CD1 and iNKT cells mediate immune responses against the GBS hemolytic lipid toxin induced by a non-toxic analog.

Although hemolytic lipids have been discovered from many human pathogens including Group B Streptococcus (GBS), strategies that neutralize their function are lacking. GBS is a leading cause of pregnancy-associated neonatal infections, and adult GBS infections are on the rise. The GBS hemolytic lipid toxin or granadaene, is cytotoxic to many immune cells including T and B cells. We previously showed that mice immunized with a synthetic nontoxic analog of granadaene known as R-P4 had reduced bacterial dissemination during systemic infection. However, mechanisms important for R-P4 mediated immune protection was not understood. Here, we show that immune serum from R-P4-immunized mice facilitate GBS opsonophagocytic killing and protect naïve mice from GBS infection. Further, CD4+ T cells isolated from R-P4-immunized mice proliferated in response to R-P4 stimulation in a CD1d- and iNKT cell-dependent manner. Consistent with these observations, R-P4 immunized mice lacking CD1d or CD1d-restricted iNKT cells exhibit elevated bacterial burden. Additionally, adoptive transfer of iNKT cells from R-P4 vaccinated mice significantly reduced GBS dissemination compared to adjuvant controls. Finally, maternal R-P4 vaccination provided protection against ascending GBS infection during pregnancy. These findings are relevant in the development of therapeutic strategies targeting lipid cytotoxins.

A Recombinant Alpha-Like Protein Subunit Vaccine (GBS-NN) Provides Protection in Murine Models of Group B Streptococcus Infection.

BACKGROUND: Group B Streptococcus (GBS) transmission during pregnancy causes preterm labor, stillbirths, fetal injury, or neonatal infections. Rates of adult infections are also rising. The GBS-NN vaccine, engineered by fusing N-terminal domains of GBS Alpha C and Rib proteins, is safe in healthy, nonpregnant women, but further assessment is needed for use during pregnancy. Here, we tested GBS-NN vaccine efficacy using mouse models that recapitulate human GBS infection outcomes. METHODS: Following administration of GBS-NN vaccine or adjuvant, antibody profiles were compared by ELISA. Vaccine efficacy was examined by comparing infection outcomes in GBS-NN vaccinated versus adjuvant controls during systemic and pregnancy-associated infections, and during intranasal infection of neonatal mice following maternal vaccination. RESULTS: Vaccinated mice had higher GBS-NN-specific IgG titers versus controls. These antibodies bound alpha C and Rib on GBS clinical isolates. Fewer GBS were recovered from systemically challenged vaccinated mice versus controls. Although vaccination did not eliminate GBS during ascending infection in pregnancy, vaccinated dams experienced fewer in utero fetal deaths. Additionally, maternal vaccination prolonged neonatal survival following intranasal GBS challenge. CONCLUSIONS: These findings demonstrate GBS-NN vaccine efficacy in murine systemic and perinatal GBS infections and suggest that maternal vaccination facilitates the transfer of protective antibodies to neonates.

Virulence, phenotype and genotype characteristics of invasive group B Streptococcus isolates obtained from Swedish pregnant women and neonates.

Group B streptococci (GBS) are bacteria that can cause preterm birth and invasive neonatal disease. Heterogeneous expression of virulence factors enables GBS to exist as both commensal bacteria and to become highly invasive. A molecular epidemiological study comparing GBS bacterial traits, genotype and host characteristics may indicate whether it is possible to predict the risk of perinatal invasive GBS disease and more accurately target intrapartum antibiotic prophylaxis. A total of 229 invasive GBS isolates from Swedish pregnant women or neonates were assessed for virulence and phenotypic traits: hemolysis zone, hemolytic pigment (Granada agar), Streptococcus B Carrot Broth (SBCB) assay, CAMP factor, and hyaluronidase activity. Genes regulating hemolytic pigment synthesis (covR/covS, abx1, stk1, stp1) were sequenced. Of the virulence factors and phenotypes assessed, a Granada pigment or SBCB score ≥ 2 captured more than 90% of EOD isolates with excellent inter-rater reliability. High enzyme activity of hyaluronidase was observed in 16% (36/229) of the invasive GBS isolates and notably, in one case of stillbirth. Hyaluronidase activity was also significantly higher in GBS isolates obtained from pregnant/postpartum individuals versus the stillbirth or neonatal invasive isolates (p < 0.001). Sequencing analysis found that abx1 (g.T106I), stk1 (g.T211N), stp1 (g.K469R) and covS (g.V343M) variants were present significantly more often in the higher (Granada pigment score ≥ 2) versus lower pigmented isolates (p < 0.001, each variant). Among the 203 higher Granada pigment scoring isolates, 22 (10.8%) isolates had 3 of the four sequence variants and 10 (4.9%) had 2 of the four sequence variants. Although heterogeneity in GBS virulence factor expression was observed, the vast majority were more highly pigmented and contained several common sequence variants in genes regulating pigment synthesis. High activity of hyaluronidase may increase risk for stillbirth and invasive disease in pregnant or postpartum individuals. Our findings suggest that testing for GBS pigmentation and hyaluronidase may, albeit imperfectly, identify pregnant people at risk for invasive disease and represent a step towards a personalized medical approach for the administration of intrapartum antibiotic prophylaxis.

