ABSTRACT
Objective To investigate the role of cyclic GMP-AMP synthase (cGAS),a cytosolic DNA sensor,in regulating innate immune responses induced by reverse transcription intermediate of human T cell leukemia virus type 1 (HTLV-1). Methods (1)ssDNA90,the reverse transcription intermediate of HTLV-1,was transfected into HeLa cells to observe changes in the expression pattern of cGAS in transfected-HeLa cells with immunoblot assay. (2) HeLa cells were firstly transfected with cGAS-encoding plasmid and then ssDNA90 24 hours later. Real-time PCR was used to measure the expression of interferon ( IFN)-β, IFN-gamma-inducible protein 10 ( IP-10 ), regulated on activation, normal T cell expressed and secreted (RANTES) and tumor necrosis factor (TNF)-α. Immunoblot assay was performed to measure phosphorylated interferon regulatory factor 3 (IRF3) and p65. (3)cGAS expression was silenced by siRNA in HeLa and phorbol-12-myristate-13-acetate (PMA)-treated THP1 (PMA-THP1) cells and then ssDNA90 was transfect-ed into these cells 24 hours later. Real-time PCR was used to measure the expression of IFN-β,IP-10,RAN-TES and TNF-α. Immunoblot assay was performed to measure phosphorylated IRF3 and p65. Results Ex-pression of cGAS was increased in HeLa cells after ssDNA90 transfection. Compared with control cells, cGAS-transfected HeLa cells showed increased expression of IFN-β, IP-10, RANTES and TNF-α and en-hanced phosphorylation of IRF3 and p65 after ssDNA90 transfection. Compared with control cells,both HeLa and PMA-THP1 cells with silenced expression of cGAS showed impaired production of IFN-β,IP-10,RAN-TES and TNF-α after ssDNA90 transfection. Moreover,ssDNA90-induced phosphorylation of IRF3 and p65 were decreased after cGAS gene-knockdown. Conclusion cGAS might promote HTLV-1 RTI ssDNA90-in-duced innate immune responses.
ABSTRACT
Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.
ABSTRACT
Objective To prepare and study the immunogenicity of hepatitis B virus surface anti-gen (HBsAg)-tetanus toxoid (TT) conjugate vaccine. Methods Tr was activated by cyangen bromide and reacted with adipic acid dihydrazide, then HBsAg-TT conjugate was prepared by carbediimide. Conjugate, HBsAg or hepatitis B vaccine was injected subcutaneously into mice. Anti-HBsAg and HBsAg-specific T cell response elicited by these immunogens were assayed. Results New HBsAg-TT conjugate elicited higher levels of anti-HBsAg and HBsAg positive conversion rates after the immunization than did HBsAg alone or hepatitis B vaccine. Conjugate induced mesdy antibodies of the IgG2a subclass, while HBsAg alone or hepa-titis B vaccine mainly elicited anti-HBsAg in the IgG1 subclass. The number of IFN-γand IL-2 secreting T cells induced by conjugate was also significantly higher than that did by HBsAg or hepatitis B vaccine. Con-clusion This study indicated new HBsAg-TT conjugate can induce both stronger humoral and TH1 type of cellular immune response.