Human herpesvirus 6A induces apoptosis of primary human fetal astrocytes via both caspase-dependent and -independent pathways.

BACKGROUND: Human herpesvirus 6 (HHV-6) is a T-lymphtropic and neurotropic virus that can infect various types of cells. Sequential studies reported that apoptosis of glia and neurons induced by HHV-6 might act a potential trigger for some central nervous system (CNS) diseases. HHV-6 is involved in the pathogenesis of encephalitis, multiple sclerosis (MS) and fatigue syndrome. However, the mechanisms responsible for the apoptosis of infected CNS cells induced by HHV-6 are poorly understood. In this study, we investigated the cell death processes of primary human fetal astrocytes (PHFAs) during productive HHV-6A infection and the underlying mechanisms. RESULTS: HHV-6A can cause productive infection in primary human fetal astrocytes. Annexin V-PI staining and electron microscopic analysis indicated that HHV-6A was an inducer of apoptosis. The cell death was associated with activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP), which is known to be an important substrate for activated caspase-3. Caspase-8 and -9 were also significantly activated in HHV-6A-infected cells. Moreover, HHV-6A infection led to Bax up-regulation and Bcl-2 down-regulation. HHV-6A infection increased the release of Smac/Diablo, AIF and cytochrome c from mitochondria to cytosol, which induced apoptosis via the caspase-dependent and -independent pathways. In addition, we also found that anti-apoptotic factors such as IAPs and NF-κB decreased in HHV-6A infected PHFAs. CONCLUSION: This is the first demonstration of caspase-dependent and -independent apoptosis in HHV-6A-infected glial cells. These findings would be helpful in understanding the mechanisms of CNS diseases caused by HHV-6.

Generation of cytotoxic T cell against HBcAg using retrovirally transduced dendritic cells.

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 granulocyte-macrophage 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 one-week interval. Intracellular IFN-gamma and IL-4 of immunized mice lymphocytes were analyzed. Generation of CTLs in lymphocytes 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 3 x 10(5) 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 DCs in vitro in the presence of rmGM-CSF 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.

[Anti-nasopharyngeal carcinoma effect in vivo and in vitro of cytotoxicity T lymphocyte induced by Ad5-latent membrane protein 2A].

OBJECTIVE: To study the biological characteristics of cytotoxicity T lymphocyte (CTL) induced by dendritic cell (DC) transfected with the Epstein-Barr virus latent membrane protein 2A (EBV-LMP2A) recombinant adenovirus. To establish nasopharyngeal carcinoma (NPC) animal models expressing LMP2A and investigate the anti-tumor effect of LMP2A specific CTL in vivo. METHODS: The mononuclear cells were isolated from human peripheral blood mononuclear cells (PBMC) and cultured with the cytokines [granulocyto-monocyte colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor-alpha TNF-alpha]. The expression of surface markers on mature DC was detected by fluorescence activated cell sorter FACS. Mature DC were transfected with LMP2A recombinant adenovirus. Under the help of interleukin-2 (IL-2), LMP2A specific CTL were induced by coculturing LMP2A-transfected DC with autologous PBMC. The population of CTL was detected by FACS. NPC animal models were constructed by implanting CNE cells expressing LMP2A subcutaneously into BALB/c nude mice. After intra-tumoral injection of LMP2A specific CTL, the size of tumor was measured. The tumors were removed after 30 d and subjected to histological examination. RESULTS: Mature DC displaying typical characteristics of morphology and phenotype were obtained from monocytes cultured in the medium containing GM-CSF, IL-4 and TNF-alpha. The LMP2A specific CTL induced by transfected DC were composed of mainly CD4+ and CD8+ T cells. The NPC animal models were constructed three weeks after implanting CNE cells. The study in vivo indicated that the tumors treated with LMP2A specific CTL grew slowly compared with control. Tumor volume of treated groups was significantly smaller than that of controls. The histological sections showed local necrosis and infiltration of lymphocyte in tumor tissue. CONCLUSIONS: Typically mature DC could be generated in vitro by culturing monocytes with the cytokines. LMP2A-specific CTL could be induced by LMP2A transfected DC in vitro. NPC mice models could be constructed by implanting CNE cells. LMP2A specific CTL could inhibit the growth of implanted tumor and produce an anti-tumor effect in vivo.

Human herpesvirus-6-specific interleukin 10-producing CD4+ T cells suppress the CD4+ T-cell response in infected individuals.

Human herpesvirus-6 (HHV-6) infection normally persists for the lifetime of the host and may reactivate with immunosuppression. The mechanism behind HHV-6 latent infection is still not fully understood. In this study, we observed that decreased proliferation of CD4+ T cells and PBMCs but not CD8+ T cells from HHV-6-infected individuals was stimulated with HHV-6-infected cell lysates. Moreover, HHV-6-stimulated CD4+ T cells from HHV-6-infected individuals have suppressive activity on naïve CD4+ T and CD8+ T cells from HHV-6-uninfected individuals. However, no increased proportion of CD4+ CD25+ Treg cells from HHV-6-infected individuals contributed to the suppressive activity of the HHV-6-stimulated CD4+ T cells from HHV-6-infected individuals. Transwell experiments, ELISA and anti-IL-10 antibody blocking experiment demonstrated that IL-10 may be the suppressive cytokine required for suppressive activity of CD4+ T cells from HHV-6-infected individuals. Results of intracellular interleukin (IL)-10 and IL-4 further implicated the HHV-6-specific IL-10-producing CD4+ T cells in the suppressive activity of CD4+ T cells from HHV-6-infected individuals. Results of intracellular interferon (IFN)-gamma demonstrated a decreased frequency of HHV-6-specific IFN-gamma-producing CD4+ T, but not CD8+ T cells in HHV-6-infected individuals, indicating that it was the CD4+ Th1 responses in HHV-6-infected individuals that were selectively impaired. Our findings indicated that HHV-6-specific IL-10-producing CD4+ T cells from HHV-6-infected individuals possess T regulatory type 1 cell activity: immunosuppression, high levels of IL-10 production, with a few cells expressing IFN-gamma, but none expressing IL-4. These cells may play an important role in latent HHV-6 infection.

