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2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  Inflammation biomarkers in vaginal fluid and preterm delivery 
Which inflammation biomarkers detected in the vaginal fluid are most informative for identifying preterm delivery (PTD) risk?
Elevated interleukin (IL)-6 at mid-trimester was associated with increased odds of spontaneous PTD at <35 weeks and with PTD plus histologic chorioamnionitis (HCA), and had the greatest sensitivity for detecting these two PTD subtypes.
Maternal and/or fetal inflammation play a role in some preterm deliveries, therefore inflammation biomarkers might help to identify women at greater risk.
We examined 1115 women from the Pregnancy Outcomes and Community Health Study, a cohort study conducted from September 1998 through June 2004, for whom data were available on mid-pregnancy inflammatory biomarkers.
At enrollment at 16–27 weeks gestation, vaginal fluid samples were collected from a swab and 15 eluted biomarkers were measured using the Meso Scale Discovery multiplex electrochemiluminescence platform. Associations of biomarkers with PTD were examined, according to clinical circumstance, week at delivery and presence/absence of HCA. Weighted logistic regression was used to determine odds ratios (OR) and 95% confidence intervals (CI) adjusted for race. Sensitivity and specificity were compared between individual and multiple biomarkers, identified by a bootstrapping method.
Elevated IL-6 (>75th percentile) displayed the strongest association with spontaneous PTD <35 weeks (OR 2.3; CI 1.3–4.0) and PTD with HCA (OR 2.8; CI 1.4–6.0). The sensitivity of IL-6 to detect spontaneous PTD <35 weeks or PTD with HCA was 0.43 and 0.51, respectively, while specificity was 0.74 and 0.75, respectively. IL-6 plus IL1β, IL-6r, tumor necrosis factor-alpha or granulocyte-macrophage colony-stimulating factor increased specificity (range 0.84–0.88), but decreased sensitivity (range 0.28–0.34) to detect both PTD subtypes. Results were similar when a combination of IL-6 and bacterial vaginosis (BV) was explored. Thus, the use of multiple biomarkers did not detect PTD subtypes with a greater sensitivity than IL-6 alone, and IL-6 is a specific but non-sensitive marker for the detection of spontaneous PTD.
Our ability to find small effect size associations between PTD and inflammation biomarkers (OR <2.0) might have been limited by the modest number of less common PTD subtypes in our population (e.g. spontaneous delivery <35 weeks, PTD accompanied by HCA) and by relatively higher variability for some cytokines, for example tumor necrosis factor-α, IL-12p70, IL-10 and granulocyte-macrophage colony-stimulating factor, that are less stable and commonly undetectable or detectable at low levels in human vaginal secretions.
Larger studies are needed to further explore a role of inflammation biomarkers in combination with other risk factors, including specific BV-associated organisms, for the prediction of PTD subtypes.
This work was supported by the National Institute of Child Health and Human Development, National Institute of Nursing, March of Dimes Foundation, Thrasher Research Foundation and Centers for Disease Control and Prevention. The authors have no conflicts of interest.
PMCID: PMC3600841  PMID: 23416276
biomarker; histologic chorioamnionitis; inflammation; preterm birth
12.  Inflammation-initiating illnesses, inflammation-related proteins, and cognitive impairment in extremely preterm infants 
Brain, behavior, and immunity  2013;29:104-112.
Neonatal inflammation is associated with perinatal brain damage. We evaluated to what extent elevated blood levels of inflammation-related proteins supplement information about the risk of impaired early cognitive function provided by inflammation-related illnesses. From 800 infants born before the 28th week of gestation, we collected blood spots on days 1, 7 and 14, for analysis of 25 inflammation-related proteins, and data about culture-positive bacteremia, necrotizing enterocolitis (Bell stage IIIb), and isolated perforation of the intestine, during the first two weeks, and whether they were ventilated on postnatal day 14. We considered a protein to be persistently or recurrently elevated if its concentration was in the top quartile (for gestational age and day blood was collected) on two separate days one week apart. We assessed the children at 2 years of age with the Bayley Mental Development Index (MDI). The combinations of NEC and ventilation on day 14, and of bacteremia and ventilation on day 14 consistently provided information about elevated risk of MDI <55, regardless of whether or not a variable for an elevated protein concentration was included in the model. A variable for a persistently or recurrently elevated concentration of each of the following proteins provided additional information about an increased risk of MDI <55: CRP, SAA, IL-6, TNF-alpha, IL-8, MIP-1beta, ICAM-1, E-SEL, and IGFBP-1. We conclude that elevated blood concentrations of inflammation-related proteins provide information about the risk of impaired cognitive function at age 2 years that supplements information provided by inflammation-associated illnesses.
