Mouse Vaginal Epithelial Cells Research Articles

Sublingual immunisation with GBS serotype III capsular polysaccharide-tetanus toxoid conjugate vaccine induces systemic and mucosal antibody responses which are opsonophagocytic and inhibit GBS colonisation of vaginal epithelial cells

No vaccines are currently licensed against Group B streptococcus (GBS), an important cause of morbidity and mortality in babies and adults. Using a mouse model, and in vitro opsonophagocytosis and colonisation assays, we evaluated the potential of a sublingually-administered polysaccharide-conjugate vaccine against GBS serotype III. Sublingual immunisation of mice with 10 µg of GBS conjugate vaccine once a week for 5 weeks induced a substantial systemic IgG anti-polysaccharide response which was similar to the level induced by subcutaneous immunsation. In addition, sublingual immunisation also induced mucosal (IgA) antibody responses in the mouth, intestines and vagina. Immune sera and intestinal washes were functionally active at mediating killing of the homologous GBS serotype III in an opsonophagocytosis assay. In addition, intestinal and vaginal washes inhibited the colonisation of mouse vaginal epithelial cells by the vaccine homologous strain. These results suggest that, in addition to the induction of high levels of IgG antibodies that could be transduced from the immunised mother to the foetus to protect the newborn against GBS infection, sublingual immunisation can elicit a substantial mucosal antibody response which might play an important role in the prevention of GBS colonisation in immunised women, thereby eliminating the risk of GBS transmission from the mother to the baby during pregnancy or at birth.

Inhibition of CXCR4 by MicroRNA-1192 Reduces the Activation of Th17 Cells and Expression of Inflammation Factors in a Mouse Model of Vulvovaginal Candidiasis

Vulvovaginal candidiasis (VVC) is a disease commonly occurring in sexually active women. The involvement of microRNAs in several kinds of infectious diseases has been highlighted in a number of researches. Therefore, we conducted the present study in order to investigate whether microRNA-1192 (miR-1192) would significantly target CXCR4 in Th17 cells as well as inflammatory factors in mouse models suffering from VVC. Seventy-five mice were selected as test subjects for this study, of which twenty-five were used as the normal control, while the rest were treated with estradiol or oil-treated in order to establish VVC mouse models (each n = 25). Protein expressions of CXCR4, IL-6, IL-17, and IL-23 were all measured using both an immunohistochemistry and ELISA. The Th17 cell percentage in peripheral blood and the expression of RORγt in Th17 cells were detected using a flow cytometry. Mouse vaginal epithelial cells were isolated from normal mice, after which the mice were treated with estradiol to regulate their estrogen, followed by treatments involving the miR-1192 mimic, miR-1192 inhibitor, siRNA-CXCR4, and miR-1192 inhibitor + si-CXCR4. The cell cycle, apoptosis, and proliferation were all examined by using an additional flow cytometry as well as the employment of the MTT assay. The miR-1192, CXCR4, IL-6, IL-17, and IL-23 expressions in tissues and cells were both measured using both RT-qPCR and western blot assay techniques. The mice treated with either estradiol or oil had presented to us lowered levels in miR-1192 expression as well as higher levels in both Th17 cell percentage and expression of RORγt in Th17 cells, along with mRNA and protein expressions of CXCR4, IL-6, IL-17, and IL-23. In cell experiments, the mouse vaginal epithelial cells that had been treated with miR-1192 inhibitor had shown us a decreased cell proliferation rate and contrarily increased expressions of CXCR4, IL-6, IL-17, and IL-23 mRNA, protein, and cell apoptosis rate; these results were opposite to the ones found in the mice treated with miR-1192 mimic. Our results provided significant evidence that miR-1192 could directly development and progression of VVC by restraining the CXCR4 gene in the VVC mice.

