primary human vaginal epithelial cells  (Lifeline Cell Technology)

Journal: Microorganisms

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

doi: 10.3390/microorganisms12081634

Figure Lengend Snippet: Evaluation of C. albicans (Ca) adhesion capacity to a vaginal epithelial cell monolayer in the presence of B. coagulans (Bc) ( A ). The histogram graph shows the average % ± SEM of fungal adhesion inhibition exerted by B. coagulans (Bc). Data are from three independent experiments. ( B ) Assessment of B. coagulans (Bc) capacity to co-aggregate with C. albicans (Ca) after 1 h of co-incubation. Boxes in the heatmap represent the score assigned to each sample in three independent experiments—0: no aggregation; 1: aggregates with small clusters; 2: aggregates with larger numbers of yeasts; 3: clumps visible with the naked eye containing large numbers of yeast cells; 4: maximum score for large clumps visible with the naked eye in the well center. ( C – E ) Effect of B. coagulans (Bc) on C. albicans (Ca) hyphal formation upon 4 h of co-incubation. Hyphal fragments were optically counted by fluorescent microscopy imaging. The fungal cell wall was stained with Uvitex 2B fluorescent dye. ( C ) The bars chart reports the mean percentage ± SEM of hyphal fragments counted in three different fields from three independent experiments. Statistical analysis was performed by the unpaired, two-tailed Student t -test. Ca vs. Bc + Ca * p < 0.05. ( D ) The heatmap shows the % of hyphal fragments counted in each field; the squares’ color indicates the abundance of hyphae in the field (red: high % hyphal fragments; green: low % hyphal fragments). ( E ) Representative images from fluorescence microscopy analysis are shown from C. albicans (Ca) or C. albicans plus B. coagulans (Bc + Ca) taken at 40× magnification.

Article Snippet: Two different human epithelial cell lines were employed: the A-431 cell line from vaginal epithelial squamous cell carcinoma (ATCC CLR-1555) and the CaCo-2 cell line from colon–rectal adenocarcinoma (ATCC HTB-37).

Techniques: Inhibition, Incubation, Microscopy, Imaging, Staining, Two Tailed Test, Fluorescence

Journal: Microorganisms

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

doi: 10.3390/microorganisms12081634

Figure Lengend Snippet: ( A ) Percentage of vaginal cell damage pre-colonized or not by B. coagulans (Bc) for 6 h and infected for further 18 h with C. albicans (Ca). The chart reports the average percentage of cell damage ± SEM of triplicate samples from three different experiments. Statistical analysis was performed by the unpaired, two-tailed Student t -test. Ca vs. Bc + Ca * p < 0.05. ( B ) Production of β-defensin-2 by vaginal epithelial cells pre-colonized or not by B. coagulans (Bc) for 6 h and infected for further 18 h with C. albicans (Ca). Uninfected cells (Ctrl) and cells colonized by the bacterium without C. albicans were also included in the experiments. The graph reports the mean ± SEM from three independent experiments. Statistical analysis was performed by the one-way ANOVA test followed by the uncorrected Fisher’s LSD test. Untreated cells vs. Bc pre-colonized cells * p < 0.05.

Article Snippet: Two different human epithelial cell lines were employed: the A-431 cell line from vaginal epithelial squamous cell carcinoma (ATCC CLR-1555) and the CaCo-2 cell line from colon–rectal adenocarcinoma (ATCC HTB-37).

Techniques: Infection, Two Tailed Test

Journal: Microorganisms

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

doi: 10.3390/microorganisms12081634

Figure Lengend Snippet: Evaluation of antifungal effect permanency upon B. coagulans removal. C. albicans (Ca) ( A ) and C. parapsilosis (Cp) ( B ) metabolic activity quantification after being incubated with B. coagulans or sterile medium for 24 h and subsequent fungal isolation and cultivation for 24 h in the lack of bacteria. The graphs show the mean OD at 492 nm wavelength ± SEM from triplicate sample of three different experiments. Statistical analysis was performed by the one-way ANOVA test followed by the uncorrected Fisher’s LSD test. ns = not significant. ( C ) Capacity of B. coagulans spores to germinate on intestinal epithelial cells CaCo-2. Bacterial spores were seeded on an intestinal epithelial cell monolayer of CaCo-2 and incubated at 37 °C + 5% CO 2 for 24 h. After incubation, monolayers were photographed (upper images) and subsequently lysed. A Gram staining was then performed to visualize the presence of germinated B. coagulans (Bc) (lower images).

