Candidate gene DNA methylation associations with breast cancer characteristics and tumor progression.

AIM: We examined methylation patterns with aggressive tumor phenotypes and investigated demographic, socioeconomic and reproductive predictors of gene methylation. MATERIALS & METHODS: Pyrosequencing quantified methylation of BRCA1, EGFR, GSTM2, RASSF1, TFF1 and Sat 2. We used quantile regression models to calculate adjusted median methylation values by estrogen and progesterone receptor (ER/PR) status. Bivariate associations between participant characteristics and methylation were examined. RESULTS: Higher percent methylation of GSTM2 was observed in ER/PR-negative compared with ER/PR-positive tumors in ductal carcinoma in situ (14 vs 2%) and invasive (35 vs 3%) tissue components. Trends in aberrant GSTM2 methylation across tissue components were stronger among ER/PR-negative tumors (p-interaction <0.001). Black women were more likely to have ER/PR-negative tumors (p = 0.01) and show hypermethylation of GSTM2 compared with other women (p = 0.05). CONCLUSION: GSTM2 promoter hypermethylation may serve as a potential biomarker of aggressive tumor development and a mechanism for ER/PR-negative tumor progression.

Multiplex immunoassay of lower genital tract mucosal fluid from women attending an urban STD clinic shows broadly increased IL1ß and lactoferrin.

BACKGROUND: More than one million new cases of sexually transmitted diseases (STDs) occur each day. The immune responses and inflammation induced by STDs and other frequent non-STD microbial colonizations (i.e. Candida and bacterial vaginosis) can have serious pathologic consequences in women including adverse pregnancy outcomes, infertility and increased susceptibility to infection by other pathogens. Understanding the types of immune mediators that are elicited in the lower genital tract by these infections/colonizations can give important insights into the innate and adaptive immune pathways that are activated and lead to strategies for preventing pathologic effects. METHODOLOGY/PRINCIPAL FINDINGS: 32 immune mediators were measured by multiplexed immunoassays to assess the immune environment of the lower genital tract mucosa in 84 women attending an urban STD clinic. IL-3, IL-1ß, VEGF, angiogenin, IL-8, ß2Defensin and ß3Defensin were detected in all subjects, Interferon-α was detected in none, while the remaining mediators were detected in 40% to 93% of subjects. Angiogenin, VEGF, FGF, IL-9, IL-7, lymphotoxin-α and IL-3 had not been previously reported in genital mucosal fluid from women. Strong correlations were observed between levels of TNF-α, IL-1ß and IL-6, between chemokines IP-10 and MIG and between myeloperoxidase, IL-8 and G-CSF. Samples from women with any STD/colonization had significantly higher levels of IL-8, IL-3, IL-7, IL-1ß, lactoferrin and myeloperoxidase. IL-1ß and lactoferrin were significantly increased in gonorrhea, Chlamydia, cervicitis, bacterial vaginosis and trichomoniasis. CONCLUSIONS/SIGNIFICANCE: These studies show that mucosal fluid in general appears to be an environment that is rich in immune mediators. Importantly, IL-1ß and lactoferrin are biomarkers for STDs/colonizations providing insights into immune responses and pathogenesis at this mucosal site.

Positive association between HIV RNA and IL-6 in the genital tract of Rwandan women.

Infections and inflammation in the genital tract can influence HIV expression or HIV susceptibility. The goal of this study was to determine if significant relationships exist between cytokines and HIV in genital tract secretions from 57 HIV-seropositive Rwandan women. Genital tract secretions were obtained by cervicovaginal lavage (CVL). Ten different cytokines in CVL were measured by multiplex cytometric bead arrays. HIV RNA in CVL and plasma were measured by quantitative PCR. In univariate analysis, genital tract HIV RNA was significantly associated with plasma HIV RNA and several of the cytokines, while in multivariate analysis, genital tract HIV RNA was significantly associated only with plasma HIV RNA and IL-6. This association of IL-6 with HIV RNA levels suggests that IL-6 is an indicator for conditions that induce HIV expression and that IL-6 may contribute to induction of HIV expression in the genital tract.

Utility of Amsel criteria, Nugent score, and quantitative PCR for Gardnerella vaginalis, Mycoplasma hominis, and Lactobacillus spp. for diagnosis of bacterial vaginosis in human immunodeficiency virus-infected women.

