Cleavage of IL-8 in genital mucosal fluids.

<p>A. Recombinant IL-8 was added to either saline (negative control) or genital fluids (diluted 1:4) from 10 HIV-seropositive subjects and incubated at either 4°C or 37°C for 20 h. Levels of IL-8 were then measured by ELISA. The mean ± SD are shown. B. Recombinant IL-8 was added to genital fluid from subject 26 diluted 1:4 or 1:40 and incubated for 4 or 24 h before detection by ELISA.</p

Total MMP levels in mucosal fluid samples.

<p>The concentration of MMPs 1, 3, 7, 8, 9, 10 and 12 were measured by Luminex immunoassay in mucosal fluid samples. 22 samples positive for IL-8 cleavage (square symbols) and 5 samples negative for IL-8 cleavage (x symbols) were evaluated.</p

The effect of protease inhibitors on the IL-8-cleaving activity in genital tract fluid.

<p>A. Two samples positive for IL-8 cleavage (samples 26 and 56) were incubated with recombinant IL-8 for 20 h at 37°C in the presence or absence (No I) of EDTA or a cocktail of protease inhibitors (general protease inhibitor, GPI). B. MMP inhibitors Marimastat and C471474 were tested. For both A and B, control is IL-8 incubated without genital fluid or inhibitors. Samples were analyzed at 20 h by IL-8 ELISA. Shown are the mean values ± SD. * p<0.05, **p<0.1, t test compared to no inhibitor.</p

Relationship of active MMPs and IL-8 cleavage.

<p>A. The amount of MMP activity in genital fluids 22 samples positive for IL-8 cleavage and 5 negative for IL-8 cleavage was measured using a fluorogenic substrate and plotted against the % cleavage of IL-8 for each sample. B. The amount of active MMP-9 in the 27 samples was plotted against the % cleavage of IL-8 for each sample.</p

Subjects’ characteristics at index visit, n = 7.

<p>Subjects’ characteristics at index visit, n = 7.</p

Glycogen Levels in Undiluted Genital Fluid and Their Relationship to Vaginal pH, Estrogen, and Progesterone

<div><p>Background</p><p>Colonization of the female lower genital tract with <i>Lactobacillus</i> provides protection against STIs and adverse pregnancy outcomes. Growth of genital <i>Lactobacillus</i> is postulated to depend on epithelial cell-produced glycogen. However, the amount of cell-free glycogen in genital fluid available for utilization by <i>Lactobacillus</i> is not known.</p><p>Methods</p><p>Eighty-five genital fluid samples from 7 pre-menopausal women taken over 4–6 weeks were obtained using the Instead SoftCup^® (EvoFem, Inc., San Diego, CA, USA) by consented donors. Cell-free glycogen and glucose in genital fluids and estrogen and progesterone in blood were quantified.</p><p>Findings</p><p>Glycogen ranged from 0.1–32 μg/μl. There were significant differences between women in glycogen over the observation period. There was a strong negative correlation between glycogen and vaginal pH (r = -0.542, p<0.0001). In multivariable analysis, free glycogen levels were significantly negatively associated with both vaginal pH and progesterone (p < 0.001 and p = 0.004, respectively). Estrogen, glucose, age, sexual intercourse 24 hours prior to visit, and days after the initial visit were not significantly associated with free glycogen levels.</p><p>Conclusion</p><p>Cell-free glycogen concentrations can be very high, up to 3% of genital fluid, and are strongly associated with acidic vaginal pH. However, the fluctuations in glycogen levels in individuals and differences between individuals do not appear to be associated with estrogen.</p></div

Factors Associated with <i>Lactobacillus</i> ≥85%, N = 177 visits.

<p>OR = Odds Ratio; CI = Confidence Interval; Ref. indicates reference category.</p><p>*Odds Ratios generated from complementary log-log function.</p><p>Multivariable model is adjusted for variables for which estimates are presented.</p

Free Glycogen in Vaginal Fluids Is Associated with <i>Lactobacillus</i> Colonization and Low Vaginal pH

<div><p>Objective</p><p><i>Lactobacillus</i> dominates the lower genital tract microbiota of many women, producing a low vaginal pH, and is important for healthy pregnancy outcomes and protection against several sexually transmitted pathogens. Yet, factors that promote <i>Lactobacillus</i> remain poorly understood. We hypothesized that the amount of free glycogen in the lumen of the lower genital tract is an important determinant of <i>Lactobacillus</i> colonization and a low vaginal pH.</p><p>Methods</p><p>Free glycogen in lavage samples was quantified. Pyrosequencing of the 16S rRNA gene was used to identify microbiota from 21 African American women collected over 8–11 years.</p><p>Results</p><p>Free glycogen levels varied greatly between women and even in the same woman. Samples with the highest free glycogen had a corresponding median genital pH that was significantly lower (pH 4.4) than those with low glycogen (pH 5.8; p<0.001). The fraction of the microbiota consisting of <i>Lactobacillus</i> was highest in samples with high glycogen versus those with low glycogen (median = 0.97 vs. 0.05, p<0.001). In multivariable analysis, having 1 vs. 0 male sexual partner in the past 6 months was negatively associated, while BMI ≥30 was positively associated with glycogen. High concentrations of glycogen corresponded to higher levels of <i>L. crispatus</i> and <i>L. jensenii,</i> but not <i>L. iners</i>.</p><p>Conclusion</p><p>These findings show that free glycogen in genital fluid is associated with a genital microbiota dominated by <i>Lactobacillus,</i> suggesting glycogen is important for maintaining genital health. Treatments aimed at increasing genital free glycogen might impact <i>Lactobacillus</i> colonization.</p></div

Longitudinal vaginal pH, serum estrogen and progesterone.

<p>Vaginal pH (A) was determined utilizing a sterile pH probe as described in Methods. Blood was drawn from the seven subjects shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153553#pone.0153553.g001" target="_blank">Fig 1</a>. Serum levels of estrogen (B) and progesterone (C) were measured by ELISA.</p

Longitudinal cell-free glycogen and glucose in undiluted vaginal secretions.

<p>Vaginal fluid was collected from seven pre-menopausal subjects using the Instead SoftCup^® as described in Methods. Glycogen (A) and glucose (B) levels were measured in undiluted vaginal fluid.</p