Targeting Both Autophagy and Immunotherapy in Breast Cancer Treatment.

As clinical efforts towards breast-conserving therapy and prolonging survival of those with metastatic breast cancer increase, innovative approaches with the use of biologics are on the rise. Two areas of current focus are cancer immunotherapy and autophagy, both of which have been well-studied independently but have recently been shown to have intertwining roles in cancer. An increased understanding of their interactions could provide new insights that result in novel diagnostic, prognostic, and therapeutic strategies. In this breast cancer-focused review, we explore the interactions between autophagy and two clinically relevant immune checkpoint pathways; the programmed cell death-1 receptor with its ligand (PD-L1)/PD-1 and the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)/CD80 and CD86 (B7-1 and B7-2). Furthermore, we discuss emerging preclinical and clinical data supporting targeting both immunotherapy and autophagy pathway manipulation as a promising approach in the treatment of breast cancer.

Alterations of the 70 kDa heat shock protein (HSP70) and sequestosome-1 (p62) in women with breast cancer.

Peripheral blood mononuclear cells (PBMCs) respond to altered physiological conditions to alleviate the threat. Production of the 70 kDa heat shock protein (HSP70) is up-regulated to protect proteins from degradation. Sequestosome-1 (p62) binds to altered proteins and the p62-protein complex is degraded by autophagy. P62 is also a regulator of intracellular kinase activity and cell differentiation. We hypothesized that the PBMC response to a malignant breast mass involves elevated production of HSP70 and a decrease in intracellular p62. In this study 46 women had their breast mass excised. PBMCs were isolated and intracellular levels of HSP70 and p62 were quantitated by ELISA. Differences between women with a benign or malignant breast mass were determined. A breast malignancy was diagnosed in 38 women (82.6%) while 8 had a benign lesion. Mean intracellular HSP70 levels were 79.3 ng/ml in PBMCs from women with a malignant lesion as opposed to 44.2 ng/ml in controls (p = 0.04). The mean PBMC p62 level was 2.3 ng/ml in women with a benign breast lesion as opposed to 0.6 ng/ml in those with breast cancer (p < 0.001). Mean p62 levels were lowest in women with invasive carcinoma and a positive lymph node biopsy when compared to those with in-situ carcinoma or absence of lymphadenopathy, respectively. Intracellular HSP70 and p62 levels in PBMCs differ between women with a malignant or benign breast lesion. These measurements may be of value in the preoperative triage of women with a breast mass.

Soluble T cell immunoglobulin mucin domain 3 (sTim-3) in maternal sera: a potential contributor to immune regulation during pregnancy.

OBJECTIVE: The immune checkpoint inhibitor, membrane-bound T cell immunoglobulin mucin domain 3 (Tim-3), binds to galectin-9 (gal-9) and promotes immune tolerance during pregnancy. Soluble Tim-3 (sTim-3) competes with Tim-3 for binding to gal-9 and modulates its activity. Our objective was to evaluate the influence of sTim-3 on immune responses and outcome in pregnant women. STUDY DESIGN: Peripheral blood from 71 pregnant women was separated into mononuclear cell (PBMC) and plasma fractions. The PBMCs were lysed and tested for Tim-3 by ELISA. Plasma was assayed for sTim-3, gal-9, tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10) and the stress-inducible 70 kDa heat shock protein (hsp70) by enzyme-linked immunosorbent assay (ELISA). Correlations were analyzed by the Spearman rank correlation test. RESULTS: The higher the sTim-3 level in plasma the lower was the PBMC Tim-3 concentration (p = .0135), suggesting that sTim-3 results from the release of membrane-bound Tim-3. Plasma sTim3 levels were positively correlated with levels of gal-9 (p < .0001), TNF-α (p = .0071) and hsp70 (p = .0144), but not with IL-10. The sTim-3 level was positively associated (p = .0276) with gestational age at delivery. There was no association between sTim-3 and gestational age at sample collection, maternal age, gravidity, parity or body mass index. CONCLUSION: The release of Tim-3 from membranes and sTim-3 reacting with gal-9 may increase proinflammatory immunity and the stress response. The release of sTim-3 from lymphoid cells into the circulation and its binding to gal-9 may modulate Tim-3-mediated activity and help optimize immune regulation during pregnancy.

