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-␥) 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-␥, 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.Natural infection with Treponema pallidum subsp. pallidum, the causative agent of syphilis, results in the formation of a primary lesion, or chancre, at the site of infection. A vigorous immune response develops early during infection and results in local clearance of the majority of treponemes and resolution of the primary lesions. The cell phenotypes and cytokines present in early syphilitic lesions during human infection and experimental infection in the rabbit models suggest that the early immune response to T. pallidum is characteristic of a delayedtype hypersensitivity or Th1 response. A large number of mononuclear cells, consisting mostly of T cells and macrophages, infiltrate early lesions prior to bacterial clearance and lesion healing (15,23,24,28). CD4 ϩ helper T cells are believed to mediate bacterial clearance primarily through the production of cytokines, such as gamma interferon (IFN-␥), which activate macrophages (14, 30). Macrophages then engulf and kill opsonized treponemes (2, 17). There is also evidence that activated cytolytic (CD8 ϩ ) T lymphocytes participate in the local immune response within lesions, although their role in clearance remains unclear (18, 31). An important role for antibody in bacterial clearance has also been demonstrated (3, 4, 16); however, reports vary on the relative abundance of B cells within the le...

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 TM 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.

BACKGROUND: Prostate cancer (PCa) is the second most common cancer in men and one of the leading causes of cancer death. Resistance to current PCa therapies, including androgen deprivation, occurs in almost all patients leading to development of castration resistant prostate cancer (CRPC). Resistance is associated with expression of splice variants of the androgen receptor (AR-Vs) that are constitutively active. The intrinsically disordered N-terminus of the AR/AR-Vs makes targeting the AR-Vs difficult. Thus, therapies that indirectly target the AR by inhibiting factors important in regulating AR levels and activity may be the most successful against inhibiting the constitutively active AR variants. Kinases have been shown to be important in regulating the AR, thus kinase inhibitors have the potential to inhibit androgen receptor signaling and function. OBJECTIVE: To demonstrate that our lead kinase inhibitor targets and inhibits AR positive CRPC. RESULTS: Bumped kinase inhibitors designed at the University of Washington are based on a pyrazolopyrimidine backbone, with R1 and R2 groups that confer a “bump” thereby preventing the BKIs from binding to most human kinase ATP binding pockets. While the majority of BKIs do not have activity against CRPC, a subset have activity against AR+ PCa lines, including CRPC lines expressing AR-Vs, but with no activity against AR negative PCa lines or a broad range of other human cell lines. We next examined the effects of our lead BKI compound (1553) on AR expression, phosphorylation, and function. BKI-1553 blocked transactivation activity of AR as judged by the reporter assays using an ARE-luciferase construct. Expression of canonical AR target genes was downregulated in LNCaP cells treated with BKI-1553. Westerns demonstrated a decrease in total AR 4 and 24 hours after treatment with BKI-1553. Levels of phospho-Serine81on the AR, which is critical for AR activity, were decreased even further than total AR by 4 hours after treatment. Thus, AR signaling appears to constitute BKI-sensitive components. We then decided to examine the effect of BKI-1553 on in vivo tumor growth. Toxicity studies demonstrated that it was non-toxic in mice at oral doses that achieved therapeutic levels. In vivo treatment with BKI-1553 significantly decreased growth of a castrate-sensitive patient-derived PCa xenograft and a castration-resistant PCa cell line (LuCaP35, p < 0.01 and LNCaP95, p < 0.005, respectively). SUMMARY: Our lead bumped kinase inhibitor, BKI-1553, selectively inhibits the growth of prostate cancer cells that express full-length AR and/or AR-Vs. BKI-1553 reduced not only cell proliferation, but decreased levels of AR and AR variants, decreased pSer81 levels, and decreased activation of AR-regulated promoters. Since the majority of CRPCs are AR driven, our targeted BKIs could be a novel therapy for cancers that are resistant to currently available therapies. Citation Format: Takuma Uo, Shihua Sun, Kathleen Haugk, Kathryn Soriano Epilepsia, Mamatha Damodarasamy, Matthew Hulverson, Wesley VanVoorhis, Kayode K. Ojo, RamaSubbaRao Vidadala, Dustin Maly, Stephen Plymate, Cynthia C. Sprenger. Bumped kinase inhibitor 1553 selectively inhibits androgen receptor positive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3837.

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