| The adherence of Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) adherence to intestinal epithelial cells is the first step for the development of this disease. TTSS is an essential constituent for virulence of these pathogens, which links the bacterium to the host cell and forms a pore in the host plasma membrane. The EspA (E. coli secreted proteinA) protein makes up a filament structure being part of the type III secretion system (TTSS) that delivers effector proteins to the host epithelial cell. Actin polymerization and pedestal formation involve the recruitment of several cytoskeletal proteins to the site of EHEC attachment. The EspA filament is a polymer of the translocator protein EspA, which is likely to be the sole constituent of these hollow filamentous conduits. Polymerization of the EspA filaments is mediated by coiled-coil interactions between EspA subunits. EspA belongs to multimer, contains genic length 579bp and its molecular weight is 25kDa. The EspA protein plays an important role in the formations of actin polymerization and A/E (attaching/effacing) lesion.At previous, we have report an monoclone antibody 1H10 and a linear epitope E07 (100Lys-120Val), the presence of which is recognized by the novel monoclonal antibody 1H10. The next, we will analyse the immune function of E07 including the EHEC-induced actin polymerization and confers protection in mice. These findings provide a better understanding of E07-induced immune responses and could lead to epitope-based vaccines and antibodybased therapies.Methods:1. Analysis of E07 antigenicity1) The immunogenicity of E07 was determined and identified by ELISA, when BALB/c mice were immunized with E07.2) Western botting and immune fluorescence were analyzed the immune response of E07, where anti-E07 antibody was from immunized mice.3) Anti-O157 sera including mice and humen were detected to the E07 response by ELISA.2. Analysis of EspA biologic function during A/E and aggregation of actin1) To detect whether E07 induced immunity was protective, BALB/c mice were divided into PBS, BSA, E07, E07 conjugated BSA and full-length recombinant EspA, then mice were challenged with EHEC O157:H7 EDL933. The number of survival and CFU in fecuswas recorded.2) EHEC O157:H7 EDL933 and E. coli K12 at were used as a positive and negative control, respectively. Immune fluorescence assay was performed to review the ability of anti-E07 sera blocking aggregation of actin. The E07 antibody was co-incubated with HeLa cell to detect whether E07 antibody was inhibited the actin polymerization.3) The E07 immunoreactivity was identified by pathological assay. Mice protect assay was showed the E07 immuno-protection.3. The Basic Local Alignment Search Tool (BLAST) was used to analyze the dentified epitope, and CLC Workbench was used to exhibit the Blast result.Results:1. Anti-E07 antibody was shown to bind specifically to recombinant EspA and native EspA in the EHEC bacteria cell lysate by western blotting.2. FAS analysis demonstrated that Polyclonal sera of epitope E07 dramaticly inhibited EHEC O157-induced actin polymerization and tissue lesion, which were exhibited using FAS and H&E analyses. EHEC O157 induced actin polymerization, but it was not observed actin aggregation in cells when O157 incubated with anti-E07 in advance.3. E07 and EspA induced immunity is protective, E07 conjugated BSA were challenged with EHEC EDL933. But epitope E07 display a partial protection compared to the full-length reEspA.4. Sequence analysis of the identified epitope by BLAST demonstrated that epitope E07 is well conserved in E. coli O157:H7 and O55:H7, and its highly homologous motif was found within a wide range of E. coli serotypes.Conclusion:We identified the epitope E07 in the protective antigen EHEC O157, When mice were treated with E07 alone or E07 conjugated to a carrier, significant protective immunity were detected. Anti-E07 antibody, which recognizes E07, inhibited EHEC-induced actin polymerization in vitro. In addition, it conferred modest protection and decreased bacterial shedding in mice. These data indicate that E07 is immunogenic and protective. The protective effect of E07 vaccination observed in this study should be verified in mice human and large animals. Nevertheless, the identification of E07 provides a foundation for further development of epitope-based vaccines and antibody-based therapies against EHEC. H.pylori infection could