Bacillus coagulans LMG S-24828 Impairs Candida Virulence and Protects Vaginal Epithelial Cells against Candida Infection In Vitro.

Probiotics are living microbes that provide benefits to the host. The growing data on health promotion, following probiotics administration, increased interest among researchers and pharmaceutical companies. Infections of the lower genital tract in females, caused by a wide range of pathogens, represent one of the main areas for the use of probiotics and postbiotics. Vulvovaginal candidiasis (VVC) affects 75% of women of reproductive age at least once during their lifetime, with 5-8% developing the recurrent form (RVVC). The disease is triggered by the overgrowth of Candida on the vaginal mucosa. Here, in order to establish its probiotic potential in the context of VVC, we evaluated the anti-fungal effects of the spore-producing Bacillus coagulans LMG S-24828 against C. albicans and C. parapsilosis as well as its beneficial effects in counteracting Candida vaginal infection in vitro. Our results show that both live B. coagulans and its Cell-Free Supernatant (CFS) exerted antifungal activity against both fungi. Moreover, live B. coagulans reduced hyphal formation, inhibited C. albicans adhesion to vaginal epithelial cells, showed co-aggregation capacity, and exerted a protective effect on vaginal epithelial cells infected with C. albicans. These data suggest that B. coagulans LMG S-24828 may provide benefits in the context of Candida vaginal infections.

[Mechanism of protective effect of n-butanol extract of Pulsatilla Decoction on vaginal epithelial cells under Candida albicans stimulation through EGFR/MAPK pathway based on transcriptomics].

This study aimed to investigate the protective effect and its underlying mechanism of n-butanol extract of Pulsatilla Decoction(BEPD) containing medicinal serum on vaginal epithelial cells under Candida glabrata stimulation via the epidermal growth factor receptor/mitogen activated protein kinase( EGFR/MAPK) pathway based on transcriptomics. A vulvovaginal candidiasis(VVC) mouse model was established first and transcriptome sequencing was performed for the vaginal mucosa tissues to analyze the gene expression differences among the control, VVC model, and BEPD intervention groups. Simultaneously, BEPD-containing serum and fluconazole-containing serum were prepared. A431 cells were divided into the control, model, blank serum, fluconazole-containing serum, BEPD-containing serum, EGFR agonist and EGFR inhibitor groups. Additionally, in vitro experiments were conducted using BEPD-containing serum, fluconazole-containing serum, and an EGFR agonist and inhibitor to investigate the intervention mechanisms of BEPD on C. glabrata-induced vaginal epithelial cell damage. Cell counting kit-8(CCK-8) assay was utilized to determine the safe concentrations of C. glabrata, drug-containing serum, and compounds on A431 cells. Enzyme-linked immunosorbent assay(ELISA)was employed to measure the expression levels of interleukin(IL)-1ß, IL-6, granulocyte-macrophage colony-stimulating factor(GMCSF), granulocyte CSF(G-CSF), chemokine(C-X-C motif) ligand 20(CCL20), and lactate dehydrogenase(LDH). Gram staining was used to evaluate the adhesion of C. glabrata to vaginal epithelial cells. Flow cytometry was utilized to assess the effect of C.glabrata on A431 cell apoptosis. Based on the transcriptomics results, immunofluorescence was performed to measure the expressions of p-EGFR and p-ERK1/2 proteins, while Western blot validated the expressions of p-EGFR, p-ERK1/2, p-C-Fos, p-P38, Bax and Bcl-2 proteins. Sequencing results showed that compared with the VVC model, BEPD treatment up-regulated 1 075 genes and downregulated 927 genes, mainly enriched in immune-inflammatory pathways, including MAPK. Mechanistically, BEPD significantly reduced the expression of p-EGFR, p-ERK1/2, p-C-Fos and p-P38, as well as the secretion of IL-1ß, IL-6, GM-CSF, G-CSF and CCL20, LDH release induced by C. glabrata, and the adhesion of C. glabrata to A431 cells, suggesting that BEPD exerts a protective effect on vaginal epithelial cells damaged by C. glabrata infection by modulating the EGFR/MAPK axis. In addition, BEPD downregulated the pro-apoptotic protein Bax expression and up-regulated the anti-apoptotic protein Bcl-2 expression, leading to a reduction in C. glabrata-induced cell apoptosis. In conclusion, this study reveals that the intervention of BEPD in C. glabrata-induced VVC may be attributed to its regulation of the EGFR/MAPK pathway, which protects vaginal epithelial cells.