Bacterial vaginosis (BV) is a clinical syndrome presenting with a malodorous vaginal discharge and increased vaginal pH. Diagnosis has been based on clinical Amsel criteria and direct Gram stain of vaginal secretions. Human immunodeficiency virus (HIV)-infected participants in the Women's Interagency HIV Study contributed cervicovaginal lavage (CVL) samples. Lactobacilli, Gardnerella vaginalis, and Mycoplasma hominis in cervicovaginal lavage samples were quantified by PCR. Gynecologic evaluation included Nugent score and Amsel criterion assessment. We compared the gold standard Nugent score to Amsel criteria and quantitative bacterial PCR for diagnosing BV in 203 CVL samples from women with Nugent scores of 7 to 10 (BV group) and 203 samples from women with BV Nugent scores of 0 to 3 ("No-BV" group). Only 75 of the 203 CVL samples from women with Nugent scores of 7 to 10 met positive Amsel criteria. Increasing levels of G. vaginalis and M. hominis and decreasing levels of lactobacilli were significantly associated with BV by Nugent score. Of the group with Nugent scores of 7 to 10, 83% and 81% had log(10) G. vaginalis counts and log(10) M. hominis counts greater than 6.81 and 4.82, respectively, while only 30% and 31% of the group with Nugent scores of 0 to 3 were above these thresholds, respectively. There was significant overlap in the log(10) lactobacillus counts between the two groups. Utilizing all three log(10) bacterial counts (G. vaginalis, M. hominis, and lactobacilli) in our model improved the sensitivity and specificity to 83% and 78%, respectively, in comparison with Nugent score. In this cohort, Amsel criteria were poorly predictive of BV. PCR quantification of G. vaginalis and M. hominis from CVL is significantly more sensitive than Amsel criteria for diagnosing BV.

Relationship of U1 cell HIV-stimulatory activity to bacterial vaginosis and HIV genital tract virus load.

Bacterial vaginosis (BV) has been associated with HIV sexual transmission and increased levels of genital tract HIV RNA. We postulated that BV induces the appearance of substances in the genital tract that stimulate HIV expression locally. To test this, we measured HIV RNA levels in genital mucosal fluid from women with or without BV (defined by Nugent score) and compared them with the ability of those fluids to stimulate HIV expression in the chronically HIV-infected monocytic line U1. The U1 activity was significantly higher in women with BV (median = 1320 pg/ml p24) than in women with normal flora (median = 103 pg/ml p24, p = 0.0001). However, levels of the U1 activity were not significantly associated with levels in the genital tract of HIV RNA. Levels of the U1 activity were also not associated with levels of Gardnerella vaginalis or Mycoplasma hominis in genital fluids, suggesting these bacteria were not the source of the activity. Thus, while these data show a strong association of U1 stimulatory activity with BV, no influence of the U1 activity on genital tract HIV expression was observed.

Female genital-tract HIV load correlates inversely with Lactobacillus species but positively with bacterial vaginosis and Mycoplasma hominis.

BACKGROUND: Bacterial vaginosis (BV) is associated with human immunodeficiency virus (HIV) acquisition. We examined the association between BV and BV-associated bacteria and expression of HIV in the female genital tract. METHODS: HIV RNA, lactobacilli, Gardnerella vaginalis, and Mycoplasma hominis in cervicovaginal lavage (CVL) samples were quantified by polymerase chain reaction (PCR). Gynecologic evaluation included Nugent score assessment, Amsel criteria assessment, detection of other genital-tract infections, and dysplasia grading. CD4 cell count, plasma HIV RNA level, and antiretroviral history were obtained. RESULTS: A total of 203 CVL samples from women with Nugent scores of 7-10 (BV group) and 203 samples from women with Nugent scores of 0-3 (no-BV group) were matched by plasma HIV RNA level and analyzed. After controlling for plasma HIV RNA level and Nugent score in univariate analyses, we found that G. vaginalis and M. hominis bacterial counts, Candida vaginitis, and herpes simplex virus (HSV) were positively associated with CVL HIV RNA levels. In multivariate analysis, only lactobacilli bacterial counts (P=.006; inverse association), M. hominis bacterial counts (P=.0001; positive association), Candida vaginitis (P=.007), and HSV (P=.03) were significantly associated with CVL HIV RNA levels. CONCLUSION: Bacteria associated with BV increase genital-tract HIV RNA levels. Quantitative bacterial counts for lactobacilli and M. hominis are better correlates of CVL HIV RNA than are Nugent score or Amsel criteria. Since plasma virus and CD4 cell levels did not differ between the BV and no-BV groups, these data suggest that the bacterial flora associated with BV influence genital-tract HIV shedding.