PMCID: PMC3582030  PMID: 23295265
cognitive impairment; necrotizing enterocolitis; extreme prematurity; systemic inflammatory response; neonatal chronic lung disease; neonatal sepsis
13.  Systemic inflammation associated with mechanical ventilation among extremely preterm infants 
Cytokine  2012;61(1):315-322.
Little evidence is available to document that mechanical ventilation is an antecedent of systemic inflammation in preterm humans. We obtained blood on postnatal day 14 from 726 infants born before the 28th week of gestation and measured the concentrations of 25 inflammation-related proteins. We created multivariable models to assess the relationship between duration of ventilation and protein concentrations in the top quartile. Compared to newborns ventilated for fewer than 7 days (N=247), those ventilated for 14 days (N=330) were more likely to have elevated blood concentrations of pro-inflammatory cytokines (IL-1β, TNF-α), chemokines (IL-8, MCP-1), an adhesion molecule (ICAM-1), and a matrix metalloprotease (MMP-9), and less likely to have elevated blood concentrations of two chemokines (RANTES, MIP-1β), a matrix metalloproteinase (MMP-1), and a growth factor (VEGF). Newborns ventilated for 7-13 days (N=149) had systemic inflammation that approximated the pattern of newborns ventilated for 14 days. These relationships were not confounded by chorioamnionitis or antenatal corticosteroid exposure, and were not altered appreciably among infants with and without bacteremia. These findings suggest that two weeks of ventilation are more likely than shorter durations of ventilation to be accompanied by high blood concentrations of pro-inflammatory proteins indicative of systemic inflammation, and by low concentrations of proteins that might protect from inflammation-mediated organ injury.
PMCID: PMC3518391  PMID: 23148992
inflammation; ventilation; preterm infant; cytokine; chemokine
14.  Two-hit model of brain damage in the very preterm newborn: small for gestational age and postnatal systemic inflammation 
Pediatric research  2012;73(3):362-370.
We sought to disentangle the contributions of perinatal systemic inflammation and small for gestational age (SGA) to the occurrence of low Bayley Mental Development Indices (MDIs) at age 2 years.
We measured the concentration of 25 inflammation-related proteins in blood obtained during the first 2 postnatal weeks from 805 infants who were born before the 28th week of gestation and who had MDI measurements at age 2 years and were able to walk independently.
SGA newborns who did not have systemic inflammation (a concentration of an inflammation-related protein in the top quartile for gestational age on 2 days a week apart) were at greater risk of an MDI < 55, but not 55–69, than their peers who had neither SGA nor systemic inflammation. SGA infants who had elevated blood concentrations of IL-1beta, TNF-alpha, or IL-8 during the first two postnatal weeks were at even higher risk of an MDI < 55 than their SGA peers without systemic inflammation and of their non-SGA peers with systemic inflammation.
SGA appears to place very preterm newborns at increased risk of a very low MDI. Systemic inflammation adds considerably to the increased risk.
PMCID: PMC3642985  PMID: 23364171
15.  Fetal-Placental Inflammation, but not Adrenal Activation, is Associated with Extreme Preterm Delivery 
Spontaneous term delivery involves activation of placental corticotropin-releasing hormone and the fetal adrenal axis, but the basis for extreme preterm delivery is unknown. Our objective was to determine whether placental corticotropin-releasing hormone is activated in extreme spontaneous preterm delivery.
Study Design
1506 mothers delivering at less than 28 weeks gestation were enrolled. Each mother/infant pair was assigned to the category that described the primary reason for hospitalization. Observers who had no knowledge of patient categorization assessed placenta microbiology, histology, and CRH expression. These were correlated with the primary reason for hospitalization.
Among infants delivered at <28 weeks gestation, spontaneous (versus induced) delivery was associated with less placental corticotropin-releasing hormone expression and more frequent signs of placental inflammation and infection.
Inflammation and infection, rather than premature activation of the fetal adrenal axis, should be the major focus of research to prevent extremely preterm human birth.
PMCID: PMC3696892  PMID: 22264652
preterm delivery; corticotropin-releasing hormone; inflammation
16.  Trichomonasvirus: a new genus of protozoan viruses in the family Totiviridae 
Archives of virology  2010;156(1):171-179.
