Cholecystokinin1 Receptors

Reighard SD, Sweet RL, Vicetti Miguel C, Vicetti Miguel RD, Chivukula M, Krishnamurti U, Cherpes TL

Reighard SD, Sweet RL, Vicetti Miguel C, Vicetti Miguel RD, Chivukula M, Krishnamurti U, Cherpes TL. human 3-D EEC model is usually a robust tool for studying host-microbe interactions and bacterial pathogenesis in the upper FRT. during its ascension from the vagina through the cervical canal (13). Bacterial vaginosis (BV) is the most common vaginal disorder worldwide and is characterized as a depletion of commensal lactobacilli and the overgrowth of and other anaerobic and microaerophilic bacteria (14). The role of in BV is still controversial. Schwebke and colleagues hypothesize that is sexually transmitted and initiates polymicrobial biofilm formation (15, 16). Importantly, BV has a high recurrence rate and is difficult to treat (17). Furthermore, BV is usually associated with serious reproductive sequelae, including endometritis and PID (1, 2). Mitchell et al. tested for the presence of vaginal bacterial species, including commensal lactobacilli (i.e., spp., spp., spp., BVAB1, BVAB2, and BVAB3), in endometrial samples collected from patients undergoing hysterectomy using quantitative PCR assays (18). The study revealed that uteri from 90% of enrolled patients were colonized SR 59230A HCl with at least one vaginal species. Another study tested for the presence of BV-associated bacteria in endometrial specimens using fluorescence hybridization (FISH) (19) and found that 50% of pregnant and nonpregnant women with BV had an endometrium covered with a and are common etiological brokers of sexually transmitted infections (STI) and are associated with chronic endometritis (20). The Centers for Disease Control and Prevention guidelines include gonococcal endometritis, which is usually well-characterized syndrome, as a manifestation of PID ( Wiesenfeld et al. confirmed the association of gonococcal contamination with PID by detecting subclinical PID in 26% of women with cervical gonococcal infections but only 11% of women without gonorrhea. Coinfection with and increased the rate of subclinical PID to 39%, whereas the rate of subclinical PID was 9% for women not infected (21). To study host-microbe interactions in the endometrium, we developed and characterized a three-dimensional (3-D) human endometrial epithelial cell (EEC) culture model, using the rotating wall vessel (RWV) bioreactor technology. Previous 3-D cell culture models have been shown to faithfully recapitulate many morphological and physiological characteristics of tissue microenvironment (22,C27). To our knowledge, this is the first report describing a bioreactor-derived 3-D EEC culture model. In this study, we characterized the morphology and structure of the 3-D EEC model as well as the innate immune responses after microbial challenge. For infection studies, we used a reductionist approach and utilized three SR 59230A HCl species individually, each of which has been shown to ascend from the lower FRT (vagina and cervix) to the upper FRT (uterus): efficiently colonize the 3-D SR 59230A HCl EEC aggregates. We observed that gonococci induce dramatic morphological changes to Rabbit polyclonal to GNMT the cortex of epithelial cells. Additionally, this is the first report showing that bacterial colonies interact with multiple adjacent 3-D cuboidal cells, mostly at their crevices and folds. These phenomena could not be detected in traditional monolayer cultures. We also observed that this 3-D EEC model produced significant levels of proinflammatory immune mediators following contamination with pathogenic gonococci but not following infection with the other tested vaginal bacteria. Moreover, using a well-characterized mutant, which produces nonretractible pilus fibers, we showed that host-microbe conversation is usually strain dependent. Overall, the studies highlighted in this report demonstrate that this human 3-D EEC model is usually a robust tool to study the host innate immune responses to microbial challenges in the upper FRT. RESULTS Morphological characteristics of the human 3-D EEC model. To construct the human 3-D endometrial epithelial cell (EEC) model, we used the rotating wall vessel (RWV) bioreactor technology (28) and the previously established endometrial epithelial cell line HEC-1A. The HEC-1A cell line was isolated in 1968 by H. Kuramoto from a patient with stage IA endometrial cancer (29). These cells were initially produced as monolayers in a tissue culture flask and then transferred to an RWV bioreactor along with collagen-coated porous microcarrier beads. The rotation of the bioreactor creates a low-fluid-shear microgravity environment that maintains the cells in free fall. In this environment, cells utilize the beads as a growth scaffold and form 3-D cellular aggregates. The progression of aggregate formation was periodically monitored by bright-field light and electron microscopy. The formation of aggregates, consisting of microcarrier beads completely covered with endometrial epithelial cells, occurred by day 21 in culture (Fig. 1A). Transmission electron microscopy (TEM) and scanning electron SR 59230A HCl microscopy (SEM) images of these bioreactor-generated human EEC aggregates showed several hallmarks of cellular differentiation and polarization (Fig. 1). First, SEM examination revealed that HEC-1A cells in 3-D culture adhered to collagen-coated beads and grew as.