhances NK killing activity to facilitate HCV clearance via CD100. Methods: Expression of CD100 on NK cells was evaluated by flow cytometry in patients with chronic HCV infection, with or without pegylated interferon-α-based therapy. NK cell cytotoxicity and interferon (IFN)-γ production were measured by flow cytometry upon culturing the NK cells with K562 and Huh7.5 or HCV JFH-1-infected Huh7.5 cells. Results: The frequency of CD100+ NK cells in HCV-infected individuals was slightly suppressed compared to healthy subjects. IFN-α treatment could significantly upregulate CD100 expression, which was confirmed by in vitro studies using peripheral blood mononuclear cells cocultured with HCV-expressing Huh7.5 cells or IFN-α. Importantly, the expression of CD100 on NK cells from HCV patients was inversely associated with the HCV-RNA levels in the early phase of IFN-α therapy, and the IFN-α upregulated CD100 led to an enhanced NK killing activity through ligations with its receptors plexin-B1/B2 on target cells. Conclusion: These results implied a novel mechanism by which IFN-α enhanced CD100/Plexin-B1/B2 interaction plays an important role in promoting NK functions in patients with chronic hepatitis C.

9.
Front Physiol ; 8: 596, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28871230

RESUMO

Background: Chronic infection with HBV (CHB) or HCV (CHC) is the most common chronic viral hepatitis that can lead to cirrhosis and hepatocellular carcinoma in humans, their infections have distinct pathogenic processes, however, little is known about the difference of glycoprotein glycopatterns in serum between hepatitis B virus (HBV)- and hepatitis C virus (HCV)-infected patients. Methods: A method combining the lectin microarrays, letin-mediated affinity capture glycoproteins, and MALDI-TOF/TOF-MS was employed to analyze serum protein glycopatterns and identify the glycan structures from patients with CHB (n = 54) or CHC(n = 47), and healthy volunteers (HV, n = 35). Lectin blotting was further utilized to validate and assess the expression levels of their serum glycopatterns. Finally, the differences of the glycoprotein glycopatterns were systematically compared between CHB and CHC patients. Conclusions: As a result, there were 11 lectins (e.g., HHL, GSL-II, and EEL) exhibited significantly increased expression levels, and three lectins (LCA, VVA, and ACA) exhibited significantly decreased expression levels of serum protein glycopatterns only in the CHB patients. However, DBA exhibited significantly decreased expression levels, and two lectins (WGA and SNA) exhibited significantly increased expression levels of serum glycopatterns only in the CHC patients. Furthermore, LEL and MAL-I showed a coincidentally increasing trend in both CHC and CHB patients compared with the HV. The individual analysis demonstrated that eight lectins (MPL, GSL-I, PTL-II, UEA-I, WGA, LEL, VVA, and MAL-I) exhibited a high degree of consistency with the pooled serum samples of HV, CHB, and CHC patients. Besides, a complex-type N-glycans binder PHA-E+L exhibited significantly decreased NFIs in the CHB compared with HV and CHC subjects (p < 0.01). The MALDI-TOF/TOF-MS results of N-linked glycans from the serum glycoproteins isolated by PHA-E+L-magnetic particle conjugates showed that there was an overlap of 23 N-glycan peaks (e.g., m/z 1419.743, 1663.734, and 1743.581) between CHB, and CHC patients, 5 glycan peaks (e.g., m/z 1850.878, 1866.661, and 2037.750) were presented in virus-infected hepatitis patients compared with HV, 3 glycan peaks (1460.659, 2069.740, and 2174.772) were observed only in CHC patients. Our data provide useful information to find new biomarkers for distinguishing CHB and CHC patients based on the precision alteration of their serum glycopatterns.

