We previously developed an in vitro transwell chamber assay that mimics HIV-1 transcytosis through primary genital epithelial cells . Specifically, PGEC were seeded onto polycarbonate membrane transwells and cultured until formation of tight junctions is achieved. The monolayer on the filter effectively Everolimus inquirer divides the well into an apical compartment and a basolateral compartment. To ensure the integrity of the PGEC barrier, the elevated transepithelial electrical resistance of each monolayer was monitored as well as the paracellular passage of the extracellular marker inulin. We found that TER was low at day 1 around 100 Ohm?cm2, peaked at day 2 up to 400 Ohm?cm2 and remained constant for 2 additional days. HIV-1 was added to the apical surface of PGEC in the upper chamber of the transwell system for 8 h at 37uC and the CYT387 JAK inhibitor amount of transcytosed virus was quantified by p24 ELISA in the lower chamber medium in contact with the basal PGEC surface. We previously determined that HIV-1 transmigration through PGEC monolayers is maximal after 8 h. Infectivity of transcytosed HIV-1 after 8 h was analyzed using TZM reporter target cells. Wild-type, but not envelope deficient HIV-1, transmigrated through PGEC, demonstrating that the PGEC layer does not allow nonspecific transmigration of viruses. Although HIV-1 crosses PGEC as infectious particles, the efficiency of transcytosis is extremely poor , suggesting that the genital epithelium serves as a major barrier against HIV-1. We measured 39.8% of viral input present in lower chamber in a control well with no cells so that one could determine how much HIV-1 crosses in the setting of ����complete disruption����. After establishing the HIV-1 transmigration assay, we defined conditions that would allow us to examine the enhancing effect of HSV infection of PGEC on HIV-1 transmigration. To address this issue, PGEC monolayers were exposed to a low HSV MOI. After 2 days, PGEC were washed and exposed this time to HIV-1. HIV-1 transmigration was quantified 8 h post-HIV-1 exposure. We first found that 55�C85% of PGEC were infected 2 days post-HSV exposure. The integrity of the PGEC barrier was monitored before and after HSV exposure. HSV enhanced the passage of FITC-inulin and decreased the TER of the monolayer, whereas ACV ) abolished these effects. These data confirm that HSV can diminish the impermeability of the PGEC barrier. It is important to note that despite the fact that a significant amount of PGEC were infected with the low HSV MOI, the integrity of the epithelial barrier was still partially preserved 2 days post-HSV infection. However, 3�C4 days post-HSV infection the PGEC barrier was severely disrupted. Moreover, we found that higher MOI totally disrupt the integrity of the PGEC barrier already after 2 days. Based on these observations, we chose for all subsequent experiments to use a low HSV MOI for the initial PGEC infection, and to subsequently expose HSV-infected PGEC to HIV-1 2 days post- HSV infection.