Torquetenovirus Titer in Vaginal Secretions from Pregnant and Postpartum Women: Association with Absence of Lactobacillus crispatus and Levels of Lactic Acid and Matrix Metalloproteinase-8.

Vaginal samples from women with term deliveries were tested for torquetenovirus (TTV) by gene amplification, matrix metalloproteinase (MMP)-8 and D- and L-lactic acid by ELISA, and microbiome composition by analysis of the bacterial 16S ribosomal RNA gene. TTV was detected in 43.2%, 31.5%, and 41.4% of first trimester, third trimester, and postpartum samples, respectively. The viral titer was higher in postpartum than in the first (p = 0.0018) or third (p = 0.0013) trimester. The mean gestational age at delivery was lower in women positive for TTV in their first trimester (p = 0.0358). In the first and third trimester, the MMP-8 level was higher if TTV was also present (p < 0.0091). The D-lactic acid level was lower in first trimester samples if TTV was present (p = 0.0334). Lactobacillus crispatus dominance in first and third trimester samples was higher when TTV was absent (p < 0.0033). We conclude that TTV is present in the vagina in many women with normal pregnancy outcomes and that its occurrence is associated with a lack of L. crispatus dominance, an increase in vaginal MMP-8 and a decrease in D-lactic acid.

Catechol-O-methyltransferase gene polymorphism and vulvar pain in women with vulvodynia.

BACKGROUND: The underlying causes of vulvar pain in women with vulvodynia remain poorly understood. Catechol-O-methyltransferase, an enzyme that metabolizes catecholamines, is a neuromodulator that is involved with perception and sensitivity to pain. The catechol-O-methyltransferase gene is polymorphic, and a single nucleotide polymorphism is associated with low activity and heightened pain sensitivity. The variant allele that encodes this polymorphism commonly is called the "L allele" because of its low enzyme activity as opposed to the normal H (high activity) allele. OBJECTIVE: The methionine-containing catechol-O-methyltransferase protein coded by the L allele results in elevated catecholamine levels, reduced inactivation of the dopaminergic and adrenergic systems, and increased sensitivity to pain. This polymorphism not only may decrease the pain threshold in response to acute pain but also may facilitate the development of chronic pain. Therefore, the objective of our study was to assess whether a variation in the catechol-O-methyltransferase genotype is involved in increased pain sensitivity in women with vulvodynia. STUDY DESIGN: We conducted a prospective cohort study. METHODS: Buccal swabs were collected from 167 white women with vulvodynia and 107 control subjects; the DNA was tested for a single nucleotide polymorphism at position 158 (rs4680) in the catechol-O-methyltransferase gene. RESULTS: Women with vulvodynia had a marginally increased, yet not significant, prevalence of the catechol-O-methyltransferase genotype that is associated with high activity of the coded protein: 32.9% in the women with vulvodynia, as opposed to 21.5% in the control subjects (odds ratio, 1.80; 95% confidence interval, 1.02-3.15). Subgrouping the cases based on pain frequency revealed that the elevated occurrence of this catechol-O-methyltransferase genotype was present in 40.6% of the subset of women who experienced pain only with sexual intercourse vs only 21.5% of control subjects (odds ratio, 2.50; 95% confidence interval, 1.27-4.93). Also, women with primary vulvodynia had a significantly higher prevalence of the H allele than did the control subjects (62.9% vs 48.1%; odds ratio, 1.82; 95% confidence interval, 1.05-3.17). CONCLUSION: Increased pain sensitivity in women with vulvodynia is not due to a genetically determined low catechol-O-methyltransferase enzyme activity. Other mechanisms may account for alterations in catechol-O-methyltransferase activity in women with pain that is limited to intercourse or primary vulvodynia that contributes to pain sensitivity.

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, ß-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.

Association between neurotrophin 4 and long-chain polyunsaturated fatty acid levels in mid-trimester amniotic fluid.

