Ann Marie Bongiovanni

Interleukin-1β gene polymorphism in women with vulvar vestibulitis syndrome

OBJECTIVE The pathogenesis of vulvar vestibulitis syndrome remains unknown but may be related to a localized chronic inflammation. The relation between this syndrome and a polymorphism at position +3953 in the interleukin-1beta gene was examined. Allele 2 of this gene has been associated with increased pro-inflammatory immunity. STUDY DESIGN Buccal or vestibular swabs from 59 women with strictly defined vulvar vestibulitis and from 48 healthy women were tested by polymerase chain reaction for the presence of two alleles at the +3953 interleukin-1beta locus. RESULTS Allele 2 of the interleukin-1beta gene was identified in 27 (46%) women with vulvar vestibulitis as opposed to 12 (25%) control women (P=0.03). The interleukin-1beta 1,1 genotype was present in 36 (75%) controls as opposed to 32 (54%) vulvar vestibulitis syndrome patients (P=0.02). All subjects had been previously tested for induced interleukin-1beta production in response to bacterial lipopolysaccharide. In both patients and controls, possession of allele 2 was associated with a small but non-statistically significant increase in induced interleukin-1beta production. CONCLUSION Allele 2 in the interleukin-1beta gene is more common in women with vulvar vestibulitis syndrome than in other women. Susceptibility to vulvar vestibulitis syndrome might be influenced by carriage of this polymorphism.

IgA ANTIBODIES TO THE 27-kDa HEAT-SHOCK PROTEIN IN THE GENITAL TRACTS OF WOMEN WITH GYNECOLOGIC CANCERS

Heat‐shock proteins promote cell survival under adverse environmental conditions. Synthesis of the 27‐kDa (HSP27), 70‐kDa (HSP70), and 90‐kDa (HSP90) heat‐shock proteins is increased in malignantly transformed cells and has been associated with tumor proliferation, metastasis, and resistance to chemotherapeutic agents. The increased expression of heat‐shock proteins and their association with tumor‐specific antigens may result in local immunity to the heat‐shock proteins. We examined the occurrence of IgA antibodies to HSP27, HSP70, and HSP90 in the lower genital tracts of women with possible gynecologic cancers. Cervical samples were obtained from 119 consecutive women being evaluated for a gynecologic malignancy or returning for a follow‐up examination following cancer treatment. Aliquots were tested for IgA anti‐heat‐shock protein antibodies by ELISA. Aliquots were also tested for IgG antibodies to HSP27 as well as for human papillomavirus. Anti‐HSP27 IgA was detected in 85.7% of 21 women with endometrial cancer tested prior to diagnosis and in 41.1% of 17 women tested after treatment. In women with ovarian cancer, 77.8% of 9 women tested prior to diagnosis and 75.0% of 24 women evaluated after treatment were anti‐HSP27 IgA‐positive. Of 6 women with cervical cancer tested prior to diagnosis, 5 were positive for this antibody. None of 25 women with benign diagnoses or 46 healthy women were cervical IgA anti‐HSP27‐positive (P < 0.0001). In contrast, anti‐HSP27 IgG was not associated with a gynecologic malignancy. HSP27 cervical antibodies were not associated with the presence of human papillomavirus. Cervical IgA antibodies to HSP90 were associated with ovarian cancer; antibodies to HSP70 were not cancer‐associated. We conclude that cervical IgA antibodies to HSP27 may be indicators of a gynecologic malignancy. Int. J. Cancer 87:824–828, 2000.

Antibodies to spermatozoa and seminal plasma antigens detected by various enzyme-linked immunosorbent (ELISA) assays

Various ELISA methods have been applied by different research centers to test the efficiency of this approach for the diagnosis of sperm-immune infertility cases. The antigens used were either whole spermatozoa or solubilized spermatozoal membrane preparations and were immobilized on microtiter plates, except in one case where plastic beads were employed. Polyvalent second antibodies or protein-A labelled with enzymes served as tracers. A high frequency of positive sera was found in all groups including fertile controls with tests using whole spermatozoa as antigen. The methods using solubilized antigen preparations showed fewer positives on the whole and correlated better with the various clinical categories of the WHO sera. Whilst there was some agreement in the results between the various laboratories on a few sera, most of the positive sera found by one laboratory were reported as negative by others. More investigative work is needed to improve reproducibility between different laboratories and to reduce non-specific reactions with normal controls. A more precise definition of the proper cut-off levels for positives and negatives is also needed. Despite these short-comings, the development of an ELISA for the diagnosis of sperm-immune infertility cases seems to be justified in the long term.