Article Snippet: Two different human epithelial cell lines were employed: the A-431 cell line from vaginal epithelial squamous cell carcinoma (ATCC CLR-1555) and the CaCo-2 cell line from colon–rectal adenocarcinoma (ATCC HTB-37).

Techniques: Activity Assay, Incubation, Sterility, Isolation, Bacteria, Staining

Journal: bioRxiv

Article Title: Chlamydial YAP activation in host endocervical epithelial cells mediates pro-fibrotic paracrine stimulation of fibroblasts

doi: 10.1101/2023.05.30.542940

Figure Lengend Snippet: (A, B) Volcano plot of differential gene expression of Chlamydia trachomatis serovar L2-infected (A) or mock-infected (B) primary human cervical epithelial cells (HCECs) relative to equivalent human vaginal epithelial cells (HVEs) at 24 hpi (see also Supplementary Table 1). n = 3, with a minimum of 3×10 7 unstranded single reads per replicate with a mean length of 150 bp. All fold changes are relative to the mock-infected control; red dots: false discovery rate p-value (FDRP) ≤ 0.05, labels: top 20 genes with lowest FDRP values. (C) Heatmap of cytokine and EMT-associated gene expression in mock-infected (left columns) and C. trachomatis serovar L2-infected (right columns) HCECs. All fold changes are relative to an equivalent infection in HVEs; only genes differentially expressed (FDRP ≤ 0.05) in at least one comparison are shown.

Article Snippet: Primary human vaginal epithelial cells (HVEs, ATCC PCS-480-010, Lot 80924222) were cultured at 37° C with 5% atmospheric CO 2 in Vaginal Epithelial Cell Basal Medium (VECBM, ATCC PCS-480-030) supplemented with all contents of a Vaginal Epithelial Cell Growth Kit (ATCC PCS-480-040), per manufacturer’s instructions.

Techniques: Gene Expression, Infection, Control, Comparison

Journal: bioRxiv

Article Title: Chlamydial YAP activation in host endocervical epithelial cells mediates pro-fibrotic paracrine stimulation of fibroblasts

doi: 10.1101/2023.05.30.542940

Figure Lengend Snippet:

Article Snippet: Primary human vaginal epithelial cells (HVEs, ATCC PCS-480-010, Lot 80924222) were cultured at 37° C with 5% atmospheric CO 2 in Vaginal Epithelial Cell Basal Medium (VECBM, ATCC PCS-480-030) supplemented with all contents of a Vaginal Epithelial Cell Growth Kit (ATCC PCS-480-040), per manufacturer’s instructions.

Techniques: Comparison, Infection

Journal: bioRxiv

Article Title: Chlamydial YAP activation in host endocervical epithelial cells mediates pro-fibrotic paracrine stimulation of fibroblasts

doi: 10.1101/2023.05.30.542940

Figure Lengend Snippet: (A, B) Heatmap of cytokine (A) and collagen/EMT-associated (B) gene expression in mock-infected HCECs relative to equivalent HVEs (left columns), Ct L2-infected HCECs relative to equivalent HVEs (center columns), and Ct L2-infected HCECs relative to mock-infected HCECs (right columns). Only genes differentially expressed (FDRP ≤ 0.05) in at least one comparison are shown. (C, D) Expression of epithelial/mesenchymal differentiation markers (C) and EMT-associated transcription factors (D) at 24 hpi in mock- and Ct L2-infected End1/E6E7 immortalized endocervical epithelial cells, as measured by RT-qPCR. n = 3 biological replicates; fold changes are relative to mean expression of the mock-infected and untreated control. Whiskers: minimum to maximum; asterisks: p-values ≤ 0.05, using pairwise Student’s t-tests and Bonferroni’s correction for multiple comparisons.

Article Snippet: Primary human vaginal epithelial cells (HVEs, ATCC PCS-480-010, Lot 80924222) were cultured at 37° C with 5% atmospheric CO 2 in Vaginal Epithelial Cell Basal Medium (VECBM, ATCC PCS-480-030) supplemented with all contents of a Vaginal Epithelial Cell Growth Kit (ATCC PCS-480-040), per manufacturer’s instructions.

