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<p><b>OBJECTIVE</b>To study the immunoregulatory effect of hepatitis B virus (HBV) e antigen (HBeAg) on peripheral blood monocytes (PBMCs).</p><p><b>METHODS</b>PBMCs were isolated from patients with chronic hepatitis B (CHB; both HBeAg- and HBeAg+) and healthy controls, and cultured with recombinant HBeAg. The HBeAg-induced changes in expression of PD-1/PD-L1 were measured by flow cytometry of the cells and in secreted cytokines were measured by enzyme-linked immunosorbent assay of the supernatants. Comparisons between two groups were made by the independent-samples t-test; the relationship between PD-1/B7-H1 level and HBV DNA copy number was evaluated by Spearman's correlation analysis.</p><p><b>RESULTS</b>Exposure to HBeAg led to a significant decrease in CD3+CD4+ T lymphocyte-specific expression of IFNa for both the CHB patients' and healthy controls' samples (t = 2.382 and -4.190 respectively, P less than 0.01). For the HBeAg- CHB patients' and healthy controls' samples, the HBeAg exposure led to increased levels of secreted cytokines IL-6, IL-10 and TNFa (t = 2.504, 3.583 and 4.324, P less than 0.01 and t = 3.542, 6.246 and 5.273, P less than 0.01 respectively) and of CD14+ PBMC-specific expression of PD-L1 (t = 4.815 and 3.454, P less than 0.05 respectively). Compared to the HBeAg-negative CHB patients' and healthy controls' samples, the HBeAg+ CHB patients' samples had significantly lower CD3+CD4+ T cell-specific expression of IFNa (t = -3.177 and -4.541, P less than 0.01 respectively), but significantly higher levels of secreted IL-4 (t = 3.382 and 4.393, P less than 0.01 respectively), of CD3+ T cells-specific expression of PD-1/PD-L1 (t = 4.755, 2.942 and 4.518, 4.595, P less than 0.01 respectively), and of CD14+ T cells-specific expression of PD-L1 (t = 5.092 and 5.473, P less than 0.01 respectively). The CD3+ T cells-specific expression of PD-L1 was significantly higher in the samples from HBeAg- CHB patients than from the healthy controls (t = 3.214, P less than 0.01).</p><p><b>CONCLUSION</b>HBeAg was able to down-regulate the production of Th1-type cytokines (IFNgamma), and up-regulate the secretion of Th2-type cytokines (IL-6, IL-10) and the expression of PD-1/PD-L1on monocytes. These changes are conducive to the formation of immune tolerance to HBV. Therefore, HBeAg may play an important role in immune tolerance to chronic HBV infection.</p>
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Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos de Casos e Controles , Células Cultivadas , Antígenos E da Hepatite B , Genética , Alergia e Imunologia , Hepatite B Crônica , Sangue , Alergia e Imunologia , Interferon gama , Alergia e Imunologia , Interleucina-10 , Alergia e Imunologia , Interleucina-6 , Alergia e Imunologia , Leucócitos Mononucleares , Alergia e Imunologia , Metabolismo , Proteínas Recombinantes , Alergia e Imunologia , Células Th1 , Alergia e Imunologia , Equilíbrio Th1-Th2 , Células Th2 , Alergia e ImunologiaRESUMO
BACKGROUND: Hepatitis B virus (HBV) is still one of the serious infectious risks for the blood transfusion safety in China. One plausible reason is the emergence of the variants in the major antigenic alpha determinant within the major hydrophilic region (MHR) of hepatitis B surface antigen (HBsAg), which have been assumed to evade the immune surveillance and pose a challenge to the disease diagnosis. It is well documented that some commercial ELISA kits could detect the wild-type but not the mutant viruses. The high prevalence of HBV in China also impaired the application of nucleic acid testing (NAT) in the improvement of blood security. Molecular epidemiological study of HBsAg variations in China is still limited. This study was designed to identify the prevalence of mutations in the HBsAg in voluntary blood donors in Nanjing, China. METHODS: A total of 20,326 blood units were enrolled in this study, 39 donors were positive for HBV S gene in the nested-PCR. Mutations in the major hydrophilic region (MHR; aa 99-169) were identified by direct sequencing of S region. RESULTS: Among of 20,326 blood units in the Red Cross Transfusion Center of Nanjing from October 2008 to April 2009, 296 samples (1.46%, 296/20,326) were HBsAg positive in the 2 successive rounds of the ELISA test. In these HBsAg positive units, HBV S gene could be successfully amplified from 39 donors (13.18%, 39/296) in the nested-PCR. Sequence analysis revealed that 32 strains (82.1%, 32/39) belong to genotype B, 7 strains (17.9%, 7/39) to genotype C. Besides well known G145R, widely dispersed variations in the MHR of S region, were observed in 20 samples of all the strains sequenced. CONCLUSIONS: HBV/B and HBV/C are dominant in Nanjing, China. The mutations in the MHR of HBsAg associated with disease diagnosis are common.