Licochalcone A Protects Vaginal Epithelial Cells Against Candida albicans Infection Via the TLR4/NF-κB Signaling Pathway.

Vulvovaginal candidiasis (VVC) is a prevalent condition affecting a significant portion of women worldwide. Licochalcone A (LA), a natural compound with diverse biological activities, holds promise as a protective agent against Candida albicans (C. albicans) infection. This study aims to investigate the potential of LA to safeguard vaginal epithelial cells (VECs) from C. albicans infection and elucidate the underlying molecular mechanisms. To simulate VVC in vitro, VK2-E6E7 cells were infected with C. albicans. Candida albicans biofilm formation, C. albicans adhesion to VK2-E6E7 cells, and C. albicans-induced cell damage and inflammatory responses were assessed by XTT reduction assay, fluorescence assay, LDH assay, and ELISA. CCK-8 assay was performed to evaluate the cytotoxic effects of LA on VK2-E6E7 cells. Western blotting assay was performed to detect protein expression. LA dose-dependently hindered C. albicans biofilm formation and adhesion to VK2-E6E7 cells. Furthermore, LA mitigated cell damage, inhibited the Bax/Bcl-2 ratio, and attenuated the secretion of pro-inflammatory cytokines in C. albicans-induced VK2-E6E7 cells. The investigation into LA's impact on the Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway revealed that LA downregulated TLR4 expression and inhibited NF-κB activation in C. albicans-infected VK2-E6E7 cells. Furthermore, TLR4 overexpression partially abated LA-mediated protection, further highlighting the role of the TLR4/NF-κB pathway. LA holds the potential to safeguard VECs against C. albicans infection, potentially offering therapeutic avenues for VVC management.

Candida albicans Infection Disrupts the Metabolism of Vaginal Epithelial Cells and Inhibits Cellular Glycolysis.

Vulvovaginal candidiasis (VVC) is a common gynecologic disorder caused by fungal infections of the vaginal mucosa, with the most common pathogen being Candida albicans (C. albicans). Exploring metabolite changes in the disease process facilitates further discovery of targets for disease treatment. However, studies on the metabolic changes caused by C. albicans are still lacking. In this study, we used C. albicans-infected vaginal epithelial cells to construct an in vitro model of VVC, analyzed the metabolites by UHPLC-Q-Exactive MS, and screened the potential metabolites based on metabolomics. The results showed that C. albicans infection resulted in significant up-regulation of D-arabitol, palmitic acid, adenosine, etc.; significant down-regulation of lactic acid, nicotinamide (NAM), nicotinate (NA), etc.; and disruption of amino acid metabolism, and that these significantly altered metabolites might be potential therapeutic targets of VVC. Further experiments showed that C. albicans infection led to a decrease in glycolytic enzymes in damaged cells, inhibiting glycolysis and leading to significant alterations in glycolytic metabolites. The present study explored the potential metabolites of VVC induced by C. albicans infection based on metabolomics and verified the inhibitory effect of C. albicans on vaginal epithelial cell glycolysis, which is valuable for the diagnosis and treatment of VVC.

Validation of vaginal microbiome proxies for in vitro experiments that biomimic Lactobacillus-dominant vaginal cultures.