Transcriptome Adaptation of Group B Streptococcus to Growth in Human Amniotic Fluid

Background Streptococcus agalactiae (group B Streptococcus) is a bacterial pathogen that causes severe intrauterine infections leading to fetal morbidity and mortality. The pathogenesis of GBS infection in this environment is poorly understood, in part because we lack a detailed understanding of the adaptation of this pathogen to growth in amniotic fluid. To address this knowledge deficit, we characterized the transcriptome of GBS grown in human amniotic fluid (AF) and compared it with the transcriptome in rich laboratory medium. Methods GBS was grown in Todd Hewitt-yeast extract medium and human AF. Bacteria were collected at mid-logarithmic, late-logarithmic and stationary growth phase. We performed global expression microarray analysis using a custom-made Affymetrix GeneChip. The normalized hybridization values derived from three biological replicates at each growth point were obtained. AF/THY transcript ratios representing greater than a 2-fold change and P-value exceeding 0.05 were considered to be statistically significant. Principal Findings We have discovered that GBS significantly remodels its transcriptome in response to exposure to human amniotic fluid. GBS grew rapidly in human AF and did not exhibit a global stress response. The majority of changes in GBS transcripts in AF compared to THY medium were related to genes mediating metabolism of amino acids, carbohydrates, and nucleotides. The majority of the observed changes in transcripts affects genes involved in basic bacterial metabolism and is connected to AF composition and nutritional requirements of the bacterium. Importantly, the response to growth in human AF included significant changes in transcripts of multiple virulence genes such as adhesins, capsule, and hemolysin and IL-8 proteinase what might have consequences for the outcome of host-pathogen interactions. Conclusions/Significance Our work provides extensive new information about how the transcriptome of GBS responds to growth in AF, and thus new leads for pathogenesis research.

Association between primary vulvar vestibulitis syndrome, defective induction of tumor necrosis factor-α, and carriage of the mannose-binding lectin codon 54 gene polymorphism

OBJECTIVE We evaluated whether women with vulvar vestibulitis syndrome (VVS) could be subdivided on the basis of genotyping the polymorphic mannose-binding lectin (MBL) gene. STUDY DESIGN DNA from 123 women with VVS was tested for a single nucleotide polymorphism at codon 54 of the MBL gene. Blood samples from 86 of the women were evaluated for ex vivo tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 receptor antagonist (IL-1ra) production in response to Candida albicans, gram-positive peptidoglycan, and gram-negative lipopolysaccharide. Associations between laboratory findings and clinical characteristics were analyzed. RESULTS The variant MBL*B allele was identified in 33 subjects (26.8%). This polymorphism was more prevalent in women whose symptoms developed at their first act of sexual intercourse (primary VVS, 40.9%), as opposed to women with secondary VVS (16.3%; P = .01). Ex vivo TNF-alpha production, but not IL-1ra production, was reduced in MBL*B carriers as compared with MBL*A homozygotes (P < or = .03). CONCLUSION The MBL gene polymorphism is associated with the development of primary VVS and a reduced capacity for TNF-alpha production in response to microbial components.

Lactic acid stimulates interleukin-23 production by peripheral blood mononuclear cells exposed to bacterial lipopolysaccharide

Lactic acid is the predominant acid present in the vagina. We evaluated the consequences of lactic acid, at physiological levels present in the vagina, on cytokine responses of peripheral blood mononuclear cells (PBMCs) obtained from 10 individuals in the presence or absence of bacterial lipopolysaccharide. Preincubation of PBMCs in 15 mM lactic acid before the addition of lipopolysaccharide resulted in a 246% mean increase in interleukin-23 (IL-23) secretion over that released in the presence of lipopolysaccharide alone (P=0.0068). The lipopolysaccharide-induced production of tumor necrosis factor-α, IL-6, IL-10 and IL-12 was unaffected by lactic acid. IL-23 stimulation was not observed if the lactic acid was neutralized before its addition to the culture medium or if hydrochloric acid was substituted for lactic acid. In the absence of lipopolysaccharide, lactic acid did not stimulate the production of IL-23 or any of the other cytokines. The increase in IL-23 production was proportional to the lactic acid concentration over a 15-60 mM range. We conclude that at body sites characterized by lactic acid accumulation, such as in the human vagina, exposure to gram-negative bacteria results in selective IL-23 production, leading to a subsequent preferential stimulation of the Th17 T lymphocyte pathway.

Influence of lactic acid on endogenous and viral RNA-induced immune mediator production by vaginal epithelial cells.