Hemolytic Membrane Vesicles of Group B Streptococcus Promote Infection.

BACKGROUND: Group B streptococci (GBS) are ß-hemolytic, Gram-positive bacteria associated with fetal injury, preterm birth, spontaneous abortion, and neonatal infections. A key factor promoting GBS virulence is the ß-hemolysin/cytolysin, a pigmented ornithine rhamnolipid (also known as granadaene) associated with the bacterial surface. METHODS: A previous study indicated that GBS produce small structures known as membrane vesicles (MVs), which contain virulence-associated proteins. In this study, we show that GBS MVs are pigmented and hemolytic, indicating that granadaene is functionally active in MVs. RESULTS: In addition, MVs from hyperhemolytic GBS induced greater cell death of neutrophils, T cells, and B cells compared with MVs from isogenic nonhemolytic GBS, implicating MVs as a potential mechanism for granadaene-mediated virulence. Finally, hemolytic MVs reduced oxidative killing of GBS and aggravated morbidity and mortality of neonatal mice infected with GBS. CONCLUSIONS: These studies, taken together, reveal a novel mechanism by which GBS deploy a crucial virulence factor to promote bacterial dissemination and pathogenesis.

Amniotic fluid interleukin 6 and interleukin 8 are superior predictors of fetal lung injury compared with maternal or fetal plasma cytokines or placental histopathology in a nonhuman primate model.

BACKGROUND: Intra-amniotic infection or inflammation is common in early preterm birth and associated with substantial neonatal lung morbidity owing to fetal exposure to proinflammatory cytokines and infectious organisms. Amniotic fluid interleukin 8, a proinflammatory cytokine, was previously correlated with the development of neonatal bronchopulmonary dysplasia, but whether amniotic fluid cytokines or placental pathology more accurately predicts neonatal lung pathology and morbidity is unknown. We have used a pregnant nonhuman primate model of group B Streptococcus infection to study the pathogenesis of intra-amniotic infection, bacterial invasion of the amniotic cavity and fetus, and microbial-host interactions. In this nonhuman primate model, we have studied the pathogenesis of group B Streptococcus strains with differing potential for virulence, which has resulted in a spectrum of intra-amniotic infection and fetal lung injury that affords the opportunity to study the inflammatory predictors of fetal lung pathology and injury. OBJECTIVE: This study aimed to determine whether fetal lung injury is best predicted by placental histopathology or the cytokine response in amniotic fluid or maternal plasma. STUDY DESIGN: Chronically catheterized pregnant monkeys (Macaca nemestrina, pigtail macaque) at 116 to 125 days gestation (term at 172 days) received a choriodecidual inoculation of saline (n=5), weakly hemolytic group B Streptococcus strain (n=5, low virulence), or hyperhemolytic group B Streptococcus strain (n=5, high virulence). Adverse pregnancy outcomes were defined as either preterm labor, microbial invasion of the amniotic cavity, or development of the fetal inflammatory response syndrome. Amniotic fluid and maternal and fetal plasma samples were collected after inoculation, and proinflammatory cytokines (tumor necrosis factor alpha, interleukin beta, interleukin 6, interleukin 8) were measured by a multiplex assay. Cesarean delivery was performed at the time of preterm labor or within 1 week of inoculation. Fetal necropsy was performed at the time of delivery. Placental pathology was scored in a blinded fashion by a pediatric pathologist, and fetal lung injury was determined by a semiquantitative score from histopathology evaluating inflammatory infiltrate, necrosis, tissue thickening, or collapse scored by a veterinary pathologist. RESULTS: The principal findings in our study are as follows: (1) adverse pregnancy outcomes occurred more frequently in animals receiving hyperhemolytic group B Streptococcus (80% with preterm labor, 80% with fetal inflammatory response syndrome) than in animals receiving weakly hemolytic group B Streptococcus (40% with preterm labor, 20% with fetal inflammatory response syndrome) and in controls (0% preterm labor, 0% fetal inflammatory response syndrome); (2) despite differences in the rate of adverse pregnancy outcomes and fetal inflammatory response syndrome, fetal lung injury scores were similar between animals receiving the weakly hemolytic group B Streptococcus strains and animals receiving the hyperhemolytic group B Streptococcus strains; (3) fetal lung injury score was significantly correlated with peak amniotic fluid cytokines interleukin 6 and interleukin 8 but not tumor necrosis factor alpha or interleukin 1 beta; and (4) fetal lung scores were poorly correlated with maternal and fetal plasma cytokine levels and placental pathology. CONCLUSION: Amniotic fluid interleukin 6 and interleukin 8 levels were superior predictors of fetal lung injury than placental histopathology or maternal plasma cytokines. This evidence supports a role for amniocentesis in the prediction of neonatal lung morbidity owing to intra-amniotic infection, which cannot be provided by cytokine analysis of maternal plasma or placental histopathology.

