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Objective To investigate the effects of different Leptospira strains on phagocytosis of peritoneal macrophages of guinea pigs,and explore the role of innate immune in the pathogenesis of leptospirosis. Methods Peritoneal macrophages of guinea pigs were infected in vitro by three different Leptospira strains,the virulent Leptospira interrogans serovar Lai type strain Lai,the avirulent L.interrogans serovar Lai type strain IPAV,and the nonpathogenic L.biflexa serovar Patoc type strain PatocⅠ,respectively,and heat inactivated Staphylococcus epidermidis was added 0.5,1.5,3 and 6 h after infection and incubated for 30 min.The effect of Leptospira on the phagocytosis of macrophage was evaluated by the inactivated Staphylococcus epidermidis phagocytosis rate and phagocytosis index.Phagocytosis and ultrastructure of peritoneal macrophages were observed by transmission electron microscopy 3 h after infection,and changes of cytoskeleton of the macrophages were observed by laser scanning confocal microscopy. Results The phagocytic rates and phagocytic indexes of strain Lai,strain IPAV and strain PatocⅠinfection groups were significantly lower than those of control group 3 h and 6 h after infection(P
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<p><b>OBJECTIVE</b>To increase the immune effect of gene vaccine, T7 RNA polymerase was used to establish a system of cytoplasmic expression.</p><p><b>METHODS</b>(1) The plasmid pT7 EMCVP1, including T7 promoter sequence, 5'-untranslated sequence of encephalomyocarditis (EMC) virus, VP1 sequence of coxsackievirus B3 (CVB3), was cotransfected with the plasmid pAR 3132, which codes for the T7 RNA polymerase, into HeLa cells and murine peritoneal macrophages. (2) The plasmid pT7 EMCVP1 and pAR 3132 were respectively transformed into the attenuated Salmonella typhimurium SL 7207. The two kinds of transformed bacteria were coinfected into murine peritoneal macrophages.</p><p><b>RESULTS</b>(1) The target antigen VP1 in the cytoplasm was about 2-4-fold higher than that of pcDNA3 VP1 singly transfected. (2) After the murine peritoneal macrophages were coinfected by two kinds of transformed bacteria, the target antigen VP1 could also be detected.</p><p><b>CONCLUSION</b>The pT7 EMCVP1 and pAR 3132 could be expressed in the cytoplasm of HeLa cells and murine peritoneal macrophages and the amount of the antigen VP1 increased remarkably as compared with that of pcDNA3 VP1 singly transfected.</p>
Assuntos
Animais , Humanos , Camundongos , Bacteriófago T7 , Genética , Proteínas do Capsídeo , Genética , Metabolismo , Citoplasma , Metabolismo , Eletroforese em Gel de Poliacrilamida , Enterovirus Humano B , Genética , Expressão Gênica , Células HeLa , Macrófagos Peritoneais , Biologia Celular , Metabolismo , Plasmídeos , Genética , Regiões Promotoras Genéticas , Genética , Salmonella typhimurium , Genética , TransfecçãoRESUMO
<p><b>OBJECTIVE</b>To study whether the live attenuated AroA-auxotrophic mutant of Salmonella (S.) typhimurium (SL7207) could be used as DNA delivery vehicle to induce more efficient immune response by using the eukaryotic expression plasmid pCMV-beta as report gene.</p><p><b>METHODS</b>Murine peritoneal macrophages were infected with SL7207(pCMV-beta) in vitro, then the expression of the beta-gal were detected by X-gal staining or RT-PCR. After mice were orally immunized with SL7207(pCMV-beta), the expression of beta-gal in the lymphoid tissue were tested by RT-PCR, humoral responses were tested by ELISA, splenic lymphocyte proliferation were tested by 3H-TdR incorporation and cytotoxic T lymphocyte reaction were tested by JAM test.</p><p><b>RESULTS</b>The results indicated that the plasmid pCMV-beta could be delivered by SL7207 into the nucleus of the murine macrophages efficiently and expressed well in vitro; after mice received oral immunizations with attenuated S.typhimurium SL7207 harboring plasmid pCMV-beta mice, the expression of beta-gal could be detected in the spleen, mesenteric lymph nodes and Peyers patches of the mice. Furthermore, the experiments demonstrated that specific humoral immune responses and cell-mediated immune responses were successfully induced in these immunized mice. Compared with the naked DNA vaccination, SL7207 (pCMV-beta) oral immunization were more efficient in inducing cellular immune responses.</p><p><b>CONCLUSIONS</b>Attenuated Salmonella typhimurium SL7207 could be used as DNA delivery vehicle for oral immunization, which have the ability to deliver the antigen-encoding DNA specifically to APC directly for inducing the specific immune response being dominant with cellular immune response.</p>