Human Genital Epithelial Cells Research Articles

Susceptibility of Human Female Primary Genital Epithelial Cells to Herpes Simplex Virus, Type-2 and the Effect of TLR3 Ligand and Sex Hormones on Infection1

Genital epithelial cells (ECs) are the first line of defense that sexually transmitted viruses encounter. The mechanism of viral pathogenesis in these cells is not well understood. Here, we show that a primary cell culture model from human reproductive tract tissues can be used as a novel ex vivo model in examining the interaction of herpes simplex virus, type 2 (HSV-2), with female genital mucosa. Confluent, polarized primary cultures of human endometrial and cervical ECs were established and shown to be free from any significant contamination of any other cell type. Both endometrial and cervical ECs were found to be highly susceptible to HSV-2 infection. The kinetic of infection was similar to in vivo infection, with the earliest viral shedding seen at 18 h postinfection. Primary EC monolayers could be infected both apically and basolaterally, but preferential viral shedding was seen on the apical side of cells. Following treatment of the monolayers with poly (I:C), an innate immune activator that acts via TLR3, viral shedding was reduced significantly, comparable to levels seen when an antiviral formulation, acyclovir, was used. Treatment of epithelial and stromal co-cultures with estradiol increased HSV-2 infection in endometrial ECs, but viral shedding decreased following treatment with progesterone. To the best of our knowledge, this is the first study that examines the interaction of primary human female genital ECs with HSV-2, using an ex vivo culture model. The results provide valuable information regarding the susceptibility of women's genital ECs to HSV-2 and the ability of innate immunity and hormones to modify this susceptibility.

Expression and Function of Bactericidal/Permeability‐Increasing Protein in Human Genital Tract Epithelial Cells

Genital tract epithelia regularly encounter and adapt to the existence of bacterial pathogens. This study provides evidence that the endocervical and ectocervical epithelia of the human female genital tract express bactericidal/permeability-increasing protein (BPI). The constitutive expression of BPI was restricted to cell-bound protein and unaffected by human papillomavirus type 16/E6E7 immortalization and proinflammatory cytokine stimulation. Epithelial BPI was, in part, responsible for killing a commensal strain of Escherichia coli. The results of the present study suggest that BPI is tightly regulated and functionally expressed by epithelial cells in the female reproductive tract and may play a role in regulating bacterial colonization in the genital mucosa.

Selective sequestration of X4 isolates by human genital epithelial cells: Implication for virus tropism selection process during sexual transmission of HIV

X4 and R5 HIV strains are present in the semen of men infected with HIV but R5 isolates are transmitted preferentially. The role of human epithelial cells in this selection is addressed. Three human cervical cell lines-CaSki, SiHa, and HEC1A-and normal human vaginal cells from HIV-negative donors were characterized for HIV receptor expression and incubated with X4 and R5 laboratory-adapted strains or primary isolates. The infection was assessed by detection of intracellular HIV DNA. The three cell lines were shown to express on their surface the CXCR4 and GalCer molecules, but not the CD4 and CCR5 ones. The three cell lines and normal human vaginal cells were found to be selectively permissive to X4 HIV entry; the preincubation of the cell lines with rhSDF-1 inhibited this infection. The detection of the intracellular proviral DNA in the cell lines and in normal human vaginal cells demonstrated a selective integration of X4 strains. Additional experiments showed that no extracellular RNA was detected in the supernatants of HEC1A cells infected by X4 isolates either after 18 days of culture or after incubation with PHA-stimulated PBMCs and that no transmission occurred after co-culture between infected HEC1A cells and PHA-stimulated PBMCs. These results suggest specific sequestration of X4 strains by genital epithelial cells, which could explain, at least in part, the HIV tropism selection process during sexual intercourse.

Stampidine is a potential nonspermicidal broad-spectrum anti-human immunodeficiency virus microbicide

Objective Stampidine (2,′3′-didehydro-3′-deoxythymidine-5′-( p-bromophenyl methoxy alaninyl phosphate) is a novel aryl phosphate derivative of stavudine/d4T with broad-spectrum anti-HIV activity in vitro and in vivo. This study investigated the potential utility of stampidine as a nonspermicidal microbicide. Design Prospective, controlled study. Setting Center for Advanced Preclinical Sciences and Reproductive Biology Department. Patient(s) Seven sperm donors. Animal(s) Fifty-two sexually mature, female and twenty-four male New Zealand white rabbits. Intervention(s) Human semen and genital tract epithelial cells were exposed to stampidine (up to 1 mM). Ovulated does in subgroups of 12 were artificially inseminated with rabbit semen pretreated with stampidine (1 mM) or vehicle. Does in subgroups of four and three, respectively, were exposed intravaginally to a gel or a thermoreversible ovule formulation with and without 0.5%, 1.0%, or 2.0% stampidine (9 to 36 mM) for 14 days. Main outcome measure(s) Effect of stampidine on human sperm motility, kinematics, penetration through cervical mucus, and epithelial cell viability. Reproductive parameters on gestation day 8. Vaginal tissues were histologically scored 24 hours after dosing. Result(s) Exposure of human sperm to stampidine even at a concentration 10 6-times higher than its in vitro anti-HIV-1 activity (50% inhibitory concentration = 1 nM) had no adverse effect on sperm motility, kinematics, cervical mucus penetrability, or the viability of vaginal and endocervical epithelial cells. Reproductive indices of pregnancy rate, embryo implantation, and preimplantation losses were not affected by pretreatment of rabbit semen with stampidine. Gel formulations of 0.5% to 2.0% stampidine (9 to 36 mM) lacked mucosal toxicity. Conclusion(s) The broad-spectrum anti-HIV agent stampidine had no adverse effect on sperm functions, was not cytotoxic, and did not induce mucosal toxicity. Stampidine has clinical potential as a prophylactic microbicide without contraceptive activity.

