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1.  Blood protein concentrations in the first two postnatal weeks associated with early postnatal blood gas derangements among infants born before the 28th week of gestation. The ELGAN Study 
Cytokine  2011;56(2):392-398.
To explore the relationships between blood gas derangements and blood concentrations of inflammation-related proteins shortly after preterm birth.
Observational cohort
14 neonatal intensive care units
734 infants born before the 28th week of gestation who were classified by their blood gas derangements during the first three postnatal days and by the concentrations of 25 proteins in their blood on days 1, 7, and 14. We classified these newborns by whether or not they had a highest or lowest PaO2, PCO2, and lowest pH in the most extreme quartile, and by whether or not they had a protein concentration in the highest quartile.
Blood gas derangements on two days were much more likely to be accompanied or followed by sustained or recurrent systemic inflammation than a derangement on only one day. This was most evident for acidemia, and slightly less so for hypercapnia.
Our finding that protein concentration patterns indicative of systemic inflammation are associated with several blood gas derangements raises the possibility that organ damage attributed to these derangements might be accompanied by or involve an inflammatory response.
PMCID: PMC4747654  PMID: 21821429
preterm infant; blood; cytokine; inflammation; acidemia; hypercapnia
3.  Bacterial Vaginosis and Subclinical Markers of Genital Tract Inflammation and Mucosal Immunity 
AIDS Research and Human Retroviruses  2015;31(11):1139-1152.
Bacterial vaginosis (BV) has been linked to an increased risk of human immunodeficiency virus (HIV) acquisition and transmission in observational studies, but the underlying biological mechanisms are unknown. We measured biomarkers of subclinical vaginal inflammation, endogenous antimicrobial activity, and vaginal flora in women with BV and repeated sampling 1 week and 1 month after completion of metronidazole therapy. We also compared this cohort of women with BV to a healthy control cohort without BV. A longitudinal, open label study of 33 women with a Nugent score of 4 or higher was conducted. All women had genital swabs, cervicovaginal lavage (CVL) fluid, and cervicovaginal biopsies obtained at enrollment and received 7 days of metronidazole treatment. Repeat sampling was performed approximately 1 week and 1 month after completion of therapy. Participant's baseline samples were compared to a healthy, racially matched control group (n=13) without BV. The CVL from women with resolved BV (Nugent 0–3) had significantly higher anti-HIV activity, secretory leukocyte protease inhibitor (SLPI), and growth-related oncogene alpha (GRO-α) levels and their ectocervical tissues had significantly more CD8 cells in the epithelium. Women with persistent BV after treatment had significantly higher levels of interleukin-1β, tumor necrosis factor alpha (TNF-α), and intercellular adhesion molecule 1 (ICAM-1) in the CVL. At study entry, participants had significantly greater numbers of CCR5+ immune cells and a higher CD4/CD8 ratio in ectocervical tissues prior to metronidazole treatment, compared to a racially matched cohort of women with a Nugent score of 0–3. These data indicate that BV is associated with changes in select soluble immune mediators, an increase in HIV target cells, and a reduction in endogenous antimicrobial activity, which may contribute to the increased risk of HIV acquisition.
PMCID: PMC4651020  PMID: 26204200
4.  The Contribution of Cervicovaginal Infections to the Immunomodulatory Effects of Hormonal Contraception 
mBio  2015;6(5):e00221-15.
