AIM: Cytotoxic T lymphocytes (CTLs) play an important role in resolving HBV infection. In the present study, we attempted to evaluate the efficiency of bone marrow-derived dendritic cells (DCs) transduced with recombi...AIM: Cytotoxic T lymphocytes (CTLs) play an important role in resolving HBV infection. In the present study, we attempted to evaluate the efficiency of bone marrow-derived dendritic cells (DCs) transduced with recombinant retroviral vector bearing hepatitis B virus (HBV) core gene and the capability of generating CTLs against HBcAg by genetically modified DCs in vivo.METHODS: A retroviral vector containing HBV core gene was constructed. Replicating DC progenitor of C57BL/6 mice was transduced by retroviral vector and continually cultured in the presence of recombinant mouse granulocytemacrophage colony-stimulating factor (rmGM-CSF) and interleukin-4(IL-4) for 6 days. LPS was added and cultured for additional two days. The efficiency of gene transfer was determined by PCR, Western blot and FACS. Transduced DCs immunized C57BL/6 mice subcutaneously 2 times at an oneweek interval. Intracellular IFN-γ and IL-4 of immunized mice lymphocytes were analyzed. Generation of CTLs in lymphooytes stimulated with mitomycin C-treated EL4-C cell which stably expresses HBcAg was determined by LDH release assays.RESULTS: Recombinant retroviral expression vector (pLCSN) was positively detected by PCR as well as enzyme digestion with EcoRI and BamH I. Retroviruses were generated by pLCSN transfection packing cell and the virus titer was 3x10s CFU/ml. Indirect immunofluorescence and FACS showed that HBV core gene was expressed in murine fibroblasts. Transduced bone marrow cells had capability of differentiating into DCsc in vitro in the presence of rmGMCSF and rmIL-4. The result of PCR showed that HBV core gene was integrated into the genome of transduced DCs.Western blot analysis showed that HBV core gene was expressed in DCs. The transduction rate was 28 % determined by FACS. Retroviral transduction had no influence on DCs expressions of CD80 and MHC class Ⅱ. HBcAg specific CTLs and Th1 type immune responses could be generated in the mice by using transduced DCs as antigen presenting cells (APCs).CONCLUSION: Retroviral transduction of myeloid DCs progenitors expresses efficiently HBcAg, and genetically modified DCs evoke a higher CTLs response than HBcAg in vivo.展开更多
AIM: To investigate the epidemiology of hepatitis B virus (HBV) strains with a mutation at nt551 in surface gene among hepatitis B patients in Nanjing and its neighbourhood.METHODS: By using mutation-specific polymera...AIM: To investigate the epidemiology of hepatitis B virus (HBV) strains with a mutation at nt551 in surface gene among hepatitis B patients in Nanjing and its neighbourhood.METHODS: By using mutation-specific polymerase chain reaction (msPCR) established by our laboratory for amplifying HBV DNAs with a mutation at nt551, 117 serum samples taken from hepatitis B patients were detected.RESULTS: The results showed that 112 samples were positive for nt551A, 4 samples were positive for nt551G.One sample was positive for nt551T. No nt551C of HBV DNA was found. The incidence of HBsAg mutants with G,C, T, A at nt551 among 117 samples was 3.42%, 0%, 0.85%,95.73%, respectively.CONCLUSION: In Nanjing and its neighbourhood, hepatitis B patients are mainly infected with wild genotype HBV.The incidence of mutants with a mutation at nt551 in HBV genome is significantly lower than that in wild genotype HBV DNA (P＜0.01). The necessity of adding components of HBsAg mutants to HBV vaccine needs further investigation.展开更多
To investigate the biological and immunological characteristics of the Nanjing local strains of HHV-7,four strains of herpesvirus were isolated from saliva specimens of one healthy individual and three children suffer...To investigate the biological and immunological characteristics of the Nanjing local strains of HHV-7,four strains of herpesvirus were isolated from saliva specimens of one healthy individual and three children suffering from a kidney disease in Nanjing.The viruses were identified by transmission electron microscopy (EM),indirect immunofluorescence assay(IFA) with a specific monoclonal antibody;nested polymerase chain reaction,restriction mapping and DNA sequencing.The virus-infected cells showed the typical cytophathic effect(CPE) under microscopy and could be detected by IFA with the human herpesvirus-7(HHV-7) specific antibody.Under EM,herpesvirus-like and virions capsids could be found in their cytoplasm or nucleoplasm. HHV-7 DNA fragments amplified from infected cells by nested PCR were confirmed by restriction mapping and DNA sequencing.Similarly to DC strain,an known HHV-7 strain used in the present study as the positive control,the virus could be inactivated by ultraviolet irradiation for 10 min,heated at 45℃ for 30 min,pH<5 or>9 at 4℃ for 2 h and ether or chloroform for 10 h.The virus induced the production of TNF-α,IL-10 and IL-12p70 while inhibited IFN-γ secretion,increased the percentage of CD2^+ cells while decreased that of CD4^+ or CD45RA^+ cells.The results indicate that the viruses isolated in Nanjing are HHV-7,which has similar biological characteristic to the known HHV-7 strain,DC.Infection with HHV-7 in vitro could affect immune function of lymphocytes by disturbing cytokine production and CD antigen expression.Cellular & Molecular Immunology.2004;1(5):367-372.展开更多