Icotinib inhibits EGFR signaling and alleviates psoriasis-like symptoms in animal models

To investigate the effects of icotinib hydrochloride and a derivative cream on epidermal growth factor receptor (EGFR) signaling and within animal psoriasis models, respectively. The effect of icotinib on EGFR signaling was examined in HaCaT cells, while its effect on angiogenesis was tested in chick embryo chorioallantoic membranes (CAM). The effectiveness of icotinib in treating psoriasis was tested in three psoriasis models, including diethylstilbestrol-treated mouse vaginal epithelial cells, mouse tail granular cell layer formation, and propranolol-induced psoriasis-like features in guinea pig ear skin. Icotinib treatment blocked EGFR signaling and reduced HaCaT cell viability as well as suppressed CAM angiogenesis. Topical application of icotinib ameliorated psoriasis-like histological characteristics in mouse and guinea pig psoriasis models. Icotinib also significantly inhibited mouse vaginal epithelium mitosis, promoted mouse tail squamous epidermal granular layer formation, and reduced the thickness of the horny layer in propranolol treated auricular dorsal surface of guinea pig. We conclude that icotinib can effectively inhibit psoriasis in animal models. Future clinical studies should be conducted to explore the therapeutic effects of icotinb in humans.

Effects of chinese formula jueyin granules on psoriasis in an animal model.

Although Traditional Chinese medicine (TCM) is known to be effective for psoriasis patients, the responsible mechanisms still remain poorly understood. In this study, we aimed to evaluate the effect of one formula, named Jueyin granules (JYG) in the mouse model of the vaginal epithelium and tail epidermis. Additionally, we also determined the anti-inflammatory effects of JYG in an imiquimod- (IMQ-) induced psoriasis-like skin mouse model. Our results show that JYG can attenuate the IMQ-induced psoriasis-like inflammation, accompanied with increased epidermal hyperplasia. We also measured estrogenic stage mitosis of vaginal epithelial cells and the formation of granular cell layers in male mouse tails per 100 scales, as well as the tissue nitric oxide (NO) and malondialdehyde (MDA) levels using the ELISA method. The results suggest that JYG significantly inhibited mitosis in mouse vaginal epithelial cells, promoted the formation of the squamous epidermal granular layer in mice tails, and reduced the levels of NO and MDA in an imiquimod-induced psoriasis-like skin mouse model after 14 d (P < 0.05). These results demonstrate that JYG might be an effective clinical treatment for psoriasis and the effects may be related to inhibited keratinocytes proliferation, improved parakeratotic epidermal cells, and reduced expression of NO and MDA.

Tissue-specific expression of Clec2g in mice

Estrogens regulate the proliferation and differentiation of mouse vaginal epithelial cells. We examined the temporal and spatial expression of DDV10, a novel C-type lectin during stratification and cornification of the vaginal epithelium. DDV10 was expressed in vagina but not uterus in ovariectomized mice treated with 17 β-estradiol (E2). In mouse stomach, the expression of DDV10 was detected in pars proventricularis but not in pars glandularis. Furthermore, the DDV10 gene was found to possess two transcripts, a long form (DDV10) and a short form (OCILrP1, osteoclast inhibitory lectin-related protein 1). DDV10 mRNA but not OCILrP1 mRNA was expressed in the stratified and cornified epithelial tissues. DDV10 mRNA was first detected between 12 and 18 h after E2 treatment in the vaginal epithelium, and was detected in the vagina of the neonatally diethylstilbestrol (DES)-treated mouse. Recently, a unified name was registered in GenBank (C-type lectin domain family 2, member g; Clec2 g). Taken together, these data suggest that DDV10 is the long form of Clec2 g (Clec2g-L), and DDV10/Clec2g-L may play a role in the stratification and/or cornification of epithelial cells during differentiation.

Expression of a novel C-type lectin in the mouse vagina.

Estrogens regulate the proliferation and differentiation of mouse vaginal epithelial cells. However, the molecular mechanisms underlying estrogen-induced changes have not been elucidated. The goal of this study was to identify estrogen-responsive genes related to the proliferation and differentiation of mouse vaginal epithelial cells. We used differential display to reveal specific genes regulated by estrogens and identified a transcript that was designated DDV10. DDV10 encodes a membrane protein with a C-type lectin domain in the carboxyl-terminal region; thus, we inferred that it belongs to the C-type lectin family. We analyzed the temporal and spatial expression of DDV10 using RT-PCR, quantitative real-time RT-PCR, and in situ hybridization. Ovariectomy decreased DDV10 mRNA levels, whereas 17beta-estradiol treatment increased expression of DDV10 mRNA in vaginas of ovariectomized mice. DDV10 mRNA was first detected between 20 and 30 d after birth and was found in eye, tongue, stomach, and stratified and cornified vaginal epithelial cells, but not in stromal cells or uterus. DDV10 transcripts were not detected in vaginas of estrogen receptor alpha knockout mice. Taken together, these data suggest that DDV10 encodes a novel, 17beta-estradiol-regulated, C-type lectin in the mouse vagina. DDV10 may play a role in the stratification and/or cornification of epithelial cells during differentiation.

