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Dissertation
Using an in vitro model of the polarized cervicovaginal epithelium to investigate the immunomodulatory effects of human semen and impact on the risk of human immunodeficiency virus 1 (HIV-1) transmission
Doctor of Philosophy (Ph.D.), Drexel University
Nov 2013
DOI:
https://doi.org/10.17918/00007853
Abstract
Factors in seminal fluid (SF) that have integral roles in reproductive biology may alter the ability of the human immunodeficiency virus type 1 (HIV-1) to penetrate the cervicovaginal epithelium and infect target immune cells. We hypothesized that the consequences of SF exposure would be (i) direct alterations in cervicovaginal epithelial tissue tight junction integrity, resulting in changes in epithelial barrier function with respect to HIV-1 passage, and (ii) polarized release of specific immunomodulatory factors by the epithelium that will modulate the local immune response. To address this possibility, human cell lines derived from three different regions of the female reproductive tract - vagina, ectocervix, and endocervix - were cultured in a transwell cell culture system to model the polarized cervicovaginal epithelial barrier. These polarized monolayers were then exposed apically to SF from healthy donors. Changes in barrier integrity were measured over time by monitoring transepithelial electrical resistance (TEER) and permeability, while protein expression in conditioned media was quantified using a multiplex bead-based immunoassay (Luminex). Apical exposure to SF caused a rapid increase in epithelial integrity across all cell lines by 30 min post-exposure, and a sustained peak in TEER by 3-4 h that persisted for approximately 48 h post-exposure. Epithelial TEER responses to SF and whole semen were similar, indicating that sperm cells did not contribute to this effect. Additional studies demonstrated that a combination of calcium, lipids, and heat labile proteins in SF were responsible for the observed changes in resistance. Measurements of immunomodulatory factors secreted by epithelial cell monolayers after SF exposure demonstrated that factor release varied in a cell-type and time-dependent manner. Considerable cell type-dependent differences were also noted. In preliminary experiments using a HIV-1-responsive reporter cell line, SF exposure also resulted in a reduction in HIV-1 long terminal repeat (LTR) activity, suggesting that SF may reduce the risk of HIV-1 infection during virus transmission. Ongoing studies are investigating changes in epithelial permeability to HIV-1 following SF exposure, the mechanism(s) by which SF affects epithelial cell tight junction integrity, and the downstream effects of epithelium-derived immunomodulatory factors on immune cell recruitment, activation, differentiation, and susceptibility to HIV-1 infection.
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Details
- Title
- Using an in vitro model of the polarized cervicovaginal epithelium to investigate the immunomodulatory effects of human semen and impact on the risk of human immunodeficiency virus 1 (HIV-1) transmission
- Creators
- Shawn Keogan
- Contributors
- Fred C. Krebs (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xvii, 282 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991021888742004721