10.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 33(8): 1056-1061, 2017 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-28871946

RESUMO

Objective To investigate the effect of iron overload on biological activity and apoptosis in Huh7.5 cells. Methods Huh7.5 cells were cultured in the medium supplemented with 50, 100, 200 µmol/L ferric ammonium citrate (FAC). Fluorescence microscopy was employed to determine cell iron load labeled by Phen Green FL; proliferation activity of Huh7.5 cells was evaluated by MTT assay; protein and mRNA levels of transferrin receptor (TfR1), TfR2, divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) in Huh7.5 cells were detected by Western blotting and real-time PCR, respectively; cell reactive oxygen species (ROS) labeled by dichlorofluorescin diacetate (DCFH-DA) and cell apoptosis labeled by annexinV-FITC/PI were analyzed by flow cytometry. Results FAC treatment increased intracellular iron load in a dose-dependent manner. Compared with control group, mRNA and protein expressions of TfR1, TfR2 and DMT1 were down-regulated, while mRNA and protein expression of FPN1 was significantly up-regulated in FAC treated groups. With the increasing dose of FAC, intracellular ROS level increased significantly and cell proliferation activity decreased significantly. The cell apoptosis rate in FAC treated groups were remarkably higher than that in control group, but after antioxidant N-acetylcysteine (NAC) was added, the cell apoptosis in FAC treated group was inhibited obviously. Conclusion Iron overload can inhibit the proliferation and promote the apoptosis of Huh7.5 cells through oxidative stress.


Assuntos
Apoptose , Hepatócitos/patologia , Sobrecarga de Ferro/patologia , Acetilcisteína/farmacologia , Proteínas de Transporte de Cátions/análise , Linhagem Celular Tumoral , Proliferação de Células , Hepatite C Crônica/terapia , Hepatócitos/metabolismo , Humanos , Sobrecarga de Ferro/metabolismo , Estresse Oxidativo , Receptores da Transferrina/análise
11.
Sci Rep ; 7: 45957, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28383031

RESUMO

Acute-on-chronic hepatitis B liver failure (ACHBLF) is an increasingly recognized distinct disease entity encompassing an acute deterioration of liver function in patients with cirrhosis, so little is known about the alterations of protein glycopatterns in serum with its development. We aimed to identify the alterations of serum glycopatterns in ACHBLF and probe the possibility of them as novel potential biomarkers for diagnosis of ACHBLF. As a result, there were 18 lectins (e.g., WFA, GSL-II, and PNA) to give significantly alterations of serum glycopatterns in ACHBLF compared with healthy controls (HC) (all p ≤ 0.0386). Meanwhile, among these lectins, there were 12 lectins (e.g., WFA, GAL-II, and EEL) also exhibited significantly alterations of serum glycopatterns in ACHBLF compared with HBV-infected chronic hepatitis (cHB) (all p ≤ 0.0252). The receiver-operating characteristic (ROC) curve analysis indicated there were 5 lectins (PHA-E + L, BS-I, ECA, ACA, and BPL) had the greatest discriminatory power for distinguishing ACHBLF and HC or cHB, respectively (all p ≤ 0.00136). We provided a new basic insight into serum glycopatterns in ACHBLF and investigated the correlation of alterations in serum glycopatterns as novel potential biomarkers for diagnosis of ACHBLF.


Assuntos
Insuficiência Hepática Crônica Agudizada/sangue , Insuficiência Hepática Crônica Agudizada/diagnóstico , Biomarcadores Tumorais/sangue , Glicoproteínas/sangue , Hepatite B Crônica/sangue , Hepatite B Crônica/diagnóstico , Adulto , Estudos de Casos e Controles , Feminino , Glicosídeos/metabolismo , Humanos , Lectinas/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Análise em Microsséries , Análise de Componente Principal , Curva ROC , Reprodutibilidade dos Testes
12.
Front Microbiol ; 8: 303, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28293227

RESUMO

Japanese encephalitis virus (JEV) is the most prevalent cause of viral encephalitis in Asia and the western Pacific. Neuronal death caused by JEV infection and inflammation induced cytotoxicity leads to progression and deterioration of Japanese encephalitis (JE). Mixed-lineage kinase domain-like protein (MLKL) mediated necroptosis is a newly discovered pathway of programmed cell death and participates in many inflammatory diseases. In this study, we demonstrated for the first time that necroptosis was involved in the neuronal loss during JE via immune-electron microscopy and immunochemistry. The expression of MLKL in neurons was upregulated in presence of JEV infection in vitro and in vivo. Deletion of MLKL alleviated the progression of JE and decreased the level of inflammatory cytokines in mice model. Taken together, this study provides evidence for the participation of necroptosis in the pathogenesis of JEV infection.