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Variação Genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B/epidemiologia , Adulto , Doadores de Sangue , China/epidemiologia , DNA Viral/química , DNA Viral/genética , Feminino , Genótipo , Hepatite B/diagnóstico , Hepatite B/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Mutação de Sentido Incorreto , Prevalência , Análise de Sequência de DNARESUMO
This report aims to investigate the dynamical changes of HBcAg18-27 epitope specific cytotoxic T lymphocytes(CTL), alanine aminotransferase (ALT), HBV DNA and HBsAg in peripheral blood of acute hepatitis B patients, and to explore the roles of HBcAg18-27-specific CTLs in virus clearance and liver injury. Acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients were divided into two groups according to results of HLA-A0201. Patients with positive HLA-A0201 were classified into HBcAg-specific CTL group and those with negative HLA-A0201 were referred as control group. The specific CTLs were stained with HLA-A0201 limited HBcAg18-27 epitope MHC-Pentamer and the frequencies of CTLs, T, B, NK and NKT cells were detected by flow cytometry (FCM). The serum ALT, HBV DNA and HBsAg were examined using speed analysis, quantitative PCR and abbott chemiluminescent technology. The frequencies of HBcAg18-27-specific CTLs in AHB patients were higher in the early three weeks as compared to the late three weeks. The apex time of HBV-specific CTL frequencies lagged behind those of HBV DNA, HBsAg and ALT. The loss of HBsAg in patients with high frequencies of HBV-specific CTL was earlier than that in patients with low frequencies (t = 2.018, P value is less than 0.05). In the second week the peak frequencies of CD3+CD8+ cells overlapped with that of HBcAg18-27-specific CTLs and with a positive correlation between (r = 0.420, P value is less than 0.05). During the early stages of AHB, the frequencies of NK and NKT cells were found significantly lower than that of control group and CHB group and the levels were back to normal after recovery. Moreover, a negative correlation existed between the frequencies of NK cells and the dynamic changes of HBcAg18-27-specific CTLs (r = -0.435, P value is less than 0.01) in AHB group. The frequencies of HBcAg18-27-specific CTLs were significantly higher as compared to CHB group in the first three weeks (z = -3.258, -4.04, and -3.259, P value is less than 0.01). The early loss of HBsAg was closely related to the high frequencies of HBcAg18-27 specific CTLs in AHB patients. HBcAg-specific CTL frequencies in peripheral blood could be used to predict clinical outcome after HBV infection. The frequencies of CD8+ T cells can reflect the changes of frequencies of HBcAg-specific CTL during acute HBV infection.
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Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Estudos de Casos e Controles , Antígeno HLA-A2 , Alergia e Imunologia , Hepatite B , Alergia e Imunologia , Antígenos do Núcleo do Vírus da Hepatite B , Sangue , Alergia e Imunologia , Células Matadoras Naturais , Alergia e Imunologia , Contagem de Linfócitos , Linfócitos T Citotóxicos , Biologia Celular , Alergia e ImunologiaRESUMO
<p><b>OBJECTIVE</b>The aim of this study was to investigate the changes in mTOR activity and survivin expression in liver cancer cell line HepG2 cells treated with tamoxifen.</p><p><b>METHODS</b>Survivin transcription level and p70S6K was demonstrated by PCR, dual-luciferase reporter assay and Western blot analysis, respectively, and the apoptosis in the HepG2 cells was detected by flow cytometry.</p><p><b>RESULTS</b>Tamoxifen leads to apoptosis of the cells and reduction in survivin expression, as well as a dramatic reduction in the activated form of p70S6K. Treating HepG2 cells with rapamycin, a specific mTOR inhibitor, significantly reduced the survivin protein level but not affected the survivin transcription, indicating that tamoxifen and rapamycin were synergistic in regards to down-regulation of survivin expression in hepatocellular carcinoma cells.</p><p><b>CONCLUSIONS</b>Our results suggest that tamoxifen down-regulates survivin expression in HepG2 cells and it is mediated by transcriptional and post-transcriptional level via PI3K/Akt/mTOR pathway to induce apoptosis.</p>
