The Impact of BKI-1294 Therapy in Mice Infected With the Apicomplexan Parasite Neospora caninum and Re-infected During Pregnancy.

Exposure of Neospora caninum tachyzoites to BKI-1294 in vitro results in the formation of long-lived multinucleated complexes (MNCs). However, in vivo treatment of BALB/c mice with BKI-1294 shortly after N. caninum infection during pregnancy was safe and profoundly reduced pup mortality and vertical transmission. We hypothesized that the formation of MNCs could trigger immune responses that contribute to BKI efficacy in vivo. In this study, mice were first vaccinated with a sublethal dose of N. caninum tachyzoites and were treated with BKI-1294. We then investigated the effects of these treatments after mating and re-infection during pregnancy. Effects on fertility, pup survival, vertical transmission, and parasite load in dams were evaluated. Cytokines in sera or splenocyte culture supernatants were assessed by either ELISA or the Luminex™ 200 system, and humoral immune responses against tachyzoite and MNC antigens were compared by ELISA, Western blotting and immunoproteomics. Our results showed that BKI-1294 treatment of live-vaccinated mice reduced the cerebral parasite load in the dams, but resulted in higher neonatal pup mortality and vertical transmission. In live-vaccinated mice, cytokine levels, most notably IFN-y, IL-10, and IL-12, were consistently lower in BKI-1294 treated animals compared to non-treated mice. In addition, comparative Western blotting identified two protein bands in MNC extracts that were only recognized by sera of live-vaccinated mice treated with BKI-1294, and were not found in tachyzoite extracts. We conclude that treatment of live-vaccinated mice with BKI-1294 influenced the cellular and humoral immune responses against infection, affected the safety of the live-vaccine, and decreased protection against re-infection and vertical transmission during pregnancy.

CD4+ lymphocytes and gamma interferon predominate in local immune responses in early experimental syphilis.

The clearance of Treponema pallidum subsp. pallidum from early syphilis lesions involves infiltration of a large number of mononuclear cells and is characteristic of a cell-mediated immune response. In the present study, we sought to determine the relative abundance of different T-lymphocyte populations and Th1/Th2-associated cytokines present in testicular lesions following experimental infection with the Chicago strain of T. pallidum. Using flow cytometry, we examined the proportion of CD4(+) and CD8(+) T cells present throughout the progression and resolution of primary syphilis in the rabbit model. We related these findings to the results of real-time reverse transcription-PCR quantification of treponemal and cytokine mRNA levels. Treponemal mRNA levels reached peak values on day 18 postinfection, coincident with an initial peak in the level of T cells, which were primarily CD4(+) T cells. T-cell levels increased again during resolution of orchitis, and there was an increased proportion of CD8(+) T cells. The maximum gamma interferon (IFN-gamma) and interleukin-10 (IL-10) mRNA levels were observed on days 11 and 18, respectively, while only negligible amounts of IL-4 and IL-2 were detected throughout the infection. In addition to showing the temporal relationship between treponemal burden and T-cell responses during lesion progression, our results also demonstrate that the composition of the T-cell population changes during lesion resolution. The presence of the mRNA for IFN-gamma, but not IL-4, is consistent with cytokine expression in human syphilis and provides further support for the hypothesis that there is a Th1 predominance during the early immune response to T. pallidum.

Expression of rabbit interleukin-4 and characterization of its biologic activity on T and B-cells.

The purpose of the current study was to express recombinant rabbit IL-4 (rRbIL-4) and to characterize its biological activity. The cDNA of RbIL-4 was cloned into an insect cell expression vector that allowed for constitutive expression in Sf9 cells and incorporated a 6-histidine tag on the recombinant protein for purification. The purified protein corresponded to the predicted size of rRbIL-4 and was recognized by an anti-human IL-4 antibody in immunoblotting. As shown for IL-4 from other species, a dose-dependent proliferative response was observed in T-lymphoblasts cultured with rRbIL-4. rRbIL-4 also induced increased expression of MHC class II molecules on the surface of rabbit B-cells in a dose-dependent manner. These results indicate that we have produced recombinant rabbit IL-4 that exhibits expected biological activity on rabbit B and T-cells.