Journal: Frontiers in Immunology

Article Title: Vitamin D boosts HIV-1 resistance in female genital epithelial cells by enhancing antiviral cathelicidin expression

doi: 10.3389/fimmu.2026.1758656

Figure Lengend Snippet: Transwell assay model. Monolayers of endocervical (End1), ectocervical (Ect1) and Vaginal (Vk2) epithelial cells grown in 24-well inserts for 24 hours in a liquid-liquid interface to obtain functional monolayers. The functional monolayers were treated with calcitriol at 1x10 -8 M or EtOH 0.1% at the basal side for 24 hours to resemble uptake of this hormone from circulation. Transmigration assays were performed for three hours, adding 1ng/mL of free-cell R5-tropic HIV-1 (Bal) to monolayers of epithelial cells at the upper chamber (Apical side). Activated CD4 + T cells were used as targets of infection at the lower chamber (Basal side) for evaluating infectiveness of transmigrated virions (a ratio of 1.5 epithelial cells to 1 CD4 T cells was used during the 3 hours co-culture of these cells); the remaining virions, at the upper chamber (untransmigrated), were also evaluated for their infectiveness of activated CD4 + T cells. The infection cultures for transmigrated and untransmigrated virions were maintained by 84 hours and infection was determined by detection of p24 by flow cytometry.

Article Snippet: Immortalized endocervical (End1) (ATCC CRL-2615 TM ), ectocervical (Ect1) (ATCC CRL-2614 TM ) and Vaginal (Vk2) (ATCC CRL-2616 TM ) epithelial cells were used in this study.

Techniques: Transwell Assay, Functional Assay, Transmigration Assay, Infection, Co-Culture Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Vitamin D boosts HIV-1 resistance in female genital epithelial cells by enhancing antiviral cathelicidin expression

doi: 10.3389/fimmu.2026.1758656

Figure Lengend Snippet: Comparison of the percentage of HIV-1 infection of CD4 + T cells with virions from the apical or basal sides of End1, Ect1 and Vk2 epithelial monolayers in the absence of treatment. Comparison of the frequency of infected p24 + CD4 + T cells with virions from the basal side (lower chamber) and apical side (upper chamber) of transwell inserts containing untreated End1 (basal, light blue dots; apical, dark blue dots), Ect1 (basal, light green dots; apical, dark green dots) or Vk2 monolayers (basal, light pink dots; apical, dark pink dots). Infection of CD4 + T cells in absence of epithelial cells (No-EpCs) included as a positive control of viral infectious capacity and cell susceptibility to infection (gray dots). Comparison between infectiveness of HIV-1 virions from the apical and basal sides of each epithelial monolayer were made using the Ratio paired t-test, and comparisons among the three cell types for the HIV-1 infection with virions from the apical or basal compartments were done using One-way ANOVA. Each paired points represent one blood donor for the source of CD4 T cells, for a total of six biological replicates (i.e., 6 independent experiments).

Article Snippet: Immortalized endocervical (End1) (ATCC CRL-2615 TM ), ectocervical (Ect1) (ATCC CRL-2614 TM ) and Vaginal (Vk2) (ATCC CRL-2616 TM ) epithelial cells were used in this study.

Techniques: Comparison, Infection, Positive Control

Journal: Frontiers in Immunology

Article Title: Vitamin D boosts HIV-1 resistance in female genital epithelial cells by enhancing antiviral cathelicidin expression

doi: 10.3389/fimmu.2026.1758656

Figure Lengend Snippet: Frequency of HIV-1-infected CD4 + T cells and MFI of p24 in CD4 + T cells following the culture with virions retained at the apical side (upper chamber) of the VitD- or EtOH-conditioned genital epithelial monolayers. Comparison of the percentage of p24 + CD4 + T cells following contact with untransmigrated HIV-1 Bal (1ng of p24) exposed to VitD- or EtOH-treated End1 (A) , Ect1 (C) or Vk2 monolayers (E) . Comparison of the MFI of expression of p24 in CD4 + T cells following contact of virions with calcitriol- or EtOH-treated End1 (B) , Ect1 (D) or Vk2 monolayers (F) . All the epithelial cells were treated with VitD or EtOH for 24 hours before co-cultures. Each paired points represent one blood donor for the source of CD4 T cells, for a total of six biological replicates (i.e., 6 independent experiments). The percentage of HIV-1 reduction by VitD compared to EtOH treatment is depicted in each figure. Comparison between treatments were made using the Ratio paired t-test, *p≤ 0.05; **p≤ 0.01; ***p≤ 0.001.