The vaginal microbiome includes diverse microbiota dominated by Lactobacillus [L.] spp. that protect against infections, modulate inflammation, and regulate vaginal homeostasis. Because it is challenging to incorporate vaginal microbiota into in vitro models, including organ-on-a-chip systems, we assessed microbial metabolites as reliable proxies in addition to traditional vaginal epithelial cultures (VECs). Human immortalized VECs cultured on transwells with an air-liquid interface generated stratified cell layers colonized by transplanted healthy microbiomes (L. jensenii- or L. crispatus-dominant) or a community representing bacterial vaginosis (BV). After 48-h, a qPCR array confirmed the expected donor community profiles. Pooled apical and basal supernatants were subjected to metabolomic analysis (untargeted mass spectrometry) followed by ingenuity pathways analysis (IPA). To determine the bacterial metabolites' ability to recreate the vaginal microenvironment in vitro, pooled bacteria-free metabolites were added to traditional VEC cultures. Cell morphology, viability, and cytokine production were assessed. IPA analysis of metabolites from colonized samples contained fatty acids, nucleic acids, and sugar acids that were associated with signaling networks that contribute to secondary metabolism, anti-fungal, and anti-inflammatory functions indicative of a healthy vaginal microbiome compared to sterile VEC transwell metabolites. Pooled metabolites did not affect cell morphology or induce cell death (∼5.5%) of VEC cultures (n = 3) after 72-h. However, metabolites created an anti-inflammatory milieu by increasing IL-10 production (p = .06, T-test) and significantly suppressing pro-inflammatory IL-6 (p = .0001), IL-8 (p = .009), and TNFα (p = .0007) compared to naïve VEC cultures. BV VEC conditioned-medium did not affect cell morphology nor viability; however, it induced a pro-inflammatory environment by elevating levels of IL-6 (p = .023), IL-8 (p = .031), and TNFα (p = .021) when compared to L.-dominate microbiome-conditioned medium. VEC transwells provide a suitable ex vivo system to support the production of bacterial metabolites consistent with the vaginal milieu allowing subsequent in vitro studies with enhanced accuracy and utility.

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

BACKGROUND: Farnesol 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). METHODS: The 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. RESULTS: Only 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). CONCLUSIONS: Farnesol 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.

Anti-Candida and Anti-Inflammatory Properties of a Vaginal Gel Formulation: Novel Data Concerning Vaginal Infection and Dysbiosis.

Vaginal ecosystem is a unique environment where, in physiological conditions, lactobacilli dominate. However, pathogenic microbial species responsible for vaginitis and vaginosis can also harbor vaginal microbiota. To extend our previously published data, we analyzed here both the anti-Candida and anti-inflammatory properties of the vaginal gel formulation, Respecta® Balance Gel (RBG), commercialized as an adjuvant to treat vaginitis and vaginosis. We evaluated its activity by an in vitro model where a monolayer of A-431 vaginal epithelial cells was infected by Candida albicans in the presence of RBG or the placebo formulation (pRBG). Specifically, we tested the RBG capacity to counteract C. albicans virulence factors and their anti-inflammatory properties. Our results show that, unlike the placebo, RBG reduces C. albicans adhesion, its capacity to form hyphae and C. albicans-induced vaginal cell damage. Interestingly, both RBG and pRBG reduce LPS-induced IL-8 secretion (with RBG being the most effective), demonstrating that also the placebo retains anti-inflammatory properties. From our experimental approach, we highlighted the possible role of farnesol on such effects, but we would like to point out that lactic acid, polydextrose and glycogen too must be relevant in the actual application. In summary, our results show that RBG impairs C. albicans virulence and is able to reduce the inflammation in the vaginal environment, ultimately allowing the establishment of a balanced vaginal ecosystem.

Isolation of Vaginal Epithelial Cells: In Preparation of Autologous Vaginal Tissue Lining for Congenital Absence of Vagina Patients.

Infertility is a condition affecting women who are born with an underdeveloped or absent vagina, a birth defect known as congenital absence of the vagina. It is a rare disorder where the development of the Mullerian duct is obstructed by unidentified causes. The case is seldom reported due to the low prevalence and sparse epidemiology studies worldwide. A potential solution for the disorder is neovaginal creation with in vitro cultured vaginal mucosa. Limited studies have reported its application, but none are reproducible or specific regarding the established processes for acquiring vaginal epithelial cells from vaginal biopsies. These research gaps were adequately answered with an epidemiology study of inpatient details in Hospital Canselor Tuanku Muhriz, Malaysia, established methods and outcomes of vaginal tissue processing and isolation, and characterization of vaginal epithelial cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and immunofluorescence assays. The reported evidence and speculation that the disorder arises because of a cellular transition event between epithelial and mesenchymal cells during the development of the Mullerian duct could be key in the creation of neovaginas using established culture procedures to improve surgical results and restore fertility.

In Vitro Screen of Lactobacilli Strains for Gastrointestinal and Vaginal Benefits.

