Vaginal Epithelial Cell Line Research Articles

Xenobiotic metabolites modify immune responses of the cervicovaginal epithelium: potential mechanisms underlying barrier disruption.

Xenobiotic metabolites are exogenous biochemicals that can adversely impact reproductive health. We previously identified xenobiotics in cervicovaginal fluid during pregnancy in association with short cervix. In other organ systems, xenobiotics can modify epithelial barrier function. We hypothesise that xenobiotics dysregulate epithelial cell and macrophage immune responses as a mechanism to disrupt the cervicovaginal barrier. In vitro cell culture system. Laboratory within academic institution. Vaginal, ectocervical and endocervical epithelial cell lines and primary macrophages. Cells were treated with diethanolamine (2.5 mM), ethyl glucoside (5 mM) or tartrate (2.5 mM) for 24 h. Cytokines and matrix metalloproteinases were measured in cell supernatants (n = 3 per condition). One-way analysis of variance (ANOVA) with Dunnett's test for multiple comparisons was performed. Diethanolamine induces inflammatory cytokines, whereas ethyl glucoside and tartrate generally exert anti-inflammatory effects across all cells. Diethanolamine increases interleukin 6 (IL-6), IL-8, interferon γ-induced protein 10 kDa (IP-10), growth-regulated oncogene (GRO), fractalkine, matrix metalloproteinase 1 (MMP-1), MMP-9 and MMP-10 (p < 0.05 for all), factors involved in acute inflammation and recruitment of monocytes, neutrophils and lymphocytes. Ethyl glucoside and tartrate decrease multiple cytokines, including RANTES and MCP-1 (p < 0.05 for all), which serve as chemotactic factors. Vaginal cells exhibit heightened inflammatory tone compared with cervical cells and macrophages, with a greater number of differentially expressed analytes after xenobiotic exposure. Xenobiotic metabolites present in the cervicovaginal space during pregnancy modify immune responses, unveiling potential pathways through which environmental exposures may contribute to the pathogenesis of cervical remodelling preceding preterm birth. Future work identifying xenobiotic sources and routes of exposure offers the potential to modify environmental risks to improve pregnancy outcomes.

The fungal quorum-sensing molecule, farnesol, regulates the immune response of vaginal epithelial cells against Candida albicans

BackgroundFarnesol is a Candida-secreted quorum-sensing molecule of great interest as a potential antifungal agent for serious and hardly curable infections—candidiasis, especially vulvovaginal candidiasis (VVC).MethodsThe effect of farnesol on cellular morphology and viability and evaluated the production of Th1 (IL-2), Th2 (IL-4), proinflammatory (IL-6), chemotactic (IL-8), and Th17 (IL-17) cytokines in the culture supernatants of vaginal epithelial cell line (VK2) were evaluated. Moreover, we tested the inhibitory effect of farnesol on C. albicans adhesion. Scanning electron microscopy was conducted to observe any VK2 cell ultrastructural changes.ResultsOnly low concentrations (≤ 50 µmol/L) of farnesol did not affect the morphology and viability of the VK2 cells (P > 0.05). Farnesol reduced the adhesion of C. albicans to the VK2 cells. When treated with farnesol, statistical elevated levels of both IL-4 and IL-17 secreted by the infected VK2 cells were present in the culture supernatants (P < 0.05).ConclusionsFarnesol acts as a stimulator to up-regulate the Th17-type innate immune response, as well as Th2-type humoral immunity following C. albicans infection. Further research is required to select the optimal therapeutic dose to develop efficacious and safe mucosal immune adjuvant for treating VVCs.

Induction of TLR5, IRAK1, and NF-κB expression by Trichomonas vaginalis in cervical cancer cell (HeLa) and normal human vaginal epithelial cell (HVECs) lines.

