Stimulation of Caco-2 cells with recombinant lactobacilli or purified flagellin induced the release of IL-8 in a dose-dependent manner (Fig. 2). Because bacterial cells were not inactivated but lyophilized once, antibiotics were included in the culture, and the incubation time was relatively short, and growth of bacterial cells was not observed during this assay. The relatively high levels of IL-8 were detected only in the culture exposed to agents including FliC. Despite

Caco-2 cells being stimulated with the same amount of bacterial cells, LCF induced much less IL-8 production than LCFS or LCSF. In particular, the amount of IL-8 evoked by 1000 μg/ml LCF was almost same as that by 100 μg/ml LCFS or LCSF. These concentrations of LCF, LCFS, and LCSF, exhibited nearly equal activity in IL-8 induction as 10 ng/ml of purified flagellin. The specific IgG titers against cSipC and FliC were measured High Content Screening by ELISA, as shown in Fig. 3. cSipC-specific IgG was produced by mice immunized with LCS, LCSF, LCFS,

purified cSipC, and a mixture of purified cSipC and flagellin. The flagellin-specific IgG was detected in sera from mice that received LCF, LCSF, LCFS, or the mixture of purified cSipC and flagellin. No significant difference was shown for NVP-BKM120 research buy cSipC-specific IgG titer between the groups immunized with cSipC-producing lactobacilli (LCS, these LCSF, and LCFS). On the other hand, the flagellin-specific IgG titers of the LCF- or LCFS-immunization groups were significantly higher than that of the LCSF-immunization group. Immunization with purified soluble antigens without adjuvant also evoked specific IgG. In addition, the titer of cSipC-specific IgG induced by inoculation with a mixture of cSipC and flagellin was higher than that of cSipC only. SE antigen-specific IgG was not detected from the immunized groups of LCN and PBS. In order to determine the IgG1/2a ratio, which represents the Th2/Th1 response, the same ELISA but using anti-IgG1 and anti-IgG2a antibodies for detection was

performed. For both anti-cSipC and anti-FliC IgG1/2a ratios, the groups immunized with soluble antigens showed greater values than the groups that received antigens exposed on the bacterial surface (Table 2). No significant difference was observed between the groups immunized with soluble antigens or between groups that received recombinant lactobacilli expressing SE antigens. Eight kinds of cytokine in spleen cell cultures, which were stimulated with SE antigens, were measured using a Bio-Plex suspension array system. Stimulation with ConA induced non-specific proliferation of splenocytes and the production of high levels of various cytokine, while poor cell-proliferation and cytokine production were observed in spleen cells incubated with PBS (data not shown).