Particular types of hormonal contraceptives (HCs) and genital tract infections have been independently associated with risk of HIV-1 acquisition. We examined whether immunity in women using injectable depot medroxyprogesterone acetate (DMPA), combined oral contraceptives (COC), or no HCs differs by the presence of cervicovaginal infections. Immune mediators were quantified in cervical swabs from 832 HIV-uninfected reproductive-age Ugandans and Zimbabweans. Bacterial infections and HIV were diagnosed by PCR, genital herpes serostatus by enzyme-linked immunosorbent assay (ELISA), altered microflora by Nugent score, and Trichomonas vaginalis and Candida albicans infection by wet mount. Generalized linear models utilizing Box-Cox-Power transformation examined associations between levels of mediators, infection status, and HCs. In no-HC users, T. vaginalis was associated with broadest spectrum of aberrant immunity (higher interleukin 1β [IL-1β], IL-8, macrophage inflammatory protein 3α [MIP-3α], β-defensin 2 [BD2], and IL-1 receptor antigen [IL-1RA]). In women with a normal Nugent score and no genital infection, compared to the no-HC group, COC users showed higher levels of IL-1β, IL-6, IL-8, and IL-1RA, while DMPA users showed higher levels of RANTES and lower levels of BD2, both associated with HIV seroconversion. These effects of COC were blunted in the presence of gonorrhea, chlamydia, trichomoniasis, candidiasis, and an abnormal Nugent score; however, RANTES was increased among COC users with herpes, chlamydia, and abnormal Nugent scores. The effect of DMPA was exacerbated by lower levels of IL-1RA in gonorrhea, chlamydia, or herpes, SLPI in gonorrhea, and IL-1β, MIP-3α, and IL-1RA/IL1β ratio in trichomoniasis. Thus, the effects of HC on cervical immunity depend on the genital tract microenvironment, and a weakened mucosal barrier against HIV may be a combined resultant of genital tract infections and HC use.
In this article, we show that in young reproductive-age women most vulnerable to HIV, hormonal contraceptives are associated with altered cervical immunity in a manner dependent on the presence of genital tract infections. Through altered immunity, hormones may predispose women to bacterial and viral pathogens; conversely, a preexisting specific infection or disturbed vaginal microbiota may suppress the immune activation by levonorgestrel or exacerbate the suppressed immunity by DMPA, thus increasing HIV risk by their cumulative action. Clinical studies assessing the effects of contraception on HIV susceptibility and mucosal immunity may generate disparate results in populations that differ by microbiota background or prevalence of undiagnosed genital tract infections. A high prevalence of asymptomatic infections among HC users that remain undiagnosed and untreated raises even more concerns in light of their combined effects on biomarkers of HIV risk. The molecular mechanisms of the vaginal microbiome’s simultaneous interactions with hormones and HIV remain to be elucidated.
PMCID: PMC4556810  PMID: 26330510
5.  Effects of Female Sex Hormones on Susceptibility to HSV-2 in Vaginal Cells Grown in Air-Liquid Interface 
Viruses  2016;8(9):241.
The lower female reproductive tract (FRT) is comprised of the cervix and vagina, surfaces that are continuously exposed to a variety of commensal and pathogenic organisms. Sexually transmitted viruses, such as herpes simplex virus type 2 (HSV-2), have to traverse the mucosal epithelial lining of the FRT to establish infection. The majority of current culture systems that model the host-pathogen interactions in the mucosal epithelium have limitations in simulating physiological conditions as they employ a liquid-liquid interface (LLI), in which both apical and basolateral surfaces are submerged in growth medium. We designed the current study to simulate in vivo conditions by growing an immortalized vaginal epithelial cell line (Vk2/E6E7) in culture with an air-liquid interface (ALI) and examined the effects of female sex hormones on their growth, differentiation, and susceptibility to HSV-2 under these conditions, in comparison to LLI cultures. ALI conditions induced Vk2/E6E7 cells to grow into multi-layered cultures compared to the monolayers present in LLI conditions. Vk2 cells in ALI showed higher production of cytokeratin in the presence of estradiol (E2), compared to cells grown in progesterone (P4). Cells grown under ALI conditions were exposed to HSV-2-green fluorescent protein (GFP) and the highest infection and replication was observed in the presence of P4. Altogether, this study suggests that ALI cultures more closely simulate the in vivo conditions of the FRT compared to the conventional LLI cultures. Furthermore, under these conditions P4 was found to confer higher susceptibility to HSV-2 infection in vaginal cells. The vaginal ALI culture system offers a better alternative to study host-pathogen interactions.
PMCID: PMC5035955  PMID: 27589787
herpes simplex virus type 2; HSV-2; female genital tract; vaginal cells; air-liquid interface; female sex hormones; estradiol; progesterone; medroxyprogesterone acetate; Vk2/E6E7 cells
6.  Cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract 
Immunity  2015;42(5):965-976.
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 may affect a woman's reproductive health, including her risk of acquiring HIV.