MicroRNA Signature of Epithelial-Mesenchymal Transition in Group B Streptococcal Infection of the Placental Chorioamniotic Membranes.

BACKGROUND: Infection-induced preterm birth is a major cause of neonatal mortality and morbidity and leads to preterm premature rupture of placental chorioamniotic membranes. The loss of amniotic epithelial cells and tensile strength preceding membrane rupture is poorly understood. We hypothesized that intrauterine bacterial infection induces changes in microRNA (miRNA) expression, leading to amniotic epithelial cell loss and membrane weakening. METHODS: Ten pregnant pigtail macaques received choriodecidual inoculation of either group B Streptococcus (GBS) or saline (n = 5/group). Placental chorioamniotic membranes were studied using RNA microarray and immunohistochemistry. Chorioamniotic membranes from women with preterm premature rupture of membranes (pPROM) and normal term pregnancies were studied using transmission electron microscopy. RESULTS: In our model, an experimental GBS infection was associated with changes in the miRNA profile in the chorioamniotic membranes consistent with epithelial to mesenchymal transition (EMT) with loss of epithelial (E-cadherin) and gain of mesenchymal (vimentin) markers. Similarly, loss of desmosomes (intercellular junctions) was seen in placental tissues from women with pPROM. CONCLUSIONS: We describe EMT as a novel mechanism for infection-associated chorioamniotic membrane weakening, which may be a common pathway for many etiologies of pPROM. Therapy based on anti-miRNA targeting of EMT may prevent pPROM due to perinatal infection.

Zika virus and the nonmicrocephalic fetus: why we should still worry.

Zika virus is a mosquito-transmitted flavivirus and was first linked to congenital microcephaly caused by a large outbreak in northeastern Brazil. Although the Zika virus epidemic is now in decline, pregnancies in large parts of the Americas remain at risk because of ongoing transmission and the potential for new outbreaks. This review presents why Zika virus is still a complex and worrisome public health problem with an expanding spectrum of birth defects and how Zika virus and related viruses evade the immune response to injure the fetus. Recent reports indicate that the spectrum of fetal brain and other anomalies associated with Zika virus exposure is broader and more complex than microcephaly alone and includes subtle fetal brain and ocular injuries; thus, the ability to prenatally diagnose fetal injury associated with Zika virus infection remains limited. New studies indicate that Zika virus imparts disproportionate effects on fetal growth with an unusual femur-sparing profile, potentially providing a new approach to identify viral injury to the fetus. Studies to determine the limitations of prenatal and postnatal testing for detection of Zika virus-associated birth defects and long-term neurocognitive deficits are needed to better guide women with a possible infectious exposure. It is also imperative that we investigate why the Zika virus is so adept at infecting the placenta and the fetal brain to better predict other viruses with similar capabilities that may give rise to new epidemics. The efficiency with which the Zika virus evades the early immune response to enable infection of the mother, placenta, and fetus is likely critical for understanding why the infection may either be fulminant or limited. Furthermore, studies suggest that several emerging and related viruses may also cause birth defects, including West Nile virus, which is endemic in many parts of the United States. With mosquito-borne diseases increasing worldwide, there remains an urgent need to better understand the pathogenesis of the Zika virus and related viruses to protect pregnancies and child health.