Article Snippet: Immortalized endocervical (End1) (ATCC CRL-2615 TM ), ectocervical (Ect1) (ATCC CRL-2614 TM ) and Vaginal (Vk2) (ATCC CRL-2616 TM ) epithelial cells were used in this study.

Techniques: Infection, Comparison, Expressing

Journal: Frontiers in Immunology

Article Title: Vitamin D boosts HIV-1 resistance in female genital epithelial cells by enhancing antiviral cathelicidin expression

doi: 10.3389/fimmu.2026.1758656

Figure Lengend Snippet: Frequency of HIV-1-infected CD4 + T cells and MFI of p24 in CD4 + T cells following the culture with virions transmigrated through the VitD- or EtOH-conditioned epithelial monolayers (lower chamber). Comparison of the percentage of p24 + CD4 + T cells following culture with HIV-1 Bal (1ng of p24) transmigrated through VitD- or EtOH-treated End1 (A) , Ect1 (C) or Vk2 monolayers (E) . Comparison of the MFI of expression of p24 in CD4 + T cells following the transmigration of virions through VitD- or EtOH-treated End1 (B) , Ect1 (D) or Vk2 monolayers (F) . Comparison of the frequency of p24 + CD4 + T cells infected with virions from the basal side (lower chamber) and apical side (upper chamber) of VitD-treated End1(light blue dots represent the basal compartment; dark blue dots represent the apical compartment), Ect1 (light green dots, basal; and dark green dots, apical) or Vk2 (light pink dots, basal; and dark pink dots, apical) monolayers (G) . All the epithelial cells were treated with VitD or EtOH for 24 hours before co-cultures. A ratio of 1.5 epithelial cells to 1 CD4 T cells was used during the 3 hours co-culture of these cells. Each paired points represent one blood donor for the source of CD4 T cells, for a total of six biological replicates (i.e., 6 independent experiments). The percentage of HIV-1 reduction by VitD compared to EtOH treatment is depicted in each figure. Comparison between VitD and EtOH treatments and comparison between infectious virions at the apical and basal sides of each epithelial monolayer were made using the Ratio paired t-test, *p≤ 0.05; **p≤ 0.01; ***p≤ 0.001; ****p<0.0001.

Article Snippet: Immortalized endocervical (End1) (ATCC CRL-2615 TM ), ectocervical (Ect1) (ATCC CRL-2614 TM ) and Vaginal (Vk2) (ATCC CRL-2616 TM ) epithelial cells were used in this study.

Techniques: Infection, Comparison, Expressing, Transmigration Assay, Co-Culture Assay

Journal: Frontiers in Immunology

Article Title: Vitamin D boosts HIV-1 resistance in female genital epithelial cells by enhancing antiviral cathelicidin expression

doi: 10.3389/fimmu.2026.1758656

Figure Lengend Snippet: Transcriptional expression of VitD pathway genes in genital epithelial cells after VitD treatment. Fold changes of expression of CYP24A1 at 6h (A) and 24 h (B) of VitD or EtOH treatments of End1, Ect1 and Vk2 cells, and comparison between 6h and 24h time points (C) . Fold changes of expression of VDR at 6h (D) and 24 h (E) of VitD treatment of End1, Ect1 and Vk2, and comparison between 6h and 24h time points (F) . Fold-change in transcript (2 -ΔΔCt (effects) ) was calculated using ΔΔC t(effects) = ΔC t(target gene of treatment group) - ΔC t (target gene of untreated group) . Average threshold cycle (C t ) of 3 technical PCR replicates of the target gene was standardized against the average C t of the 18S rRNA (internal input reference) of the corresponding sample, termed ΔC t (target gene) . Shown was the data of 4 independent experiments (i.e., 4 transwell filters per treatment group, per cell line). Ratio paired t test was used. *p≤ 0.05; **p≤ 0.01.

Article Snippet: Immortalized endocervical (End1) (ATCC CRL-2615 TM ), ectocervical (Ect1) (ATCC CRL-2614 TM ) and Vaginal (Vk2) (ATCC CRL-2616 TM ) epithelial cells were used in this study.

