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02438 DEVELOPMENT OF AN IN VITRO DUAL-CHAMBER MODEL FOR EVALUATION OF CANDIDATE MICROBICIDES Van Herrewege, Yven * A dual-chamber system of an endocervical epithelial cell line ME-180 (in the apical chamber) and co-cultures of monocyte-derived dendritic cells (MO-DC)/CD4 T cells (in the basal chamber) was used to model sexual HIV transmission. For microbicide evaluation, a confluent layer of ME-180 cells was cultured in a laminin coated apical chamber, of which the bottom consisted of a microporous membrane (pore size:3 µm). ME-180 cells were pre-treated with compound (1 h). The apical chamber was inserted into the basal chamber and cell-associated HIV-1 Ba-L was added to the apical chamber, which was removed 24 hours later. Cells of the basal chamber were cultured for 14 days, without compound or added cytokines. Culture supernatants were analysed by ELISA for the presence of HIV antigen. Several compounds were evaluated, including non-nucleoside reverse transcriptase inhibitors (NNRTIs) (UC-781, TMC120) and polyanionic entry inhibitors (PRO2000, DS-5000, Cellulose Sulphate). Toxicity of the compounds towards the epithelial layer was analysed both microscopically and by FACS. Infection was prevented with 0.1 µM TMC120 or 1 µM UC-781. Surprisingly, none of the entry inhibitors completely blocked infection at a concentration of 100µg/ml. Cell death was induced above 1 µM TMC120 or 10 µM UC-781. No cell death was induced by the entry inhibitors. However, intercellular contacts between epithelial cells were broken and cells detached from the culture plate after a 24-hours treatment with 250 µg/ml or more of an entry inhibitor . These results indicate NNRTIs might be potent candidate microbicides. Van Herrewege Yven |
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