The fetal origins of mental illness.

The impact of infections and inflammation during pregnancy on the developing fetal brain remains incompletely defined, with important clinical and research gaps. Although the classic infectious TORCH pathogens (ie, Toxoplasma gondii, rubella virus, cytomegalovirus [CMV], herpes simplex virus) are known to be directly teratogenic, emerging evidence suggests that these infections represent the most extreme end of a much larger spectrum of injury. We present the accumulating evidence that prenatal exposure to a wide variety of viral and bacterial infections-or simply inflammation-may subtly alter fetal brain development, leading to neuropsychiatric consequences for the child later in life. The link between influenza infections in pregnant women and an increased risk for development of schizophrenia in their children was first described more than 30 years ago. Since then, evidence suggests that a range of infections during pregnancy may also increase risk for autism spectrum disorder and depression in the child. Subsequent studies in animal models demonstrated that both pregnancy infections and inflammation can result in direct injury to neurons and neural progenitor cells or indirect injury through activation of microglia and astrocytes, which can trigger cytokine production and oxidative stress. Infectious exposures can also alter placental serotonin production, which can perturb neurotransmitter signaling in the developing brain. Clinically, detection of these subtle injuries to the fetal brain is difficult. As the neuropsychiatric impact of perinatal infections or inflammation may not be known for decades after birth, our construct for defining teratogenic infections in pregnancy (eg, TORCH) based on congenital anomalies is insufficient to capture the full adverse impact on the child. We discuss the clinical implications of this body of evidence and how we might place greater emphasis on prevention of prenatal infections. For example, increasing uptake of the seasonal influenza vaccine is a key strategy to reduce perinatal infections and the risk for fetal brain injury. An important research gap exists in understanding how antibiotic therapy during pregnancy affects the fetal inflammatory load and how to avoid inflammation-mediated injury to the fetal brain. In summary, we discuss the current evidence and mechanisms linking infections and inflammation with the increased lifelong risk of neuropsychiatric disorders in the child, and how we might improve prenatal care to protect the fetal brain.

Mast cell chymase decreases the severity of group B Streptococcus infections.

BACKGROUND: Group B Streptococcus (GBS) or Streptococcus agalactiae are ß-hemolytic gram-positive bacteria that colonize the lower genital tracts of women and are frequently associated with infections during pregnancy. Innate immune defenses are critical for controlling GBS dissemination and systemic infection. Mast cells are resident sentinel cells that come into contact with pathogens early during colonization and infection. OBJECTIVE: We aimed to investigate the contribution of chymase to systemic GBS infection and rates of preterm birth. METHODS: Pharmacologic and genetic approaches using mice deficient in mast cell protease (MCPT) 4, the mouse functional homologue of human chymase, were used. RESULTS: Our studies show that mast cells release a protease with chymotrypsin-like cleavage specificity in response to GBS. Additionally, increased GBS systemic infection and preterm births were observed in MCPT4-deficient mice versus MCPT4-sufficient mice. Furthermore, we observed that proteolytic cleavage of the host extracellular matrix protein fibronectin by peritoneal cell-derived mast cell lysates diminished GBS adherence. Consistent with this observation, the increase in GBS dissemination and preterm births observed in MCPT4-deficient mice was abolished when GBS was deficient in expression of the fibronectin-binding protein SfbA. CONCLUSIONS: Taken together, our results suggest that the protective effect of MCPT4 against GBS dissemination and preterm labor can be attributed in part to MCPT4-mediated proteolysis of fibronectin. Our studies reveal a novel role of mast cells in defense against bacterial infections.

