Genital Chlamydia trachomatis serovars D-K are responsible for epidemic sexually transmitted infections in the USA, affecting over 4 million men, women and infants per year. In women, without treatment, chlamydiae may spread canalicularly from the endocervical canal to the endometrium (endometritis), and the fallopian tubes (salpingitis)- the constellation of which is defined as pelvic inflammatory disease. As a result of tubal scarring and/or impaired ovum transportation, ectopic pregnancy and infertility can occur. Clearly, chlamydial diseases constitute significant primary, secondary and tertiary health care concerns in which women bear a special burden because of their increased risk of adverse reproductive consequences. Effective topical microbicides are urgently needed to help prevent and control sexually transmitted infections. The purpose of this grant is to evaluate the action of the topical microbicide C316 (and its derivatives B58A and B64D) versus nonoxynol-9 at pH 5.7 and 7.0 on Chlamydia trachomatis serovar E and its target host cell. The concentrations of the microbicides will be quantitated for compatibility and cytotoxicity with polarized human genital epithelial cells (squamocolumnar, columnar) using fluorescein diacetate and propidium iodide in Specific Aim 1 and with isolated chlamydiae in Specific Aim 2. Both radiolabeled infectious elementary bodies (EB) and metabolically active reticulate bodies will be analyzed for loss of antigens by SDS-PAGE, Western blots, autoradiography, and immunoelectron microscopy and for viability: microbicide-exposed EB will also be assayed for attachment to host epithelial cells and inclusion development.
In Specific Aim 3, the effect of microbicide exposure on polarized human genital epithelial cells (a) normally infected and (b) persistently infected with chlamydiae will be examined by fluorescence and transmission electron microscopy, and modulation of the effect by estrogen and estrogen plus progesterone will be assessed in Specific Aim 4. Unexposed and microbicide-exposed infected polarized human epithelial cells will be compared for increased premature chlamydial antigen release from inclusions and antigen secretion to host cell surface - by post-embedding immunolabeling of electron microscopy samples processed in Lowicryl (Specific Aim 5). Finally, we shall determine wether or not inflammatory response cell migration to chlamydiae- infected polarized epithelial cells is modulated by microbicide exposure.
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