The omega-3 long-chain polyunsaturated fatty acid (LCPUFA) docosahexaenoic acid (DHA) and the omega-6 LCPUFA arachidonic acid (AA) are essential nervous system components that increase in concentration throughout gestation. The neurotrophins, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4) are small basic peptides crucial for fetal brain development. The DHA supplementation during pregnancy has been suggested to enhance neural development. We evaluated whether amniotic fluid DHA and AA concentrations correlated with intra-amniotic neurotrophin levels. Amniotic fluid, obtained at 15 to 19 weeks gestation from 62 women, was tested for BDNF, NGF, NT3, and NT4 by enzyme-linked immunosorbent assay. Concentrations of DHA and AA, and saturated and monounsaturated fatty acids, were determined by gas chromatography. Associations were analyzed by the Spearman rank correlation test. Median levels of AA and DHA were 2.3% and 1.3% of the total intra-amniotic fatty acids, respectively. Median neurotrophin levels (pg/mL) were 36.7 for NT3, 26.8 for BDNF, 5.2 for NT4, and 0.8 for NGF. Intra-amniotic NT4 and BDNF levels were correlated (P = .0016), while NT3 and NGF levels were unrelated to each other or to BDNF or NT4. Only NT4 was positively correlated with amniotic fluid DHA (P < .0001) and AA (P = .0003) concentrations. There were no associations between DHA, AA, or any neurotrophin and maternal age, gestational age at time of amniocentesis, amniocentesis indication, parity, or gestational age at delivery. Elevations in intra-amniotic NT4 with increasing levels of DHA and AA suggest that these LCPUFAs may specifically influence the extent of NT4-mediated fetal brain neurogenesis.

Unique variation in genetic selection among Black North American women and its potential influence on pregnancy outcome.

We hypothesize that variations in the frequency of genetic polymorphisms, reflecting ancestral differences in living conditions and exposure to microorganisms, increase susceptibility to adverse pregnancy outcome among present day Black North American women. Striking differences were observed in the frequency of genetic variants between Black and White or Hispanic women in 5 genes (IL1RN, MBL2, PPARA, ATG16L1, CIAS1) associated with inflammation and anti-microbial immunity. The CIAS1 and IL1RN polymorphisms were associated with altered interleukin-1ß serum levels; the MBL2 polymorphism resulted in a decreased serum mannose-binding lectin concentration. Gene polymorphisms associated with an alteration in innate immunity were most frequent in Black women. This may reflect an evolutionary selection in response to an ancient environment containing a high multitude of microorganisms, and may increase susceptibility of Black women to infection-associated preterm birth in the current North American environment.

Inhibition of autophagy by sera from pregnant women.

Autophagy is a process that maintains homeostasis by eliminating senescent or damaged intracellular organelles and proteins. Its role in pregnancy has been scarcely studied. We compared the influence of sera from pregnant and nonpregnant women on autophagy induction. Peripheral blood mononuclear cells (PBMCs) were incubated with sera from 35 pregnant or nonpregnant women in the presence or absence of the autophagy inducer, rapamycin. After 48 hours, the cells were assayed for p62, a cytoplasmic protein essential for autophagy induction. Its concentration in the cytoplasm is inversely proportional to the level of autophagy induction. Sera were tested for immune mediators by enzyme-linked immunosorbent assay. Median (range) p62 concentrations were 6.7 ng/mL (1.1-22.7) for PBMCs incubated with pregnancy sera versus 2.5 ng/mL (0.8-7.7) for nonpregnant sera (P < .0001). In the presence of rapamycin, median p62 levels were 1.3 ng/mL (<0.1-4.9) with pregnancy sera, when compared to 0.6 ng/mL (<0.1-3.3) with control sera (P = .0191). Among the pregnant patients, the p62 level was inversely proportional to the results of a 50-g glucose challenge test (r = -.5630, P = .0005). Sera from pregnant women had elevated levels of insulin-like growth factor 1 (IGF-1), interleukin 13 (IL-13), and transforming growth factor ß1 (TGF-ß1). Autophagy during pregnancy may be inhibited by IGF-1, IL-13, and/or TGF-ß1 and may influence insulin resistance.

Influence of mid-trimester amniotic fluid on endogenous and lipopolysaccharide-mediated responses of mononuclear lymphoid cells.