Diminished Capsule Exacerbates Virulence, Blood-Brain Barrier Penetration, Intracellular Persistence, and Antibiotic Evasion of Hyperhemolytic Group B Streptococci.

Group B streptococci (GBS) are encapsulated, ß-hemolytic bacteria that are a common cause of infections in human newborns and certain adults. Two factors important for GBS virulence are the sialic acid capsular polysaccharide that promotes immune evasion and the hemolytic pigment that induces host cell cytotoxcity. These virulence factors are often oppositely regulated by the CovR/CovS two-component system. Clinical GBS strains exhibiting hyperhemolysis and low capsule due to pathoadaptive covR/S mutations have been isolated from patients. Given the importance of capsule to GBS virulence, we predicted that a decrease or loss of capsule would attenuate the virulence of covR/S mutants. Surprisingly, hyperhemolytic GBS with low or no capsule exhibit increased virulence, intracellular persistence, and blood-brain barrier penetration, which was independent of a Trojan horse mechanism of barrier penetration. Additionally, intracellular persistence enabled both hemolytic and hyperhemolytic GBS to evade antibiotics routinely used to treat these infections. The finding that diminished capsule expression promotes GBS virulence, intracellular persistence, and antibiotic evasion has important implications for sustained antibiotic therapy and efficacy of capsule-based vaccines.

Human Cervical Mucus Plugs Exhibit Insufficiencies in Antimicrobial Activity Towards Group B Streptococcus.

Preterm birth is a leading cause of neonatal mortality and lacks an effective therapy. Ascending microbial infections from the lower genital tract lead to infection of the placenta, amniotic fluid, and fetus causing preterm birth or stillbirth. Directly in the path of an ascending infection is the cervical mucus plug (CMP), a dense mucoid structure in the cervical canal with potential antimicrobial properties. In this study, we aimed to define the components of CMP responsible for antimicrobial activity against a common lower genital tract organism associated with preterm birth and stillbirths, namely, group B streptococcus (GBS). Using a quantitative proteomic approach, we identified antimicrobial factors in CMPs that were collected from healthy human pregnancies. However, we noted that the concentration of antimicrobial peptides present in the human CMPs were insufficient to directly kill GBS, and antimicrobial activity, when observed, was due to antibiotics retained in the CMPs. Despite this insufficiency, CMP proteins were able to activate leukocytes in whole blood resulting in increased rates of bacterial killing, suggesting a role for the CMP in enhancing complement-mediated killing or leukocyte activation. This study provides new insight into how the human CMP may limit ascending bacterial infection.

A Nonhemolytic Group B Streptococcus Strain Exhibits Hypervirulence.

Group B streptococci (GBS) are Gram-positive bacteria that are a leading cause of neonatal infections. Most invasive isolates are ß-hemolytic, and hemolytic activity is critical for GBS virulence. Although nonhemolytic GBS strains are occasionally isolated, they are often thought to be virulence attenuated. In this study, we show that a nonhemolytic GBS strain (GB37) isolated from a septic neonate exhibits hypervirulence. Substitution of tryptophan to leucine (W297L) in the sensor histidine kinase CovS results in constitutive kinase signaling, leading to decreased hemolysis and increased activity of the GBS hyaluronidase, HylB. These results describe how nonpigmented and nonhemolytic GBS strains can exhibit hypervirulence.

Femur-sparing pattern of abnormal fetal growth in pregnant women from New York City after maternal Zika virus infection.