Trichomoniasis is the most common non-viral sexually transmitted infection that increases the risk of cervical cancer. Trichomonas vaginalis (T. vaginalis) can regulate the pro-inflammatory cytokine production in the host cells. Toll-like receptors (TLRs) are a family of the pattern recognition receptors (PRRs) of mammalian cells, expressed in various host cells and have an important role in recognizing pathogens, and pro-inflammatory responses. The aim of the present study is to investigate the role of TLR5 in cervical cancer cells (HeLa) and human vaginal epithelial cells (HVECs) exposed to T. vaginalis. First, the cells and parasites were cultured in RPMI and trypticase yeast extract maltose (TYM), respectively. After adaption of parasite and epithelial cells by RPMI-TYM medium co-culture (9:1 vol/vol), HVECs and HeLa cells were stimulated with T. vaginalis trophozoites (24-hour incubation at 37 °C, 5% CO2). Following RNA extraction and cDNA synthesis, the gene expression levels of TLR5, IRAK1, and NF-κB were assessed using real-time PCR. Besides, the protein levels were measured using western blotting. All tests and controls were normalized using β-actin as a housekeeping control. Real-time PCR results showed an increased gene expression of TLR5, IRAK1, and NF-κB in T. vaginalis exposed HVECs and HeLa cells compared to the control group (p < 0.05). Additionally, western blot analysis showed a statistically significant increase in TLR5, and NF-κB proteins in both groups after exposure to the parasite (p < 0.05). These findings provide insight into the host-parasite interaction, and the results indicated that T. vaginalis could stimulate TLR5 and activate related pathways.

Alterations in the Level of Ergosterol in Candida albicans’ Plasma Membrane Correspond with Changes in Virulence and Result in Triggering Diversed Inflammatory Response

Opportunistic pathogen Candida albicans possesses multiple virulence factors which enable colonization and infection of host tissues. Candida-related infections frequently occur in immunocompromised patients, which is related to an insufficient inflammatory response. Furthermore, immunosuppression and multidrug resistance of C. albicans clinical isolates make the treatment of candidiasis a challenge for modern medicine. The most common resistance mechanism of C. albicans to antifungals includes point mutations in the ERG11 gene, which encodes target protein for azoles. We investigated whether the mutations or deletion of the ERG11 gene influence the pathogen-host interactions. We prove that both C. albicans erg11∆/∆ and ERG11K143R/K143R exhibit increased cell surface hydrophobicity. Additionally, C. albicans KS058 has an impaired ability of biofilm and hyphae formation. Analysis of the inflammatory response of human dermal fibroblasts and vaginal epithelial cell lines revealed that altered morphology of C. albicans erg11∆/∆ results in a significantly weaker immune response. C. albicans ERG11K143R/K143R triggered stronger production of pro-inflammatory response. Analysis of genes encoding adhesins confirmed differences in the expression pattern of key adhesins for both erg11∆/∆ and ERG11K143R/K143R strains. Obtained data indicate that alterations in Erg11p consequence in resistance to azoles and affect the key virulence factors and inflammatory response of host cells.

LAMP3/CD63 Expression in Early and Late Endosomes in Human Vaginal Epithelial Cells Is Associated with Enhancement of HSV-2 Infection.

Herpes simplex virus 2 (HSV-2) is a lifelong sexually transmitted virus that disproportionately infects women through heterosexual transmission in the vaginal tract. The vaginal epithelium is known to be highly susceptible to HSV-2 infection; however, the cellular mechanism of HSV-2 uptake and replication in vaginal epithelium has not been extensively studied. Previously, we observed that lysosomal-associated membrane protein-3 (LAMP3/CD63) was among the highly upregulated genes during HSV-2 infection of human vaginal epithelial cell line VK2, leading us to posit that LAMP3/CD63 may play a role in HSV-2 infection. Consequently, we generated two gene-altered VK2-derived cell lines, a LAMP3-overexpressed (OE) line and a LAMP3 knockout (KO) line. The wild-type VK2 and the LAMP3 OE and KO cell lines were grown in air-liquid interface (ALI) cultures for 7 days and infected with HSV-2. Twenty-four hours postinfection, LAMP3 OE cells produced and released significantly higher numbers of HSV-2 virions than wild-type VK2 cells, while virus production was greatly attenuated in LAMP3 KO cells, indicating a functional association between LAMP3/CD63 expression and HSV-2 replication. Fluorescence microscopy of HSV-2-infected cells revealed that HSV-2 colocalized with LAMP3 in both early endosomes and lysosomal compartments. In addition, blocking endosomal maturation or late endosomal/lysosomal fusion using specific inhibitors resulted in reduced HSV-2 replication in VK2 cells. Similarly, LAMP3 KO cells exhibited very low viral entry and association with endosomes, while LAMP3 OE cells demonstrated large amounts of virus that colocalized with LAMP3/CD63 in endosomes and lysosomes. IMPORTANCE Collectively, these results showed that HSV-2 is taken up by human vaginal epithelial cells through an endosomal-lysosomal pathway in association with LAMP3, which plays a crucial role in the enhancement of HSV-2 replication. These findings provide the basis for the future design of antiviral agents for prophylactic measures against HSV-2 infection.

