((same orientation and magnification), but displayed as a 3D topographical reconstruction. surface. In quantitative assays, pretreatment with a blocking antibody directed against 1 integrin reduced HIV virion binding. Collectively, these results highlight a continuum of complex interactions that occurs when natural sources of HIV infectivity are deposited onto mucosal surfaces in the female reproductive tract. Keywords: confocal microscopy, female reproductive tissue, HIV transmission, organ culture, tissue surface reconstruction Today women account for more than half of the newly HIV-infected adults worldwide, and most women acquire HIV through heterosexual exposure (1C6). There is, however, only limited information available concerning the specific cellular and molecular events that lead to HIV transmission through sexual activity. Semen contains either cell-free virus or cell-associated virus (7C12), and each source of HIV infectivity may require different mechanisms to establish infection. Clear understanding of the molecular interactions between sources of infectivity and mucosal surfaces will be crucial in developing topical agents to prevent sexual HIV transmission. The current view of sexual HIV transmission has largely been developed from simian immunodeficiency virus (SIV) infections of female nonhuman primates (reviewed in ref. 4). When macaques were exposed to cell-free SIV intravaginally, infected resident cells were consistently detected in reproductive tract tissues before being detected in the draining lymph nodes (13). Taken together, the data from macaque infections indicate that SIV (100% infectious dose of cell-free virus) readily traverses an epithelial surface and establishes focal infections within susceptible resident cells in female reproductive tissues and then the infection spreads systemically. For heterosexual HIV transmission, the properties of the infectious inoculum are highly variable as cell-free HIV virions (12) or HIV-infected cells may be shed in semen (9). Furthermore, both soluble and cellular constituents of semen may induce transient changes in exposed epithelial surfaces, resulting in increased susceptibility to primary HIV infection (14, 15). The complex nature of mucosal surfaces presents a major challenge for comprehensive cellular and molecular studies of microbial infections (16). The development of organ cultures with human mucosal tissues has provided some insight into HIV infection at these sites. Although methods for organ culture have varied, previous studies with human cervical tissue have shown that cells in the submucosa are susceptible to HIV infection (17C19). Additionally, organ cultures have been used to evaluate inhibitors of HIV infection and replication (19C21). We have previously described binding of HIV virions and seminal cells to the mucosal surface of palatine tonsil in a reconstruction of HIV transmission after oral exposure (22). Here, we set out to study sexual transmission of HIV at mucosal surfaces of the cervix. The selection of human cervical tissue allowed the study of both Rabbit Polyclonal to S6K-alpha2 the stratified epithelium of the ectocervix, which closely resembles the vaginal epithelial surface, and the columnar epithelium of the endocervix. Each epithelial surface may interact differently with sources of HIV infectivity. Our organ culture methods have the advantage of using human materials for the target mucosal tissue and biologically CX-6258 relevant sources of infectivity: semen from HIV-seropositive donors and a cell-free CX-6258 stock of HIV virions obtained by limited passage of a primary patient isolate. Methods Tissue Samples. Cervical tissues from premenopausal women with conditions not involving the cervix were processed in the laboratory within 1C3 h of completion of surgery. The experimental protocols had full Institutional Review Board approval. Cervical tissue with external epithelial CX-6258 surfaces (10C25 mm2) was mounted in agarose medium such that the intact epithelial surface remained exposed and all cut surfaces were covered with agarose (22). The stratified epithelium of the ectocervix remained largely intact for up to 48 h, then squamous epithelial cells sloughed away but the basal epithelial cell layer remained intact for 7C10 days. An intact endocervical columnar epithelium was retained for 4C6 days and continued to secrete mucus. Tissue pieces in agarose were infected and processed as described (22, 23). Alternatively, in some infections, cervical tissue was submerged in cell-free virus and then the tissue was cultured on collagen sponges at the gas-medium interface. In all experiments reported here, infections were performed with a dual tropic primary patient isolate (HIV 96-480; refs. 22 and 23), and binding studies were performed with a noninfectious X4 tropic virion preparation, HIV-GFP (ref. 22; see and and cellular activation signals. Collectively, the results shown in Fig. 1 validate the infection of cervical organ cultures with a primary isolate of HIV and CX-6258 provide specific information from a human experimental.