Techniques: Gene Expression, Infection, Comparison, Expressing, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: Chlamydial YAP activation in host endocervical epithelial cells mediates pro-fibrotic paracrine stimulation of fibroblasts

doi: 10.1101/2023.05.30.542940

Figure Lengend Snippet: Coculture with infected endocervical epithelial cells alters the putative myofibroblast transcriptome. (A) Representative micrographs of mock-(left) or C. trachomatis serovar L2-infected (right) End1/E6E7 endocervical epithelial cells cocultured with KCO2 uterine fibroblasts for 23 h (starting at 1 hpi); green: actin expression (phalloidin), blue: DAPI, arrowheads: chlamydial inclusions, scale bar: 20 μm. (B) Uniform manifold approximation and projection (UMAP) feature plot of KRT19 (top) and VIM (bottom) expression in same-well coculture of mock- or Ct serovar L2-infected End1 endocervical epithelial cells with KCO2 fibroblasts for 23 h (starting at 1 hpi). n = 2 biological replicates, 1000-cell libraries per sample, approx. 5-7 x 10 4 reads/cell. (C) (top) UMAP plot of fibroblast ( VIM +) subclustering in same-well coculture of mock- or Ct serovar L2-infected End1 endocervical epithelial cells with KCO2 fibroblasts. Colors: cluster identity, all percentages relative to total fibroblast population of the respective mock-/ Ct L2-infected coculture. (bottom) Violin plot of fibroblast ( VIM +) per-cluster expression of α-SMA/ ACTA2 (left) and TIMP1 (right) in same-well coculture of mock- or Ct serovar L2-infected End1 endocervical epithelial cells with KCO2 fibroblasts. Colors: cluster identity, dots: per-cell expression values. (D) Per-cell expression heatmap of genes differentially expressed (p ≤ 0.05) by cluster F3 fibroblasts of Ct serovar L2-infected cocultures (right), relative to cluster F3 fibroblasts of mock-infected cocultures (left).

Article Snippet: Primary human vaginal epithelial cells (HVEs, ATCC PCS-480-010, Lot 80924222) were cultured at 37° C with 5% atmospheric CO 2 in Vaginal Epithelial Cell Basal Medium (VECBM, ATCC PCS-480-030) supplemented with all contents of a Vaginal Epithelial Cell Growth Kit (ATCC PCS-480-040), per manufacturer’s instructions.

Techniques: Infection, Expressing

Journal: bioRxiv

Article Title: Chlamydial YAP activation in host endocervical epithelial cells mediates pro-fibrotic paracrine stimulation of fibroblasts

doi: 10.1101/2023.05.30.542940

Figure Lengend Snippet: (A) Schematic representation of the transwell-mediated approach of coculturing C. trachomatis -infected endocervical epithelial cells (top compartment) with uninfected uterine fibroblasts (bottom compartment). (B) Representative micrographs of collagen I (green) produced by KCO2 fibroblasts cocultured with mock-infected, Ct L2-infected, or TGF-β1-treated (20 ng/mL) End1 cells for 24 h, starting at 1 hpi. Scale bar: 20 μm. (C) Quantification of mean fluorescence intensity (MFI) of collagen I in (B). n = 3 biological replicates, 5 fields measured per sample. Black bars: group means; asterisks: p-values ≤ 0.05, using pairwise Wilcoxon rank sum tests and Bonferroni’s correction for multiple comparisons. (D) COL1A1 and ACTA2 expression as measured by RT-qPCR in KCO2 fibroblasts cocultured for 24 h with siRNA-transfected (10 nM of YAP1-targeting or non-targeting siRNA, for 24 h prior to infection), Ct L2-infected End1 cells (coculture starting at 1 hpi). n = 3 biological replicates; fold changes are relative to mean expression of the equivalent mock-infected control. Whiskers: minimum to maximum; asterisks: p-values ≤ 0.05, using Student’s t-tests and Bonferroni’s correction for multiple comparisons.

Article Snippet: Primary human vaginal epithelial cells (HVEs, ATCC PCS-480-010, Lot 80924222) were cultured at 37° C with 5% atmospheric CO 2 in Vaginal Epithelial Cell Basal Medium (VECBM, ATCC PCS-480-030) supplemented with all contents of a Vaginal Epithelial Cell Growth Kit (ATCC PCS-480-040), per manufacturer’s instructions.

Techniques: Infection, Produced, Fluorescence, Expressing, Quantitative RT-PCR, Transfection, Control