induce chronic gastric inflammation that progresses to gastritis, peptic ulcer, or even gastric mucosa associated lymphoid tissue lymphoma and gastric cancer. Several studies indicate that the host immmue reponse induced by H.pylori infection is sufficient for the persistence of H. pylori infection and inflammation.Considerable evidences from both human and animal studies indicate that CD4~+ T cell responses play critical roles in the immunopathogenesis of H. pylori infection. It is generally accepted that Th17/Th1 cell responses contribute to H. pylori colonization and induction of gastric inflammation. Notably, a new CD4~+ T subset—Th9 is driven by na?ve CD4~+ T cells, and it is also named IL-9-producing CD4~+ T cell because of expressing a large number of IL-9. But, Th9 cells have not any defined transcription factors like T-bet, GATA3, ROR?t, and Foxp3, thus it need to be more investigation unlike Th1, Th17, and Treg cells.In this study, we attempted to investigate the correlation of IL-9 and CD4~+IL-9~+ T cell response to H.pylori infection. The expression of cytokine IL-9 at the levels of mRNA and protein in H. pylori-infected individuals and mice model had been measured by quantitative analyses using real-time PCR and ELISA, and also CD4~+IL-9~+ T cells response were investigated by flow cytometric analysis. Taken into consideration the role of IL-9 producing CD4~+ T cell in host immune to H. pylori infection.Methods:1. H. pylori culture and BABL/c mice infection. BABL/c and C56BL mice were challenged four times in two day by orogastric route with 1×109 CFU of H. pylori B5 per time. To establishment the model of infected mice, histology and Real time PCR were used th ensured the infected effect.2. Quantitative analyses for mRNA of cytokines. IFN-??IL-17?IL-4 and IL-9 were analyzed by real time PCR,?2-microglobulin (?2-M) was used as reference gene. Gene expression was calculated with relative quantification at each time point, using the??CT method.3. Expression of IL-9 mRNA and protein. In gastric tissues, spleen and PLN, Total RNA was extracted from these tissues of H. pylori-infected and time-matched uninfected mice, what were subjected to real-time PCR and ELISA.4. The change of CD4~+IL-9~+ cells responses and IL-9 contents from supernatants of cultured splenic lymphocytes in mice. CD4~+ T cells were isolated from spleen of H. pylori-infected and uninfected mice at the appointed time, and the resultes were detected by FCS and ELISA.5. The content of IL-9 in serum which came from PBMC of clinical H. pylori negative and positive individuals were examed by ELISA. (B) CD4~+T cells were isolated from PBMC including 16 H. pylori-positive and 24 H. pylori-negative individuals and examined by intracellular staining.Resultes:1. The result confirmed that a reliable animal model was established by infecting BALB/c mice with H. pylori. The inflammation of gastric tissue in the H. pylori-infected group was significantly higher than uninfected group. There was showed apparent inflammatory cells infiltration in the gastric lamina propria with H. pylori treated mice.2. The inflammation and high level of colonization in stomach were all started on the day 21 p.i, so we detected the expression of IL-9?IL-17 and IFN-?at this time point. Our results indicated that these cytokines significantly increased at the level of protein and mRNA in H. pylori infected mice.3. The mRNA level of IL-9 expression reached a peak value at day 35 post-infection in stomach tissue as well as PLN, while at day 28 in spleen. Furthermore, the protein level of IL-9 expression in gastric mucosa detected by ELISA also significantly increased.4. FCS results indicated that H. pylori induced IL-9-producing CD4~+ T cell responses in the process of H. pylori infection.5. The contents of IL-9 and percentage of CD4~+IL-9~+T cells in positive serum were significant more compare with it in negative serum.Conclusion:There are tremendous advances about the elucidation of IL-9 and CD4~+IL-9~+ T cells that are activated by recognition of H. pylori-associated inflammatory factors. Our investigations have more helpful to understand the multiple mechanisms of H. pylori pathogenesis, and to appropriately focus diagnostic testing and eradication therapy. The role of IL-9-producing CD4~+ T cell pathway in the host immune response of H. pylori infection needs further investigation. |
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