15-Deoxy-Delta-12,14-prostaglandin J2 modulates pro-labour and pro-inflammatory responses in human myocytes, vaginal and amnion epithelial cells.

BackgroundPrematurity is the leading cause of childhood death under the age of five. The aetiology of preterm birth is multifactorial; however, inflammation and infection are the most common causal factors, supporting a potential role for immunomodulation as a therapeutic strategy. 15-Deoxy-Delta-12,14-prostaglandin J2 (15dPGJ2) is an anti-inflammatory prostaglandin and has been shown to delay lipopolysaccharide (LPS) induced preterm labour in mice and improve pup survival. This study explores the immunomodulatory effect of 15dPGJ2 on the transcription factors NF-κB and AP-1, pro-inflammatory cytokines, and contraction associated proteins in human cultured myocytes, vaginal epithelial cell line (VECs) and primary amnion epithelial cells (AECs).MethodsCells were pre-incubated with 32µM of 15dPGJ2 and stimulated with 1ng/mL of IL-1β as an in vitro model of inflammation. Western immunoblotting was used to detect phosphorylated p-65 and phosphorylated c-Jun as markers of NF-κB and AP-1 activation, respectively. mRNA expression of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α was examined, and protein expression of COX-2 and PGE2 were detected by western immunoblotting and ELISA respectively. Myometrial contractility was examined ex-vivo using a myograph.Results15dPGJ2 inhibited IL-1β-induced activation of NF-κB and AP-1, and expression of IL-6, IL-8, TNF-α, COX-2 and PGE2 in myocytes, with no effect on myometrial contractility or cell viability. Despite inhibiting IL-1β-induced activation of NF-κB, expression of IL-6, TNF-α, and COX-2, 15dPGJ2 led to activation of AP-1, increased production of PGE2 and increased cell death in VECs and AECs.ConclusionWe conclude that 15dPGJ2 has differential effects on inflammatory modulation depending on cell type and is therefore unlikely to be a useful therapeutic agent for the prevention of preterm birth.

Trichomonas vaginalis infection impairs anion secretion in vaginal epithelium

Trichomonas vaginalis is a common protozoan parasite, which causes trichomoniasis associated with severe adverse reproductive outcomes. However, the underlying pathogenesis has not been fully understood. As the first line of defense against invading pathogens, the vaginal epithelial cells are highly responsive to environmental stimuli and contribute to the formation of the optimal luminal fluid microenvironment. The cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel widely distributed at the apical membrane of epithelial cells, plays a crucial role in mediating the secretion of Cl− and HCO3−. In this study, we investigated the effect of T. vaginalis on vaginal epithelial ion transport elicited by prostaglandin E2 (PGE2), a major prostaglandin in the semen. Luminal administration of PGE2 triggered a remarkable and sustained increase of short-circuit current (ISC) in rat vaginal epithelium, which was mainly due to Cl− and HCO3− secretion mediated by the cAMP-activated CFTR. However, T. vaginalis infection significantly abrogated the ISC response evoked by PGE2, indicating impaired transepithelial anion transport via CFTR. Using a primary cell culture system of rat vaginal epithelium and a human vaginal epithelial cell line, we demonstrated that the expression of CFTR was significantly down-regulated after T. vaginalis infection. In addition, defective Cl− transport function of CFTR was observed in T. vaginalis-infected cells by measuring intracellular Cl− signals. Conclusively, T. vaginalis restrained exogenous PGE2-induced anion secretion through down-regulation of CFTR in vaginal epithelium. These results provide novel insights into the intervention of reproductive complications associated with T. vaginalis infection such as infertility and disequilibrium in vaginal fluid microenvironment.

Expression, Activity, and Regulation of Phosphorylating Enzymes in Tissues and Cells Relevant to HIV-1 Sexual Transmission.