PMCID: PMC4461369  PMID: 25992865
7.  Effects of feminine hygiene products on the vaginal mucosal biome 
Microbial Ecology in Health and Disease  2013;24:10.3402/mehd.v24i0.19703.
Over-the-counter (OTC) feminine hygiene products come with little warning about possible side effects. This study evaluates in-vitro their effects on Lactobacillus crispatus, which is dominant in the normal vaginal microbiota and helps maintain a healthy mucosal barrier essential for normal reproductive function and prevention of sexually transmitted infections and gynecologic cancer.
A feminine moisturizer (Vagisil), personal lubricant, and douche were purchased OTC. A topical spermicide (nonoxynol-9) known to alter the vaginal immune barrier was used as a control. L. crispatus was incubated with each product for 2 and 24h and then seeded on agar for colony forming units (CFU). Human vaginal epithelial cells were exposed to products in the presence or absence of L. crispatus for 24h, followed by epithelium-associated CFU enumeration. Interleukin-8 was immunoassayed and ANOVA was used for statistical evaluation.
Nonoxynol-9 and Vagisil suppressed Lactobacillus growth at 2h and killed all bacteria at 24h. The lubricant decreased bacterial growth insignificantly at 2h but killed all at 24h. The douche did not have a significant effect. At full strength, all products suppressed epithelial viability and all, except the douche, suppressed epithelial-associated CFU. When applied at non-toxic dose in the absence of bacteria, the douche and moisturizer induced an increase of IL-8, suggesting a potential to initiate inflammatory reaction. In the presence of L. crispatus, the proinflammatory effects of the douche and moisturizer were countered, and IL-8 production was inhibited in the presence of the other products.
Some OTC vaginal products may be harmful to L. crispatus and alter the vaginal immune environment. Illustrated through these results, L. crispatus is essential in the preservation of the function of vaginal epithelial cells in the presence of some feminine hygiene products. More research should be invested toward these products before they are placed on the market.
PMCID: PMC3758931  PMID: 24009546
Lactobacillus; L. crispatus; cytokines; Interleukin-8; Nonoxynol-9; vaginal microbiota; vaginal epithelial colonization; mucosal immunity
8.  Clinical Isolates of Trichomonas vaginalis Concurrently Infected by Strains of Up to Four Trichomonasvirus Species (Family Totiviridae)▿† 
Journal of Virology  2011;85(9):4258-4270.
Trichomonas vaginalis, which causes the most common nonviral sexually transmitted disease worldwide, is itself commonly infected by nonsegmented double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae. To date, cDNA sequences of one or more strains of each of three trichomonasvirus species have been reported, and gel electrophoresis showing several different dsRNA molecules obtained from a few T. vaginalis isolates has suggested that more than one virus strain might concurrently infect the same parasite cell. Here, we report the complete cDNA sequences of 3 trichomonasvirus strains, one from each of the 3 known species, infecting a single, agar-cloned clinical isolate of T. vaginalis, confirming the natural capacity for concurrent (in this case, triple) infections in this system. We furthermore report the complete cDNA sequences of 11 additional trichomonasvirus strains, from 4 other clinical isolates of T. vaginalis. These additional strains represent the three known trichomonasvirus species, as well as a newly identified fourth species. Moreover, 2 of these other T. vaginalis isolates are concurrently infected by strains of all 4 trichomonasvirus species (i.e., quadruple infections). In sum, the full-length cDNA sequences of these 14 new trichomonasviruses greatly expand the existing data set for members of this genus and substantiate our understanding of their genome organizations, protein-coding and replication signals, diversity, and phylogenetics. The complexity of this virus-host system is greater than has been previously well recognized and suggests a number of important questions relating to the pathogenesis and disease outcomes of T. vaginalis infections of the human genital mucosa.
PMCID: PMC3126235  PMID: 21345965
9.  Novel Vaginal Microflora Colonization Model Providing New Insight into Microbicide Mechanism of Action 
mBio  2011;2(6):e00168-11.
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.
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.
PMCID: PMC3202752  PMID: 22027006
10.  The relationship between TSH and systemic inflammation in extremely-preterm newborns 
Endocrine  2014;48(2):595-602.