Traditional probiotics comprise mainly lactic acid bacteria that are safe for human use, tolerate acid and bile, and adhere to the epithelial lining and mucosal surfaces. In this study, one hundred commercial and non-commercial strains that were isolated from human feces or vaginal samples were tested with regards to overall growth in culture media, tolerance to acid and bile, hydrogen peroxide (H2O2) production, and adhesion to vaginal epithelial cells (VECs) and to blood group antigens. As a result, various of the tested lactobacilli strains were determined to be suitable for gastrointestinal or vaginal applications. Commercial strains grew better than the newly isolated strains, but tolerance to acid was a common property among all tested strains. Tolerance to bile varied considerably between the strains. Resistance to bile and acid correlated well, as did VEC adhesion and H2O2 production, but H2O2 production was not associated with resistance to bile or acid. Except for L. iners strains, vaginal isolates had better overall VEC adhesion and higher H2O2 production. Species- and strain-specific differences were evident for all parameters. Rank-ordered clustering with nine clusters was used to identify strains that were suitable for gastrointestinal or vaginal health, demonstrating that the categorization of strains for targeted health indications is possible based on the parameters that were measured in this study.

Ureaplasma parvum infection induces inflammatory changes in vaginal epithelial cells independent of sialidase.

BACKGROUND: Ureaplasma, a genus of the order Mycoplasmatales and commonly grouped with Mycoplasma as genital mycoplasma is one of the most common microbes isolated from women with infection/inflammation-associated preterm labor (PTL). Mycoplasma spp. produce sialidase that cleaves sialic acid from glycans of vaginal mucous membranes and facilitates adherence and invasion of the epithelium by pathobionts, and dysregulated immune response. However, whether Ureaplasma species can induce the production of sialidase is yet to be demonstrated. We examined U. parvum-infected vaginal epithelial cells (VECs) for the production of sialidase and pro-inflammatory cytokines. METHODS: Immortalized VECs were cultured in appropriate media and treated with U. parvum in a concentration of 1 × 105 DNA copies/ml. After 24 h of treatment, cells and media were harvested. To confirm infection and cell uptake, immunocytochemistry for multi-banded antigen (MBA) was performed. Pro-inflammatory cytokine production and protein analysis for sialidase confirmed pro-labor pathways. RESULTS: Infection of VECs was confirmed by the presence of intracellular MBA. Western blot analysis showed no significant increase in sialidase expression from U. parvum-treated VECs compared to uninfected cells. However, U. parvum infection induced 2-3-fold increased production of GM-CSF (p = 0.03), IL-6 (p = 0.01), and IL-8 (p = 0.01) in VECs compared to controls. CONCLUSION: U. parvum infection of VECs induced inflammatory imbalance associated with vaginal dysbiosis but did not alter sialidase expression at the cellular level. These data suggest that U. parvum's pathogenic effect could be propagated by locally produced pro-inflammatory cytokines and, unlike other genital mycoplasmas, may be independent of sialidase.

Interferon Epsilon Signaling Confers Attenuated Zika Replication in Human Vaginal Epithelial Cells.

Zika virus (ZIKV) is an emerging flavivirus that causes congenital birth defects and neurological compilations in the human host. Although ZIKV is primarily transmitted through infected mosquitos, recent studies reveal sexual contact as a potential transmission route. In vagina-bearing individuals, the vaginal epithelium constitutes the first line of defense against viruses. However, it is unclear how ZIKV interacts with the vaginal epithelium to initiate ZIKV transmission. In this study, we demonstrate that exposing ZIKV to human vaginal epithelial cells (hVECs) resulted in de novo viral RNA replication, increased envelope viral protein production, and a steady, extracellular release of infectious viral particles. Interestingly, our data show that, despite an increase in viral load, the hVECs did not exhibit significant cytopathology in culture as other cell types typically do. Furthermore, our data reveal that the innate antiviral state of hVECs plays a crucial role in preventing viral cytopathology. For the first time, our data show that interferon epsilon inhibits ZIKV replication. Collectively, our results in this study provide a novel perspective on the viral susceptibility and replication dynamics during ZIKV infection in the human vaginal epithelium. These findings will be instrumental towards developing therapeutic agents aimed at eliminating the pathology caused by the virus.

Divergent EGFR/MAPK-Mediated Immune Responses to Clinical Candida Pathogens in Vulvovaginal Candidiasis.