Techniques: Expressing, Comparison

Journal: Frontiers in Immunology

Article Title: Vitamin D boosts HIV-1 resistance in female genital epithelial cells by enhancing antiviral cathelicidin expression

doi: 10.3389/fimmu.2026.1758656

Figure Lengend Snippet: Expression of cathelicidin (CAMP) gene and protein in genital epithelial cells following VitD treatment. Fold changes of the CAMP RNA transcripts at 6h (A) and 24 h (B) following VitD- or EtOH- treatment of End1, Ect1 and Vk2, and comparison between 6h and 24h time points (C) . Fold-change in transcript (2 -ΔΔCt (effects) ) was calculated using ΔΔC t(effects) = ΔC t(target gene of treatment group) - ΔC t (target gene of untreated group) , as described in Figure 6 . Shown was the data of 4 independent experiments (i.e., 4 transwell filters per treatment group, per cell line). Ratio paired t tests were used. *p≤ 0.05; **p≤ 0.01. The amount of cathelicidin protein released into the apical side of End1, Ect1 and Vk2 monolayers was assessed using ELISA (D) . Three independent experiments (3 transwell inserts) for each cell type were performed. The supernatant from the apical side of the monolayers were pooled and concentrated using speed vacuum; concentrations shown were adjusted according to concentration factor, to nanogram of cathelicidin per ml of supernatant.

Article Snippet: Immortalized endocervical (End1) (ATCC CRL-2615 TM ), ectocervical (Ect1) (ATCC CRL-2614 TM ) and Vaginal (Vk2) (ATCC CRL-2616 TM ) epithelial cells were used in this study.

Techniques: Expressing, Comparison, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Open Forum Infectious Diseases

Article Title: Induction of Human β-Defensin-2 by Vaginal Lactobacillus crispatus Strains in Vaginal Epithelial Cells Correlates With Their Adhesion Abilities

doi: 10.1093/ofid/ofag193

Figure Lengend Snippet: Vaginal Lactobacillus crispatus strains differed in their ability to enhance human β-defensin-2 (HβD-2) mRNA expression and production. L. crispatus was incubated with VK2/E6E7 cells at a multiplicity of infection (MOI) of 50 for ( A ) 6 or ( B ) 24 h. A , After RNA extraction from VK2/E6E7 cells, cDNA was prepared, and quantitative PCR was used to measure HβD2 or GAPDH mRNA levels. Each value was normalized to GAPDH mRNA levels. Normalized value in Dulbecco's phosphate-buffered saline (DPBS)-treated cells was set to 1. Vertical axis indicates relative changes in HβD2 mRNA compared with the DPBS control, while the horizontal axis represents the different L. crispatus strains. B , HβD-2 concentration in cell supernatant was quantified by ELISA. Vertical bars represent mean ± standard deviations. Statistically significant differences (*; P < .05, ***; P < .001, ****; P < .0001) are presented based on one-way analysis of variance followed by Tukey's multiple comparison test (A: DPBS vs MV-1A-US; P = .0315, DPBS vs HMS-115; P < .0001, DPBS vs HMS-122; P = .0498, MV-1A-US vs HMS-115; P = .0114, B: DPBS vs MV-1A-US; P = .0002, DPBS vs HMS-115; P < .0001, DPBS vs HMS-122; P = .0007). All experiments were conducted in at least three independent trials.

Article Snippet: VK2/E6E7 (ATCC CRL-2616), primary immortalized human vaginal epithelial cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Incubation, Infection, RNA Extraction, Real-time Polymerase Chain Reaction, Saline, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Open Forum Infectious Diseases

Article Title: Induction of Human β-Defensin-2 by Vaginal Lactobacillus crispatus Strains in Vaginal Epithelial Cells Correlates With Their Adhesion Abilities

doi: 10.1093/ofid/ofag193

Figure Lengend Snippet: Ability of Lactobacillus crispatus and Lactobacillus gasseri to enhance human β-defensin-2 ( HβD2 ) mRNA expression was associated with its binding capacity to vaginal epithelial cells. L. crispatus or L. gasseri was incubated with VK2/E6E7 at a multiplicity of infection (MOI) of 50 for ( A ) 4 or ( B ) 6 h. A , Nuclei of both bacterial and host cells were stained blue with DAPI. White scale bar represents 10 μm. Microscopic bacterial cell counts were performed on nine images per sample. B , A correlation is highlighted between induced HβD2 mRNA expression and the number of L. crispatus strains that adhere to vaginal epithelial cells. Dotted lines show the 95% confidence bands of the best-fit lines. All experiments were conducted in at least three independent trials. Each dot indicates the mean values. The correlation was evaluated using Spearman's correlation coefficient; **** indicates statistically significant differences ( P < .0001).

