CD4＋CD25＋FoxP3 ＋ regulatory T ceils （Tregs） are increased in patients with chronic hepatitis C, which may contribute to the sustained suppression of hepatitis C virus （HCV）-specific T-cell responses and viral p...CD4＋CD25＋FoxP3 ＋ regulatory T ceils （Tregs） are increased in patients with chronic hepatitis C, which may contribute to the sustained suppression of hepatitis C virus （HCV）-specific T-cell responses and viral persistence in HCV-infected individuals. We postulated that HCV core protein （HCVc） directly contributes to the expansion of Tregs in HCV-infected patients, and we provide evidence to support this hypothesis in the report. Peripheral blood mononuclear cells （PBMCs） and sera were collected from 87 treatment-naive chronic HCV-infected patients, CD4＋CD25＋ Tregs were measured by flow cytometry, and HCV RNA and HCVc levels were detected using qPCR and enzyme-linked immunosorbent assay （ELISA）, respectively. CD4＋, CD8＋, CD4＋CD25＋ and CD4＋CD25- T cells were purified from healthy donors and cultured with recombinant HCVc and Toll-like receptor （TLR） ligands. Flow cytometry was used to analyze cell proliferation, and ELISA was performed to measure cytokine production. In the 87 chronic HCV-infected patients, HCVc showed a significant correlation with HCV RNA and CD4＋CD25＋Tregs. Mechanistic studies showed that HCVc, together with anti-CD3 antibody, augmented CD4＋CD25＋ Treg proliferation, but inhibited CD4＋CD25- T-cell proliferation and IFN-γ production, in a dose-dependent and Treg-dependent manner. Moreover, unlike the TLR3 ligand （poly hC） and the TLR4 ligand （lipopolysaccharide, LPS）, the TLR2 ligand （lipoteichoic acid, LTA） and HCVc both inhibited TCR-induced CD4＋ T-cell proliferation and IFN-γ secretion in a Treg-dependent manner. These data indicate that HCVc, like other TLR2 ligands, triggers CD4＋CD25＋ Treg activation and expansion to inhibit host immune responses, which may play a critical role in viral persistence in HCV-infected patients.展开更多
AIM: To investigate a specific association between hepatic steatosis and hepatitis C virus (HCV) core. METHODS: HeLa cells and primary mouse hepatocytes were transfected with HCV core plasmid, and conditional transgen...AIM: To investigate a specific association between hepatic steatosis and hepatitis C virus (HCV) core. METHODS: HeLa cells and primary mouse hepatocytes were transfected with HCV core plasmid, and conditional transgenics in which hepatic over-expression of HCV core is regulated by the tetracycline-off system, were developed. The expression of the HCV core was assessed over one to six months after withdrawal of doxycycline (dox) by immunohistochemistry (IHC) and Western blotting and by sequential liver biopsy. Hepatic steatosis was evaluated using oil red stain. 8-hydroxydeoxyguanosine (8-OHdG) stains and caspase levels were conducted to clarify hepatic oxidative stress and apoptosis rate. Serum aminotransferase was checked. RESULTS: The transfected hepatocytes had globular cores under the lipid vesicles. In transgenic mice on control diet, core expression was robust, localized to the cytoplasmic vesicle membrane and strongly associatedwith microvesicular steatosis, which was gradually replaced by macrovesicular steatosis. However, both steatosis and core positive hepatocytes diminished with time. Increases in aminotransferase, caspase and 8-OHdG were associated with peak core expression. CONCLUSION: The core protein was readily detected and morphologically associated with steatosis in individual hepatocytes both in vitro and in vivo. In vivo, oxidative stress caused by the core potentially reduced the number of core positive hepatocytes and in parallel the level of steatosis. To our knowledge, this is the f irst animal model that directly shows topological relationship between HCV core and hepatic lipid vesicles.展开更多
丙型肝炎病毒(Hepatitis C virus,HCV)是引起慢性肝炎的重要病因之一,严重危害公众健康。目前临床HCV感染常采用干扰素联合病毒唑进行治疗,然而应答率不高且易反复。因此,探索新型抗HCV治疗策略及药物显得尤为迫切。针对HCV核心基因的序...丙型肝炎病毒(Hepatitis C virus,HCV)是引起慢性肝炎的重要病因之一,严重危害公众健康。目前临床HCV感染常采用干扰素联合病毒唑进行治疗,然而应答率不高且易反复。因此,探索新型抗HCV治疗策略及药物显得尤为迫切。针对HCV核心基因的序列,设计与之互补的引导序列(Guide Sequence,GS),通过PCR的方法将其共价连接于大肠杆菌核糖核酸酶P(RNase P)催化性亚基(M1 RNA)的3?末端,成功构建了一种靶向性M1GS核酶(M1GS-HCV/C141)。经体外切割试验、胞内反义效应及胞内毒性研究,结果表明:M1GS-HCV/C141核酶不仅能够在体外对靶RNA片段产生特异性切割,在HCV感染的Huh7.5.1细胞中,也能显著抑制病毒核心蛋白的表达,进而使HCV RNA的拷贝数减少约1 000倍。因此,文中构建的M1GS-HCV/C141核酶在体外具有显著的抗HCV活性,这为HCV治疗研究提供了一条新的潜在途径。展开更多
目的构建丙肝病毒核心基因克隆载体pGEM-HCV/core,为进一步研究外部引导序列(external guide sequence,EGS)引导核糖核酸酶P在RNA水平阻断丙肝病毒核心基因的表达做好前期基础。方法将含HCV全长基因的pBRTM/HCV1-301l质粒于大肠杆菌JMl0...目的构建丙肝病毒核心基因克隆载体pGEM-HCV/core,为进一步研究外部引导序列(external guide sequence,EGS)引导核糖核酸酶P在RNA水平阻断丙肝病毒核心基因的表达做好前期基础。方法将含HCV全长基因的pBRTM/HCV1-301l质粒于大肠杆菌JMl09内扩增;提取pBRTM/HCV1-301l质粒;从pBRTM/HCV1-301l质粒中PCR扩增出HCV核心基因片段并将其插入pGEM-3Z克隆载体;以得到重组的pGEM-HCV/core克隆载体;最后EcoR I and Hind III双酶切鉴定HCV核心基因克隆载体。结果pGEM-HCV/core克隆载体经双酶切、凝胶电泳证明插入片段与HCV核心基因片段大小相符;经测序证明其插入序列与HCV core基因序列一致。结论成功构建了HCV核心基因的克隆载体pGEM-HCV/core。展开更多
基金This work was supported by the Natural Science Foundation of China （81373143, to ZT） and by NIH （AI095097, to LS）. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the paper.