OBJECTIVE: To evaluate the influence of lactic acid on immune mediator release from vaginal epithelial cells. METHODS: The human vaginal epithelial cell line, VK2/E6E7, was cultured in the presence or absence of physiological concentrations of lactic acid, and in the presence or absence of the viral Toll-like receptor 3 agonist, poly (inosinic acid:cytidylic acid). Supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) for interleukin (IL)-1&bgr;, IL-6, IL-8, IL-23, transforming growth factor (TGF)-&bgr; and secretory leukocyte protease inhibitor. RESULTS: Vaginal epithelial cells spontaneously released IL-1&bgr; (25.9 pg/mL), IL-8 (1.0 ng/mL), TGF-&bgr; (175 pg/mL), and secretory leukocyte protease inhibitor (33.8 ng/mL). Only TGF-&bgr; production was marginally enhanced (49%) by addition of lactic acid alone. Poly (inosinic acid:cytidylic acid) by itself stimulated the release of IL-6 (305 pg/mL) and enhanced IL-8 production (2.8 ng/mL). The combination of poly (inosinic acid:cytidylic acid) and lactic acid markedly increased IL-8 production (5.0 ng/mL) and induced the release of IL-1&bgr; (96.2 pg/mL). The poly (inosinic acid:cytidylic acid)-mediated lactic acid effect on IL-1&bgr; and IL-8 release was abrogated when the lactic acid was neutralized or if acetic acid was substituted for lactic acid. CONCLUSION: Lactic acid enhances the release of selective mediators from vaginal epithelial cells and stimulates antiviral immune responses.

Antibody to the Chlamydia trachomatis 60 kDa heat shock protein in follicular fluid and in vitro fertilization outcome.

The association between 60 kDa Chlamydia trachomatis heat shock protein (CHSP60) antibodies and the etiology and outcomes of in vitro fertilization (IVF) outcomes is not well known.

Differential vaginal expression of interleukin-1 system cytokines in the presence of Mycoplasma hominis and Ureaplasma urealyticum in pregnant women.

OBJECTIVE: The genital mycoplasmas, Ureaplasma urealyticum and Mycoplasma hominis, are commonly identified in the vagina of healthy pregnant women. However, these microorganisms are the most common isolates from the amniotic fluids of women in preterm labor. The mechanisms responsible for vaginal colonization and ascent to the uterus remain undetermined. We evaluated the association between U. urealyticum and M. hominis vaginal colonization and the presence of pro-inflammatory and anti-inflammatory interleukin-1 system components in asymptomatic pregnant women of different ethnicities. METHODS: Vaginal specimens, obtained from 224 first trimester pregnant women, were assayed for interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1ra) concentrations by ELISA. U. urealyticum and M. hominis vaginal colonization were identified by polymerase chain reaction (PCR). RESULTS: Vaginal colonization with M. hominis was identified in 37 (16.5%) women, and was more prevalent in black (18.9%) and Hispanic (20.9%) than in white (4.2%) women (p = 0.01). U. urealyticum was present in 84 (37.5%) women and there was no ethnic disparity in its detection. M. hominis colonization was associated with elevated median vaginal IL-1beta concentrations in both black women (p = 0.02) and Hispanic women (p = 0.04), and was unrelated to vaginal IL-1ra concentrations. In marked contrast, U. urealyticum colonization was associated with elevations in vaginal IL-1ra levels, but not with IL-1beta concentrations, in black women (p = 0.02) and Hispanic women (p < 0.0001) and marginally in white women (p = 0.06). CONCLUSION: M. hominis colonization in healthy pregnant women is associated with localized pro-inflammatory immune activation, while U. urealyticum colonization is associated with immune suppression.

α-Amylase in Vaginal Fluid: Association With Conditions Favorable to Dominance of Lactobacillus.

Vaginal glycogen is degraded by host α-amylase and then converted to lactic acid by Lactobacilli. This maintains the vaginal pH at ≤4.5 and prevents growth of other bacteria. Therefore, host α-amylase activity may promote dominance of Lactobacilli. We evaluated whether the α-amylase level in vaginal fluid is altered in women with bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) and whether its concentration was associated with levels of lactic acid isomers and host mediators. Vaginal fluid was obtained from 43 women with BV, 50 women with VVC, and 62 women with no vulvovaginal disorders. Vaginal fluid concentrations of α-amylase, secretory leukocyte protease inhibitor (SLPI), hyaluronan, hyaluronidase-1, β-defensin, and elafin were measured by enzyme-linked immunosorbent assay (ELISA). Vaginal concentrations of neutrophil gelatinase-associated lipocalin (NGAL), matrix metalloproteinase (MMP) 8, and d- and l-lactic acid levels in these patients were previously reported. The median vaginal fluid α-amylase level was 1.83 mU/mL in control women, 1.45 mU/mL in women with VVC, and 1.07 mU/mL in women with BV. Vaginal levels of α-amylase were correlated with d-lactic acid (P = .003) but not with l-lactic acid (P > .05) and with SLPI (P < .001), hyaluronidase-1 (P < .001), NGAL (P = .001), and MMP-8 (P = .005). The exfoliation of glycogen-rich epithelial cells into the vaginal lumen by hyaluronidase-1 and MMP-8 may increase glycogen availability and promote α-amylase activity. The subsequent enhanced availability of glycogen breakdown products would favor proliferation of Lactobacilli, the primary producers of d-lactic acid in the vagina. Concomitant production of NGAL and SLPI would retard growth of BV-related bacteria.