13.
Stem Cell Res Ther ; 8(1): 38, 2017 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-28209182

RESUMO

BACKGROUND: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. Japanese encephalitis (JE) caused by JEV is characterized by extensive inflammatory cytokine secretion, microglia activation, blood-brain barrier (BBB) breakdown, and neuronal death, all of which contribute to the vicious cycle of inflammatory damage. There are currently no effective treatments for JE. Mesenchymal stem cells (MSCs) have been demonstrated to have a therapeutic effect on many central nervous system (CNS) diseases by regulating inflammation and other mechanisms. METHODS: In vivo, 8- to 10-week-old mice were infected intraperitoneally with JEV and syngeneic bone marrow MSCs were administered through the caudal vein at 1 and 3 days post-infection. The mortality, body weight, and behavior were monitored daily. Brains from each group were harvested at the indicated times for hematoxylin and eosin staining, immunohistochemical observation, flow cytometric analysis, TUNEL staining, Western blot, quantitative real-time polymerase chain reaction, and BBB permeability assays. In vitro, co-culture and mixed culture experiments of MSCs with either microglia or neurons were performed, and then the activation state of microglia and survival rate of neurons were tested 48 h post-infection. RESULTS: MSC treatment reduced JEV-induced mortality and improved the recovery from JE in our mouse model. The inflammatory response, microglia activation, neuronal damage, BBB destruction, and viral load (VL) were significantly decreased in the MSC-treated group. In co-culture experiments, MSCs reprogrammed M1-to-M2 switching in microglia and improved neuron survival. Additionally, the VL was decreased in Neuro2a cells in the presence of MSCs accompanied by increased expression of interferon-α/ß. CONCLUSION: MSC treatment alleviated JEV-induced inflammation and mortality in mice.


Assuntos
Encéfalo/patologia , Encefalite Japonesa/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Microglia/patologia , Neurônios/patologia , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/virologia , Encéfalo/metabolismo , Encéfalo/virologia , Permeabilidade Capilar , Sobrevivência Celular , Técnicas de Cocultura , Modelos Animais de Doenças , Vírus da Encefalite Japonesa (Espécie)/crescimento & desenvolvimento , Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Encefalite Japonesa/mortalidade , Encefalite Japonesa/patologia , Encefalite Japonesa/virologia , Feminino , Humanos , Interferon-alfa/biossíntese , Interferon beta/biossíntese , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Microglia/metabolismo , Microglia/virologia , Neurônios/metabolismo , Neurônios/virologia , Cultura Primária de Células , Análise de Sobrevida
14.
Immunology ; 150(3): 301-311, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27809352

RESUMO

Hepatitis C virus (HCV) induces a high rate of chronic infection via dysregulation of host immunity. We have previously shown that T-cell immunoglobulin and mucin domain protein-3 (Tim-3) is up-regulated on monocyte/macrophages (M/Mφ) during chronic HCV infection; little is known, however, about the transcription factor that controls its expression in these cells. In this study, we investigated the role of transcription factor, T-box expressed in T cells (T-bet), in Tim-3 expression in M/Mφ in the setting of HCV infection. We demonstrate that T-bet is constitutively expressed in resting CD14+ M/Mφ in the peripheral blood. M/Mφ from chronically HCV-infected individuals exhibit a significant increase in T-bet expression that positively correlates with an increased level of Tim-3 expression. Up-regulation of T-bet is also observed in CD14+ M/Mφ incubated with HCV+ Huh7.5 cells, as well as in primary M/Mφ or monocytic THP-1 cells exposed to HCV core protein in vitro, which is reversible by blocking HCV core/gC1qR interactions. Moreover, the HCV core-induced up-regulation of T-bet and Tim-3 expression in M/Mφ can be abrogated by incubating the cells with SP600125 - an inhibitor for the c-Jun N-terminal kinase (JNK) signalling pathway. Importantly, silencing T-bet gene expression decreases Tim-3 expression and enhances interleukin-12 secretion as well as signal transducer and activator of transcription 1 phosphorylation. These data suggest that T-bet, induced by the HCV core/gC1qR interaction, enhances Tim-3 expression via the JNK pathway, leading to dampened M/Mφ function during HCV infection. These findings reveal a novel mechanism for Tim-3 regulation via T-bet during HCV infection, providing new targets to combat this global epidemic viral disease.