文摘CD4＋CD25＋FoxP3 ＋ regulatory T ceils （Tregs） are increased in patients with chronic hepatitis C, which may contribute to the sustained suppression of hepatitis C virus （HCV）-specific T-cell responses and viral persistence in HCV-infected individuals. We postulated that HCV core protein （HCVc） directly contributes to the expansion of Tregs in HCV-infected patients, and we provide evidence to support this hypothesis in the report. Peripheral blood mononuclear cells （PBMCs） and sera were collected from 87 treatment-naive chronic HCV-infected patients, CD4＋CD25＋ Tregs were measured by flow cytometry, and HCV RNA and HCVc levels were detected using qPCR and enzyme-linked immunosorbent assay （ELISA）, respectively. CD4＋, CD8＋, CD4＋CD25＋ and CD4＋CD25- T cells were purified from healthy donors and cultured with recombinant HCVc and Toll-like receptor （TLR） ligands. Flow cytometry was used to analyze cell proliferation, and ELISA was performed to measure cytokine production. In the 87 chronic HCV-infected patients, HCVc showed a significant correlation with HCV RNA and CD4＋CD25＋Tregs. Mechanistic studies showed that HCVc, together with anti-CD3 antibody, augmented CD4＋CD25＋ Treg proliferation, but inhibited CD4＋CD25- T-cell proliferation and IFN-γ production, in a dose-dependent and Treg-dependent manner. Moreover, unlike the TLR3 ligand （poly hC） and the TLR4 ligand （lipopolysaccharide, LPS）, the TLR2 ligand （lipoteichoic acid, LTA） and HCVc both inhibited TCR-induced CD4＋ T-cell proliferation and IFN-γ secretion in a Treg-dependent manner. These data indicate that HCVc, like other TLR2 ligands, triggers CD4＋CD25＋ Treg activation and expansion to inhibit host immune responses, which may play a critical role in viral persistence in HCV-infected patients.
基金grants from National Science Council, Taiwan, China. No. NSC 93-2314-B-182A-148, 94-2314-B-182A-185 and 95-3112-B-182A-002 Chang Gung Memorial Hospital, Taoyuan, Taiwan, China. No. CMRPG 33014, CMRPG 340341 and SMRPG350081
文摘AIM: To investigate a specific association between hepatic steatosis and hepatitis C virus (HCV) core. METHODS: HeLa cells and primary mouse hepatocytes were transfected with HCV core plasmid, and conditional transgenics in which hepatic over-expression of HCV core is regulated by the tetracycline-off system, were developed. The expression of the HCV core was assessed over one to six months after withdrawal of doxycycline (dox) by immunohistochemistry (IHC) and Western blotting and by sequential liver biopsy. Hepatic steatosis was evaluated using oil red stain. 8-hydroxydeoxyguanosine (8-OHdG) stains and caspase levels were conducted to clarify hepatic oxidative stress and apoptosis rate. Serum aminotransferase was checked. RESULTS: The transfected hepatocytes had globular cores under the lipid vesicles. In transgenic mice on control diet, core expression was robust, localized to the cytoplasmic vesicle membrane and strongly associatedwith microvesicular steatosis, which was gradually replaced by macrovesicular steatosis. However, both steatosis and core positive hepatocytes diminished with time. Increases in aminotransferase, caspase and 8-OHdG were associated with peak core expression. CONCLUSION: The core protein was readily detected and morphologically associated with steatosis in individual hepatocytes both in vitro and in vivo. In vivo, oxidative stress caused by the core potentially reduced the number of core positive hepatocytes and in parallel the level of steatosis. To our knowledge, this is the f irst animal model that directly shows topological relationship between HCV core and hepatic lipid vesicles.
文摘目的构建丙肝病毒核心基因克隆载体pGEM-HCV/core,为进一步研究外部引导序列(external guide sequence,EGS)引导核糖核酸酶P在RNA水平阻断丙肝病毒核心基因的表达做好前期基础。方法将含HCV全长基因的pBRTM/HCV1-301l质粒于大肠杆菌JMl09内扩增;提取pBRTM/HCV1-301l质粒;从pBRTM/HCV1-301l质粒中PCR扩增出HCV核心基因片段并将其插入pGEM-3Z克隆载体;以得到重组的pGEM-HCV/core克隆载体;最后EcoR I and Hind III双酶切鉴定HCV核心基因克隆载体。结果pGEM-HCV/core克隆载体经双酶切、凝胶电泳证明插入片段与HCV核心基因片段大小相符;经测序证明其插入序列与HCV core基因序列一致。结论成功构建了HCV核心基因的克隆载体pGEM-HCV/core。