Growth inhibition of Candida by human oral epithelial cells.

Oropharyngeal candidiasis (OPC) caused by Candida albicans is a significant problem in human immunodeficiency virus (HIV)-infected persons. Recognizing the paucity of information on innate and/or adaptive mucosal host defenses against C. albicans, we recently reported that human and nonhuman primate and mouse vaginal epithelial cells inhibit the growth of C. albicans in vitro. In the present study, oral epithelial cells collected from saliva of healthy volunteers and a purified oral epithelial cell line were found to inhibit blastoconidia and/or hyphal growth of several Candida species. Cell contact was a strict requirement for the epithelial cell anti-Candida activity; neither saliva nor culture supernatants alone inhibited Candida growth, and addition of saliva to the coculture did not modulate the epithelial cell activity. Finally, epithelial cell anti-Candida activity was significantly lower in HIV-infected persons with OPC. Together, these results suggest that oral epithelial cells may play a role in innate resistance against OPC.

Involvement of Fas in regression of vaginal epithelia after ovariectomy and during an estrous cycle.

Fas, also called APO-1, belongs to the tumor necrosis factor/nerve growth factor receptor family and transmits an apoptotic signal within the cell by binding to the Fas ligand. Fas has been implicated in the activation-induced suicide of T cells and cytotoxic T cell activity in the immune system. Non-immune cells such as those in liver, lung and ovary also express Fas, but its role in these cells remains unclear. Ovariectomy has been used to study homeostasis of female reproductive organs, which is regulated by sex hormones. Here we analyzed Fas function in the ovariectomy-induced regression of mouse vaginal epithelial cells. Fas expression was detected in vagina and was elevated after ovariectomy. Fas-deficient lpr and lpr(cg) mice did not exhibit ovariectomy-induced regression of vaginal epithelia, whereas uterine regression induced by ovariectomy was not affected in these mice. The vaginas of lpr and lpr(cg) mice were in a persistent estrous stage with cornification of vaginal epithelia, as judged from the cell types in the vaginal fluid. Thus, Fas appears to be involved directly in the regression of vaginal epithelia induced by ovariectomy and during the estrous cycle, suggesting that the physiological role of this receptor extends beyond that exerted on immune cells. This is the first evidence of a role for Fas inducing physiological apoptosis in non-immune cells.

Proliferation and differentiation of prepubertal mouse vaginal epithelial cells in vitro and the specificity of estrogen-induced growth retardation

Mouse vaginal epithelial cells were isolated from intact 21-day-old BALB/cCrgl mice and cultured in a serum-free medium (SF20: basal medium supplemented with insulin, epidermal growth factor, transferrin, and bovine serum albumin--fraction V) to examine the proliferation, differentiation, and specificity of estrogen-induced growth retardation in vitro. Histologic and ultrastructural studies showed that vaginal epithelial cells undergo differentiative changes in vitro in the absence of estrogen, and that these changes are similar to those induced in vivo by estrogen. Addition of 17 beta-estradiol inhibited cellular proliferation in a dose-dependent manner. Whereas other estrane derivatives (17 alpha-estradiol and estriol) also significantly retarded cellular proliferation, cholesterol, testosterone, and progesterone had no effect. Keoxifene, an antiestrogen, significantly reversed estrogen-induced growth inhibition, resulting in proliferation of estrogen-treated cells equivalent to that of the untreated control. The results suggest that both proliferation and differentiation of prepubertal mouse vaginal epithelial cells in vitro are estrogen-independent, and that the growth inhibition is a specific estrogen-induced response.

Growth of mouse vaginal epithelial cells in culture: effect of sera and supplemented serum-free media.