Phosphorylating enzymes (PEs) are responsible for activating nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) such as tenofovir (TFV) and are critical for their conversion to obtain intracellular antiviral activity. However, there are limited data available regarding the expression of PEs and their activity in the female genital tract. This work compared the messenger RNA (mRNA) expression levels of PEs in human female genital tissue, immune cells, and animal models that are commonly used in human immunodeficiency virus (HIV) research. Furthermore, the effect of contraceptive hormones and proinflammatory cytokines on tenofovir diphosphate (TFV-DP) formation and efficacy in human vaginal, epithelial, and immune cells was also evaluated. We found that human vaginal and ectocervical tissues had similar mRNA expression for seven PEs tested. Polymerase chain reaction results revealed that creatine kinase brain (CKB), mitochondrial creatine kinase 1 (CKMT1), mitochondrial creatine kinase 2 (CKMT2), adenylate kinase AK3L1 (AK4), and nucleoside diphosphate kinase 1 (NME1) exhibited a 10- to 10,000-fold higher expression level in a vaginal epithelial cell line, VK2, compared with CD4+ T cells (p < .05). Medroxyprogesterone acetate (MPA)/progesterone (P4) and IL-1β/IL-8 treatment resulted in altered TFV-DP levels in VK2 and PM1 cells. MPA and P4 at concentrations above 0.1 μM, as well as IL-1β and IL-8 at concentrations above 10 ng/mL, significantly decreased HIV-1BaL inhibition in PM1 cells when 1 μM TFV was added. However, this observed effect of hormones and cytokines was abrogated when TFV concentration was raised to 1 mM. These in vitro results elucidate the role of PEs in TFV metabolism and provide information regarding differences in PE tissue expression for animal models commonly used in HIV testing. This information can be applied to better understand and interpret data obtained using these models.

TRIM26 Facilitates HSV-2 Infection by Downregulating Antiviral Responses through the IRF3 Pathway.

Herpes simplex virus type 2 (HSV-2) is the primary cause of genital herpes which results in significant morbidity and mortality, especially in women, worldwide. HSV-2 is transmitted primarily through infection of epithelial cells at skin and mucosal surfaces. Our earlier work to examine interactions between HSV-2 and vaginal epithelial cells demonstrated that infection of the human vaginal epithelial cell line (VK2) with HSV-2 resulted in increased expression of TRIM26, a negative regulator of the Type I interferon pathway. Given that upregulation of TRIM26 could negatively affect anti-viral pathways, we decided to further study the role of TRIM26 in HSV-2 infection and replication. To do this, we designed and generated two cell lines derived from VK2s with TRIM26 overexpressed (OE) and knocked out (KO). Both, along with wildtype (WT) VK2, were infected with HSV-2 and viral titres were measured in supernatants 24 h later. Our results showed significantly enhanced virus production by TRIM26 OE cells, but very little replication in TRIM26 KO cells. We next examined interferon-β production and expression of two distinct interferon stimulated genes (ISGs), MX1 and ISG15, in all three cell lines, prior to and following HSV-2 infection. The absence of TRIM26 (KO) significantly upregulated interferon-β production at baseline and even further after HSV-2 infection. TRIM26 KO cells also showed significant increase in the expression of MX1 and ISG15 before and after HSV-2 infection. Immunofluorescent staining indicated that overexpression of TRIM26 substantially decreased the nuclear localization of IRF3, the primary mediator of ISG activation, before and after HSV-2 infection. Taken together, our data indicate that HSV-2 utilizes host factor TRIM26 to evade anti-viral response and thereby increase its replication in vaginal epithelial cells.

The Role of 17β-Estrogen in Candida albicans Adhesion on Human Vaginal Epithelial Cells via FAK Phosphorylation.