The family Totiviridae includes a number of viruses with monosegmented dsRNA genomes and isometric virions that infect either fungi or a number of medically important protozoan parasites such as Leishmania and Giardia. A new genus, Trichomonasvirus, was recently proposed for this family. Its name is based on the genus of its host organism, Trichomonas vaginalis, a protozoan parasite that colonizes the human genitourinary mucosa and is the most common non-viral sexually transmitted infection in the world. The type species of this new genus is Trichomonas vaginalis virus 1. Distinguishing characteristics of the new genus include infection of a human sexually transmitted parasite, stable mixed infection with more than one distinct Trichomonasvirus species, and sequence-based phylogenetic divergence that distinguishes it from all other family members.
PMCID: PMC3659425  PMID: 20976609
17.  Structure of a Protozoan Virus from the Human Genitourinary Parasite Trichomonas vaginalis 
mBio  2013;4(2):e00056-13.
The flagellated protozoan Trichomonas vaginalis is an obligate human genitourinary parasite and the most frequent cause of sexually transmitted disease worldwide. Most clinical isolates of T. vaginalis are persistently infected with one or more double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae, which appear to influence not only protozoan biology but also human disease. Here we describe the three-dimensional structure of Trichomonas vaginalis virus 1 (TVV1) virions, as determined by electron cryomicroscopy and icosahedral image reconstruction. The structure reveals a T = 1 capsid comprising 120 subunits, 60 in each of two nonequivalent positions, designated A and B, as previously observed for fungal Totiviridae family members. The putative protomer is identified as an asymmetric AB dimer consistent with either decamer or tetramer assembly intermediates. The capsid surface is notable for raised plateaus around the icosahedral 5-fold axes, with canyons connecting the 2- and 3-fold axes. Capsid-spanning channels at the 5-fold axes are unusually wide and may facilitate release of the viral genome, promoting dsRNA-dependent immunoinflammatory responses, as recently shown upon the exposure of human cervicovaginal epithelial cells to either TVV-infected T. vaginalis or purified TVV1 virions. Despite extensive sequence divergence, conservative features of the capsid reveal a helix-rich fold probably derived from an ancestor shared with fungal Totiviridae family members. Also notable are mass spectrometry results assessing the virion proteins as a complement to structure determination, which suggest that translation of the TVV1 RNA-dependent RNA polymerase in fusion with its capsid protein involves −2, and not +1, ribosomal frameshifting, an uncommonly found mechanism to date.
Trichomonas vaginalis causes ~250 million new cases of sexually transmitted disease each year worldwide and is associated with serious complications, including premature birth and increased transmission of other pathogens, including HIV. It is an extracellular parasite that, in turn, commonly hosts infections with double-stranded RNA (dsRNA) viruses, trichomonasviruses, which appear to exacerbate disease through signaling of immunoinflammatory responses by human epithelial cells. Here we report the first three-dimensional structure of a trichomonasvirus, which is also the first such structure of any protozoan dsRNA virus; show that it has unusually wide channels at the capsid vertices, with potential for releasing the viral genome and promoting dsRNA-dependent responses by human cells; and provide evidence that it uses −2 ribosomal frameshifting, an uncommon mechanism, to translate its RNA polymerase in fusion with its capsid protein. These findings provide both mechanistic and translational insights concerning the role of trichomonasviruses in aggravating disease attributable to T. vaginalis.
PMCID: PMC3622925  PMID: 23549915
18.  Systemic responses of preterm newborns with presumed or documented bacteremia 
To compare the frequency of elevated concentrations of inflammation-related proteins in the blood of infants born before the 28th week of gestation who had documented bacteremia to those who had presumed (antibiotic-treated but culture-negative) bacteremia to those who neither.
The subjects of this study are the 868 infants born at 14 institutions for whom information about protein measurements on at least two of the three protocol days (days 1, 7, and 14) was available and who did not have Bell stage 3 necrotizing enterocolitis or isolated bowel perforation, which were strongly associated with bacteremia in this sample.
Newborns with presumed early (week 1) bacteremia had elevated concentrations of only a few inflammation-related proteins, while those who had presumed late (weeks 2–4) bacteremia did not have any elevations. In contrast, newborns who had documented early bacteremia had a moderately strong signal, while those who had documented late bacteremia had a stronger signal with more protein concentrations elevated on two separate occasions a week apart.
Culture-confirmed early and late bacteremia are accompanied/followed by systemic inflammatory responses not seen with presumed early and late bacteremia.