Assuntos
Hepacivirus/fisiologia , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Hepatite C Crônica/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Proteínas com Domínio T/metabolismo , Adulto , Linhagem Celular , Feminino , Receptor Celular 2 do Vírus da Hepatite A/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/virologia , Masculino , Pessoa de Meia-Idade , Monócitos/virologia , RNA Interferente Pequeno/genética , Transdução de Sinais/imunologia , Proteínas com Domínio T/genética , Regulação para Cima , Proteínas do Core Viral/imunologia , Adulto Jovem
15.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 32(11): 1522-1526, 2016 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-27774948

RESUMO

Objective To observe the alterations of innate immunity related long non-coding RNAs (lncRNAs) in exosomes extracted from the plasma of hemorrhagic fever with renal syndrome (HFRS) patients, and analyze their relationship with the disease stage and severity. Methods Exosomes were extracted from the plasma samples of HFRS patients, healthy controls and recovered HFRS patients. Transmission electronic microscopy and Western blotting were performed to confirm the efficiency of the extraction. lncRNA profiles in the different groups were determined by high-throughput sequencing. The contents of several innate immunity related lncRNAs were detected by quantitative real-time PCR, and their relationship with the disease stage and severity was analyzed. Results Exosomes from the plasma were accurately extracted. Innate immunity related lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1), negative regulator of interferon response (NRIR), negative regulator of antiviral response (NRAV) were found in exosomes. NEAT1 content was significantly reduced in the exosomes from HFRS patients compared with healthy controls and it was significantly restored in recovered HFRS patients. The exosome NEAT1 content was correlated with the epidemic of HFRS but had no relationship with the stage and severity of the disease. Conclusion Several innate immunity related lncRNAs exist in the exosome from HFRS patients, among which NEAT1 content significantly decreases in HFRS patients compared with healthy controls and recovered HFRS patients. The reduced NEAT1 level is correlated with the epidemic of HFRS.


Assuntos
Exossomos/metabolismo , Febre Hemorrágica com Síndrome Renal/genética , Febre Hemorrágica com Síndrome Renal/metabolismo , Imunidade Inata/fisiologia , RNA Longo não Codificante/genética , Adulto , Exossomos/ultraestrutura , Feminino , Humanos , Imunidade Inata/genética , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , RNA Longo não Codificante/metabolismo
16.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 32(6): 746-9, 2016 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-27371839

RESUMO

Objective To express core region of HCV1b (Hebei strain) E2 protein (E2c) by eukaryotic system, and establish the detection method of specific anti-HCV E2 antibody in the sera from hepatitis C patients. Methods Based on the literature, the E2c gene was modified from the HCV1b gene and synthesized via overlapping PCR. Thereafter, the E2c gene including tissue-type plasminogen activator (tPA) signal peptide was cloned into the pCI-neo eukaryotic expression vector, and the product was named pCI-tpa-1bE2c. After HEK293T cells were transfected with pCI-tpa-1bE2c, the supernatant was collected, condensed and purified. Its specificity was identified by Western blotting. Galanthus nivalis agglutinin (GNA)-based ELISA was used to detect the antibody against HCVE2 in the sera from hepatitis C patients. Results Modified HCV E2c protein was successfully expressed in HEK293T cells and the GNA-based ELISA was developed for detecting the antibody against HCV E2 in the sera from hepatitis C patients. Conclusion HCV-1bE2c protein can be effectively expressed in HEK293T cells and applied clinically.