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Humanos , Antibióticos Antineoplásicos , Farmacologia , Antineoplásicos Hormonais , Farmacologia , Apoptose , Proliferação de Células , Regulação para Baixo , Sinergismo Farmacológico , Células Hep G2 , Proteínas Inibidoras de Apoptose , Genética , Metabolismo , Fosfatidilinositol 3-Quinases , Metabolismo , Proteínas Proto-Oncogênicas c-akt , Metabolismo , RNA Mensageiro , Metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa , Metabolismo , Transdução de Sinais , Sirolimo , Farmacologia , Serina-Treonina Quinases TOR , Metabolismo , Tamoxifeno , FarmacologiaRESUMO
<p><b>OBJECTIVES</b>In order to investigate the relationship among the toll-like receptor 2 (TLR2), hepatitis B e antigen and HBV DNA, the expression levels of TLR2 on peripheral blood monocytes of chronic hepatitis B (CHB) patients as well as on their monocytes stimulated by ligand of TLR2 (Pam3CSK4) and HBeAg were analyzed.</p><p><b>METHODS</b>Sixty-eight adults with CHB were enrolled, including 37 HBeAg-positive patients, 17 HBeAg-negative and HBV DNA negative patients, and 14 HBeAg-negative and HBV DNA positive patients. Sixteen healthy volunteers were also studied as controls. TLR2 expression levels on their peripheral blood monocytes stimulated with Pam3CSK4 or not stimulated were analyzed by FACS Caliber. The relationship of the expression levels of TLR2, HBeAg and HBV DNA were also analyzed. The level of TLR2 on peripheral blood monocytes of healthy volunteers and HBeAg-negative CHB patients stimulated by HBeAg was examined for six hours.</p><p><b>RESULTS</b>The TLR2 expression levels on CD14+ cells were significantly reduced in HBeAg-positive patients (47.57%+/-21.40 %) compared to both healthy volunteers (76.51%+/-7.46%) and HBeAg-negative patients (HBV DNA positive group 73.2%+/-14.2%, HBV DNA negative group 75.2%+/-11.3%); but there was no difference between those of the HBeAg-negative patients and the healthy volunteers. Expression levels of TLR2 on monocytes stimulated by TLR2 ligand in HBeAg-positive patients were obviously increased, and reached the basic levels of the healthy volunteers and the HBeAg-negative patients. The expression levels of TLR2 on monocytes stimulated by HBeAg of the healthy volunteers and the HBeAg-negative patients were markedly reduced.</p><p><b>CONCLUSIONS</b>In the presence of HBeAg, HBV down-regulates the expressions of TLR2 on CD14+ cells from peripheral blood, and there is no correlation between HBV-DNA and TLR2. Pam3CSK4 can boost the TLR2 expression in HBeAg-positive patients. The proposed interaction between HBV and TLR2 may provide an important clue to explain the reasons of the establishment of persistent HBV infection.</p>
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Humanos , Estudos de Casos e Controles , DNA Viral , Sangue , Antígenos E da Hepatite B , Sangue , Hepatite B Crônica , Sangue , Alergia e Imunologia , Metabolismo , Receptores de Lipopolissacarídeos , Metabolismo , Monócitos , Metabolismo , Receptor 2 Toll-Like , MetabolismoRESUMO
<p><b>OBJECTIVE</b>To determine the expression level changes of survivin, a inhibitor of apoptosis protein, followed by activation of insulin receptors in human hepatocellular carcinoma HepG2 cell line, and to investigate the signalling pathway involved in the regulation.</p><p><b>METHODS</b>Human hepatocellular carcinoma HepG2 cells were treated with insulin alone or pre-treated with LY294002, a specific inhibitor of PI3K signalling pathway, to determine whether blocking PI3K signaling can attenuate the up-regulation of survivin expression. Real time RT-PCR and Western blot analysis were used to measure survivin mRNA and protein changes before and after treatment, respectively.</p><p><b>RESULTS</b>Without serum supplement, HepG2 cells expressed a small amount of survivin. Insulin induced survivin expression in a dose- and time-dependent fashion. Survivin expression was blocked if cells were pre-treated with LY294002 prior to insulin stimulation.</p><p><b>CONCLUSION</b>Insulin induces survivin expression via PI3K signalling pathway, suggesting that to interfere the key gene in this signalling pathway may block survivin expression, therefore, promoting apoptosis in hepatocellular carcinoma cells.</p>
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Humanos , Apoptose , Cromonas , Farmacologia , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Proteínas Inibidoras de Apoptose , Insulina , Farmacologia , Proteínas Associadas aos Microtúbulos , Genética , Metabolismo , Morfolinas , Farmacologia , Fosfatidilinositol 3-Quinases , RNA Mensageiro , Metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