PROBLEM We evaluated the influence of amniotic fluid (AF) on immune mediator production by mononuclear leukocytes. METHOD OF STUDY Thirty mid-gestation AFs were incubated with peripheral blood mononuclear cells (PBMCs) in the presence or absence of lipopolysaccharide (LPS). Supernatants were tested for interleukin (IL) - 6, 10, 12, 23, tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein (MCP)-1. RESULTS Endogenous mediator production was minimal or non-detectable. AF stimulated endogenous MCP-1, IL-6 and TNF-α release. In the presence of LPS, production of MCP-1 and IL-10 by PBMCs was enhanced eight- to ninefold by AF. Release of IL-6 and IL-23 was enhanced less than twofold by the addition of AF while TNF-α production was unchanged. AF-stimulated mediator production was similar irrespective of pregnancy outcome. CONCLUSION Selective AF stimulation of LPS-mediated MCP-1 and IL-10 release may be a mechanism to promote antibody production and the influx of phagocytic cells to engulf pathogens while downregulating the production of pro-inflammatory cytokines.

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)-1ß, IL-6, IL-8, IL-23, transforming growth factor (TGF)-ß and secretory leukocyte protease inhibitor. RESULTS: Vaginal epithelial cells spontaneously released IL-1ß (25.9 pg/mL), IL-8 (1.0 ng/mL), TGF-ß (175 pg/mL), and secretory leukocyte protease inhibitor (33.8 ng/mL). Only TGF-ß 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-1ß (96.2 pg/mL). The poly (inosinic acid:cytidylic acid)-mediated lactic acid effect on IL-1ß 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.

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.

An interleukin-23 binding protein in mid-trimester amniotic fluid.

PROBLEM: The binding of mid-trimester amniotic fluid to cytokines was evaluated. METHOD OF STUDY: Purified tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-10, IL-12, and IL-23 were incubated with amniotic fluid from 25 women undergoing a mid-trimester amniocentesis, or with bovine serum albumin or saline, and cytokine binding to monoclonal antibodies was quantitated by ELISA. Aliquots of amniotic fluid were heated to 95 degrees C for 15 min and then retested for IL-23 binding. The effect of amniotic fluid dilution on IL-23 quantitation was evaluated. RESULTS: All amniotic fluids had a negligible effect on TNF-alpha, IL-10, and IL-12 detection. In marked contrast, pre-incubation with amniotic fluid from each subject reduced the subsequent ability to detect IL-23 by >50%. The extent of inhibition was directly proportional to the amniotic fluid dilution and was markedly reduced following heating at 95 degrees C for 15 min. Amniotic fluids from White, Black, Asian, East Indian, and Hispanic women were equally effective. CONCLUSION: Interleukin-23 and IL-12 share a common p40 subunit and no inhibition of IL-12 was apparent. It appeared that a component of mid-trimester amniotic fluid specifically interacts with the p19 subunit unique to IL-23. Mid-trimester amniotic fluid reactivity with IL-23 may be a mechanism to limit intra-amniotic neutrophil-derived inflammation.

Hyaluronan modulates pro-inflammatory immune activity in the mid-trimester amniotic cavity.

Hyaluronan (HA), which comprises repeating disaccharides of D-glucuronic acid and N-acetyl-glucosamine, is a component of the extracellular matrix. In response to infection or tissue injury HA is released into the extracellular milieu where it modulates immune activity. We hypothesized that HA is present in mid-trimester amniotic fluid and contributes to immune regulation at that site. Amniotic fluid from 392 women undergoing a mid-trimester amniocentesis were tested for HA by ELISA. Amniotic fluids from 41 women were also cultured ex vivo in the presence or absence of lipopolysaccharide (LPS). Supernatants were collected after 24h and tested for tumor necrosis factor-alpha (TNFalpha) and interleukin (IL)-10 by ELISA. Clinical parameters were obtained after completion of laboratory testing. All amniotic fluids were positive for HA. The median (range) concentration was 3.2 (0.6-91.7) microg/mg amniotic fluid protein. Women with at least 2 prior pregnancies and a history of > or =2 spontaneous abortions had a higher median HA concentration than did previously pregnant women with 0-1 prior abortions. Women who conceived following in vitro fertilization also had an elevated median amniotic fluid HA compared to women with spontaneous conceptions. Both endogenous and LPS-induced TNFalpha production by ex vivo cultured amniotic fluid cells, but not IL-10 production, was inversely proportional to the amniotic fluid HA concentration. In conclusion, intraamniotic HA levels are elevated in pregnancies at risk for adverse outcome and HA may be a component of the fetal response to immune alterations that threaten gestation.

Hyaluronan in vaginal secretions: association with recurrent vulvovaginal candidiasis.