Assuntos
Células Eucarióticas/metabolismo , Hepacivirus/metabolismo , Hepatite C/imunologia , Proteínas do Envelope Viral/metabolismo , Western Blotting , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Células HEK293 , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C/sangue , Hepatite C/virologia , Anticorpos Anti-Hepatite C/sangue , Anticorpos Anti-Hepatite C/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
17.
PLoS One ; 11(6): e0155934, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27276081

RESUMO

BACKGROUND: Little is known on the cost-effectiveness of novel regimens for hepatitis C virus (HCV) compared with standard-of-care with pegylated interferon (pegIFN) and ribavirin (RBV) therapy in developing countries. We evaluated cost-effectiveness of sofosbuvir/ledipasvir for 12 weeks compared with a 48-week pegIFN-RBV regimen in Chinese patients with genotype 1b HCV infection by economic regions. METHODS: A decision analytic Markov model was developed to estimate quality-adjusted-life-years, lifetime cost of HCV infection and incremental cost-effectiveness ratios (ICERs). SVR rates and direct medical costs were obtained from real-world data. Parameter uncertainty was assessed by one-way and probabilistic sensitivity analyses. Threshold analysis was conducted to estimate the price which can make the regimen cost-effective and affordable. RESULTS: Sofosbuvir/ledipasvir was cost-effective in treatment-experienced patients with an ICER of US$21,612. It varied by economic regions. The probability of cost-effectiveness was 18% and 47% for treatment-naive and experienced patients, and it ranged from 15% in treatment-naïve patients in Central-China to 64% in treatment-experienced patients in Eastern-China. The price of 12-week sofosbuvir/ledipasvir treatment needs to be reduced by at least 81% to US$18,185 to make the regimen cost-effective in all patients at WTP of one time GDP per capita. The price has to be US$105 to make the regimen affordable in average patients in China. CONCLUSION: Sofosbuvir/ledipasvir regimen is not cost-effective in most Chinese patients with genotype 1b HCV infection. The results vary by economic regions. Drug price of sofosbuvir/ledipasvir needs to be substantially reduced when entering the market in China to ensure the widest accessibility.


Assuntos
Benzimidazóis/economia , Fluorenos/economia , Hepacivirus , Hepatite C/economia , Modelos Econômicos , Sofosbuvir/economia , Grupo com Ancestrais do Continente Asiático , Benzimidazóis/administração & dosagem , China/epidemiologia , Custos e Análise de Custo , Feminino , Fluorenos/administração & dosagem , Hepatite C/tratamento farmacológico , Hepatite C/epidemiologia , Humanos , Masculino , Cadeias de Markov , Sofosbuvir/administração & dosagem
18.
J Ginseng Res ; 40(2): 196-202, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27158241

RESUMO

BACKGROUND: Ginsenoside Rd (GSRd), a main component of the root of Panax ginseng, exhibits anti-inflammation functions and decreases infarct size in many injuries and ischemia diseases such as focal cerebral ischemia. M1 Macrophages are regarded as one of the key inflammatory cells having functions for disease progression. METHODS: To investigate the effect of GSRd on renal ischemia/reperfusion injury (IRI) and macrophage functional status, and their regulatory role on mouse polarized macrophages in vitro, GSRd (10-100 mg/kg) and vehicle were applied to mice 30 min before renal IRI modeling. Renal functions were reflected by blood serum creatinine and blood urea nitrogen level and histopathological examination. M1 polarized macrophages infiltration was identified by flow cytometry analysis and immunofluorescence staining with CD11b(+), iNOS(+)/interleukin-12/tumor necrosis factor-α labeling. For the in vitro study, GSRd (10-100 µg/mL) and vehicle were added in the culture medium of M1 macrophages to assess their regulatory function on polarization phenotype. RESULTS: In vivo data showed a protective role of GSRd at 50 mg/kg on Day 3. Serum level of serum creatinine and blood urea nitrogen significantly dropped compared with other groups. Reduced renal tissue damage and M1 macrophage infiltration showed on hematoxylin-eosin staining and flow cytometry and immunofluorescence staining confirmed this improvement. With GSRd administration, in vitro cultured M1 macrophages secreted less inflammatory cytokines such as interleukin-12 and tumor necrosis factor-α. Furthermore, macrophage polarization-related pancake-like morphology gradually changed along with increasing concentration of GSRd in the medium. CONCLUSION: These findings demonstrate that GSRd possess a protective function against renal ischemia/reperfusion injury via downregulating M1 macrophage polarization.