Objective To evaluate the expression of Toll-like receptor(TLR)on the monocyte- derived dendritic cells(DC)from chronic hepatitis B(CHB)patients and to analyze the expression pro- file and significance of the TLR such as TLR3,TLR4,TLR?,TLR8 and TLRg,which are associat- ed with immune response to viral infection.Methods Peripheral blood mononuclear cell(PBMC) centrifugated by the hydroxyethyl starch(HES)centrifugation were cultured and induced into DC by granulocyte-maerophage colony stimulating factor(GM-CSF)and interleukin-4(IL-4),and their mor- phology and phenotype were detected by the inverted microscope and flow cytometry respectively. Monocyte-derived DC were obtained from 10 chronically hepatitis B virus(HBV)-infected patients and 15 healthy volunteers.TLR3,TLR4,TLR7,TLRS,TLR9 expression on immature and mature DC were analyzed by FACS Calibur.DC was pulsed with HBcAg on day 3 and 5,then DC maturation and ability to process HBcAg and to stimulate autogeneic T cells were evaluated.Results Monocyte- derived DC developed different TLR expression patterns as they went through different maturation stages.TLR7,TLR8 expressions on immature DC and TLR3,TLR7 expressions on mature DC were lower in CHB than in control(for TLR7,TLR8 expression on immature DC:75.9%,1.0%vs 98.4%,15.4%,P
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<p><b>OBJECTIVES</b>To observe immunogenicity of new DNA vaccine encoding for hepatitis B virus core antigen (HBcAg).</p><p><b>METHODS</b>A new DNA vaccine (pSW3891/HBc) encoding for hepatitis B virus core antigen was constructed using plasmid pSW3891 which can be used in human. Control and experiment groups of Balb/c mice were immunized with pSW3891 or pSW3891/HBc by gene gun. Anti-HBc in sera of mice was tested by ELISA (enzyme linked immune sorbent assay). Specific cytotoxicity of cytotoxic T lymphocyte (CTL) of mice was detected by LDH release assay.</p><p><b>RESULTS</b>pSW3891/HBc can express in 293T cell line effectively. Mice immunized with pSW3891/HBc showed strong anti-HBc response and specific high cytotoxicity of CTL.</p><p><b>CONCLUSION</b>pSW3891/HBc induced significantly humoral and cellular immune responses in Balb/c mice.</p>
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Animais , Camundongos , Anticorpos Anti-Hepatite B , Sangue , Antígenos do Núcleo do Vírus da Hepatite B , Genética , Alergia e Imunologia , Vacinas contra Hepatite B , Alergia e Imunologia , Imunidade Celular , Camundongos Endogâmicos BALB C , Linfócitos T Citotóxicos , Alergia e Imunologia , Vacinas de DNA , Alergia e ImunologiaRESUMO
Objective To establish a method for detecting HBV cccDNA in hepatocytes of chronic hepatitis B patients.Method 21 liver biopsies from the hepatic operation patients in the hospital of jiangsu province,concluding 19 HBV chronic infected patients (10 HBeAg positive patients and 9 HBeAg negative patients) and 4 uninfected patients,HBV DNA(+) serum of hepatitis B patients was thought as rcDNA.To use proteinase K to release HBV cccDNA and genomic DNA,then divide the cell lysis solution into two parts,one for detecting HBV cccDNA,the other for detecting the number of ?-Globin as internal control. Nucleic acid for detecting HBV cccDNA extracted by phenol-chloroform was digested by plasmid-safe ATP dependent DNase which was applied to digest the single strand DNA in rcDNA and ssDNA,then was quantitated by the primers spanning across the nick and SYBR Green Ⅰ dye.The specifity of PCR production was confirmed by the sequence analysis and rcDNA comparison.The significance of the difference of HBV cccDNA level between HBeAg(+) and HBeAg(-) group was analyzed by two group t test.Results The agarose gelelectrophoresis showed the molecular weight of the PCR production was about 350bp.The coincidence rate of PCR production and goal fragement was nearly 99% by sequence analysis.The result of PCR detection of rcDNA group was negative.The positive rate of HBV cccDNA of liver biopsies of HBeAg (+) patients detected by this method was 100%,the level of HBV cccDNA in the liver biopsies of HBeAg (+) patients was higher than HBeAb(+) patients.Conclusions The specificity of the method is proved by agarose electrophoresis,gene sequencing of the PCR product and rcDNA comparison.The quantitative method that use SYBR Green Ⅰ dye and ?-Globin as internal control is more specific,sensitive and economical,and more suitable for clinical purpose.