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Pooled sera from the Group RhoC 2 and Group 3 mice reacted strongly to both f-Tp and u-Tp sonicates at all time points, confirming that these sera recognize native Tp antigens (Fig.?2). Because Tp was derived from testicular tissue of experimentally infected rabbits, the pooled mouse sera were tested by ELISA with sonicate of NRTE. Group 2 and Group 3 sera did not exhibit significant reactivity over that of Group 1 sera to NRTE at any time point, indicating that the IgG antibody response of the Group 2 and Group 3 mice is specifically directed against Tp rather than contaminating rabbit cells (Fig.?2). IgG isotyping indicated that both IgG1 and IgG2c antibodies to Tp were present in pooled sera from the Group 2 and Group 3 mice (see Supporting Information, Fig. S1). The IgG1 level was higher than the IgG2c level for the Group 2 mice at all time points and for the Group 3 mice at 4 and 8?weeks after SLN implantation. find more However, IgG1 and IgG2c levels were nearly equivalent for the Group 3 mice at 12?weeks after SLN implantation. Western blot analysis with pooled sera from the three groups of mice showed that only the Group 2 and Group 3 mice exhibited strong reactivity to Tp (Fig.?3). IgG antibodies in the sera of these mice recognized several Tp proteins, most of which were in the range of c. 55�C31?kDa, at all time points after SLN implantation. The latter results are very similar to those of Saunders & Folds (1985) for Western blots performed with sera of mice that had been infected intradermally with virulent Tp for 12�C18?weeks. Interestingly, these investigators reported that sera from mice that were given a single intradermal injection of 1?��?107 heat-inactivated Tp cells did not show any reactivity to Tp when tested by Western blot. Secretion of IFN�� (Th1 cytokine) by pooled splenocytes from the three groups of mice was examined at 4, 8, and 12?weeks after SLN implantation. Incubation of splenocytes Galunisertib ic50 with medium only resulted in an undetectable level of IFN�� (i.e. below the assay detection limit of 31.25?pg?mL?1) in the supernatants for all groups of mice at all time points (data not shown). In contrast, stimulation of splenocytes with f-Tp cells resulted in secretion of a significantly higher level of IFN�� for Group 2 (Tp SLN) and Group 3 (Tp+CpG SLN) mice compared with Group 1 (empty SLN) mice at all time points (P?