Article Snippet: VK2/E6E7 (ATCC CRL-2616), primary immortalized human vaginal epithelial cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Binding Assay, Incubation, Infection, Staining

Journal: Open Forum Infectious Diseases

Article Title: Induction of Human β-Defensin-2 by Vaginal Lactobacillus crispatus Strains in Vaginal Epithelial Cells Correlates With Their Adhesion Abilities

doi: 10.1093/ofid/ofag193

Figure Lengend Snippet: No significant differences were observed in human β-defensin-2 ( HβD 2) mRNA expression and production between live and heat-killed bacterial cells, but heat-killed cells showed higher adhesion abilities than live cells. Live or heat-killed L. crispatus was incubated with VK2/E6E7 cells at a multiplicity of infection (MOI) of 50 for ( A ) 6, ( B ) 24, or ( C ) 4 h. A , After RNA extraction from VK2/E6E7 cells, cDNA was prepared, and quantitative PCR was conducted to measure mRNA levels of HβD2 or GAPDH . Each value was normalized to GAPDH mRNA levels. Normalized value in Dulbecco's phosphate-buffered saline (DPBS)-treated cells was set to 1. Vertical axis indicates the relative changes compared with the DPBS control of HβD2 mRNA, while the horizontal axis represents the live or heat-killed L. crispatus HMS-115. B , The amount of HβD-2 in the cell supernatant was measured by ELISA. C , Microscopic bacterial cell counts were conducted on nine images per sample. Vertical bars represent mean ± standard deviations. Statistically significant differences (**; P < .01, ***; P < .001, ****; P < .0001) are presented based on unpaired t -test or one-way analysis of variance followed by Tukey's multiple comparison test (A: DPBS vs HMS-115 Live; P = .0034, DPBS vs HMS-115 Heat-killed; P = .0033, B: DPBS vs HMS-115 Live; P = .0003, DPBS vs HMS-115 Heat-killed; P < .0001, C: Live vs Heat-killed; P = .0009). The experiment was conducted in at least three independent trials.

Article Snippet: VK2/E6E7 (ATCC CRL-2616), primary immortalized human vaginal epithelial cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Incubation, Infection, RNA Extraction, Real-time Polymerase Chain Reaction, Saline, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Open Forum Infectious Diseases

Article Title: Induction of Human β-Defensin-2 by Vaginal Lactobacillus crispatus Strains in Vaginal Epithelial Cells Correlates With Their Adhesion Abilities

doi: 10.1093/ofid/ofag193

Figure Lengend Snippet: Impact of Lactobacillus crispatus bacterial viability on vaginal epithelial cells. L. crispatus was incubated with VK2/E6E7 cells at a multiplicity of infection (MOI) of 50 for 6 h. After extracting RNA from VK2/E6E7 cells, cDNA was prepared, and quantitative PCR was performed to measure mRNA levels for IL8 ( A ), TNFα ( B ), and MUC1 ( C ). Each value was normalized to GAPDH mRNA levels, with the value in Dulbecco's phosphate-buffered saline (DPBS)-treated cells set to 1. Vertical axis represents the relative changes for each target mRNA compared with the DPBS control, while the horizontal axis shows the live or heat-killed L. crispatus HMS-115. All experiments were conducted in three independent trials. Vertical bars represent mean ± standard deviations. Statistically significant differences (**; P < .01, ***; P < .001, ****; P < .0001) are presented based on one-way analysis of variance followed by Tukey's multiple comparison test (A: DPBS vs HMS-115 Live; P = .0002, HMS-115 Live vs Heat-killed; P < .0001, B: DPBS vs HMS-115 Live; P < .0001, HMS-115 Live vs Heat-killed; P < .0001, C: DPBS vs HMS-115 Heat-killed; P = .0034, HMS-115 Live vs Heat-killed; P = .0061).

Article Snippet: VK2/E6E7 (ATCC CRL-2616), primary immortalized human vaginal epithelial cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Incubation, Infection, Real-time Polymerase Chain Reaction, Saline, Control, Comparison

Journal: Open Forum Infectious Diseases

Article Title: Induction of Human β-Defensin-2 by Vaginal Lactobacillus crispatus Strains in Vaginal Epithelial Cells Correlates With Their Adhesion Abilities

doi: 10.1093/ofid/ofag193

Figure Lengend Snippet: Ability of Lactobacillus crispatus and Lactobacillus gasseri to enhance human β-defensin-2 ( HβD2 ) mRNA expression was associated with its binding capacity to vaginal epithelial cells. L. crispatus or L. gasseri was incubated with VK2/E6E7 at a multiplicity of infection (MOI) of 50 for ( A ) 4 or ( B ) 6 h. A , Nuclei of both bacterial and host cells were stained blue with DAPI. White scale bar represents 10 μm. Microscopic bacterial cell counts were performed on nine images per sample. B , A correlation is highlighted between induced HβD2 mRNA expression and the number of L. crispatus strains that adhere to vaginal epithelial cells. Dotted lines show the 95% confidence bands of the best-fit lines. All experiments were conducted in at least three independent trials. Each dot indicates the mean values. The correlation was evaluated using Spearman's correlation coefficient; **** indicates statistically significant differences ( P < .0001).