BACKGROUND: Zika virus is a mosquito-transmitted flavivirus, which can induce fetal brain injury and growth restriction following maternal infection during pregnancy. Prenatal diagnosis of Zika virus-associated fetal injury in the absence of microcephaly is challenging due to an incomplete understanding of how maternal Zika virus infection affects fetal growth and the use of different sonographic reference standards around the world. We hypothesized that skeletal growth is unaffected by Zika virus infection and that the femur length can represent an internal standard to detect growth deceleration of the fetal head and/or abdomen by ultrasound. OBJECTIVE: We sought to determine if maternal Zika virus infection is associated with a femur-sparing pattern of intrauterine growth restriction through analysis of fetal biometric measures and/or body ratios using the 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project and World Health Organization Fetal Growth Chart sonographic references. STUDY DESIGN: Pregnant women diagnosed with a possible recent Zika virus infection at Columbia University Medical Center after traveling to an endemic area were retrospectively identified and included if a fetal ultrasound was performed. Data were collected regarding Zika virus testing, fetal biometry, pregnancy, and neonatal outcomes. The 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project and World Health Organization Fetal Growth Chart sonographic standards were applied to obtain Z-scores and/or percentiles for fetal head circumference, abdominal circumference, and femur length specific for each gestational week. A novel 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project standard was also developed to generate Z-scores for fetal body ratios with respect to femur length (head circumference:femur length, abdominal circumference:femur length). Data were then grouped within clinically relevant gestational age strata (<24, 24-27 6/7, 28-33 6/7, >34 weeks) to analyze time-dependent effects of Zika virus infection on fetal size. Statistical analysis was performed using Wilcoxon signed-rank test on paired data, comparing either abdominal circumference or head circumference to femur length. RESULTS: A total of 56 pregnant women were included in the study with laboratory evidence of a confirmed or possible recent Zika virus infection. Based on the Centers for Disease Control and Prevention definition for microcephaly after congenital Zika virus exposure, microcephaly was diagnosed in 5% (3/56) by both the 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project and World Health Organization Fetal Growth Chart standards (head circumference Z-score ≤-2 or ≤2.3%). Using 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project, intrauterine fetal growth restriction was diagnosed in 18% of pregnancies (10/56; abdominal circumference Z-score ≤-1.3, <10%). Analysis of fetal size using the last ultrasound scan for all subjects revealed a significantly abnormal skewing of fetal biometrics with a smaller abdominal circumference vs femur length by either 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project or World Health Organization Fetal Growth Chart (P < .001 for both). A difference in distribution of fetal abdominal circumference compared to femur length was first apparent in the 24-27 6/7 week strata (2014 International Fetal and Newborn Growth Consortium for the 21st Century Project, P = .002; World Health Organization Fetal Growth Chart, P = .001). A significantly smaller head circumference compared to femur length was also observed by 2014 International Fetal and Newborn Growth Consortium for the 21st Century Project as early as the 28-33 6/7 week strata (2014 International Fetal and Newborn Growth Consortium for the 21st Century Project, P = .007). Overall, a femur-sparing pattern of growth restriction was detected in 52% of pregnancies with either head circumference:femur length or abdominal circumference:femur length fetal body ratio <10th percentile (2014 International Fetal and Newborn Growth Consortium for the 21st Century Project Z-score ≤-1.3). CONCLUSION: An unusual femur-sparing pattern of fetal growth restriction was detected in the majority of fetuses with congenital Zika virus exposure. Fetal body ratios may represent a more sensitive ultrasound biomarker to detect viral injury in nonmicrocephalic fetuses that could impart long-term risk for complications of congenital Zika virus infection.

Evidence of cardiac involvement in the fetal inflammatory response syndrome: disruption of gene networks programming cardiac development in nonhuman primates.

