Polarized tissue constructs VEC-100™ derived from primary ectocervical/vaginal epithelial cells, previously depicted immune properties comparable to that of normal tissues of origin [37, 38] were purchased from MatTek Corporation, Ashland, MA. The VEC-100™ tissues were maintained in antibiotic-free medium provided by MatTek. Recovery of cryopreserved wild type bacteria and bioengineered derivatives Multiple aliquots from three separate batches of L. jensenii WT and derivatives were received

frozen from Osel, Inc and stored at −80°C until tested. Each batch was examined in a minimum of three independent experiments. All strains were tested simultaneously by comparison of colony forming units (CFU) before use in our epithelial colonization model.

For that purpose, one aliquot per strain from each batch was thawed, washed once in PBS by centrifugation, serially diluted in PBS and plated onto Brucella-based agar plates learn more GSK1210151A (PML Microbiologicals, Wilsonville, OR). Plates were incubated in an anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, MI) containing an atmosphere of 10% carbon dioxide, 10% hydrogen, 80% nitrogen at 37°C for 24 h-48 h (until visible colonies formed), GSK2118436 purchase followed by CFU counting. Percent recovery of viable bacteria was determined in comparison to CFU counts obtained prior to cryopreservation by Osel, Inc. Epithelial colonization L. jensenii suspensions were prepared in antibiotic-free KSFM (Invitrogen) at 7×106 CFU/ml to colonize epithelial surfaces for 24 h, 48 h and 72 h as previously described for other vaginal bacteria [20]. In the immortalized cell line model, epithelial monolayers were grown to 100% confluence in 96-well plates (Fisher Scientific, Pittsburgh, PA) and bacterial suspensions (0.1 ml) were added to achieve a multiplicity of infection of ~10:1. In the VEC-100™ model, tissue inserts were placed over 0.5 ml medium in

12-well plates (Fisher Scientific) followed by heptaminol addition of 0.156 ml bacterial suspension to the apical epithelial surface. The bacterial-epithelial cocultures were incubated for 24 h-72 h under anaerobic conditions generated by AnaeroPack System (Mitsubishi Gas Chemical Co. Inc., New York, NY), at 35°C on an orbital shaker. Cell culture supernatants from the immortalized epithelia and basal chamber culture fluids from the VEC-100 tissue model were collected in 24 h time intervals for measurement of soluble immune mediator levels and mCV-N as described below. At the end of each 24 h period the cells/tissue were washed and used for enumeration of epithelia-associated CFU (see below), or medium was reapplied and cultures were returned to anaerobic chamber for additional 24 h incubations. In some experiments, the cells were lysed for assessment of NF-κB activation or apoptosis (see sections below). Transmission electron microscopy Vk2/E6E7 cells were seeded on Aclar embedding film (Ted Pella Inc. Redding CA) and colonized with L. jensenii strains for 24 h.