A NEW MODEL FOR STUDIES ON CERVICOVAGINAL INFLAMMATION AND BARRIER DISRUPTION.

INTRODUCTION: Several microbicide candidates have failed in recent clinical trials due to the induction of vaginal inflammation and epithelial barrier disruption. New preclinical models are needed for assessing inflammation and barrier disruption of the vaginal epithelium, and for testing effects of microbicides in an inflammatory environment. OBJECTIVE: The purpose of this study was to establish a reliable and cost-effective in vitro model of vaginal inflammation, to further characterize molecular events associated with inflammation, and to determine the effects of inflammation on barrier integrity. MATERIALS AND METHODS: EpiVaginal™ organotypic cultures (MatTek Corp., Ashland, MA) were treated with various doses of the proinflammatory cytokine TNF-á for 24hours. Apical and basolateral supernatants of treated tissues were assessed for IL-6, IL-8 and RANTES by Bioplex Cytokine Immunoassay. Adhesion and junctional proteins were identified by immunofluorescence and changes in expression were moni-tored by RT-PCR. Epithelial permeability was determined by transepithelial electrical resistance (TEER), leakage of sodium fluorescein and transmigration of leukocytes from the apical to basal compartment. RESULTS: TNF-á treatment significantly increased the expression of ICAM-1 and basolateral secretion of IL-6, IL-8 and RANTES in Epivaginal cultures. TNF-á- treated cultures had significantly less electrical resistance and were more permeable to fluorescein dye and leukocyte migration. Epivaginal cultures expressed junctional molecules JAM-A, JAM-C, claudin-1, E-cadherin and ZO-1 in a pattern similar to that observed in normal vaginal tissue, and several alterations in the expression of these proteins were observed following TNF-á treatment. CONCLUSIONS: We have developed a new model to study molecular events associated with inflammation in vaginal epithelial tissue. This model could provide important insight into how vaginal permeability is regulated in order to guide future microbicide development. Further, the identification of biomarkers to detect barrier dysfunction could advance the efforts to formulate safe and effective microbicides to prevent the transmission of sexually transmitted pathogens.

Adhesion proteins, Barrier disruption, Claudin-1, Confocal microscopy, E-cadherin, Epithelial barrier disruption, ICAM-1, IL-6, IL-8, Infiltrating leukocytes, Inflammation, JAM-A, JAM-C, Junctional proteins, RANTES, Sodium fluorescein, TEER, Vaginal inflammation, ZO-1

TNF-á