Elevated thyrotropin (TSH) levels in critically ill extremely premature infants have been attributed to transient hypothyroidism of prematurity or non-thyroidal illness syndrome. We evaluated the hypothesis that relatively high TSH levels in the first two postnatal weeks follow recovery from systemic inflammation, similar to non-thyroidal illness syndrome.
The study was conducted in 14 Neonatal Intensive Care Units and approved by each individual Institutional Review Board. We measured the concentrations of TSH and 25 inflammation-related proteins in blood spots obtained on postnatal days 1, 7, and 14. We then evaluated the temporal relationships between hyperthyrotropinemia (HTT), defined as a TSH concentration in the highest quartile for gestational age and postnatal day, and elevated levels of inflammation-related proteins.
880 newborns less than 28 weeks of gestation were included. Elevated concentrations of inflammation-related proteins during the first or second week did not precede day-14 HTT. Systemic inflammation on day 7 was associated with day-14 HTT only if inflammation persisted through the end of the two week period. HTT frequently accompanied elevated concentrations of inflammation-related proteins on the same day.
The hypothesis that HTT follows recovery from severe illness, defined as preceding systemic inflammation, is weakly supported by our study. Our findings more prominently support the hypothesis that TSH conveys information about concomitant inflammation in the extremely premature newborn.
PMCID: PMC4285685  PMID: 24996532
thyroid; inflammation; prematurity; TSH; nonthyroidal illness; sick euthyroid
11.  Systemic inflammation in the extremely low gestational age newborn following maternal genitourinary infections 
Gestational genitourinary infections are associated with life-long disabilities, but it is unknown if neonatal inflammation is involved.
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.
Compared to neither UTI/CVI, mothers 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 micoplasma were more common in pregnancies with CVI.
Gestational UTI/CVI should be targeted for preventing systemic inflammation in the very preterm newborn.
PMCID: PMC4293273  PMID: 25164433
cervicitis; vaginitis; cytokines; acute phase proteins; preterm birth; placental microbiome
12.  Guiding the vaginal microbicide trials with biomarkers of inflammation 
This article discusses cytokine patterns as potential biomarkers of vaginal inflammation, which are needed for the safety evaluation of topical microbicide products for the prevention of sexually transmitted HIV-1 infection. In order to be effective, the vaginal anti-HIV-1 microbicides should avoid proinflammatory responses that facilitate transepithelial viral penetration and replication. Pro-inflammatory and anti-inflammatory cytokines play bi-directional roles in HIV-1 pathogenesis, transmission, susceptibility and resistance. Previous research has shown that many of these key mediators of mucosal barrier function (e.g. IL-1, IL-1 receptor antagonist, IL-6, TNF-α, TNF-receptor II, transforming growth factor β, IL-10, IL-12, IL-8, macrophage inhibitory protein 1, etc.) can be detected in the vaginal secretions of healthy or infected individuals using non-invasive sampling techniques. As part of two microbicide trials, we measured IL-1α, IL-1β, IL-1 receptor antagonist, TNF-α and IL-8 in 291 cervicovaginal lavage samples obtained before product use and at the seventh and 14th day after product use. We showed that vaginal formulations, temperature and matrix-specific factors in the vaginal fluids may interfere with cytokine detection, and therefore specific protocols must be validated for various collection procedures and cytokine assays. Our results suggest that combined patterns of cytokine dynamics rather than individual measurements might distinguish proinflammatory product-related effects in microbicide safety trials. More research is needed to establish cytokine mucosal baselines and modulation by genetic factors, sexual intercourse, menstrual cycle, exercise, hormones, stress and infections before guidelines can be established for clinical trial enrollment criteria, the prediction of side/adverse events and ultimately microbicide benefit prognostication.
PMCID: PMC2643374  PMID: 16419271
Cervicovaginal lavage; cytokines; HIV-1; inflammation; microbicides; safety evaluation
13.  The breadth and type of systemic inflammation and the risk of adverse neurological outcomes in extremely low gestational age newborns 
Pediatric neurology  2014;52(1):42-48.
We hypothesized that the risk of brain damage in extremely preterm neonates increases with the breadth and type of systemic inflammation, indexed by the number of elevated inflammation-related proteins and the number of functional categories of inflammation-related proteins exhibiting an elevated concentration.
In blood from 881 infants born before 28 weeks gestation, we measured the concentrations of 25 inflammation-related proteins, representing 6 functional categories (cytokines, chemokines, growth factors, adhesion molecules, metalloproteinases, and liver-produced acute phase reactant proteins) on postnatal days 1, 7, and 14 and evaluated associations between the number and type of proteins whose concentrations were elevated on two separate occasions a week apart and the diagnoses of ventriculomegaly as a neonate, and, at 2 years, microcephaly, impaired early cognitive functioning, cerebral palsy, and autism risk as assessed with the Modified Checklist for Autism in Toddlers (M-CHAT) screen, and, in a subset of these children from 12 of 14 sites (n= 826), an attention problem identified with the Child Behavior Checklist (CBCL).
The risk of abnormal brain structure and function overall was increased among children who had recurrent/persistent elevations of the 25 protein. The risk for most outcomes did not rise until at least four proteins in at least two functional categories were elevated. When we focused our analysis on 10 proteins previously found to be associated consistently with neurological outcomes, we found the risk of low Mental Development Index (MDI) on the Bayley Scales of Infant Development-II, microcephaly, and a CBCL-defined attention problem increased with higher numbers of these recurrently/persistently elevated proteins.
Increasing breadth of early neonatal inflammation, indexed by the number of protein elevations or the number of protein functional classes elevated, is associated with increasing risk of disorders of brain structure and function among infants born extremely premature.
PMCID: PMC4276530  PMID: 25459361
14.  Trichomonas vaginalis Lipophosphoglycan Exploits Binding to Galectin-1 and -3 to Modulate Epithelial Immunity* 
The Journal of Biological Chemistry  2015;291(2):998-1013.
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.
PMCID: PMC4705417  PMID: 26589797
cytokine; galectin; inflammation; interleukin; parasite; Interleukin 8 (IL-8); CCL5 (RANTES); CCL-20 (MIP-3α); sexually transmitted infection; human vagina
15.  A blinded, randomized controlled trial of high-dose vitamin D supplementation to reduce recurrence of bacterial vaginosis 
American journal of obstetrics and gynecology  2014;211(5):479.e1-479.e13.
Low serum vitamin D levels have been associated with increased prevalence of the reproductive tract condition bacterial vaginosis (BV). The objective of this trial was to evaluate the effect of high-dose vitamin D supplementation on BV recurrence.
Study design
This randomized, placebo-controlled, double-blinded trial enrolled 118 women with symptomatic BV from an urban STD clinic ( registration NCT01450462). All participants received 500mg oral metronidazole twice daily for seven days. Intervention participants (n=59) also received nine doses of 50,000 international units of cholecalciferol (vitamin D3) over 24 weeks; control women (n=59) received matching placebo. Recurrent BV was assessed via Nugent scoring after 4, 12 and 24 weeks. We assessed the effect of the intervention using an intention-to-treat approach, fitting Cox proportional hazards models to evaluate recurrent BV over the follow-up period.
Most participants (74%) were black, with a median age of 26 years. Median presupplementation serum 25-hydroxyvitamin D [25(OH)D] was similar across randomization arms: 16.6 ng/mL in the vitamin D arm and 15.8 ng/mL in the control arm. At trial completion, median 25(OH)D among women receiving vitamin D was 30.5 ng/mL, vs 17.8 ng/mL in control women; 16% of women receiving vitamin D and 57% receiving placebo remained vitamin D deficient (<20 ng/mL). BV prevalence among women randomized to vitamin D was very similar to those randomized to placebo at the 4- and 12-week visits, but by the 24-week visit, BV prevalence was 65% among women in the vitamin D arm and 48% among control women. BV recurrence was not reduced by vitamin D supplementation (intention-to-treat hazard ratio, 1.11; 95% confidence interval, 0.68-1.81). Among women experiencing recurrent BV, median time to recurrence was 13.7 weeks in the vitamin D arm and 14.3 weeks in the control arm.
Women receiving vitamin D experienced significant increases in serum 25(OH)D, but this increase was not associated with decreased BV recurrence in this high-risk STD clinic population.
PMCID: PMC4254061  PMID: 24949544
Bacterial vaginosis; randomized controlled trial; STD clinic; vitamin D
16.  Endogenous erythropoietin varies significantly with inflammation-related proteins in extremely premature newborns 
Cytokine  2014;69(1):22-28.
Erythropoietin, a pluripotent glycoprotein essential for erythropoiesis, fetal growth, and development, has recently been implicated in innate immune regulation. Data from the ELGAN Study allowed us to evaluate relationships between endogenous erythropoietin and 25 inflammation-related proteins in extremely premature newborns.
We measured the concentrations of 25 inflammation-related proteins and of erythropoietin in blood spots collected on postnatal days 1, 7, and 14 from 936 infants born before 28 weeks gestation. We calculated the odds that infants with an inflammation-related protein in the highest quartile for gestational age and collection day had an erythropoietin concentration in the highest or lowest quartile.
The proportion of children with inflammation-associated protein concentrations in the top quartile tended to increase monotonically with increasing quartile of EPO concentrations on 2 of the 3 days assessed. To a large extent, on each of the 3 days assessed, the odds ratios for an erythropoietin concentration in the top quartile were significantly elevated among those with an inflammation-related protein concentration in the top quartile.
Our findings suggest that in very preterm newborns, circulating levels of endogenous erythropoietin vary significantly with circulating levels of inflammation-related proteins. Elevation of endogenous erythropoietin might not be an epiphenomenon, but instead might contribute to subsequent events, by either promoting or reducing inflammation, or by promoting an anti-injury or repair capability.
PMCID: PMC4285695  PMID: 25022958
Infant; Premature; Protein; Acute-phase; Cytokine
17.  Gene Expression Profiling of Human Vaginal Cells In Vitro Discriminates Compounds with Pro-Inflammatory and Mucosa-Altering Properties: Novel Biomarkers for Preclinical Testing of HIV Microbicide Candidates 
PLoS ONE  2015;10(6):e0128557.
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.
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).
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.
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.
PMCID: PMC4459878  PMID: 26052926
18.  Elevated Urinary iFABP Predicts Necrotizing Enterocolitis within Seven Days Prior to Clinical Onset 
The Journal of pediatrics  2014;164(6):1486-1488.
Necrotizing enterocolitis (NEC), 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 NEC.
PMCID: PMC4120986  PMID: 24636854
19.  Elevated blood levels of inflammation-related proteins are associated with an attention problem at age 24 months in extremely preterm infants 
Pediatric research  2014;75(6):781-787.
Extremely preterm birth is associated with subsequent behavioral problems. We hypothesized that perinatal systemic inflammation, a risk factor for cerebral white matter injury and cognitive impairment, is associated with behavior problems observed at 2 years.
In a cohort of 600 children born before 28 weeks gestation, we measured 25 inflammation-related proteins in blood collected on postnatal days 1, 7, and 14, and identified behavior problems using parent responses to the Child Behavior Checklist for Ages 1.5–5 (CBCL/1.5-5) at two years of age. A persistent or recurrent protein elevation was defined as a concentration in the highest quartile (for gestational age and postnatal age) on at least two days approximately one week apart. Behavior problems were defined by CBCL/1.5-5 subscale scores at or above the 93rd percentile.
A single-day elevation of ICAM-3 was associated with an increased risk of an attention problem, as were persistent or recurrent elevations of MPO, IL-6, TNF-RI, IL-8, ICAM-3, VEGF-R1, and VEGF-R2. These associations persisted among infants without white matter injury and cognitive impairment.
Among children born extremely prematurely, recurrent or persistent elevations of inflammation-related proteins in blood during in the first two postnatal weeks are associated with an attention problem at age 2 years.
PMCID: PMC4429865  PMID: 24614800
20.  Elevated Endogenous Erythropoietin Concentrations Are Associated with Increased Risk of Brain Damage in Extremely Preterm Neonates 
PLoS ONE  2015;10(3):e0115083.
We sought to determine, in very preterm infants, whether elevated perinatal erythropoietin (EPO) concentrations are associated with increased risks of indicators of brain damage, and whether this risk differs by the co-occurrence or absence of intermittent or sustained systemic inflammation (ISSI).
Protein concentrations were measured in blood collected from 786 infants born before the 28th week of gestation. EPO was measured on postnatal day 14, and 25 inflammation-related proteins were measured weekly during the first 2 postnatal weeks. We defined ISSI as a concentration in the top quartile of each of 25 inflammation-related proteins on two separate days a week apart. Hypererythropoietinemia (hyperEPO) was defined as the highest quartile for gestational age on postnatal day 14. Using logistic regression and multinomial logistic regression models, we compared risks of brain damage among neonates with hyperEPO only, ISSI only, and hyperEPO+ISSI, to those who had neither hyperEPO nor ISSI, adjusting for gestational age.
Newborns with hyperEPO, regardless of ISSI, were more than twice as likely as those without to have very low (< 55) Mental (OR 2.3; 95% CI 1.5-3.5) and/or Psychomotor (OR 2.4; 95% CI 1.6-3.7) Development Indices (MDI, PDI), and microcephaly at age two years (OR 2.4; 95%CI 1.5-3.8). Newborns with both hyperEPO and ISSI had significantly increased risks of ventriculomegaly, hemiparetic cerebral palsy, microcephaly, and MDI and PDI < 55 (ORs ranged from 2.2-6.3), but not hypoechoic lesions or other forms of cerebral palsy, relative to newborns with neither hyperEPO nor ISSI.
hyperEPO, regardless of ISSI, is associated with elevated risks of very low MDI and PDI, and microcephaly, but not with any form of cerebral palsy. Children with both hyperEPO and ISSI are at higher risk than others of very low MDI and PDI, ventriculomegaly, hemiparetic cerebral palsy, and microcephaly.
PMCID: PMC4368546  PMID: 25793991
21.  Are preterm newborns who have relative hyperthyrotropinemia at increased risk of brain damage? 
We sought to disentangle the contributions of hyperthyrotropinemia (an indicator of thyroid dysfunction) (HTT) and intermittent or sustained systemic inflammation (ISSI) to structural and functional indicators of brain damage.
We measured the concentrations of TSH on day 14, and of 25 inflammation-related proteins in blood collected during the first 2 postnatal weeks from 786 infants born before the 28th week of gestation who were not considered to have hypothyroidism. We defined hyperthyrotropinemia (HTT) as a TSH concentration in the highest quartile for gestational age on postnatal day 14 and ISSI was defined as a concentration in the top quartile for gestational age of a specific inflammation-related protein on two separate days a week apart during the first two postnatal weeks. We first assessed the risk of brain damage indicators comparing 1) neonates who had HTT to those without (regardless of ISSI), and 2) neonates with HTT only, ISSI only, or HTT+ ISSI, to those who were exposed to neither HTT nor ISSI. HTT was defined as a TSH concentration in the highest quartile for gestational age on postnatal day 14.
In univariable models that compared those with HTT to those without, HTT was not significantly associated with any indicator of brain damage. In models that compared HTT only, ISSI only, and HTT+ISSI, to those with neither, children with ISSI only or with HTT+ISSI were at significantly higher risk of ventriculomegaly [odds ratios (OR) ranged from 2–6], while those with HTT only were at significantly reduced risk of a hypoechoic lesion [ORs ranged from 0.2–0.4]. Children with HTT only had a higher risk of quadriparesis and those with ISSI alone had a higher risk of hemiparesis [ORs ranged from 1.6–2.4]. Elevated risk of a very low mental development score was associated with both ISSI only and with HTT+ISSI while a very low motor development score and microcephaly were associated with HTT+ISSI.
The association of HTT with increased or decreased risk of indicators of brain damage depends upon the presence or absence of ISSI.
PMCID: PMC4317282  PMID: 24897395
thyroid stimulating hormone; inflammation; cerebral palsy; microcephaly
22.  Systemic inflammation, intraventricular hemorrhage, and white matter injury 
Journal of child neurology  2012;28(12):1637-1645.
To see if the systemic inflammation profile of 123 infants born before the 28th week of gestation who had intraventricular hemorrhage (IVH) without white matter injury (WMI) differed from that of 68 peers who had both IVH and WMI, we compared both groups to 677 peers who had neither. Cranial ultrasound scans were read independently by multiple readers until concordance. The concentrations of 25 proteins were measured with multiplex arrays using an electrochemiluminescence system. Infants who had IVH and WMI were more likely than others to have elevated concentrations of CRP and IL-8 on days 1, 7, and 14, and elevated concentrations of SAA and TNF-alpha on 2 of these days. IVH should probably be viewed as two entities, IVH unaccompanied by WMI, and IVH accompanied by WMI. Each entity is associated with inflammation, but IVH accompanied by WMI has a stronger inflammatory signal than IVH unaccompanied by WMI.
PMCID: PMC4166653  PMID: 23112243
Infant; premature; hemorrhage; brain injuries; acute; inflammation
23.  Puerperal Mastitis: a Reproductive Event of Importance Affecting Anti-Mucin Antibody Levels and Ovarian Cancer Risk 
Cancer causes & control : CCC  2013;24(11):1911-1923.
Test the hypothesis that puerperal mastitis may alter immunity related to the mucin (MUC) family of glycoproteins and lower risk for ovarian cancer.
In two case-control studies conducted in New England between 1998–2008, 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.
Prior mastitis was associated with a significantly lower risk for 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.
Puerperal that mastitis may produce long-lasting anti-mucin antibodies that may lower the risk for 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.
PMCID: PMC3805704  PMID: 23925696
CA125; CA15.3; Ovarian Cancer; Puerperal Mastitis
24.  Systemic Inflammation Associated with Severe Intestinal Injury in Extremely Low Gestational Age Newborns 
Fetal and pediatric pathology  2012;32(3):222-234.
To define the role of systemic inflammation in infants with intestinal perforation (IP) and necrotizing enterocolitis (NEC), we measured 25 blood protein concentrations on days 1, 7, and 14 in 939 infants born before 28 weeks’ gestation. On days 7 and 14, infants with NEC had elevated levels of CRP, serum amyloid A (SAA), IL-6, and IL-8. Infants with IP had elevated levels of CRP and insulin growth factor binding protein-1 on day 7 and elevated CRP, SAA, TNF-receptor-2, and matrix metalloproteinase-9 levels on day 14. A better understanding of systemic inflammation might help prevent and treat these disorders.
PMCID: PMC4201234  PMID: 23002960
Infant, premature; necrotizing enterocolitis; intestinal perforation; cytokines
25.  Clinical Evidence for the Role of Trichomonas vaginalis in Regulation of Secretory Leukocyte Protease Inhibitor in the Female Genital Tract 
The Journal of Infectious Diseases  2013;207(9):1462-1470.
Background. Secretory leukocyte protease inhibitor (SLPI) is responsible for regulating inflammatory damage to and innate and adaptive immune responses in the vaginal mucosa. Depressed cervicovaginal SLPI levels have been correlated with both Trichomonas vaginalis infection and poor reproductive health outcomes.
Methods. We measured levels of SLPI in 215 vaginal specimens collected from adolescent and young adult females aged 14–22 years. Log-transformed SLPI values were compared by analysis of variance or by an unpaired t test before and after adjustment for confounding effects through the propensity score method.
Results. Females receiving hormonal contraceptives and those with an abnormal vaginal pH had lower SLPI levels as compared to their peers. After propensity score adjustment for race, behavioral factors, hormonal use, and other sexually transmitted infections (STIs), SLPI levels were lower in females with a positive T. vaginalis antigen test result, a vaginal pH >4.5, vaginal leukocytosis, and recurrent (vs initial) T. vaginalis infection, with the lowest levels observed in those with the highest T. vaginalis loads.
Conclusions. The SLPI level was reduced by >50% in a T. vaginalis load–dependent manner. Future research should consider whether identifying and treating females with low levels of T. vaginalis infection (before they become wet mount positive) would prevent the loss of SLPI and impaired vaginal immunity. The SLPI level could be used as a vaginal-health marker to evaluate interventions and vaginal products.
PMCID: PMC3610423  PMID: 23355743
SLPI protein; human; Trichomonas vaginalis; Vaginosis; Bacterial; Sexually Transmitted Diseases; Adolescent

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