基金a grant from Key Lab Programs of Jiangsu Province,No.k2030
文摘AIM: Cytotoxic T lymphocytes (CTLs) play an important role in resolving HBV infection. In the present study, we attempted to evaluate the efficiency of bone marrow-derived dendritic cells (DCs) transduced with recombinant retroviral vector bearing hepatitis B virus (HBV) core gene and the capability of generating CTLs against HBcAg by genetically modified DCs in vivo.METHODS: A retroviral vector containing HBV core gene was constructed. Replicating DC progenitor of C57BL/6 mice was transduced by retroviral vector and continually cultured in the presence of recombinant mouse granulocytemacrophage colony-stimulating factor (rmGM-CSF) and interleukin-4(IL-4) for 6 days. LPS was added and cultured for additional two days. The efficiency of gene transfer was determined by PCR, Western blot and FACS. Transduced DCs immunized C57BL/6 mice subcutaneously 2 times at an oneweek interval. Intracellular IFN-γ and IL-4 of immunized mice lymphocytes were analyzed. Generation of CTLs in lymphooytes stimulated with mitomycin C-treated EL4-C cell which stably expresses HBcAg was determined by LDH release assays.RESULTS: Recombinant retroviral expression vector (pLCSN) was positively detected by PCR as well as enzyme digestion with EcoRI and BamH I. Retroviruses were generated by pLCSN transfection packing cell and the virus titer was 3x10s CFU/ml. Indirect immunofluorescence and FACS showed that HBV core gene was expressed in murine fibroblasts. Transduced bone marrow cells had capability of differentiating into DCsc in vitro in the presence of rmGMCSF and rmIL-4. The result of PCR showed that HBV core gene was integrated into the genome of transduced DCs.Western blot analysis showed that HBV core gene was expressed in DCs. The transduction rate was 28 % determined by FACS. Retroviral transduction had no influence on DCs expressions of CD80 and MHC class Ⅱ. HBcAg specific CTLs and Th1 type immune responses could be generated in the mice by using transduced DCs as antigen presenting cells (APCs).CONCLUSION: Retroviral transduction of myeloid DCs progenitors expresses efficiently HBcAg, and genetically modified DCs evoke a higher CTLs response than HBcAg in vivo.
基金Supported by the Natural Science Foundation of Jiangsu Province,No. BJ2000039
文摘AIM: To investigate the epidemiology of hepatitis B virus (HBV) strains with a mutation at nt551 in surface gene among hepatitis B patients in Nanjing and its neighbourhood.METHODS: By using mutation-specific polymerase chain reaction (msPCR) established by our laboratory for amplifying HBV DNAs with a mutation at nt551, 117 serum samples taken from hepatitis B patients were detected.RESULTS: The results showed that 112 samples were positive for nt551A, 4 samples were positive for nt551G.One sample was positive for nt551T. No nt551C of HBV DNA was found. The incidence of HBsAg mutants with G,C, T, A at nt551 among 117 samples was 3.42%, 0%, 0.85%,95.73%, respectively.CONCLUSION: In Nanjing and its neighbourhood, hepatitis B patients are mainly infected with wild genotype HBV.The incidence of mutants with a mutation at nt551 in HBV genome is significantly lower than that in wild genotype HBV DNA (P＜0.01). The necessity of adding components of HBsAg mutants to HBV vaccine needs further investigation.
基金This work was supported by grants from Natural Science Foundation of Nation(NO.30371345)Jiangsu Province(NO.BK970S1 and BK2001115).
文摘To investigate the biological and immunological characteristics of the Nanjing local strains of HHV-7,four strains of herpesvirus were isolated from saliva specimens of one healthy individual and three children suffering from a kidney disease in Nanjing.The viruses were identified by transmission electron microscopy (EM),indirect immunofluorescence assay(IFA) with a specific monoclonal antibody;nested polymerase chain reaction,restriction mapping and DNA sequencing.The virus-infected cells showed the typical cytophathic effect(CPE) under microscopy and could be detected by IFA with the human herpesvirus-7(HHV-7) specific antibody.Under EM,herpesvirus-like and virions capsids could be found in their cytoplasm or nucleoplasm. HHV-7 DNA fragments amplified from infected cells by nested PCR were confirmed by restriction mapping and DNA sequencing.Similarly to DC strain,an known HHV-7 strain used in the present study as the positive control,the virus could be inactivated by ultraviolet irradiation for 10 min,heated at 45℃ for 30 min,pH<5 or>9 at 4℃ for 2 h and ether or chloroform for 10 h.The virus induced the production of TNF-α,IL-10 and IL-12p70 while inhibited IFN-γ secretion,increased the percentage of CD2^+ cells while decreased that of CD4^+ or CD45RA^+ cells.The results indicate that the viruses isolated in Nanjing are HHV-7,which has similar biological characteristic to the known HHV-7 strain,DC.Infection with HHV-7 in vitro could affect immune function of lymphocytes by disturbing cytokine production and CD antigen expression.Cellular & Molecular Immunology.2004;1(5):367-372.