Vulvovaginal candidiasis (VVC) is characterized by symptomatic inflammatory responses in the vagina caused by Candida albicans and non-albicans Candida (NAC) species. The epidermal growth factor receptor (EGFR) -mitogen-activated protein kinase (MAPK) signaling pathway has been linked to immune responses of oral mucosa after C. albicans exposure, but whether this pathway plays a similar response in vaginal epithelial cells is not known. Here, we observed that phosphorylation of EGFR and p38 was continuously activated in vaginal epithelial cells by C. albicans strain SC5314. This differs markedly from oral epithelial cells, which respond in a biphasic manner in order to properly discriminate the morphology of C. albicans. When compared with SC5314, a highly azole-resistant C. albicans isolate 1052 can induce a stronger phosphorylated signal of EGFR and p38, while clinically-isolated NAC strains including C. tropicalis, C. glabrata, C. parapsilosis and C. auris trigger higher levels of phosphorylated ERK1/2 and c-Fos than C. albicans. Inhibition of EGFR significantly reduces inflammatory response and epithelial damage induced by C. albicans both in vitro and in vivo, while inhibition of p38 leads to significant repair of epithelial damage triggered by both C. albicans and NAC species. These results confirm the importance of the EGFR-MAPK signaling in VVC pathogenesis and highlight the remarkable immunogenic differences between C. albicans and NAC species in host-microbe interactions.

[Effect of Empagliflozin on Candida glabrata Adhesion to Vaginal Epithelial Cells].

A high incidence of genital infections, such as vulvovaginal candidiasis, has been reported in patients with diabetes treated with sodium-glucose co-transporter type 2 inhibitors. This is because Candida growth and virulence are enhanced in high glucose environments. Our previous study demonstrated that the adhesive interaction between Candida complement receptors and a ligand on vaginal epithelial cells (intracellular adhesion molecule-1: ICAM-1) is a factor for Candida albicans colonization, and the high ICAM-1 expression by vaginal epithelial cells exposed to high glucose conditions increases C. albicans adhesion. In this study, we examined the effect of a sodium-glucose co-transporter type 2 inhibitor, empagliflozin, on Candida glabrata adhesion to human cells (VK2/E6E7). There was no significant difference among four conditions that contained empagliflozin at various concentrations. We demonstrated that empagliflozin does not affect C. glabrata adhesion to VK2/E6E7 cells.

Berberine Inhibits the Adhesion of Candida albicans to Vaginal Epithelial Cells.

Vulvovaginal candidiasis (VVC) is an inflammatory disease of the vagina mainly caused by Candida albicans (C. albicans), which affects around three-quarters of all women during their reproductive age. Although some antifungal drugs such as azoles have been applied clinically for many years, their therapeutic value is very limited due to the emergence of drug-resistant strains. Previous studies have shown that the adhesion of C. albicans to vaginal epithelial cells is essential for the pathogenesis of VVC. Therefore, preventing the adhesion of C. albicans to vaginal epithelial cells may be one of the most effective strategies for the treatment of VVC. Berberine (BBR) is a biologically active herbal alkaloid that was used to treat VVC. However, so far, its mechanism has remained unclear. This study shows BBR significantly inhibits the adhesion of C. albicans to vaginal epithelial cells by reducing the expressions of ICAM-1, mucin1, and mucin4 in vaginal epithelial cells, which play the most important role in modulating the adhesion of C. albicans to host cells, and balancing IL-2 and IL-4 expressions, which play a key effect on regulating the inflammatory response caused by C. albicans infection. Hence, our findings demonstrate that BBR may be a potential therapeutic agent for VVC by interfering with the adhesion of C. albicans to vaginal epithelial cells and represents a new pathway for developing antifungal therapies agents from natural herbs.

pH Stress Mediated Alteration in Protein Composition and Reduction in Cytotoxic Potential of Gardnerella vaginalis Membrane Vesicles.

The vagina of healthy women is predominantly colonized by lactobacilli but it also harbors a limited proportion of certain anaerobes such as Gardnerella vaginalis. An increase in G. vaginalis along with other anaerobes on account of perturbation in the vaginal microbiota is associated with bacterial vaginosis (BV). Although strategies adopted by G. vaginalis for survival and pathogenesis in a conducive environment (i.e., high vaginal pH, characteristic of BV) have been previously studied, the approaches potentially employed for adaptation to the low pH of the healthy vagina are unknown. In the present study, we investigated the effect of acidic stress on the modulation of the production and function of membrane vesicles (MVs) of G. vaginalis. pH stress led to a distortion of the bacterial cell morphology as well as an altered biogenesis of MVs, as revealed by transmission electron microscopy (TEM). Both qualitative and quantitative differences in protein content of MVs produced in response to pH stress were observed by flow cytometry. A significant change in the protein composition characterized by presence of chaperones despite a reduction in number of proteins was also noted in the stress induced MVs. Further, these changes were also reflected in the reduced cytotoxic potential toward vaginal epithelial cells. Although, these findings need to be validated in the in vivo settings, the modulation of G. vaginalis MV biogenesis, composition and function appears to reflect the exposure to acidic conditions prevailing in the host vaginal mileu in the absence of vaginal infection.