[Retroviral-mediated transfection of hepatitis B virus core gene into bone marrow-derived dendritic cells].

AIM: To evaluate the transfection efficiency of recombinant retrovirus vector bearing hepatitis B virus(HBV) core gene to bone marrow-derived dendritic cells(DCs) and the capability of these DCs to induce cytotoxic T lymphocyte(CTL) response. METHODS: C57BL/6 mice bone marrow cells were stimulated with recombinant mouse granulocyte-macrophage colony-stimulating factor(rmGM-CSF) and interleukin-4(rmIL-4) for 6 days. Expanding DC progenitors were transfected by retrovirus vector containing HBV core gene. Integration and transcription of HBV core gene were determined by PCR and RT-PCR, respectively. Expression of HBcAg was analyzed by fluorescence activated cell sorter (FACS) and Western blot. Cytokines were quantified by enzyme immunoassay. The expressions of CD80 and MHC class II molecules on DCs were measured by FACS. Generation of CTLs in mixed leukocyte reaction were determined by LDH release assays. RESULTS: Transfected bone marrow cells were capable of differentiating into DCs in-vitro at the presence of rmGM-CSF and rmIL-4. The result of PCR and RT-PCR showed that the HBV core gene was integrated into the genome of infected DCs. Western blot analysis showed that HBV core gene was expressed in DCs. Transfection rate was 28% determined by FACS. Retroviral transfection had no influence on expressions of CD80 and MHC class II molecules, as well as IL-12 production. HBcAg-specific CTLs could be generated by using transfected DCs as antigen presenting cell (APC). CONCLUSION: Retroviral transfected myeloid DC progenitors could efficiently express HBcAg, without significant change on development and function of DCs, which lays a solid foundation for immunotherapy of chronic hepatitis B.

[Construction and identification of recombinant adenovirus vector containing thioredoxin reductase gene].

AIM: To construct recombinant adenovirus vector containing human thioredoxin reductase (TR) gene and to explore the correlation between antioxidant activity of TR and the degenerative neuropathy. METHODS: Full length TR cDNA was obtained from recombinant plasmid pGEM-TR via digestion with Apa I and Not I and was cloned into pShuttle vector and pShuttle-TR was recut with I-Ceu I and PI-Sce I. Fragment containing TR gene and CMV promoter was inserted into E1 and E3 deficient adeno-X virus DNA, and then the recombinant adenovirus vector was transfected into HEK 293 cells through lipofectamine and identified by PCR. The TR expression on and in cell lysate of CV1 cells infected with recombinant adenovirus was by immuno fluorescence assay and Western blot analysis. RESULTS: After replication of recombinant adenovirus Adeno-TR, the virus titer was about 4.4x10(11) pfu/L. The TR expression on CV1 cells was proved by fluorescent microscopy. Western blot analysis showed a band with relative molecular mass (M(r)) of 55,000. CONCLUSION: A recombinant adenovirus vector has been successfully constructed and TR is expressed on CV1 cells. This result lays the foundation for further study on function of TR and its correlation with degenerative neuropathy.

[Expression of HBcAg in eukaryotic cells by retroviral vector mediated gene transfer].

BACKGROUND: To construct recombinant retroviral vector expressing HBcAg in eukaryotic cells. METHODS: The HBV core gene fragment was amplified by using PCR from pADR which contains complement nucleotide sequence of HBV subtype adr and cloned into retroviral expression plasmid pLXSN, then transfected into packing cell (PT67) with lipofec AMINE. After 2-3 weeks selection with G418, large colonies were isolated and transferred to individual plates. Virus-containing medium was collected and used to infect NIH3T3, EL4 and mouse bone marrow derived dendritic cells(DC). DNA was extracted from infected cells and tested by PCR. Indirect immunofluorescence and FACS were used to detect the expression of HBcAg. Cell mediated immunity of immunized C57BL/6 mice with transduced DC was analyzed. RESULTS: The insertion of HBV core gene fragment in the recombinant retroviral plasmid was confirmed by PCR as well as enzyme digestion with EcoR1 and BamH2. The viral titer reached 3 x 10(5) CFU/ml. The result of PCR showed that the HBV core gene had been integrated into the genome of infected NIH3T3 cells. Indirect immunofluorescence and FACS analysis showed that HBcAg had been expressed in these cells. HBcAg specific CTL responses could be generated in mice immunized with retrovirus transduced DC. CONCLUSIONS: HBV core gene had been integrated into eukaryotic cells with retroviral vector and target gene had been expressed efficiently. These results may have some impact on gene therapy of chronic hepatitis B.