Article Snippet: VK2/E6E7 (ATCC CRL-2616), primary immortalized human vaginal epithelial cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Binding Assay, Incubation, Infection, Staining

Journal: Open Forum Infectious Diseases

Article Title: Induction of Human β-Defensin-2 by Vaginal Lactobacillus crispatus Strains in Vaginal Epithelial Cells Correlates With Their Adhesion Abilities

doi: 10.1093/ofid/ofag193

Figure Lengend Snippet: Lactobacillus crispatus -induced human β-defensin-2 ( HβD2 ) mRNA expression in cervical epithelial cells, but not in endometrial epithelial cells. Heat-killed L. crispatus was incubated with ( A ) A2EN and ( B ) HEC-1A cells at a multiplicity of infection (MOI) of 50 for 6 h. After extracting RNA, cDNA was prepared, and quantitative PCR was performed to measure the mRNA levels for HβD2 or GAPDH . Each value was normalized to GAPDH mRNA levels. Normalized value in Dulbecco's phosphate-buffered saline (DPBS)-treated cells was set to 1. Vertical axis indicates the relative changes compared with the DPBS control of HβD2 mRNA. All experiments were conducted in at least four independent trials. Vertical bars represent mean ± standard deviations. Statistically significant difference (*; P < .05) is presented based on unpaired t -test results (A: DPBS vs HMS-115 Heat-killed; P = .0105).

Article Snippet: VK2/E6E7 (ATCC CRL-2616), primary immortalized human vaginal epithelial cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Incubation, Infection, Real-time Polymerase Chain Reaction, Saline, Control

Journal: Open Forum Infectious Diseases

Article Title: Induction of Human β-Defensin-2 by Vaginal Lactobacillus crispatus Strains in Vaginal Epithelial Cells Correlates With Their Adhesion Abilities

doi: 10.1093/ofid/ofag193

Figure Lengend Snippet: Impact of Lactobacillus crispatus bacterial viability on vaginal epithelial cells. L. crispatus was incubated with VK2/E6E7 cells at a multiplicity of infection (MOI) of 50 for 6 h. After extracting RNA from VK2/E6E7 cells, cDNA was prepared, and quantitative PCR was performed to measure mRNA levels for IL8 ( A ), TNFα ( B ), and MUC1 ( C ). Each value was normalized to GAPDH mRNA levels, with the value in Dulbecco's phosphate-buffered saline (DPBS)-treated cells set to 1. Vertical axis represents the relative changes for each target mRNA compared with the DPBS control, while the horizontal axis shows the live or heat-killed L. crispatus HMS-115. All experiments were conducted in three independent trials. Vertical bars represent mean ± standard deviations. Statistically significant differences (**; P < .01, ***; P < .001, ****; P < .0001) are presented based on one-way analysis of variance followed by Tukey's multiple comparison test (A: DPBS vs HMS-115 Live; P = .0002, HMS-115 Live vs Heat-killed; P < .0001, B: DPBS vs HMS-115 Live; P < .0001, HMS-115 Live vs Heat-killed; P < .0001, C: DPBS vs HMS-115 Heat-killed; P = .0034, HMS-115 Live vs Heat-killed; P = .0061).

Article Snippet: VK2/E6E7 (ATCC CRL-2616), primary immortalized human vaginal epithelial cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Incubation, Infection, Real-time Polymerase Chain Reaction, Saline, Control, Comparison

Journal: Open Forum Infectious Diseases

Article Title: Induction of Human β-Defensin-2 by Vaginal Lactobacillus crispatus Strains in Vaginal Epithelial Cells Correlates With Their Adhesion Abilities

doi: 10.1093/ofid/ofag193

Figure Lengend Snippet: Schematic representation of the interaction between vaginal Lactobacillus crispatus and human β-defensin-2 (HβD-2) production. HβD-2 mRNA expression and production in vaginal epithelial cells varied significantly across L. crispatus strains. Vaginal L. crispatus with strong adhesion capability increased HβD-2 production in vaginal epithelial cells.

Article Snippet: VK2/E6E7 (ATCC CRL-2616), primary immortalized human vaginal epithelial cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing