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Background: Cancers of ductal origin often express glycoprotein mucin 1 (MUC1), also known as CA15.3, with higher levels leading to poor prognosis. Conversely, anti-MUC1 antibodies develop in some patients, leading to better prognosis. We sought to identify epidemiologic factors associated with CA15.3 antigen or antibody levels., Methods: Levels of CA15.3 antigen and anti-CA15.3 IgG antibodies were measured in archived sera from 2,302 mostly healthy women from the National Health and Nutritional Survey; and epidemiologic predictors of their levels were examined using multivariate and correlational analyses., Results: Among racial groups, Black women had the highest levels of CA15.3 antigen and lowest levels of antibodies. Increasing body mass index and current smoking were associated with low anti-CA15.3 antibody levels. Low CA15.3 antigen levels were seen in oral contraceptive users and high levels in women who were pregnant or lactating at the time of blood collection, with the latter group also having high antibody levels. Past reproductive events associated with high antigen levels included the following: later age at menarche, having given birth, and history of endometriosis. Lower antigen levels were seen with increasing duration of OC use. Anti-CA15.3 antibody levels decreased with an increasing estimated number of ovulatory years., Conclusions: Key determinants of CA.15.3 antigen or antibody levels include the following: race, body mass index, smoking, later menarche, childbirth, number of ovulatory cycles, and endometriosis., Impact: This study supports the premise that known epidemiologic factors affecting risk for or survival after MUC1-expressing cancers may, at least partially, operate through their association with CA15.3 antigen or antibody levels., (©2024 American Association for Cancer Research.)

Objective: The aim of the study was to assess intestinal inflammatory measures, urinary intestinal fatty acid-binding protein (IFABP), and fecal calprotectin (FC) by gestational age (GA) and postmenstrual age (PMA) and determine the association between intestinal inflammation and growth in preterm infants from birth to hospital discharge. We hypothesized that intestinal inflammation is associated with adverse growth in preterm infants., Methods: We assayed repeated measures of IFABP and FC in 72 hospitalized preterm infants (<34 weeks' gestation). We calculated weight and length z scores at birth and discharge using the Fenton growth reference. Associations between mean IFABP or FC, growth z scores at discharge, and growth faltering (weight or length z score difference <-0.8 from birth to discharge) were assessed using mixed linear and logistic regression models, adjusted for intrafamilial correlation and potential confounders: GA, sex, birth z score, race/ethnicity, and maternal age., Results: Mean IFABP was greater among infants born at earlier GA and decreased with increasing PMA. Mean FC did not vary by GA or PMA. Higher mean IFABP and FC were associated with lower discharge growth z scores and greater likelihood of growth faltering significant only for mean IFABP and discharge length z score (β = -0.353, 95% confidence interval [CI]: -0.704 to -0.002) and mean IFABP and length faltering (odds ratio [OR] 1.99, P = 0.018)., Conclusions: Intestinal inflammation, measured by IFABP, was associated with lower length z scores and length faltering at discharge. Interventions to prevent intestinal inflammation may improve linear growth among preterm infants., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

Co-infections with sexually transmittable pathogens are common and more likely in women with disturbed vaginal bacteriome. Among those pathogens, the protozoan parasite Trichomonas vaginalis (TV) is most common after accounting for the highly persistent DNA viruses human papillomavirus (HPV) and genital herpes. The parasitic infection often concurs with the dysbiotic syndrome diagnosed as bacterial vaginosis (BV) and both are associated with risks of superimposed viral infections. Yet, the mechanisms of microbial synergisms in evading host immunity remain elusive. We present clinical and experimental evidence for a new role of galectins, glycan-sensing family of proteins, in mixed infections. We assessed participants of the HIV Epidemiology Research Study (HERS) at each of their incident TV visits (223 case visits) matched to controls who remained TV-negative throughout the study. Matching criteria included age, race, BV (by Nugent score), HIV status, hysterectomy, and contraceptive use. Non-matched variables included BV status at 6 months before the matched visit, and variables examined at baseline, within 6 months of and/or at the matched visit e.g. HSV-2, HPV, and relevant laboratory and socio-demographic parameters. Conditional logistic regression models using generalized estimating equations calculated odds ratios (OR) for incident TV occurrence with each log 10 unit higher cervicovaginal concentration of galectins and cytokines. Incident TV was associated with higher levels of galectin-1, galectin-9, IL-1β and chemokines (ORs 1.53 to 2.91, p <0.001). Galectin-9, IL-1β and chemokines were up and galectin-3 down in TV cases with BV or intermediate Nugent versus normal Nugent scores (p <0.001). Galectin-9, IL-1β and chemokines were up in TV-HIV and down in TV-HPV co-infections. In-vitro , TV synergized with its endosymbiont Trichomonasvirus (TVV) and BV bacteria to upregulate galectin-1, galectin-9, and inflammatory cytokines. The BV-bacterium Prevotella bivia alone and together with TV downregulated galectin-3 and synergistically upregulated galectin-1, galectin-9 and IL-1β, mirroring the clinical findings of mixed TV-BV infections. P. bivia also downregulated TVV+TV-induced anti-viral response e.g. IP-10 and RANTES, providing a mechanism for conducing viral persistence in TV-BV co-infections. Collectively, the experimental and clinical data suggest that galectin-mediated immunity may be dysregulated and exploited by viral-protozoan-bacterial synergisms exacerbating inflammatory complications from dysbiosis and sexually transmitted infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fichorova, DeLong, Cu-Uvin, King, Jamieson, Klein, Sobel, Vlahov, Yamamoto and Mayer.)

Data support the notion that 40-60% of patients with bipolar disorder (BD) have neurocognitive deficits. It is increasingly accepted that functioning in BD is negatively impacted by these deficits, yet they have not been a successful target for treatment. The biomarkers that predict cognitive deficits in BD are largely unknown, however recent evidence suggests that inflammation may be associated with poorer cognitive outcomes in BD. We measured C-reactive protein (CRP), a marker of systemic inflammation and risk of inflammatory disease, in 222 euthymic BD patients and 52 healthy controls. Within the patient sample, using multivariate analyses of covariance (MANCOVA) we compared cognitive performance of those with high CRP (≥5 mg/L) versus the remaining subjects (<5 mg/L) on a battery of cognitive tests. We evaluated relationships with several other relevant clinical features. We also examined the role of CRP in cognitive decline using a proxy cognitive decline metric, defined as the difference between premorbid and current IQ estimates, in a logistic regression analysis. Approximately 80% of our sample were BD-I, and the remainder were BD-II and 42.6% of our sample had a history of psychosis. We found a statistically significant effect of CRP on cognitive performance on a broad range of tests; participants with CRP ≥ 5 mg/L had worse performance on several measures of executive functioning, MATRICS processing speed and MATRICS reasoning and problem solving relative to those with lower CRP. We also identified CRP as a significant positive predictor of proxy cognitive decline. Our results indicate that elevated CRP is associated with a broad cognitive dysfunction in affectively remitted BD patients. These results may point to a subgroup of patients who might benefit from treatments to reduce inflammation., (© 2019. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: Among healthy postmenopausal women, levels of CA125 and CA15.3 are influenced by demographic and reproductive factors, including race/ethnicity. In this study, we sought to examine the interaction between race/ethnicity and other correlates of these biomarkers and whether the racial differences observed are simply determined by other correlates with racial differences., Methods: In archived sera from 946 postmenopausal women who participated in the 2001-2002 cycle of the National Health and Nutrition Examination Survey, we measured CA125 and CA15.3 and examined their associations with health survey and examination data available in this cohort. We used multivariable linear regression to examine the association between CA125 and CA15.3 and race/ethnicity. We then calculated geometric means of these markers by demographic and reproductive factors stratified by race/ethnicity and used likelihood ratio tests to evaluate heterogeneity., Results: Non-white race was associated with lower CA125, with Non-Hispanic Black women being associated with - 29.0% (95% CI - 42.5%, - 12.2%) difference and Mexican American women being associated with - 6.4% (95% CI - 18.1%, 6.9%) difference on average compared to Non-Hispanic White women. Associations between CA125 and age and parity varied by race/ethnicity. Non-Hispanic Black women were associated with higher CA15.3 compared to Non-Hispanic White women, with 17.3% (95% CI - 0.5%, 38.3%) differences on average. Associations between CA15.3 and age, number of births, and age at natural menopause varied by race/ethnicity., Conclusions: Among postmenopausal women, Non-Hispanic Black women were associated with lower CA125 and higher CA15.3 levels compared to Non-Hispanic White women. Our results support that race/ethnicity should be considered when assigning thresholds for these biomarkers being tested for diagnostic or screening purposes.

The protozoan parasite Trichomonas vaginalis (TV), exclusively adapted to the human genital tract, is one of the most common sexually transmitted pathogens. Adding to the complexity of the host-pathogen interactions, the parasite harbors TV-specific endosymbiont viruses ( Trichomonasvirus , TVV). It was reported that small extracellular vesicles (sEVs) released by TV play a role in host immunity; however, the role of the viral endosymbiosis in this process remained unknown. We hypothesized that the virus may offer evolutionary benefit to its protozoan host at least in part by altering the immunomodulatory properties of sEVs spreading from the site of infection to non-infected immune effector cells. We infected human vaginal epithelial cells, the natural host of the parasite, with TV natively harboring TVV and an isogenic derivative of the parasite cured from the viral infection. sEVs were isolated from vaginal cell culture 24 h post TV infection and from medium where the isogenic TV strains were cultured in the absence of the human host. sEVs from TVV-negative but not TVV-positive parasites cultured alone caused NF-κB activation and increase of IL-8 and RANTES expression by uterine endocervical cells, which provide innate immune defense at the gate to the upper reproductive tract. Similarly, mononuclear leukocytes increased their IL-8, IL-6 and TNF-α output in response to sEVs from virus-negative, but not isogenic virus-positive parasites, the latter exosomes being immunosuppressive in comparison to TV medium control. The same phenomenon of suppressed immunity induced by the TVV-positive compared to TVV-negative phenotype was seen when stimulating the leukocytes with sEVs originating from infected vaginal cultures. In addition, the sEVs from the TVV-positive infection phenotype suppressed immune signaling of a toll-like receptor ligand derived from mycoplasma, another frequent TV symbiont. Quantitative comparative proteome analysis of the secreted sEVs from virus-positive versus virus-negative TV revealed differential expression of two functionally uncharacterized proteins and five proteins involved in Zn binding, protein binding, electron transfer, transferase and catalytic activities. These data support the concept that symbiosis with viruses may provide benefit to the protozoan parasite by exploiting sEVs as a vehicle for inter-cellular communications and modifying their protein cargo to suppress host immune activation., (Copyright © 2020 Govender, Chan, Yamamoto, Budnik and Fichorova.)

Sexually transmitted infections (STIs) and vaginal dysbiosis (disturbed resident microbiota presenting with abnormal Nugent score or candidiasis) have been associated with mucosal inflammation and risk of HIV-1 infection, cancer and poor reproductive outcomes. To date, the temporal relationships between aberrant cervical innate immunity and the clinical onset of microbial disturbance have not been studied in a large population of reproductive age women. We examined data from a longitudinal cohort of 934 Ugandan and Zimbabwean women contributing 3,274 HIV-negative visits who had complete laboratory, clinical and demographic data. Among those, 207 women later acquired HIV, and 584 women were intermittently diagnosed with C. trachomatis (CT), N. gonorrhoeae (NG), genital herpes (HSV-2), T. vaginalis (TV), candidiasis, and abnormal intermediate (4-6) or high (7-10) Nugent score, i.e. bacterial vaginosis (BV). Immune biomarker concentrations in cervical swabs were analyzed by generalized linear and mixed effect models adjusting for site, age, hormonal contraceptive use (HC), pregnancy, breastfeeding, genital practices, unprotected sex and overlapping infections. High likelihood ratios (1.5-4.9) denoted the values of cervical immune biomarkers to predict onset of abnormal Nugent score and candidiasis at the next visits. When controlling for covariates, higher levels of β-defensin-2 were antecedent to BV, CT and HSV-2, lower anti-inflammatory ratio IL-1RA:IL-1β-to intermediate Nugent scores and candida, lower levels of the serine protease inhibitor SLPI-to candida, lower levels of the adhesion molecule ICAM-1 -to TV, and lower levels of the oxidative stress mitigator and endothelial activation marker VEGF-to NG. Changes in innate immunity following onset of dysbiosis and infections were dependent on HC use when controlling for all other covariates. In conclusion, imminent female genital tract dysbiosis or infection can be predicted by distinct patterns of innate immunity. Future research should characterize biotic and abiotic determinants of this pre-existing innate immunity state., Competing Interests: The authors have declared that no competing interests exist.

CA125 is the best ovarian cancer early detection marker to date; however, sensitivity is limited and complementary markers are required to improve discrimination between ovarian cancer cases and non-cases. Anti-CA125 autoantibodies are observed in circulation. Our objective was to evaluate whether these antibodies (1) can serve as early detection markers, providing evidence of an immune response to a developing tumor, and (2) modify the discriminatory capacity of CA125 by either masking CA125 levels (resulting in lower discrimination) or acting synergistically to improve discrimination between cases and non-cases. We investigated these objectives using a nested case-control study within the European Prospective Investigation into Cancer and Nutrition cohort (EPIC) including 250 cases diagnosed within 4 years of blood collection and up to four matched controls. Circulating CA125 antigen and antibody levels were quantified using an electrochemiluminescence assay. Adjusted areas under the curve (aAUCs) by 2-year lag-time intervals were calculated using conditional logistic regression calibrated toward the absolute risk estimates from a pre-existing epidemiological risk model as an offset-variable. Anti-CA125 levels alone did not discriminate cases from controls. For cases diagnosed <2 years after blood collection, discrimination by CA125 antigen was suggestively higher with higher anti-CA125 levels (aAUC, highest antibody tertile: 0.84 [0.76-0.92]; lowest tertile: 0.76 [0.67-0.86]; p het  = 0.06). We provide the first evidence of potentially synergistic discrimination effects of CA125 and anti-CA125 antibodies in ovarian early detection. If these findings are replicated, evaluating CA125 in the context of its antibody may improve ovarian cancer early detection., (© 2017 UICC.)

Background: Ovarian cancer early detection markers CA125, CA15.3, HE4, and CA72.4 vary between healthy women, limiting their utility for screening., Methods: We evaluated cross-sectional relationships between lifestyle and reproductive factors and these markers among controls (n = 1910) from a nested case-control study in the European Prospective Investigation into Cancer and Nutrition (EPIC). Improvements in discrimination of prediction models adjusting for correlates of the markers were evaluated among postmenopausal women in the nested case-control study (n = 590 cases). Generalized linear models were used to calculate geometric means of CA125, CA15.3, and HE4. CA72.4 above vs. below limit of detection was evaluated using logistic regression. Early detection prediction was modeled using conditional logistic regression., Results: CA125 concentrations were lower, and CA15.3 higher, in post- vs. premenopausal women (p ≤ 0.02). Among postmenopausal women, CA125 was higher among women with higher parity and older age at menopause (p trend  ≤ 0.02), but lower among women reporting oophorectomy, hysterectomy, ever use of estrogen-only hormone therapy, or current smoking (p < 0.01). CA15.3 concentrations were higher among heavier women and in former smokers (p ≤ 0.03). HE4 was higher with older age at blood collection and in current smokers, and inversely associated with OC use duration, parity, and older age at menopause (≤ 0.02). No associations were observed with CA72.4. Adjusting for correlates of the markers in prediction models did not improve the discrimination., Conclusions: This study provides insights into sources of variation in ovarian cancer early detection markers in healthy women and informs about the utility of individualizing marker cutpoints based on epidemiologic factors.

Purpose: About 60% of ovarian cancers are diagnosed at late stage, when 5-year survival is less than 30% in contrast to 90% for local disease. This has prompted search for early detection biomarkers. For initial testing, specimens taken months or years before ovarian cancer diagnosis are the best source of information to evaluate early detection biomarkers. Here we evaluate the most promising ovarian cancer screening biomarkers in prospectively collected samples from the European Prospective Investigation into Cancer and Nutrition study., Experimental Design: We measured CA125, HE4, CA72.4, and CA15.3 in 810 invasive epithelial ovarian cancer cases and 1,939 controls. We calculated the sensitivity at 95% and 98% specificity as well as area under the receiver operator curve (C-statistic) for each marker individually and in combination. In addition, we evaluated marker performance by stage at diagnosis and time between blood draw and diagnosis., Results: We observed the best discrimination between cases and controls within 6 months of diagnosis for CA125 (C-statistic = 0.92), then HE4 (0.84), CA72.4 (0.77), and CA15.3 (0.73). Marker performance declined with longer time between blood draw and diagnosis and for earlier staged disease. However, assessment of discriminatory ability at early stage was limited by small numbers. Combinations of markers performed modestly, but significantly better than any single marker., Conclusions: CA125 remains the single best marker for the early detection of invasive epithelial ovarian cancer, but can be slightly improved by combining with other markers. Identifying novel markers for ovarian cancer will require studies including larger numbers of early-stage cases. Clin Cancer Res; 22(18); 4664-75. ©2016 AACRSee related commentary by Skates, p. 4542., (©2016 American Association for Cancer Research.)

Trichomoniasis is the most common non-viral sexually transmitted infection caused by the vaginotropic extracellular protozoan parasite Trichomonas vaginalis. The infection is recurrent, with no lasting immunity, often asymptomatic, and linked to pregnancy complications and risk of viral infection. The molecular mechanisms of immune evasion by the parasite are poorly understood. We demonstrate that galectin-1 and -3 are expressed by the human cervical and vaginal epithelial cells and act as pathogen-recognition receptors for the ceramide phosphoinositol glycan core (CPI-GC) of the dominant surface protozoan lipophosphoglycan (LPG). We used an in vitro model with siRNA galectin knockdown epithelial clones, recombinant galectins, clinical Trichomonas isolates, and mutant protozoan derivatives to dissect the function of galectin-1 and -3 in the context of Trichomonas infection. Galectin-1 suppressed chemokines that facilitate recruitment of phagocytes, which can eliminate extracellular protozoa (IL-8) or bridge innate to adaptive immunity (MIP-3α and RANTES (regulated on activation normal T cell expressed and secreted)). Silencing galectin-1 increased and adding exogenous galectin-1 suppressed chemokine responses to Trichomonas or CPI-GC/LPG. In contrast, silencing galectin-3 reduced IL-8 response to LPG. Live Trichomonas depleted the extracellular levels of galectin-3. Clinical isolates and mutant Trichomonas CPI-GC that had reduced affinity to galectin-3 but maintained affinity to galectin-1 suppressed chemokine expression. Thus via CPI-GC binding, Trichomonas is capable of regulating galectin bioavailability and function to the benefit of its parasitic survival. These findings suggest novel approaches to control trichomoniasis and warrant further studies of galectin-binding diversity among clinical isolates as a possible source for symptom disparity in parasitic infections., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients' homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE(2)RD), which addresses all these impediments on a single platform. The NE(2)RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE(2)RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE(2)RD's broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients' homes.

Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Inflammation and immune activation of the cervicovaginal mucosa are considered factors that increase susceptibility to HIV infection. Therefore, it is essential to screen candidate anti-HIV microbicides for potential mucosal immunomodulatory/inflammatory effects prior to further clinical development. The goal of this study was to develop an in vitro method for preclinical evaluation of the inflammatory potential of new candidate microbicides using a microarray gene expression profiling strategy., Methods: To this end, we compared transcriptomes of human vaginal cells (Vk2/E6E7) treated with well-characterized pro-inflammatory (PIC) and non-inflammatory (NIC) compounds. PICs included compounds with different mechanisms of action. Gene expression was analyzed using Affymetrix U133 Plus 2 arrays. Data processing was performed using GeneSpring 11.5 (Agilent Technologies, Santa Clara, CA)., Results: Microarraray comparative analysis allowed us to generate a panel of 20 genes that were consistently deregulated by PICs compared to NICs, thus distinguishing between these two groups. Functional analysis mapped 14 of these genes to immune and inflammatory responses. This was confirmed by the fact that PICs induced NFkB pathway activation in Vk2 cells. By testing microbicide candidates previously characterized in clinical trials we demonstrated that the selected PIC-associated genes properly identified compounds with mucosa-altering effects. The discriminatory power of these genes was further demonstrated after culturing vaginal cells with vaginal bacteria. Prevotella bivia, prevalent bacteria in the disturbed microbiota of bacterial vaginosis, induced strong upregulation of seven selected PIC-associated genes, while a commensal Lactobacillus gasseri associated to vaginal health did not cause any changes., Conclusions: In vitro evaluation of the immunoinflammatory potential of microbicides using the PIC-associated genes defined in this study could help in the initial screening of candidates prior to entering clinical trials. Additional characterization of these genes can provide further insight into the cervicovaginal immunoinflammatory and mucosal-altering processes that facilitate or limit HIV transmission with implications for the design of prevention strategies.

Colonization by Lactobacillus in the female genital tract is thought to be critical for maintaining genital health. However, little is known about how genital microbiota influence host immune function and modulate disease susceptibility. We studied a cohort of asymptomatic young South African women and found that the majority of participants had genital communities with low Lactobacillus abundance and high ecological diversity. High-diversity communities strongly correlated with genital pro-inflammatory cytokine concentrations in both cross-sectional and longitudinal analyses. Transcriptional profiling suggested that genital antigen-presenting cells sense gram-negative bacterial products in situ via Toll-like receptor 4 signaling, contributing to genital inflammation through activation of the NF-κB signaling pathway and recruitment of lymphocytes by chemokine production. Our study proposes a mechanism by which cervicovaginal microbiota impact genital inflammation and thereby might affect a woman's reproductive health, including her risk of acquiring HIV., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Problem: Severe preeclampsia has been independently linked to complement dysregulation and angiogenic imbalance; however, the relationship between complement and angiogenic factors in human pregnancy is unclear., Method of Study: Utilizing existing biomarkers, our study sought to better understand this relationship in active disease. We performed a case-control study, enrolling 25 cases with severe preeclampsia, 25 controls with chronic hypertension, and 25 healthy controls without hypertension. Levels of complement components (C3a, C5a, and C5b-9) and angiogenic markers [basic fibroblast growth factor (bFGF), placental growth factor (PlGF), vascular endothelial growth factor (VEGF), and soluble fms-like tyrosine kinase-1 (sFlt-1)] were measured simultaneously., Results: Compared to both hypertensive and non-hypertensive controls, severe preeclampsia was associated with increased plasma sFlt-1, decreased plasma VEGF and PlGF, decreased urinary PlGF, and increased urinary C5b-9. Urinary marker C5b-9 correlated strongly with the anti-angiogenic condition. In subjects with detectable urinary excretion of C5b-9, median plasma levels of sFlt-1 were significantly greater (32,029 versus 4556 pg/mL, P < 0.0001) and levels of PlGF (15.6 versus 226 pg/mL, P < 0.0001) and VEGF (119 versus 153 pg/mL, P = 0.001) were significantly lower., Conclusion: More so than plasma complement markers, urinary C5b-9 may a useful measure to link complement dysregulation with angiogenic imbalance in severe preeclampsia., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Problem: Gestational genitourinary infections are associated with lifelong disabilities, but it is unknown if neonatal inflammation is involved., Method: Mothers of 914 infants born before 28th gestation week reported cervical/vaginal infection (CVI), and/or urine/bladder/kidney infection (UTI), or neither. Inflammation proteins measured in baby's blood on postnatal days 1, 7, and 14 were considered elevated if in the top quartile for gestational age. Logistic regression models adjusting for potential confounders assessed odds ratios., Results: Compared to mothers with neither UTI/CVI, those with CVI were more likely to have infants with elevated CRP, SAA, MPO, IL-1β, IL-6, IL-6R, TNF-α, RANTES, ICAM-3, E-selectin, and VEGF-R2 on day 1; those with UTI were more likely to have infants with elevated MPO, IL-6R, TNF-R1, TNF-R2, and RANTES on day 7. Placental anaerobes and genital mycoplasma were more common in pregnancies with CVI., Conclusion: Gestational UTI/CVI should be targeted for preventing systemic inflammation in the very preterm newborn., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Kidney injury with proteinuria is a characteristic feature of preeclampsia, yet the nature of injury in specific regions of the nephron is incompletely understood. Our study aimed to use existing urinary biomarkers to describe the pattern of kidney injury and proteinuria in pregnancies affected by severe preeclampsia. We performed a case-control study of pregnant women from Brigham and Women's Hospital from 2012 to 2013. We matched cases of severe preeclampsia (n=25) 1:1 by parity and gestational age to 2 control groups with and without chronic hypertension. Urinary levels of kidney injury molecule-1 and complement components (C3a, C5a, and C5b-9) were measured by enzyme-linked immunosorbent assay, and other markers (albumin, β2 microglobulin, cystatin C, epithelial growth factor, neutrophil gelatinase-associated lipocalin, osteopontin, and uromodulin) were measured simultaneously with a multiplex electrochemiluminescence assay. Median values between groups were compared with the Wilcoxon signed-rank test and correlations with Spearman correlation coefficient. Analysis of urinary markers revealed higher excretion of albumin and kidney injury molecule-1 and lower excretion of neutrophil gelatinase-associated lipocalin and epithelial growth factor in severe preeclampsia compared with chronic hypertension and healthy controls. Among subjects with severe preeclampsia, urinary excretion of complement activation products correlated most closely with kidney injury molecule-1, a specific marker of proximal tubule injury (C5a: r=0.60; P=0.001; and C5b-9: r=0.75; P<0.0001). Taken together, we describe a pattern of kidney injury in severe preeclampsia that is characterized by glomerular impairment and complement-mediated inflammation and injury, possibly localized to the proximal tubule in association with kidney injury molecule-1., (© 2014 American Heart Association, Inc.)

Necrotizing enterocolitis, characterized by sudden onset and rapid progression, remains the most significant gastrointestinal disorder among premature infants. In seeking a predictive biomarker, we found intestinal fatty acid binding protein, an indicator of enterocyte damage, was substantially increased within three and seven days before the diagnosis of necrotizing enterocolitis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The complement cascade is activated in normal pregnancy, and excessive complement activation propagates the systemic inflammatory response in severe preeclampsia. Consequently, biomarkers of complement dysregulation may be useful for prediction or treatment of disease. Because renal damage with proteinuria is a characteristic pathological feature of preeclampsia, we hypothesized that complement markers in urine, rather than plasma, could better reflect complement dysregulation in disease. To investigate this, we performed a case-control study of pregnant women, enrolling 25 cases with severe preeclampsia, 25 controls with chronic hypertension, and 25 healthy controls without hypertension matched by gestational age and parity. Subjects were recruited from the Brigham and Women's Hospital from March 2012 to March 2013. Urine and blood samples were collected on the day of enrollment, with complement activation (C3a, C5a, and C5b-9) measured by ELISA. Severe preeclampsia was associated with marked elevations in urinary C5b-9 (median [interquartile range], 4.3 [1.2-15.1] ng/mL) relative to subjects with chronic hypertension (0 [0-0]) and healthy controls (0 [0-0]; P<0.0001). Urinary excretion of C5b-9 was detected in 96% of cases with severe preeclampsia, 12% of controls with chronic hypertension, and 8% of healthy controls. Cases were also notable for significantly greater urinary excretion of C3a and C5a. Plasma levels of C5a and C5b-9, but not C3a, were increased in the cases with severe preeclampsia compared with healthy controls; however, they did not distinguish preeclampsia from chronic hypertension, supporting our hypothesis that complement markers in urine, rather than plasma, better reflect complement dysregulation. Complement inhibition is an intriguing treatment option for patients with severe preeclampsia.

Purpose: Test the hypothesis that puerperal mastitis may alter immunity related to the mucin (MUC) family of glycoproteins and lower risk of ovarian cancer., Methods: In two case-control studies conducted in New England between 1998 and 2008, we examined the association between self-reported mastitis and ovarian cancer in 1,483 women with epithelial ovarian cancer and 1,578 controls. IgG1 antibodies against (MUC1) CA15.3 and (MUC16) CA125 were measured using electrochemiluminescence assays in a subset of controls (n = 200). Preoperative CA125 was recorded in 649 cases. The association between ovarian cancer and mastitis was assessed using unconditional logistic regression to calculate adjusted odds ratios, OR, and 95 % confidence intervals (CI). Associations between mastitis and anti-CA15.3 and anti-CA125 antibodies and preoperative CA125 levels were evaluated using adjusted linear regression models., Results: Prior mastitis was associated with a significantly lower risk of ovarian cancer: OR (and 95 % CI) of 0.67 (0.48, 0.94) adjusted for parity, breastfeeding, and other potential confounders. The association was strongest with 2 or more episodes of mastitis, and risk declined progressively with increasing number of children and episodes of mastitis. Among controls, prior mastitis was associated with significantly higher anti-CA15.3 and anti-CA125 antibody levels and, among cases, with significantly lower preoperative CA125 levels., Conclusion: Puerperal mastitis may produce long-lasting anti-mucin antibodies that may lower the risk of ovarian cancer, plausibly through enhanced immune surveillance. Studying immune reactions related to MUC1 and MUC16 in the 10-20 % of breastfeeding women who develop mastitis may suggest ways to duplicate its effects through vaccines based on both antigens.

Objectives: Complex interactions of vaginal microorganisms with the genital tract epithelium shape mucosal innate immunity, which holds the key to sexual and reproductive health. Bacterial vaginosis (BV), a microbiome-disturbance syndrome prevalent in reproductive-age women, occurs commonly in concert with trichomoniasis, and both are associated with increased risk of adverse reproductive outcomes and viral infections, largely attributable to inflammation. To investigate the causative relationships among inflammation, BV and trichomoniasis, we established a model of human cervicovaginal epithelial cells colonised by vaginal Lactobacillus isolates, dominant in healthy women, and common BV species (Atopobium vaginae, Gardnerella vaginalis and Prevotella bivia)., Methods: Colonised epithelia were infected with Trichomonas vaginalis (TV) or exposed to purified TV virulence factors (membrane lipophosphoglycan (LPG), its ceramide-phosphoinositol-glycan core (CPI-GC) or the endosymbiont Trichomonas vaginalis virus (TVV)), followed by assessment of bacterial colony-forming units, the mucosal anti-inflammatory microbicide secretory leucocyte protease inhibitor (SLPI), and chemokines that drive pro-inflammatory, antigen-presenting and T cells., Results: TV reduced colonisation by Lactobacillus but not by BV species, which were found inside epithelial cells. TV increased interleukin (IL)-8 and suppressed SLPI, likely via LPG/CPI-GC, and upregulated IL-8 and RANTES, likely via TVV as suggested by use of purified pathogenic determinants. BV species A vaginae and G vaginalis induced IL-8 and RANTES, and also amplified the pro-inflammatory responses to both LPG/CPI-GC and TVV, whereas P bivia suppressed the TV/TVV-induced chemokines., Conclusions: These molecular host-parasite-endosymbiont-bacteria interactions explain epidemiological associations and suggest a revised paradigm for restoring vaginal immunity and preventing BV/TV-attributable inflammatory sequelae in women.