To investigate the role of 17β-estrogen in Candida albicans (C. albicans) adhesion on human vaginal epithelial cells in vulvovaginal candidiasis (VVC). The vaginal epithelial cell line, VK2/E6E7, was used to study the estrogen-induced molecular events between C. albicans and cells. An adhesion study was performed to evaluate the involvement of the estrogen-dependent focal adhesion kinase (FAK) activation in cell adhesion. The phosphorylation status of FAK and estrogen receptor α (ERα) upon estrogen challenge was assessed by western blotting. Specific inhibitors for ERα were used to validate the involvement of ERα-FAK signaling cascade. A transient activation of ERα and FAK was observed following the stimulation with 1000nM estrogen for 48h, as well as the increased average number of C. albicans adhering to each vaginal epithelial cell. Estrogen-induced activation of ERa and FAK was inhibited by the specific inhibitor of ERα, especially when the inhibitor reached a 10μM concentration and allowed to act for 12h. Simultaneously, a decrease in the number of adherent C. albicans was observed. However, this inhibitory effect diminished as the concentration of estrogen increased. FAK and ERα signaling cascades were involved in the early interaction between the vaginal epithelial cells and C. albicans, which appeared to be linked with the enhanced cell adhesion leading to VVC and promoted by a certain concentration of estrogen.

Relationship Between Human β-Defensin 2 and the Vaginal Environment.

As one of the main antimicrobial peptides, human β-defensin 2 (HBD2) plays multiple roles in the lower genital tract. Based on the Nugent score as a diagnostic criterion for bacterial vaginosis, we sought to clarify the correlations among the Nugent score and interleukin-6 (IL-6) and HBD2 levels in vaginal secretions in association with various types of infection. Ninety-eight women were recruited for this study. Levels of HBD2 and IL-6 in vaginal wash were measured by enzyme-linked immunosorbent assays. According to the Nugent method, the number of Lactobacillus morphotypes per field of view was well correlated with the HBD2 level. The amount of HBD2 was also well correlated with the presence of Candida spp. (P < 0.01). In vitro experiments revealed that the expression of HBD2 from the human vaginal epithelial cell line, VK2/E6E7, was induced by the addition of heat-killed C. albicans (HKCA). The addition of HKCA induced expression of Dectin-1 mRNA. A luciferase assay for nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) responsive elements showed that HKCA activated NF-κB signaling. These results suggested that C. albicans induced the activation of Dectin-1 and (NF-κB) signaling, resulting in HBD2 expression. In conclusion, the expression of HBD2 positively correlated with the presence of Lactobacillus and Candida spp.

Group B Streptococcus serotypes Ia and V induce differential vaginal immune responses that may contribute to long term colonization of the female reproductive tract.

Group B Streptococcus (GBS) is a common colonizer of the female genital tract at the time of pregnancy and has been associated with severe neonatal infections. Despite trials for GBS vaccines already being underway, the factors influencing vaginal GBS colonization and clearance are currently poorly understood. Within this study, we investigated the host immune responses to GBS infections in mice that affect GBS vaginal colonization and clearance. Cervicovaginal swabs were used to measure vaginal GBS persistence, and vaginal cytokine responses were measured using the BioPlex® system. Lymphocytes isolated from spleens were stimulated with UV-killed GBS to examine systemic cellular responses. Additional in vitro cellular experiments using human vaginal epithelial cells were also performed, examining the effect pregnancy level hormones had on GBS adhesion, invasion, and cytokine responses. We observed significant differences in the ability of GBS serotype V infections to persist, compared with GBS serotype Ia vaginal infections. Vaginal cytokine response examination identified temporal changes in cytokine production (IL10, IFNγ, IL6, IL1β, and TNFα) in relation to GBS serotype and clearance or colonization. Lymphocyte proliferation assays also revealed robust cellular immune responses to GBS vaginal infections irrespective of clearance or colonization. In vitro human cellular analyses also identified that vaginal epithelial cell line cytokine production was suppressed in the presence of hormones despite no alteration in adhesion/invasion. Here, we establish previously unknown, serotype specific, temporal immune responses which may be associated with vaginal GBS colonization or clearance in the female genital tract.

Mechanisms underlying the regulation of intracellular and luminal pH in vaginal epithelium.

The vagina provides a characteristic low-Na+ and low-pH fluid microenvironment that is considered generally protective. Previous studies have shown that various types of epithelial cells harbor the capacity of intracellular pH (pHi) regulation. However, it remains elusive whether vaginal epithelium could actively regulate pHi by transporting acid-base ions. In this study, we verified that after transient exposure to NH4 Cl, the pHi values could rapidly recover from acidification via Na+ -H+ exchanger (NHE), Na+ -HCO3 - cotransporter (NBC), and carbonic anhydrase in human vaginal epithelial cell line VK2/E6E7. Positive expression of the main acid-base transporters including NHE1-2, NBCe1-2, and NBCn1 mRNA was also detected in VK2/E6E7 cells. Moreover, the in vivo study further showed that interfering with the function of V-type H+ -ATPase, NHE or NBC expressed in vagina impaired vaginal luminal pH homeostasis in rats. Taken together, our study reveals the property of pH regulation in vaginal epithelial cells, which might provide novel insights into the potential role of vaginal epithelium in the formation of the vaginal acidic microenvironment.