BACKGROUND: Most early preterm births are associated with intraamniotic infection and inflammation, which can lead to systemic inflammation in the fetus. The fetal inflammatory response syndrome describes elevations in the fetal interleukin-6 level, which is a marker for inflammation and fetal organ injury. An understanding of the effects of inflammation on fetal cardiac development may lead to insight into the fetal origins of adult cardiovascular disease. OBJECTIVE: The purpose of this study was to determine whether the fetal inflammatory response syndrome is associated with disruptions in gene networks that program fetal cardiac development. STUDY DESIGN: We obtained fetal cardiac tissue after necropsy from a well-described pregnant nonhuman primate model (pigtail macaque, Macaca nemestrina) of intrauterine infection (n=5) and controls (n=5). Cases with the fetal inflammatory response syndrome (fetal plasma interleukin-6 >11 pg/mL) were induced by either choriodecidual inoculation of a hypervirulent group B streptococcus strain (n=4) or intraamniotic inoculation of Escherichia coli (n=1). RNA and protein were extracted from fetal hearts and profiled by microarray and Luminex (Millipore, Billerica, MA) for cytokine analysis, respectively. Results were validated by quantitative reverse transcriptase polymerase chain reaction. Statistical and bioinformatics analyses included single gene analysis, gene set analysis, Ingenuity Pathway Analysis (Qiagen, Valencia, CA), and Wilcoxon rank sum. RESULTS: Severe fetal inflammation developed in the context of intraamniotic infection and a disseminated bacterial infection in the fetus. Interleukin-6 and -8 in fetal cardiac tissues were elevated significantly in fetal inflammatory response syndrome cases vs controls (P<.05). A total of 609 probe sets were expressed differentially (>1.5-fold change, P<.05) in the fetal heart (analysis of variance). Altered expression of select genes was validated by quantitative reverse transcriptase polymerase chain reaction that included several with known functions in cardiac injury, morphogenesis, angiogenesis, and tissue remodeling (eg, angiotensin I converting enzyme 2, STEAP family member 4, natriuretic peptide A, and secreted frizzled-related protein 4; all P<.05). Multiple gene sets and pathways that are involved in cardiac morphogenesis and vasculogenesis were downregulated significantly by gene set and Ingenuity Pathway Analysis (hallmark transforming growth factor beta signaling, cellular morphogenesis during differentiation, morphology of cardiovascular system; all P<.05). CONCLUSION: Disruption of gene networks for cardiac morphogenesis and vasculogenesis occurred in the preterm fetal heart of nonhuman primates with preterm labor, intraamniotic infection, and severe fetal inflammation. Inflammatory injury to the fetal heart in utero may contribute to the development of heart disease later in life. Development of preterm labor therapeutics must also target fetal inflammation to lessen organ injury and potential long-term effects on cardiac function.

Group B streptococcal infection of the choriodecidua induces dysfunction of the cytokeratin network in amniotic epithelium: a pathway to membrane weakening.

Early events leading to intrauterine infection remain poorly defined, but may hold the key to preventing preterm delivery. To determine molecular pathways within fetal membranes (chorioamnion) associated with early choriodecidual infection that may progress to preterm premature rupture of membranes (PPROM), we examined the effects of a Group B Streptococcus (GBS) choriodecidual infection on chorioamnion in a nonhuman primate model. Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118-125 days gestation (term = 172 days) received choriodecidual inoculation of either GBS (n = 5) or saline (n = 5). Cesarean section was performed in the first week after GBS or saline inoculation. RNA extracted from chorioamnion (inoculation site) was profiled by microarray. Single gene, Gene Set, and Ingenuity Pathway Analysis results were validated using qRT-PCR (chorioamnion), Luminex (amniotic fluid, AF), immunohistochemistry, and transmission electron microscopy (TEM). Despite uterine quiescence in most cases, significant elevations of AF cytokines (TNF-α, IL-8, IL-1ß, IL-6) were detected in GBS versus controls (p<0.05). Choriodecidual infection resolved by the time of cesarean section in 3 of 5 cases and GBS was undetectable by culture and PCR in the AF. A total of 331 genes were differentially expressed (>2-fold change, p<0.05). Remarkably, GBS exposure was associated with significantly downregulated expression of multiple cytokeratin (CK) and other cytoskeletal genes critical for maintenance of tissue tensile strength. Immunofluorescence revealed highly significant changes in the CK network within amniocytes with dense CK aggregates and retraction from the cell periphery (all p = 0.006). In human pregnancies affected by PPROM, there was further evidence of CK network retraction with significantly shorter amniocyte foot processes (p = 0.002). These results suggest early choriodecidual infection results in decreased cellular membrane integrity and tensile strength via dysfunction of CK networks. Downregulation of CK expression and perturbations in the amniotic epithelial cell intermediate filament network occur after GBS choriodecidual infection, which may contribute to PPROM.

The sensor histidine kinase RgfC affects group B streptococcal virulence factor expression independent of its response regulator RgfA.

