Background: HIV is the leading cause of death for reproductiveage women, with the majority of transmission events occurring via heterosexual intercourse. The first site of exposure is the vagina, and understanding its immune environment, which changes with hormone levels across the menstrual cycle, is crucial to developing effective vaccines and prevention measures. Methods: Using a menstrual cup, we developed a novel technique to concurrently recover fresh undiluted vaginal secretions (VS) and vaginal epithelial cells (VEC) at mid-proliferative, ovulatory and mid-secretory stages of the menstrual cycle. VEC were isolated and treated in 96-well plates with estradiol (E 2 : 5x10 -8 M), progesterone (P 4 : 1x10 -7 M), or a combination of both for 48hrs after which conditioned media (CM) were recovered. VEC CM and VS were analyzed for presence of anti-HIV proteins by ELISA, and anti-HIV activity using a TZM-bl assay. Results: CCL20, RANTES, elafin, HBD2, SDF-1a and IL-8 were present in VS. VS demonstrated significant inhibition of X4 (IIIB) HIV and increased infectivity of reporter cells by R5 (CH077.t) HIV. No inhibitory effect was seen for BaL and CH058. No significant differences in either antiviral protein concentration or anti-HIV activity with respect to menstrual cycle stage were measured, but marked differences were observed in both parameters over the course of the cycle between different women, and in consecutive cycles from the same woman. For VEC, E 2 significantly decreased the secretion of HBD2 and elafin over 48hrs. CM from E 2 -or P 4 -treated VEC had no anti-HIV activity. However, CM from E 2 /P 4 co-treated cells significantly inhibited both R5 and X4 HIV. Conclusions: There are multiple levels of protection in the vagina. The sensitivity of VEC to E 2 and P 4 suggests that their contribution to immune protection varies across the menstrual cycle. The variation in anti-HIV activity in VS demonstrates that immune protection is not constant and that factors in addition to hormones influence antiviral protection. P40.25 Antiviral Responses of Fibroblasts in the Female Reproductive TractBackground: The female reproductive tract (FRT) is the primary location of heterosexual transmission of HIV. However, the intricacies of immune protection at this site are not well understood, in particular the contribution of stromal fibroblasts in preventing HIV infection. Fibroblasts are the predominant cell type in the sub-epithelial layers of the FRT, and the initial stages of HIV infection occur in their presence. Methods: Benign FRT tissues from the endometrium (EM), endocervix (Cx) and ectocervix (ECx), were recovered from HIVwomen undergoing hysterectomy. Following enzymatic digestion, cells were passed through nylon filters to isolate purified populations of fibroblasts. These were maintained in culture for 4-6 days prior to treatment with estradiol (E 2 : 5x10 -8 M) for up to 72hrs in the presence or absence of viral stimuli. Conditioned media (CM) was recovered and analyzed for the presence of anti-HIV prote...
Background: HIV is the leading cause of death for reproductiveage women, with the majority of transmission events occurring via heterosexual intercourse. The first site of exposure is the vagina, and understanding its immune environment, which changes with hormone levels across the menstrual cycle, is crucial to developing effective vaccines and prevention measures. Methods: Using a menstrual cup, we developed a novel technique to concurrently recover fresh undiluted vaginal secretions (VS) and vaginal epithelial cells (VEC) at mid-proliferative, ovulatory and mid-secretory stages of the menstrual cycle. VEC were isolated and treated in 96-well plates with estradiol (E 2 : 5x10 -8 M), progesterone (P 4 : 1x10 -7 M), or a combination of both for 48hrs after which conditioned media (CM) were recovered. VEC CM and VS were analyzed for presence of anti-HIV proteins by ELISA, and anti-HIV activity using a TZM-bl assay. Results: CCL20, RANTES, elafin, HBD2, SDF-1a and IL-8 were present in VS. VS demonstrated significant inhibition of X4 (IIIB) HIV and increased infectivity of reporter cells by R5 (CH077.t) HIV. No inhibitory effect was seen for BaL and CH058. No significant differences in either antiviral protein concentration or anti-HIV activity with respect to menstrual cycle stage were measured, but marked differences were observed in both parameters over the course of the cycle between different women, and in consecutive cycles from the same woman. For VEC, E 2 significantly decreased the secretion of HBD2 and elafin over 48hrs. CM from E 2 -or P 4 -treated VEC had no anti-HIV activity. However, CM from E 2 /P 4 co-treated cells significantly inhibited both R5 and X4 HIV. Conclusions: There are multiple levels of protection in the vagina. The sensitivity of VEC to E 2 and P 4 suggests that their contribution to immune protection varies across the menstrual cycle. The variation in anti-HIV activity in VS demonstrates that immune protection is not constant and that factors in addition to hormones influence antiviral protection. P40.25 Antiviral Responses of Fibroblasts in the Female Reproductive TractBackground: The female reproductive tract (FRT) is the primary location of heterosexual transmission of HIV. However, the intricacies of immune protection at this site are not well understood, in particular the contribution of stromal fibroblasts in preventing HIV infection. Fibroblasts are the predominant cell type in the sub-epithelial layers of the FRT, and the initial stages of HIV infection occur in their presence. Methods: Benign FRT tissues from the endometrium (EM), endocervix (Cx) and ectocervix (ECx), were recovered from HIVwomen undergoing hysterectomy. Following enzymatic digestion, cells were passed through nylon filters to isolate purified populations of fibroblasts. These were maintained in culture for 4-6 days prior to treatment with estradiol (E 2 : 5x10 -8 M) for up to 72hrs in the presence or absence of viral stimuli. Conditioned media (CM) was recovered and analyzed for the presence of anti-HIV prote...
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