Pokeweed antiviral protein: a potential nonspermicidal prophylactic antiviral agent

Objective: To investigate the effects of pokeweed antiviral protein (PAP), a 29-kDa anti-human immunodeficiency virus (HIV) protein purified from the leaves of Phytolacca americana, on human sperm function. Design: Prospective, controlled study. Setting: Reproductive biology department. Patient(s): Seven sperm donors. Intervention(s): Human sperm and female genital tract epithelial cells were exposed to PAP ranging in concentration from 1 to 1,000 μg/mL. Main Outcome Measures: Effect of PAP on sperm motility, kinematics, and sperm penetration through bovine mucus, as well as binding, penetration, and fusion of zona-free hamster eggs. Results: Exposing human sperm to PAP (IC 50 p24 = 14 ± 2 nM) did not affect sperm motility and kinematics over a dose range of 1 to 1,000 μg/mL. Treating sperm with either 100 or 1,000 μg/mL of PAP had no effect on cervical mucus penetrability, nor did it affect sperm binding, penetration, and fusion of zona-free hamster eggs. PAP was noncytotoxic to genital-tract epithelial cells. Conclusions: The broad-spectrum antiviral agent PAP was nontoxic to human sperm and female genital tract epithelial cells even at a concentration 2,000 times higher than its IC 50 value against HIV-1. PAP has particular clinical usefulness both as a nonspermicidal intravaginal microbicide and as a prophylactic antiviral agent that can inactivate infective viruses and virus-infected cells in semen before assisted reproductive technology procedures are undertaken.

Thymidine kinase-independent intracellular delivery of bioactive nucleotides by aryl phosphate derivatives of bromo-methoxy zidovudine (compounds WHI-05 and WHI-07) in normal human female genital tract epithelial cells and sperm.

The compounds WHI-05 (5-bromo-6-methoxy-5, 6-dihydro-3'-azidothymidine-5'-[p-methoxyphenyl] methoxyalaninyl phosphate) and WHI-07 (5-bromo-6-methoxy-5, 6-dihydro-3'-azidothymidine-5'-[p-bromophenyl] methoxyalaninyl phosphate) are aryl phosphate derivatives of zidovudine (ZDV) with dual-function anti-human immunodeficiency virus and contraceptive activity. These drugs were rationally designed to bypass the thymidine kinase (TK) dependency of ZDV activation as well as to achieve spermicidal activity. We investigated the TK activity and intracellular metabolism of WHI-05 and WHI-07 in normal human vaginal and cervical epithelial cells as well as sperm. The time- and concentration-dependent intracellular formation of ZDV metabolites following addition of WHI-05 and WHI-07 to normal human vaginal, ectocervical, and endocervical epithelial cells as well as motile sperm was studied by analytical HPLC. Thymidine kinase activity in these cells was determined by the flow cytometric method based on intracellular phosphorylation of the fluorescent nucleoside, 5-amino-2-deoxyuridine-dansyl chloride and by the ability of cell-free extracts to convert [(3)H]thymidine to thymidine monophosphate in comparison to NALM-6, a pre-B leukemia cell line. TK activity of genital tract epithelial cells and sperm was found to be relatively low or lacking. Addition of WHI-05 and WHI-07 to vaginal and cervical epithelial cells resulted in their concentration- and time-dependent conversion to alaninyl ZDV monophosphate (Ala-ZDV-MP) and 5'-ZDV monophosphate as the major metabolites. Studies using motile human sperm also demonstrated the conversion of WHI-05 and WHI-07 to Ala-ZDV-MP. These results demonstrate that human female genital tract epithelial cells and sperm efficiently convert WHI-05 and WHI-07 to bioactive ZDV metabolites despite their TK deficiency.

Activation of the focal adhesion kinase signal transduction pathway in cervical carcinoma cell lines and human genital epithelial cells immortalized with human papillomavirus type 18.