PMCID: PMC3294175  PMID: 22085230
bacteremia; infant; premature; blood proteins
19.  Homeostatic properties of Lactobacillus jensenii engineered as a live vaginal anti-HIV microbicide 
BMC Microbiology  2013;13:4.
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.
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.
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.
PMCID: PMC3605260  PMID: 23298379
20.  Relationships among the concentrations of 25 inflammation-associated proteins during the first postnatal weeks in the blood of infants born before the 28th week of gestation 
Cytokine  2011;57(1):182-190.
Inflammation appears to be involved in processes leading to organ damage in preterm newborns, yet little is known about the relationships among elevated concentrations of inflammation-associated proteins in the blood of preterm newborns.
In this exploratory study, we used an electrochemiluminescence method to measure 25 proteins in blood obtained on postnatal day 1 (range 1–3), day 7 (range 5–8), and day 14 (range 12–15) from 939 children born before the 28th week of gestation and evaluated to what extent those whose concentration of each protein was elevated (defined as in the highest quartile for gestational age and day the specimen was obtained) also had an elevated concentration of every other protein the same day or on a day 1 or 2 weeks later (p < .0001).
On each of the 3 days assessed, elevated concentrations of 17 proteins were associated with elevated concentrations of 15 or more of the other 24 proteins. VEGF, VEGF-R1, VEGF-R2 were among these proteins, while IGFBP-1 was associated with 13 other proteins on day 7. An elevated concentration of 8 proteins on day 1 predicted an elevated concentration of 10 or more proteins on day 7, while an elevated concentration of only two proteins on day 7 were associated with elevated concentrations of 10 or more proteins on day-14. Few associations were seen between day 1 and day 14.
Inflammation is a diffuse process in ELGANs, with elevated concentrations of cytokines, chemokines, adhesion molecules, matrix metalloproteinases, a growth factor and its receptors, as well as a growth factor binding protein associated with each other the same day, as well as on subsequent days.
PMCID: PMC3246087  PMID: 22133344
Preterm newborn; Inflammation; Biomarkers
21.  Cluster analysis of placental inflammatory proteins can distinguish preeclampsia from preterm labor and premature membrane rapture in singleton deliveries less than 28 weeks gestation 
Inflammation within the preterm placenta is common and leads to adverse outcomes for premature infants. The risks of complications are different between iatrogenic (e.g. preeclampsia) and spontaneous (e.g. preterm labor and membrane rapture) causes of preterm delivery, suggesting different underlying biology contributes to these placental conditions.
Method of Study
Thirty preterm singleton placentas from the following groups were analyzed: 1) severe preeclampsia (PE), 2) preterm premature membrane rupture (pPROM) and 3) preterm labor (PL). Proinflammatory and anti-inflammatory cytokines, adhesion and angiogenic molecules were measured in placental lysates using a multiplex assay. K-means cluster analysis was used to generate patterns of protein level intensity.
Three cluster patterns were apparent. Placentas from PE had high levels of VEGF combined with low levels of acute inflammatory proteins (IL-1β, IL-18, IL-6, TNF-α), low IL-1 RA and high TGF-β. PL and pPROM had higher anti-inflammatory IL-1 RA and thrombomodulin combined with lower VEGF, regardless of proinflammatory cytokines and adhesion molecules. Half of the PL and pPROM cases had clusters of heightened inflammatory responses (lower TGF-β clustered with higher intensity of inflammatory mediators).
Discriminating protein patterns were elucidated and may serve as a foundation from which to understand the biological mechanisms underlying these pregnancy complications.
PMCID: PMC3165070  PMID: 21623999
inflammation; placenta; preterm labor; preterm premature membrane rupture; preterm delivery
22.  Endobiont Viruses Sensed by the Human Host – Beyond Conventional Antiparasitic Therapy 
PLoS ONE  2012;7(11):e48418.
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.
PMCID: PMC3492353  PMID: 23144878
23.  Searching for Lower Female Genital Tract Soluble and Cellular Biomarkers: Defining Levels and Predictors in a Cohort of Healthy Caucasian Women 
PLoS ONE  2012;7(8):e43951.
High concentrations of pro-inflammatory cytokines have been previously observed in the genital fluids of women enrolled in microbicide trials and may explain observed increased HIV transmission in some of these trials. Although the longitudinal nature of these studies allows within-subject comparisons of post-product levels to baseline levels, the fact that the physiologic variations of these cytokines and other markers of immune activation are not fully defined in different populations, makes it difficult to assess changes that can be directly attributed to microbicide use as opposed to other biological and behavioural factors.