OBJECTIVE: We evaluated whether vaginal concentrations of hyaluronan were altered in women with recurrent vulvovaginal candidiasis (RVVC). STUDY DESIGN: Lavage samples from 17 women with acute RVVC, 27 women who were receiving a maintenance antifungal regimen, and 24 control women were tested for hyaluronan and interleukin (IL)-6, IL-12, and IL-23 by enzyme-linked immunosorbent assay. RESULTS: Median vaginal hyaluronan concentrations were 33.8 ng/mL (range, 21.6-66.3 ng/mL) in women with acute RVVC, 15.0 ng/mL (range, 11.2-50.6 ng/mL) in women who were receiving maintenance therapy, and 4.2 ng/mL (range, 3.6-12.0 ng/mL) in control subjects (P

Endogenous adenosine down-modulates mid-trimester intraamniotic tumor necrosis factor-alpha production.

PROBLEM: To determine whether adenosine in amniotic fluid down-regulates pro-inflammatory cytokine production. METHOD OF STUDY: Mid-trimester amniotic fluid from 21 women was incubated ex vivo in the presence or absence of human adenosine deaminase, the enzyme that irreversibly degrades adenosine. After 24 hr, supernatants were assayed by ELISA for tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-10. Clinical parameters were obtained after completion of laboratory testing. RESULTS: Inclusion of adenosine deaminase resulted in a median increase in TNF-alpha production from 0.9 to 7.3 pg/mL (P = 0.0014). IL-6 production exhibited a non-significant median increase from <2.0 to 53.0 pg/mL (P = 0.0780). Median IL-10 production increased slightly from a median of <0.2 to 1.3 pg/mL. Adenosine deaminase-stimulated TNF-alpha production was proportional to parity and unrelated to gestational age, time of delivery, maternal age or indication for amniocentesis. CONCLUSION: Adenosine deaminase treatment increases TNF-alpha production by ex vivo-cultured amniotic fluid. Adenosine contributes to immune modulation in the amniotic cavity.

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.

Gelsolin down-regulates lipopolysaccharide-induced intraamniotic tumor necrosis factor-alpha production in the midtrimester of pregnancy.

OBJECTIVE: The purpose of this study was to identify gelsolin in midtrimester amniotic fluid and evaluate its interaction with lipopolysaccharide (LPS). STUDY DESIGN: Supernatants from 40 midtrimester amniotic fluid samples were incubated with Escherichia coli LPS, and gelsolin binding was measured by enzyme-linked immunosorbent assay. Unfractionated aliquots of 25 of the fluids were cultured ex vivo for 24 hours in the presence of LPS and supernatants tested for tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 production, and the influence of antigelsolin antibody was evaluated. RESULTS: Each amniotic fluid was positive for gelsolin that bound to LPS. LPS-induced TNF-alpha production was inversely proportional to the amniotic fluid concentrations of LPS-bound gelsolin (r = -0.5047; P = .006). Preincubation with monoclonal antibody to gelsolin led to an increase in LPS-induced TNF-alpha production (P = .01). There was no relationship between gelsolin and IL-10 production. CONCLUSION: Gelsolin is present in midtrimester amniotic fluid, binds to LPS, and inhibits the induction of TNF-alpha.

Hyaluronan in follicular fluid and embryo implantation following in vitro fertilization and embryo transfer.

PURPOSE: To determine whether the concentration of hyaluronan (HA) in follicular fluid predicts implantation success following embryo transfer. METHODS: Follicular fluids from 170 IVF patients were tested by ELISA for HA concentration. RESULTS: The mean (standard error) HA concentration in follicular fluids was 158.0 (21.9) ng/ml from women whose embryos did not implant, 220.0 (21.3) ng/ml from women in which one embryo implanted and 239.3 (40.1) ng/ml from women with 2-3 implantations (implantation vs. no implantation p = .019). The HA level was unrelated to maternal age, number of oocytes harvested or fertilized or number of embryos transferred. Follicular fluids from women with an endocrine problem had a lower mean HA level (142.0 ng/ml) as compared to women undergoing IVF due to male factor infertility (257.3 ng/ml) (P = .05). CONCLUSIONS: HA in follicular fluid is decreased in women with unsuccessful implantation or with an endocrine disorder. A woman's level of HA production may influence the potential for implantation of her embryos.

Association between primary vulvar vestibulitis syndrome, defective induction of tumor necrosis factor-alpha, 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.