25 research outputs found
Oleate lipase activity in Gardnerella vaginalis and reconsideration of existing biotype schemes
vitro and ex vivo testing of tenofovir shows it is effective as an HIV-1 microbicide
Abstract Background: Tenofovir gel has entered into clinical trials for use as a topical microbicide to prevent HIV-1 infection but has no published data regarding pre-clinical testing using in vitro and ex vivo models. To validate our findings with on-going clinical trial results, we evaluated topical tenofovir gel for safety and efficacy. We also modeled systemic application of tenofovir for efficacy
The Effects of Hormones and Vaginal Microflora on the Glycome of the Female Genital Tract: Cervical-Vaginal Fluid
<div><p>In this study, we characterized the glycome of cervical-vaginal fluid, collected with a Catamenial cup. We quantified: glycosidase levels; sialic acid and high mannose specific lectin binding; mucins, MUC1, MUC4, MUC5AC, MUC7; and albumin in the samples collected. These data were analyzed in the context of hormonal status (day of menstrual cycle, hormonal contraception use) and role, if any, of the type of the vaginal microflora present. When the Nugent score was used to stratify the subjects by microflora as normal, intermediate, or bacterial vaginosis, several important differences were observed. The activities of four of six glycosidases in the samples from women with bacterial vaginosis were significantly increased when compared to normal or intermediate women: sialidase, <i>P</i> = <0.001; α-galactosidase, <i>P</i> = 0.006; β-galactosidase, <i>P</i> = 0.005; α-glucosidase, <i>P</i> = 0.056. Sialic acid binding sites as measured by two lectins, <i>Maackia amurensis</i> and <i>Sambucus nigra</i> binding, were significantly lower in women with BV compared to women with normal and intermediate scores (<i>P</i> = <0.0001 and 0.008 respectively). High mannose binding sites, a measure of innate immunity were also significantly lower in women with BV (<i>P</i> = <0.001). Additionally, we observed significant increases in MUC1, MUC4, MUC5AC, and MUC7 concentrations in women with BV (<i>P</i> = <0.001, 0.001, <0.001, 0.02 respectively). Among normal women we found that the membrane bound mucin MUC4 and the secreted MUC5AC were decreased in postmenopausal women (<i>P</i> = 0.02 and 0.07 respectively), while MUC7 (secreted) was decreased in women using levonorgestrel-containing IUDs (<i>P</i> = 0.02). The number of sialic acid binding sites was lower in the postmenopausal group <i>(P</i> = 0.04), but the number of high mannose binding sites, measured with Griffithsin, was not significantly different among the 6 hormonal groups. The glycosidase levels in the cervical-vaginal mucus were rather low in the groups, with exception of α-glucosidase activity that was much lower in the postmenopausal group (<i>P</i><0.001). These studies present compelling evidence that the vaginal ecosystem responds to the presence of different vaginal microorganisms. These effects were so influential that it required us to remove subjects with BV for data interpretation of the impact of hormones. We also suggest that certain changes occurring in vaginal/cervical proteins are due to bacteria or their products. Therefore, the quantitation of vaginal mucins and lectin binding offers a new method to monitor bacteria-host interactions in the female reproductive tract. The data suggest that some of the changes in these components are the result of host processing, such as the increases in mucin content, while the microflora is responsible for the increases in glycosidases and the decreases in lectin binding. The methods should be considered a valid marker for insult to the female genital tract.</p></div
Summary of enzyme activity, lectin binding and mucin content of women stratified by hormonal status.