Cervicovaginal lavage samples were collected from 30 healthy Caucasian and assayed for concentrations of ten cytokines/chemokines, total protein content and two antimicrobial proteins using a multiplex immunoassay and ELISA. Cellular markers were characterized by flow cytometry on mononuclear cells collected from the endocervix using flocked swabs. Bacterial quantification was performed using quantitative PCR.
Ectopy, menstrual cycle phase, prostate-specific antigen and presence of leucocytes in endocervical cells' supernatant were associated with the concentrations of cyto-/chemokines in cervicovaginal secretions. Approximately 3% of endocervical cells collected were monocytes of which a median of 52% (SD  = 17) expressed both CD4 and CCR5 markers. Approximately 1% of the total cells were T-cells with a median of 61% (SD  = 10) CD4 and CCR5 expression. Around 5% of the monocytes and 16% of the T-cells expressed the immune activation marker HLA-DR. Higher percentages of T-cells were associated with greater quantities of IL-1RA, GM-CSF and elafin.
We demonstrate the presence of selected soluble and cellular immune activation markers and identify their predictors in the female genital tract of healthy women. Future clinical trials should consider ectopy, sexual activity, menstrual cycle phase and presence of bacterial species as possible confounders when evaluating the possible inflammatory effects of microbicide compounds.
PMCID: PMC3432048  PMID: 22952818
24.  The relationship between neonatal blood protein profiles and placenta histologic characteristics in extremely low gestation age newborns 
Pediatric research  2011;69(1):68-73.
Amniotic fluid infection with chorioamnionitis is associated with increased risks of morbidity and mortality in children born prematurely. These risks depend on the presence of a fetal inflammatory response. We measured the concentrations of 25 proteins in the blood of 871 infants born before the 28th week of gestation, and examined their placentas for acute inflammation. Newborns who had inflammatory lesions of the placenta were much more likely than their peers (p<0.01) to have elevated blood concentrations of cytokines (IL-1β, IL-6, TNF-alpha), chemokines (IL-8, MIP-1β, RANTES, I-TAC), adhesion molecules (ICAM-1, ICAM-3, E-selectin), matrix metalloproteinases (MMP1, MMP -9), the angiogenic inflammatory factor VEGF and its receptor VEGF-R2 as well as acute phase proteins (SAA and CRP) during first three days after birth. In contrast, newborns with poor placental perfusion had lower levels of inflammatory proteins (p<0.01, IL-6, RANTES, ICAM-1, ICAM-3, VCAM-1, E-selectin, MMP-1, MMP-9, MPO, VEGF). An inverse pattern was found between newborn levels of VEGF and its competitive inhibitor VEGF-R1 in both the inflamed and poorly perfused placenta categories. These results confirm the predictive value of placental histology for the presence or absence of elevated inflammatory response in the newborn.
PMCID: PMC3066075  PMID: 20921924
25.  Mumps and ovarian cancer: modern interpretation of an historic association 
Cancer causes & control : CCC  2010;21(8):1193-1201.
Epidemiologic studies found childhood mumps might protect against ovarian cancer. To explain this association, we investigated whether mumps might engender immunity to ovarian cancer through antibodies against the cancer-associated antigen MUC1 abnormally expressed in the inflamed parotid gland.
Through various health agencies, we obtained sera from 161 cases with mumps parotitis. Sera were obtained from 194 healthy controls. We used an ELISA to measure anti-MUC1 antibodies and electro-chemiluminescence assays to measure MUC1 and CA 125. Log-transformed measurements were analyzed by t-tests, generalized linear models, and Pearson or Spearman correlations. We also conducted a meta-analysis of all published studies regarding mumps and ovarian cancer.
Adjusting for assay batch, age, and sex, the level of anti-MUC1 antibodies was significantly higher in mumps cases compared to controls (p = 0.002). Free circulating levels of CA 125, but not MUC1, were also higher in cases (p = 0.02). From the meta-analysis, the pooled odds ratio estimate (and 95% CI) for the mumps and ovarian cancer association was 0.81 (0.68–0.96) (p = 0.01).
Mumps parotitis may lead to expression and immune recognition of a tumor-associated form of MUC1 and create effective immune surveillance of ovarian cancer cells that express this form of MUC1.
PMCID: PMC2951028  PMID: 20559706
Ovarian cancer; Mumps parotitis; MUC1; CA125

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