The inappropriate activation of protein-tyrosine kinases (PTKs) has been associated with initiation and progression of several types of human cancers. We therefore postulated that immortalization by DNA tumor viruses results in the induction of PTKs fundamental to these processes. An RT-PCR-based screen was thus used to identify PTKs that were abundantly expressed in HPV-18-immortalized epithelial cells and HPV-containing carcinoma cell lines. One of the genes isolated in this screen was the focal adhesion kinase (FAK; pp125FAK), a cytoplasmic protein kinase that is activated in v-src transformed cells or by stimulation with mitogenic polypeptides. FAK also becomes catalytically active upon integrin engagement with extracellular matrix proteins, such as fibronectin. We found that FAK expression and activity were significantly elevated in HPV-18 E6/E7-immortalized human genital epithelial cells relative to their primary cell counterparts. Protein expression and tyrosine phosphorylation of the putative FAK substrate, paxillin, were also notably increased upon HPV-18 immortalization of genital epithelial cells and in HPV-containing cervical carcinoma cell lines. Most significantly, these cells expressed markedly higher levels of both intracellular and extracellular fibronectin, thus providing a mechanism for activation of FAK and increased tyrosine phosphorylation of paxillin. These findings suggest a role for the integrin/FAK-mediated signaling pathway in cervical carcinogenesis and represent one of the first demonstrations of a tyrosine kinase whose activity is elevated following viral immortalization.

The microbicidal agent C31G inhibits Chlamydia trachomatis infectivity in vitro.

Safe and effective vaginal microbicidal compounds are being sought to offer women an independent method for protection against transmission of sexually acquired pathogens. The purpose of this study was to examine the efficacy of two formulations of one such compound, C31G, against Chlamydia trachomatis serovar E alone, its host epithelial cell (HEC-1B) alone, and against chlamydiae-infected HEC-1B cells. Preexposure of isolated, purified infectious chlamydial elementary bodies (EB) to C31G, at pHs 7.2 and 5.7, for 1 h at 4 degrees C resulted in reduced infectivity of EB for HEC-1B cells. Examination of the C31G-exposed 35S-EB on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs and by Western blotting revealed a C31G concentration-dependent and pH-dependent destabilization of the chlamydial envelope, resulting in the release of chlamydial lipopolysaccharide and proteins. Interestingly, when the host human genital columnar epithelial cells were infected with chlamydiae and then exposed to dilute concentrations of C31G which did not alter epithelial cell viability, chlamydial infectivity was also markedly reduced. C31G gained access to the developing chlamydial inclusion causing damage to or destruction of metabolically active reticulate bodies as well as apparent alteration of the inclusion membrane, which resulted in premature escape of chlamydial antigen to the infected epithelial surface. These studies show that the broad-spectrum antiviral and antibacterial microbicide C31G also has antichlamydial activity.

Transforming growth factors beta 1 and 2 transcriptionally regulate human papillomavirus (HPV) type 16 early gene expression in HPV-immortalized human genital epithelial cells

Human papillomavirus type 16 (HPV16) early proteins E6 and E7 have been implicated in maintenance of the malignant phenotype in cervical cancer. Transforming growth factors beta one and two (TGF betas 1 and 2), polypeptides that regulate cellular growth and differentiation, reversibly inhibited expression of the HPV16 E6 and E7 genes in several immortal genital epithelial cell lines. Loss of E6 and E7 protein expression followed a dramatic time- and dose-dependent decrease in E6 and E7 RNA levels and was accompanied by cessation of cell proliferation. TGF betas 1 and 2 inhibited HPV16 RNA expression at the transcriptional level; inhibition was dependent upon ongoing protein synthesis. TGF betas 1 and 2 also induced a six- to sevenfold increase in TGF beta 1 RNA. Cells became partially resistant to the inhibitory effects of TGF beta 1 on cell growth and HPV early gene expression after prolonged cultivation in vitro or after malignant transformation. Thus, TGF beta 1 may function as an autocrine regulator of HPV gene expression in infected genital epithelial cells.

Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P105 promoters

cis-Acting elements involved in E2 modulation of human papillomavirus type 16 (HPV-16) P97 promoter activity and HPV-18 P105 promoter activity were examined. In transfected primary human keratinocytes, each promoter had a basal activity that could be repressed by the bovine papillomavirus type 1 E2 gene product. Mutational analysis of the E2-binding sites in the long control region upstream of each promoter revealed that E2 repression was mediated through the E2-binding sites proximal to each promoter. In the context of a mutated E2-binding site at the promoter proximal position, the HPV-16 P97 and HPV-18 P105 promoters could be transactivated by E2. E2-mediated repression of HPV-18 P105 promoter activity was shown to be a transcriptional effect. The interaction of E2 with promoter-proximal E2-binding sites is likely to be important for the controlled expression of viral genes transcribed from the HPV-16 P97 promoter and the HPV-18 P105 promoter in infected human genital epithelial cells.