19.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 32(5): 666-70, 2016 May.
Artigo em Chinês | MEDLINE | ID: mdl-27126947

RESUMO

OBJECTIVE: To investigate the distribution of peripheral blood monocyte subsets of chronic hepatitis C (CHC) patients and observe the expression of negative regulators T cell immunoglobulin and mucin domain-3 (Tim-3) and programmed cell death-1 (PD-1) on the monocyte subsets. METHODS: Flow cytometry was employed to determine the distribution of three monocyte subsets as well as Tim-3 and PD-1 expression on the three monocyte subsets. Their correlations with the clinical parameters were analyzed by Spearman test. RESULTS: Compared with healthy controls, an increased distribution of CD14(+)CD16(+) monocytes, especially CD14(++)CD16(+) monocyte subset, was observed in CHC patients. Tim-3 expression was significantly elevated on CD14(++)CD16(-) and CD14(+)CD16(++) subsets in CHC patients. Obviously increased PD-1 expression was found mainly on CD14(++)CD16(-) and CD14(++)CD16(+) subsets. There were no significant correlations between monocyte subsets, PD-1, Tim-3 and the clinical parameters. CONCLUSION: The levels Tim-3 and PD-1 are different in three monocyte subsets.


Assuntos
Hepatite C Crônica/sangue , Proteínas de Membrana/sangue , Monócitos/química , Receptor de Morte Celular Programada 1/sangue , Adulto , Idoso , Feminino , Proteínas Ligadas por GPI/sangue , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Receptores de Lipopolissacarídeos/sangue , Masculino , Pessoa de Meia-Idade , Receptores de IgG/sangue
20.
Hepat Mon ; 16(1): e31278, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27110255

RESUMO

BACKGROUND: There are limited options for chronic hepatitis B (CHB) patients who have poor responses to adefovir (ADV). OBJECTIVES: The aim of this study is to evaluate the effects of adding on telbivudine (LdT) or switching to pegylated interferon alfa-2a (PEG-IFN-α2a) as alternative rescue therapies for patients with poor responses to the initial ADV treatments. PATIENTS AND METHODS: Ninety-seven CHB patients with HBV DNA > 2 log10 copies/mL 48 weeks after ADV monotherapy were included in this study. Fifty-nine of these patients were treated with a combination of LdT plus ADV (LdT + ADV) daily, while thirty-eight patients were switched to PEG-IFN-α2a subcutaneous injections weekly for 48 weeks. RESULTS: Both rescue strategies were proven to be safe and the majority of patients tolerated the therapies well. LdT + ADV led to more rapid reductions in viral loads than PEG-IFN-α2a monotherapy, with 2.14 (LdT + ADV) and 0.98 (PEG-IFN-α2a) log10 copies/mL decreases 48 weeks after rescue treatments, respectively (P < 0.00001). The rates corresponding to virological and biochemical responses were also elevated in patients who received the LdT + ADV combination therapy at the end of the observation period (88.1 vs. 68.4% for virological response, P = 0.017; 83.3 vs. 47.2%, P = 0.00045). However, the decline in the hepatitis B surface antigen (HBsAg) was more pronounced in PEG-IFN-α2a treated patients. Moreover, the cumulative rates of serological responses were higher in patients who switched to the PEG-IFN-α2a therapy. CONCLUSIONS: Both add-on LdT and switching to PEG-IFN-α2a were satisfactory and optimal treatments for CHB patients with poor responses to ADV. Both rescue strategies resulted in significant reductions in serum viral load and ALT levels, and were associated with high rate of serological outcomes in our hospital.

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