Normal mouse vaginal epithelial cells isolated from ovariectomized ca. 35-d-old BALB/cCrgl mice were grown in primary culture using collagen gel matrix and a serum-free medium composed of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 (D:H) medium supplemented with insulin (IN), epidermal growth factor (EGF), cholera toxin, transferrin, and bovine serum albumin V (BSA). Three-dimensional cellular outgrowths occurred inside the collagen gel matrix. The contribution of each factor to cell growth was examined by individual addition to the basic D:H medium and by individual deletion from the complete serum-free medium. When added individually, only IN promoted growth. Deletion of IN from the complete serum-free medium markedly diminished growth; deletion of EGF or BSA slightly diminished growth. When horse, fetal bovine, or chicken serum was added to the basal D:H medium, only with increasing doses of horse serum was there enhanced cell growth. The effect of 17 beta-estradiol and diethylstilbestrol on the growth of cells was also tested, using a suboptimal medium of D:H supplemented with BSA and IN, or a minimal medium supplemented with IN alone. During the 8-d time period, addition of estrogen did not enhance cell growth in either medium. To date, we have been unable to demonstrate a mitogenic effect of estrogen; rather a dose-dependent inhibition of proliferation is seen.

Growth of mouse vaginal epithelial cells in culture: Functional integrity of the estrogen receptor system and failure of estrogen to induce proliferation

Normal mouse vaginal epithelial cells isolated from ovariectomized ca. 40-day-old BALB cCrg1 mice were purified by Percoll density gradient centrifugation and grown in primary culture using a collagen gel matrix and a serum-free complete medium. During the 9-day culture period, a 6-fold increase in cell number was observed. Addition of estrogen to the medium did not enhance epithelial cell proliferation. In fact, all doses of estrogen (180 fM to 18 nM) were inhibitory, resulting in only a 3- to 4-fold increase in cell number by day 9 of culture. Continuous exposure to estradiol (1.8 nM) for 9 days in the serum-free complete medium resulted in a decrease in cytosolic estrogen receptors with associated nuclear accumulation of estrogen receptors. A corresponding increase of cytosolic progestin receptors was also observed, indicating that no qualitative modification of the estrogen receptor system had occurred. Thus, despite its effectiveness in specific product synthesis (progestin receptors), estrogen does not stimulate proliferation of vaginal cells in this culture system, but rather inhibits epithelial cell proliferation.

Growth of normal mouse vaginal epithelial cells in and on collagen gels.

Sustained growth in primary culture of vaginal epithelial cells from ovariectomized adult BALB/cCrgl mice embedded within or seeded on collagen gel matrix was achieved in a serum-free medium composed of Ham's F-12 medium/Dulbecco's modified Eagle's medium, 1:1 (vol/vol), supplemented with insulin, bovine serum albumin fraction V, epidermal growth factor, cholera toxin, and transferrin. Three-dimensional growth of vaginal epithelial cells occurred inside the collagen gel matrix. Cell numbers increased 4- to 8-fold in collagen gel and about 4-fold on collagen gel after 9-10 days in culture. The effect of 17 beta-estradiol (0.00018-180 nM in gel or 0.018-180 nM on gel) and diethylstilbestrol (DES; 0.0186-186 nM in gel) on the growth of vaginal epithelial cells was examined. The addition of estrogen did not enhance the growth of vaginal epithelial cells during this time period either in the complete medium or in a suboptimal medium. Cultures on floating collagen gels in the serum-free medium are composed of 1-3 cell layers with superficial cornification. Estrogen does not appear to be a direct mitogen for vaginal epithelial cells, at least in this system.

Growth of mouse vaginal epithelial cells in vitro.

Pieces of adult mouse vagina (comprising epithelium and connective tissue), when explanted onto glass coverslips, gave rise to outgrowing sheets of pure epithelium whose cells had ultrastructural features in common with the cells of origin in vivo. Epithelial outgrowths from vaginas of estradiol-primed and nonprimed ovariectomized mice were studied. After the first 5 days in vitro, in the absence of estradiol, the labeling index and length of the cell cycle were similar in both types of cultures. The values were similar to those reported by others in vivo in response to estrogen. Thus, proliferative activity of cells from nonprimed mice was stimulated merely by in vitro conditions, while proliferation of cells from primed mice continued at the high level existing prior to explantation. The high rate of proliferation wasnot associated with keratinization of any cells. In the continued absence of estrogen, cells from both kinds of cultures showed a progressive decrease in proliferative activity between 5 and 14 days, also associated with inability of cells to keratinize. Addition of estradiol didnot reverse the mitotic drop or promote keratinization. Supplementation with hydrocortisone and insulin had no effect. The results suggest that (a) vaginal epithelial cells in vitro require factors in addition to estradiol in order to maintain a high level of proliferative activity or to differentiate fully by keratinization and (b) keratinization is not dependent on rate of cell proliferation.