Article Figures & Data
Figures
- FIG 1
Colonization of human vaginal and cervical epithelial cells by vaginal bacteria showed consistent bacterial association with epithelial cells in the absence of apoptosis and cell toxicity. (A–D) Transmission electron microscopy image showing L. crispatus (A and B) and P. bivia (C and D) bacteria, visualized as electron-dense bodies, adherent to the surface of vaginal epithelial cells (Vk2/E6E7) with intact morphology after 24 h of colonization. The bars and images represent 500 nm and ×4,800 magnification (A), 500 nm and ×6,800 magnification (B), 2 µm and ×1,900 magnification (C), and 500 nm and ×13,000 magnification, respectively. (E) Caspase-3 cleavage is presented as percentages of cleaved total caspase-3 measured in vaginal epithelial cell lysates at 24 h after bacterial colonization or treatment with 1 µM staurosporine. Bars represent means and standard errors of the means (SEM) of the results determined with duplicate cultures used in two experiments. (F) Viability of vaginal epithelial cells assessed by trypan blue inclusion tests at 5 days postcolonization. Bars represent means and SEM of the results from triplicate culture experiments. (G) CFU counts per square centimeter of epithelial cell surface at 24 h and 48 h postcolonization of Vk2/E6E7 cells. Bars represent means and SEM of the results determined with triplicate cultures used in three experiments. (H) Parallel assessment of CFU counts associated with primary polarized (VEC-100) and immortalized monolayer (Ect1/E6E7) ectocervical epithelial cells at 48 h postcolonization.
- FIG 2
Proinflammatory properties of vaginal microflora strains at 24 h postcolonization of epithelial monolayers in the absence of CS and HEC. (A) NF-κB activation assessed by luciferase activity. Bars represent means and SEM of the results determined with quadruplicate cultures in one of three independent experiments. (B) IL-8 levels measured in the vaginal epithelial cell culture supernatants. Bars represent means of the results determined with duplicate cultures in one of three experiments. For comparisons of the results determined with bacterially colonized cultures to those determined with control cultures without bacteria, ++ represents P < 0.01 and +++ represents P < 0.001 (ANOVA [Dunnett’s multiple-comparison test]).
- FIG 3
Cell viability determined by the MTT assay after 24 h of compound exposure. (A) Immortalized vaginal epithelial cell monolayers (Vk2/E6E7) exposed to the same dose range of cellulose sulfate and hydroxyethylcellulose; (B) comparison of primary polarized (VEC-100) and immortalized monolayer (Ect1/E6E7) ectocervical epithelial cells exposed to cellulose sulfate. Values represent means and SEM of the results determined with duplicate cultures in one of three experiments.
- FIG 4
Effects of cellulose sulfate and hydroxyethylcellulose (HEC) on bacterial colonization assessed after 24 h bacterial-epithelial coculture followed by 24h exposure to compound test doses. Bars represent means and SEM of CFUs associated with duplicate vaginal epithelial cell cultures in two experiments with two different batches of each test compound. For comparisons of the results determined with various compound doses to those determined with control medium, * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001 (ANOVA [Dunnett’s multiple-comparison test]).
- FIG 5
Direct effects of hydroxyethylcellulose (HEC) and cellulose sulfate on planktonic bacterial growth in the absence of epithelial cells. The bacterial suspensions that were used for epithelial colonization were simultaneously mixed with equal volumes of compound doses and incubated under anaerobic conditions for 24 h, followed by agar plating for enumeration of CFUs. Note that A. vaginae did not survive well in the absence of epithelial cells, whereas it maintained a stable colonization rate in the vaginal and cervical epithelial cells, as shown in Fig. 1G and H and 4D. Bars represent means and SEM of the results determined with triplicate cultures in two independent experiments performed with two different batches of test compounds. For comparisons of the results determined with various compound doses to those determined with control medium, *** represents P < 0.001 (ANOVA [Dunnett’s multiple-comparison test]).
- FIG 6
Compound-induced innate immune responses after 24 h of colonization with vaginal bacterial strains followed by 24 h of exposure to cellulose sulfate or hydroxyethylcellulose. Bars represent means and SEM of RANTES (AB), IL-8 (CD), and SLPI (EF) levels measured in supernatants from duplicate ectocervical epithelial cell cultures in one of three experiments with three different batches of cellulose sulfate. The dashed line represents medium-control basal levels in the absence of bacterial colonization. Similar results were obtained with the vaginal epithelial cells (data not shown). For comparisons of the results determined with various compound doses to those determined with control medium within each treatment group (shown at the bottom), * represents P < 0.05 and ** represents P < 0.01; for comparisons of the results determined with bacterially colonized cultures to those determined with cultures without bacteria for each compound dose, + represents P < 0.05 and ++ represents P<0.01 (ANOVA [Dunnett’s multiple-comparison test]).
- FIG 7
Compound-induced NF-κB activation after 24 h of bacterial colonization followed by 24 h of exposure to cellulose sulfate (A) or hydroxyethylcellulose (B). Bars represent means and SEM from luciferase activity determinations performed with duplicate cultures in one of three experiments with three different batches of each test compound. For comparisons of the results determined with various compound doses to those determined with control medium within each treatment group (shown at the right), ** represents P < 0.01; for comparisons of the results determined with bacterially colonized cultures to those determined with cultures without bacteria for each compound dose, ++ represents P < 0.01 and +++ represents P < 0.001 (ANOVA [Dunnett’s multiple-comparison test]).