Exosomes derived from human umbilical cord mesenchymal stem cells accelerate growth of VK2 vaginal epithelial cells through MicroRNAs in vitro.

Could human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Ex) accelerate vaginal epithelium cell (VK2) growth? HucMSC-Ex play a significant role in promoting proliferation of VK2 cells by accelerating the cell cycle and inhibiting apoptosis through exosomal microRNAs in vitro. Numerous studies have reported that MSC-Ex play an important role in tissue injury repair. hucMSC and exosomes isolated from their conditioned medium were used to treat a vaginal epithelial cell line (VK2). Normal human fibroblasts (HFF-1) were used as negative control to hucMSC. VK2 cells were co-cultured with hucMSC whose paracrine effect on the viability, cell cycle and cell apoptosis of VK2 vaginal epithelial cells was further assessed by the CCK-8 assay and flow cytometry. HucMSC-Ex isolated from culture medium by ultracentrifuge were characterized by transmission electron microscopy, nanoparticle tracking analysis and Western blot. HucMSC-Ex at different concentrations and HFF-1 exosomes were used to treat VK2 cells. High-throughput RNA sequencing was utilized to reveal the profile of microRNAs in hucMSC, hucMSC-Ex, HFF-1 and HFF-1 exosomes and GO analysis was applied to demonstrate their functions. To evaluate the function of these specific microRNAs in hucMSC-Ex, VK2 cells were treated with RNA-interfered-hucMSC-Ex (RNAi-hucMSC-Ex) and their proliferation was measured by Label-free Real-time Cellular Analysis System. The study showed that hucMSC stimulate VK2 cell growth possibly through a paracrine route by promoting cell cycle and inhibiting apoptosis. Compared with control and low dose groups, hucMSC-Ex of high concentration (more than 1000 ng/ml) significantly increased VK2's growth after treatment in a dose-depended manner (P < 0.05). HucMSC-Ex raised the proportion of cells in S-phase and reduced the percentage of apoptotic cells in VK2 cells in comparison with the HFF-1 exosomes and control groups (P < 0.05). microRNAs, including miR-100 (16.92%), miR-146a (9.21%), miR-21 (6.67%), miR-221 (6.39%) and miR-143 (4.63%), were found to be specifically enriched (P < 0.05) in hucMSC-Ex and their functions concentrated on cell cycle, development and differentiation. Collectively, our findings indicate that hucMSC-Ex may play a significant role in accelerating VK2's proliferation by promoting cell cycle and inhibiting apoptosis through exosomal microRNAs in vitro. N/A. Our study did not confirm the function of hucMSC-Ex or specifically enriched exosomal microRNAs in vivo. miR-100 and miR-146a are well-known immunomodulatory miRNAs that participate in the regulation of inflammatory disorders and may enhance the therapeutic effect of hucMSC-Ex by promoting the surgical injury repair after vaginal reconstruction. But whether it acts through anti-inflammatory responses needs further study. This finding supports the potential use of hucMSC-Ex as a cell-free therapy of Meyer-Rokitansky-Küster-Hauser syndrome (MRKHS) after vaginoplasty. This study was supported by the Chinese National Nature Sciences Foundation (grant number 91440107, 81471416 and 81771524) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19040102). All authors state that there is no conflict of interest to disclose.

Lactobacillus crispatus accelerates re-epithelialization in vaginal epithelial cell line MS74.