The Role of 17ß-Estrogen in Escherichia coli Adhesion on Human Vaginal Epithelial Cells via FAK Phosphorylation.

Estrogen, the predominant sex hormone, has been found to be related to the occurrence of vaginal infectious diseases. However, its role in the occurrence and development of bacterial vaginitis caused by Escherichia coli is still unclear. The objective of this study was to investigate the role of 17ß-estrogen in E. coli adhesion on human vaginal epithelial cells. The vaginal epithelial cell line VK2/E6E7 was used to study the molecular events induced by estrogen between E. coli and cells. An adhesion study was performed to evaluate the involvement of the estrogen-dependent focal adhesion kinase (FAK) activation with 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. The results showed that, following stimulation with 1,000 nM estrogen for 48 h, transient activation of ERα and FAK was observed, as was an increased average number of E. coli cells adhering to vaginal epithelial cells. In addition, estrogen-induced activation of ERα and FAK was inhibited by the specific inhibitor of ERα, especially when the inhibitor reached a 10 µM concentration and acted for 1 h, and a decrease in the number of adherent E. coli cells was observed simultaneously. However, this inhibitory effect diminished as the concentration of estrogen increased. In conclusion, FAK and ERα signaling cascades were associated with the increasing E. coli adherence to vaginal epithelial cells, which was promoted by a certain concentration of estrogen.

Immunocompetent Human 3D Organ-Specific Hormone-Responding Vaginal Mucosa Model of HIV-1 Infection.

The lack of appropriate experimental models often limits our ability to investigate the establishment of infections in specific tissues. To reproduce the structural and spatial organization of vaginal mucosae to study human immunodeficiency virus type-1 (HIV-1) infection, we used the self-assembly technique to bioengineer tridimensional vaginal mucosae using human cells extracted from HIV-1-negative healthy pre- and postmenopausal donors. We produced a stroma, free of exogenous material, that can be adapted to generate near-to-native vaginal tissue with the best complexity obtained with seeded epithelial cells on the organ-specific stroma. The autologous engineered tissues had mechanical properties close to native mucosa and shared similar glycogen production, which declined in reconstructed tissues of the postmenopausal donor. The in vitro-engineered tissues were also rendered immune competent by adding human monocyte-derived macrophages (MDMs) on the epithelium or in the stroma layers. The model was infected with HIV-1, and viral replication and transcytosis were observed when immunocompetent reconstructed vaginal mucosa tissue has incorporated MDMs into the stroma and infected with free HIV-1 green fluorescent protein (GFP) viral particles. These data illustrate a natural permissiveness of immunocompetent untransformed human vaginal mucosae to HIV-1 infection. This model offers a physiological tool to explore viral load, HIV-1 transmission in an environment that may contribute to the virus propagation, and new antiviral treatments in vitro. Impact statement This study introduces an innovative immunocompetent three-dimensional human organ-specific vaginal mucosa free of exogenous material for in vitro modeling of human immunodeficiency virus type-1 (HIV-1) infection. The proposed model is histologically close to native tissue, especially by presenting glycogen accumulation in the epithelium's superficial cells, responsive to estrogen, and able to sustain a monocyte-derived macrophage population infected or not by HIV-1 during ∼2 months.

The Role of Purinergic Signaling in Trichomonas vaginalis Infection.

Trichomoniasis, one of the most common non-viral sexually transmitted infections worldwide, is caused by the parasite Trichomonas vaginalis. The pathogen colonizes the human urogenital tract, and the infection is associated with complications such as adverse pregnancy outcomes, cervical cancer, and an increase in HIV transmission. The mechanisms of pathogenicity are multifactorial, and controlling immune responses is essential for infection maintenance. Extracellular purine nucleotides are released by cells in physiological and pathological conditions, and they are hydrolyzed by enzymes called ecto-nucleotidases. The cellular effects of nucleotides and nucleosides occur via binding to purinoceptors, or through the uptake by nucleoside transporters. Altogether, enzymes, receptors and transporters constitute the purinergic signaling, a cellular network that regulates several effects in practically all systems including mammals, helminths, protozoa, bacteria, and fungi. In this context, this review updates the data on purinergic signaling involved in T. vaginalis biology and interaction with host cells, focusing on the characterization of ecto-nucleotidases and on purine salvage pathways. The implications of the final products, the nucleosides adenosine and guanosine, for human neutrophil response and vaginal epithelial cell damage reveal the purinergic signaling as a potential new mechanism for alternative drug targets.