Background: Vaginal probiotics are investigated as a binary strategy for prevention of bacterial vaginosis and HIV. We applied an innovative experimental model using primary and immortalized human cervical and vaginal epithelial cells to assess the functional properties of Lactobacillus jensenii, a predominant constituent of the healthy vaginal microbiome, engineered to express the HIV-1 entry inhibitor modified cyanovirin-N (mCV-N). In this model bacteria colonize the epithelial cells over a period of 24-72 h. Staurosporine and the Toll-like receptor 2/6 ligand macrophage-activating lipopeptide-2 (MALP-2) serve as positive controls for apoptosis and proinflammatory activation, respectively. In 24-hour intervals, the colonized epithelium is assessed microscopically, supernatants are collected for measurement of soluble immunoinflammatory mediators and production of CV-N, and cells are lysed for assessment of: 1) apoptosis by cleaved versus total caspase-3 assay; 2) NF-κB activation by a luciferase reporter assay; or 3) epithelia-associated colony forming units (CFU) in Brucella agar., Results: Wild type (WT) L. jensenii 1153 consistently colonized cervical and vaginal cells in the absence of epithelial damage and apoptosis. The bioengineered derivatives expressing mCV-N or control plasmids showed the same stable colonization pattern, which was reproducible between technologists and bacterial batches (CFU coefficient of variation <10% within and between experiments and epithelial cell types). MALP-2 activated NF-κB and caused fold-increased levels of proinflammatory mediators with clinically established significance in the cervicovaginal environment (IL-1α, IL-1β, IL-6, TNF-α, IL-8, RANTES, MIP-3α, and ICAM-1), measured by a multiplex electrochemiluminescence assay. At the same time levels of protective anti-inflammatory mediators interleukin 1 receptor antagonist (IL-1RA) and secretory leukocyte protease inhibitor (SLPI), both measured by ELISA, remained constant (IL-1RA) or moderately increased (SLPI). Similarly to MALP-2, colonization by L. jensenii WT activated NF-κB; however, unlike the synthetic TLR2/6 ligand, the live microorganisms did not induce significant changes in the secreted levels across all inflammation-associated proteins. The mCV-N production and function were confirmed by western blot and a HIV-1 gp120 binding assay, respectively. The bioengineered lactobacilli expressed mCV-N with anti-HIV activity preserved in the epithelial cell context and caused no significant immunoinflammatory changes as compared to the WT L. jensenii., Conclusions: These results highlight the translational value of the colonization model and justify further clinical investigation of the homeostatic and anti-HIV effectiveness of the L. jensenii derivates.

Wide-spread protozoan parasites carry endosymbiotic dsRNA viruses with uncharted implications to the human host. Among them, Trichomonas vaginalis, a parasite adapted to the human genitourinary tract, infects globally ∼250 million each year rendering them more susceptible to devastating pregnancy complications (especially preterm birth), HIV infection and HPV-related cancer. While first-line antibiotic treatment (metronidazole) commonly kills the protozoan pathogen, it fails to improve reproductive outcome. We show that endosymbiotic Trichomonasvirus, highly prevalent in T. vaginalis clinical isolates, is sensed by the human epithelial cells via Toll-like receptor 3, triggering Interferon Regulating Factor -3, interferon type I and proinflammatory cascades previously implicated in preterm birth and HIV-1 susceptibility. Metronidazole treatment amplified these proinflammatory responses. Thus, a new paradigm targeting the protozoan viruses along with the protozoan host may prevent trichomoniasis-attributable inflammatory sequelae.

Unlabelled: Several broad-spectrum microbicides, including cellulose sulfate (CS), have passed conventional preclinical and phase I clinical safety evaluation and yet have failed to protect women from acquiring HIV-1 in phase II/III trials. Concerns have been raised that current preclinical algorithms are deficient in addressing the complexity of the microflora-regulated vaginal mucosal barrier. We applied a novel microflora-colonized model to evaluate CS and hydroxyethylcellulose (HEC), which is used as a "universal placebo" in microbicide trials. Cervicovaginal epithelial cultures were colonized with normal vaginal microflora isolates representing common Lactobacillus species used as probiotics (L. acidophilus and L. crispatus) or Prevotella bivia and Atopobium vaginae, most prevalent in the disturbed microflora of bacterial vaginosis (BV). At baseline, all strains maintained constant epithelium-associated CFUs without inducing cytotoxicity and apoptosis. CS selectively reduced epithelium-associated CFUs and (to a lesser extent) planktonic CFUs, most significantly affecting L. crispatus. Inducing only minor changes in sterile epithelial cultures, CS induced expression of innate immunity mediators (RANTES, interleukin-8 [IL-8], and secretory leukocyte protease inhibitor [SLPI]) in microflora-colonized epithelia, most significantly potentiating effects of bacteria causing BV. In the absence of CS, all bacterial strains except L. acidophilus activated NF-κB, although IL-8 and RANTES levels were increased by the presence of BV-causing bacteria only. CS enhanced NF-κB activation in a dose-dependent manner under all conditions, including L. acidophilus colonization. HEC remained inert. These results offer insights into possible mechanisms of CS clinical failure. The bacterially colonized cervicovaginal model reveals unique aspects of microflora-epithelium-drug interactions and innate immunity in the female genital tract and should become an integral part of preclinical safety evaluation of anti-HIV microbicides and other vaginal formulations., Importance: This report provides experimental evidence supporting the concept that the vaginal microflora regulates the epithelial innate immunity in a species- and strain-specific manner and that topically applied microbicides may alter both the bacterial and epithelial components of this homeostatic interaction. Our data also highlight the importance of differentiating the effects of biomedical interventions on epithelium-associated versus conventional planktonic bacterial growth when assessing vaginal mucosal health and immunity.