The functions of vaginal lactobacilli in susceptibility to infectious diseases as regards epithelial barrier integrity and wound healing remain incompletely understood. Lactobacillus crispatus, one of the most common Lactobacillus species in the vagina and among the most protective against sexually transmitted infections, was cocultured with an immortalized human vaginal epithelial cell line (MS74), and a scratch assay was performed to evaluate re-epithelialization. The concentration of vascular endothelial growth factor A (VEGF) was measured using enzyme-linked immunosorbent assay (ELISA). An immunofluorescence assay was performed to locate the expression of VEGF and VEGF receptor (VEGFR) 1 and 2. The effects of the bacterial supernatant of L.crispatus were also evaluated. Lactobacillus crispatus significantly accelerated re-epithelialization of MS74 cells, accompanied by an increase in VEGF concentration. In contrast, heat-killed L.crispatus did not show this effect. The bacterial supernatant of L.crispatus also induced re-epithelialization. The immunoreactivity of VEGF was higher at the scratched edge, whereas VEGFR1 and 2 stained site-independently. Recombinant VEGF induced cell migration in a dose-dependent manner. The bacterial supernatant of L.crispatus also significantly accelerated re-epithelialization in MS74 cells and increased the concentration of VEGF in the culture 24hours after the scratch. These results may enhance our knowledge of the importance of L.crispatus in the healing of damaged vaginal epithelium and protection against the consequent risk of pathogenic infections, such as human immunodeficiency virus (HIV), and improve our understanding of vaginal epithelial barrier integrity maintenance by this bacterium.

Switchable On-Demand Release of a Nanocarrier from a Segmented Reservoir Type Intravaginal Ring Filled with a pH-Responsive Supramolecular Polyurethane Hydrogel.

To achieve a pH-responsive switchable on-demand release of nanoparticles (NPs) from intravaginal rings (IVR), a new pH-sensitive polyurethane (PU) bearing dimethylolpropionic acid (PEG-DMPA-HDI-PG) was synthesized to encapsulate NPs as a physically cross-linked hydrogel within a segmented reservoir-IVR. A new PEGylated polyaspartic acid-based copolymer conjugated with the fluorescent dye Orange II (PASP-PEG-Ph-Orange) was synthesized to self-assemble in aqueous solution into NPs (251-283 nm) for the release study. Chemical structures of the PEG-DMPA-HDI-PG and PASP-PEG-Ph-Orange were confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) and1H nuclear magnetic resonance (1H NMR) spectroscopy. PASP-PEG-Ph-Orange NPs showed the highest fluorescent emission at 570 nm for tracking, and PEG-DMPA-HDI-PG became a pH-responsive supramolecular hydrogel in distilled water at 20 wt %. PASP-PEG-Ph-Orange NPs were blended with the PEG-DMPA-HDI-PG hydrogel to form an inclusion complex and then filled into segmented reservoir-IVRs containing two 1/32 in. diameter holes. The segmented IVR filled with the NP encapsulated hydrogel showed continuous release of the NPs at pH 7.0 but a close-to-zero release at pH 4.2 for 12 h and, moreover, demonstrated a pH-responsive switchable on-demand NPs release. The PASP-PEG-Ph-Orange and PEG-DMPA-HDI-PG showed no and low cytotoxicity toward the human vaginal epithelial cell line VK2/E6E7, respectively. Overall, the segmented IVR filled with PEG-DMPA-HDI-PG hydrogel demonstrated its potential use for the switchable on-demand intravaginal release of nanocarriers.

Stratification of mouse vaginal epithelium. 1. Development of three-dimensional models in vitro with clonal cell lines.

The mouse vagina consists of stratified squamous epithelium and stroma and is regulated by ovarian hormones. Vaginal epithelial cells do not stratify, but rather form a monolayer and show an inconsistent responsiveness to ovarian hormones when cultured on plastic dish or matrix. To address the discrepancy between in vivo and in vitro observations, three-dimensional (3D) co-culture models are developed with clonal vaginal epithelial and stromal cell lines; stromal cells are embedded in collagen gel and epithelial cells are seeded on the gel. In the 3D models, epithelial cells express Transformation related protein 63 (Trp63) and begin to stratify when they are co-cultured with two out of three stromal cell lines, but not with the other stromal cell line. Stroma may consist of various types of cells with distinct functions.

Coassociation between Group B Streptococcus and Candida albicans Promotes Interactions with Vaginal Epithelium.