Transcriptional response of vaginal epithelial cells to medroxyprogesterone acetate treatment results in decreased barrier integrity.

Medroxyprogesterone acetate (MPA) is a frequently used hormonal contraceptive that has been shown to significantly increase HIV-1 susceptibility by approximately 40 %. However, the underlying mechanism by which this occurs remains unknown. Here, we examined the biological response to MPA by vaginal epithelial cells, the first cells to encounter HIV-1 during sexual transmission, in order to understand the potential mechanism(s) of MPA-mediated increase of HIV-1 infection. Using microarray analysis and in vitro assays, we characterized the response of vaginal epithelial cells, grown in biologically relevant air-liquid interface (ALI) cultures, to physiological levels of female sex hormones, estradiol (E2), progesterone (P4), or MPA. Transcriptional profiling of E2, P4 or MPA-treated vaginal epithelial cells indicated unique transcriptional profiles associated with each hormone. MPA treatment increased transcripts of genes related to cholesterol/sterol synthesis and decreased transcripts related to cell division and cell-cell adhesion, results not seen with E2 or P4 treatments. MPA treatment also resulted in unique gene expression indicative of decreased barrier integrity. Functional assays confirmed that MPA, but not E2 or P4 treatments, resulted in increased epithelial barrier permeability and inhibited cell cycle progression. The effects of MPA on vaginal epithelial cells seen in this study may help explain the increase of HIV-1 infection in women who use MPA as a hormonal contraceptive.

Evaluation of the Inhibitory Effects of Lactobacillus gasseri and Lactobacillus crispatus on the Adhesion of Seven Common Lower Genital Tract Infection-Causing Pathogens to Vaginal Epithelial Cells.

Background/Purpose: Lactobacillus colonization is important to maintain urogenital flora stability and prevent pathogenic infection. Different Lactobacillus species have distinct properties and effects on the urogenital flora. To select probiotics that colonize the vagina and provide protection against pathogenic infection, we evaluated the adhesion of five Lactobacillus strains and their inhibitory effects on the adhesion of pathogens to vaginal epithelial cells (VECs). Methods and Materials: (1) Lactobacillus adhesion experiments: VK2/E6E7 and primary VECs were used to evaluate the adhesion of two Lactobacillus gasseri and three Lactobacillus crispatus strains. The adhesion of these five Lactobacillus strains was compared. (2) Adhesion inhibition experiments: The inhibitory effects of the five Lactobacillus strains on the adhesion of pathogens (Gardnerella, Mobiluncus, Candida albicans, Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis) were evaluated by adhesion exclusion, displacement, and competition experiments. Results: (1) Lactobacillus adhesion was stronger in the primary VECs than in the VK2/E6E7 VECs (P < 0.05). The adhesion of the three L. crispatus strains was stronger than that of the two L. gasseri strains (P < 0.05). L. crispatus 4# showed the strongest adhesion. (2) The exclusion, displacement, and competition experiments showed that all five Lactobacillus strains significantly inhibited the adhesion of the seven pathogenic strains to the VECs (P < 0.05). The displacement effect was stronger than the exclusion and competition effects of each Lactobacillus strain. (3) The results of the exclusion, displacement, and competition experiments indicated that L. gasseri 1# showed the strongest adhesion inhibition of C. albicans and S. agalactiae. L. crispatus 3# showed the strongest adhesion inhibition of S. aureus, whereas L. crispatus 4# showed the strongest adhesion inhibition of Gardnerella, Mobiluncus, E. coli, and E. faecalis. Conclusion: The source of the VECs might not affect the selection of the most adhesive Lactobacillus strain. L. crispatus showed stronger VEC adhesion than L. gasseri. The degree of antagonism of the Lactobacillus strains toward the different pathogens varied. This result provides incentives for personalized clinical treatment.