<p>Summary of enzyme activity, lectin binding and mucin content of women stratified by hormonal status.</p
Studying the Effects of Reproductive Hormones and Bacterial Vaginosis on the Glycome of Lavage Samples from the Cervicovaginal Cavity
<div><p>The cervicovaginal fluid (CVF) coating the vaginal epithelium is an important immunological mediator, providing a barrier to infection. Glycosylation of CVF proteins, such as mucins, IgG and S-IgA, plays a critical role in their immunological functions. Although multiple factors, such as hormones and microflora, may influence glycosylation of the CVF, few studies have examined their impact on this important immunological fluid. Herein we analyzed the glycosylation of cervicovaginal lavage (CVL) samples collected from 165 women under different hormonal conditions including: (1) no contraceptive, post-menopausal, (2) no contraceptive, days 1-14 of the menstrual cycle, (3) no contraceptive, days 15-28 of the menstrual cycle, (4) combined-oral contraceptive pills for at least 6 months, (5) depo-medroxyprogesterone acetate (Depo-Provera) injections for at least 6 months, (6) levonorgestrel IUD for at least 1 month. Glycomic profiling was obtained using our lectin microarray system, a rapid method to analyze carbohydrate composition. Although some small effects were observed due to hormone levels, the major influence on the glycome was the presence of an altered bacterial cohort due to bacterial vaginosis (BV). Compared to normal women, samples from women with BV contained lower levels of sialic acid and high-mannose glycans in their CVL. The change in high mannose levels was unexpected and may be related to the increased risk of HIV-infection observed in women with BV, as high mannose receptors are a viral entry pathway. Changes in the glycome were also observed with hormonal contraceptive use, in a contraceptive-dependent manner. Overall, microflora had a greater impact on the glycome than hormonal levels, and both of these effects should be more closely examined in future studies given the importance of glycans in the innate immune system.</p></div
Effects of menstrual cycle on glycosylation of CVL.
<p>(A) Bi-plot of lectin microarray data for CVL from women in days 1–14 (x-axis) versus days 15–28 (y-axis). Graph shows average data for each lectin. Lectins and antibodies showing significant differences (p < 0.05) between the two groups are labeled with diamonds. Lectins with similar binding glycans are labeled in the same color (yellow: Gal/GalNAc; green: high mannose; purple: sialyl Lewis A). Lectins above the red dashed line showed increased expression levels during days 15–28 of the menstrual cycle compared to days 1–14. (B) Visual representation of glycans showing significant differences between the two groups.</p
Effects of vaginal microflora on sialylation.
<p>(A) Representative <i>N</i>-linked glycan before and after sialidase digestion. Lectin binding determinants are shaded in grey with lectin names indicated. (B-E) Notched boxplot representation of binding levels of (B) SNA, (C) TJA-I, (D) ECA, (E) RCA to Normal, Intermediate and BV sample cohorts. (F) Representative <i>O</i>-linked glycans before and after sialidase. (G-H) Notched boxplot representation of binding levels of (G) AIA and (H) MNA-G. For all plots, significance levels between groups indicated by lines are as follows: *, 0.01< <i>p</i> ≤0.05; **, 0.001< p ≤0.01; ***, 0.0001</p
Effects of vaginal microflora on high mannose.
<p>(A) High mannose glycan structure. Lectin binding determinants are shaded in grey. (B-C) Notched boxplot representation of binding levels of (B) GRFT and (C) SVN. Significance: *, 0.01< <i>p</i> ≤0.05; **, 0.001< p ≤0.01; ***, 0.0001</p
Effects of exogenous hormones on CVL glycome.
<p>(A) Representative <i>N</i>-linked complex glycan with bisecting GlcNAc. Lectin binding epitopes are shaded in grey. (B-C) Notched boxplot representation of binding levels of (B) PHA-E and (C) RCA for women on no hormonal contraceptives (No HC) or on hormonal contraceptives (HC). (D-G) Notched boxplot representation of detailed analysis of binding of (D) PHA-E, (E) RCA, (F) Calsepa and (G) ECA for women on oral contraceptives (Oral), Depo-Provera (Depo) or IUD in comparison to the No HC cohort. Significance: *, 0.01< <i>p</i> ≤0.05; **, 0.001< p ≤0.01; ***, 0.0001</p
Median levels of glycosidases and lectin binding stratified by hormonal group, excluding women with bacterial vaginosis.
<p><sup>1</sup>Data are presented as medians (range) in μMoles substrate hydrolyzed per min per mg protein. Griffithsin is measured as pg/ng protein.</p><p><sup>2</sup><i>P</i>-value from Kruskal-Wallis test across all groups.</p><p><sup>3</sup><i>P</i>-value <0.02 from Mann-Whitney U test comparing post-menopausal women to women of reproductive age.</p><p><sup>4</sup><i>P</i>-value = 0.02 and 0.047 from Mann-Whitney U test comparing women in days 1–14 of cycle to women in days 15–28 of the cycle for, respectively Griffithsin and α-glucosidase.</p><p>Median levels of glycosidases and lectin binding stratified by hormonal group, excluding women with bacterial vaginosis.</p