Group B Streptococcus (GBS) is a leading cause of neonatal sepsis, pneumonia, and meningitis worldwide. In the majority of cases, GBS is transmitted vertically from mother to neonate, making maternal vaginal colonization a key risk factor for neonatal disease. The fungus Candida albicans is an opportunistic pathogen of the female genitourinary tract and the causative agent of vaginal thrush. Carriage of C. albicans has been shown to be an independent risk factor for vaginal colonization by GBS. However, the nature of interactions between these two microbes is poorly understood. This study provides evidence of a reciprocal, synergistic interplay between GBS and C. albicans that may serve to promote their cocolonization of the vaginal mucosa. GBS strains NEM316 (serotype III) and 515 (serotype Ia) are shown to physically interact with C. albicans, with the bacteria exhibiting tropism for candidal hyphal filaments. This interaction enhances association levels of both microbes with the vaginal epithelial cell line VK2/E6E7. The ability of GBS to coassociate with C. albicans is dependent upon expression of the hypha-specific adhesin Als3. In turn, expression of GBS antigen I/II family adhesins (Bsp polypeptides) facilitates this coassociation and confers upon surrogate Lactococcus lactis the capacity to exhibit enhanced interactions with C. albicans on vaginal epithelium. As genitourinary tract colonization is an essential first step in the pathogenesis of GBS and C. albicans, the coassociation mechanism reported here may have important implications for the risk of disease involving both of these pathogens.

High glucose-mediated overexpression of ICAM-1 in human vaginal epithelial cells increases adhesion of Candida albicans

To investigate the involvement of ICAM-1 in the adhesion of Candida to the genitourinary epithelial cells in high glucose, we examined the adhesion of Candida albicans or Candida glabrata to human vaginal epithelial cells (VK2/E6E7) or human vulvovaginal epidermal cells (A431). These cells were cultured in 100, 500 or 3000 mg/dL glucose for three days and inoculated with Candida for 60 minutes. Followed by, adhering of Candida to the cells, which were counted. While the adhesion of Candida albicans to VK2/E6E7 significantly increased in the high glucose, A431 did not. We next examined the expression of ICAM-1 as a ligand on the epithelial cells. ICAM-1 expression was increased in VK2/E6E7 cultured in the high glucose; however, the expression level in A431 was not high compared with VK2/E6E7. This data suggested that ICAM-1 functions as one of ligands in the adhesion of Candida albicans to the vaginal epithelial cells in a high glucose environment. Impact statement What is already known on the subject: Candida’s complement receptor is involved in the adhesion to epithelial cells. The expression of this receptor has been reported to increase as glucose concentration increases. This is considered as a contributing factor to the high risk for vulvovaginal candidiasis (VVC) in diabetes. On the host side, diabetic patients have a factor that facilitates adhesion of Candida to epithelial cells. This factor has been unknown until recently. What the results of this study add: In this study, we used a vaginal epithelial cell line and showed that the adhesion of C. albicans to cells increased at higher glucose concentrations. At the same time, ICAM-1 expression of cells also increased. Thereby, it is suggested that the expression of ICAM-1 in vaginal epithelial cells is increased by glucose such as urinary sugar in diabetic patients and is a condition for facilitating adhesion of Candida. What the implications are of these findings for clinical practice and/or further research: We expect not only host immune dysfunction but also alteration in epithelial cells will be focussed on as a cause of VVC in diabetic patients.

Self-assembled nanoparticles made from a new PEGylated poly(aspartic acid) graft copolymer for intravaginal delivery of poorly water-soluble drugs

New amphiphilic PEGylated poly(aspartic acid) graft copolymer (PASP-PEG-Ph) was synthesized as a nanocarrier for intravaginal drug delivery of poorly water-soluble drugs. PASP-PEG-Ph self-assembled into negatively charged spherically shaped nanoparticles in the presence of pH 4.5 and pH 7.0 vaginal fluid simulants with a diameter of approximately 200 nm as evidenced by Zeta-potentiometer, scanning electron microscope (SEM), dynamic light scattering (DLS) analysis. A significant number of stable NPs could be maintained at pH 4.5, 37 °C for 13 days. The PASP-PEG-Ph NP showed no significant cytotoxicity toward the T-cell line SupT1 and human vaginal epithelial cell line Vk2/E6E7 up to 1 mg/mL. The highest encapsulation efficiency of the model drug coumarin 6 (C6) by PASP-PEG-Ph was 92.0 ± 5.7%. The sustained release profile of the encapsulated C6 was demonstrated by an in vitro release study. An in vitro cellular uptake study revealed strong cellular uptake of the C6 loaded NP by SupT1 cells within 2 h.