Group B streptococci (GBS; Streptococcus agalactiae) are beta-hemolytic, Gram-positive bacteria that are common asymptomatic colonizers of healthy adults. However, these opportunistic bacteria also cause invasive infections in human newborns and in certain adult populations. To adapt to the various environments encountered during its disease cycle, GBS encodes a number of two-component signaling systems. Previous studies have indicated that the TCS comprising the sensor histidine kinase RgfC and the response regulator RgfA mediate GBS binding to extracellular matrix components, such as fibrinogen. However, in certain GBS clinical isolates, a point mutation in rgfA results in premature truncation of the response regulator. The truncated RgfA protein lacks the C-terminal DNA binding domain necessary for promoter binding and gene regulation. Here, we show that deletion of rgfC in GBS strains lacking a functional RgfA increased systemic infection. Furthermore, infection with the rgfC mutant increased induction of proinflammatory signaling pathways in vivo. Phosphoproteomic analysis revealed that 19 phosphopeptides corresponding to 12 proteins were differentially phosphorylated at aspartate, cysteine, serine, threonine, or tyrosine residues in the rgfC mutant. This included aspartate phosphorylation of a tyrosine kinase, CpsD, and a transcriptional regulator. Consistent with this observation, microarray analysis of the rgfC mutant indicated that >200 genes showed altered expression compared to the isogenic wild-type strain and included transcriptional regulators, transporters, and genes previously associated with GBS pathogenesis. Our observations suggest that in the absence of RgfA, nonspecific RgfC signaling affects the expression of virulence factors and GBS pathogenesis.

Choriodecidual Group B Streptococcal Infection Induces miR-155-5p in the Fetal Lung in Macaca nemestrina.

The mechanisms underlying fetal lung injury remain poorly defined. MicroRNAs (miRNAs) are small noncoding, endogenous RNAs that regulate gene expression and have been implicated in the pathogenesis of lung disease. Using a nonhuman primate model of choriodecidual infection, we sought to determine if differentially expressed miRNAs were associated with acute fetal lung injury. After inoculating 10 chronically catheterized pregnant monkeys (Macaca nemestrina) with either group B streptococcus (GBS) at 1 × 10(6) CFU (n = 5) or saline (n = 5) in the choriodecidual space, we extracted fetal lung mRNA and miRNA and profiled the changes in expression by microarray analysis. We identified 9 differentially expressed miRNAs in GBS-exposed fetal lungs, but of these, only miR-155-5p was validated by quantitative reverse transcription-PCR (P = 0.02). Significantly elevated miR-155-5p expression was also observed when immortalized human fetal airway epithelial (FeAE) cells were exposed to proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]). Overexpression of miR-155-5p in FeAE cells in turn increased the production of IL-6 and CXCL10/gamma interferon-induced protein 10, which are implicated in leukocyte recruitment but also in protection from lung injury. Interestingly, while miR-155-5p decreased fibroblast growth factor 9 (FGF9) expression in a luciferase reporter assay, FGF9 levels were actually increased in GBS-exposed fetal lungs in vivo. FGF9 overexpression is associated with abnormal lung development. Thus, upregulation of miR-155-5p may serve as a compensatory mechanism to lessen the increase in FGF9 and prevent aberrant lung development. Understanding the complicated networks regulating lung development in the setting of infection is a key step in identifying how to prevent fetal lung injury leading to bronchopulmonary dysplasia.

Uterine overdistention induces preterm labor mediated by inflammation: observations in pregnant women and nonhuman primates.

OBJECTIVE: Uterine overdistention is thought to induce preterm labor in women with twin and multiple pregnancies, but the pathophysiology remains unclear. We investigated for the first time the pathogenesis of preterm birth associated with rapid uterine distention in a pregnant nonhuman primate model. STUDY DESIGN: A nonhuman primate model of uterine overdistention was created using preterm chronically catheterized pregnant pigtail macaques (Macaca nemestrina) by inflation of intraamniotic balloons (N = 6), which were compared to saline controls (N = 5). Cesarean delivery was performed due to preterm labor or at experimental end. Microarray, quantitative reverse transcriptase polymerase chain reaction, Luminex (Austin, TX), and enzyme-linked immunosorbent assay were used to measure messenger RNA (mRNA) and/or protein levels from monkey (amniotic fluid, myometrium, maternal plasma) and human (amniocytes, amnion, myometrium) tissues. Statistical analysis employed analysis of covariance and Wilcoxon rank sum. Biomechanical forces were calculated using the law of Laplace. RESULTS: Preterm labor occurred in 3 of 6 animals after balloon inflation and correlated with greater balloon volume and uterine wall stress. Significant elevations of inflammatory cytokines and prostaglandins occurred following uterine overdistention in an "inflammatory pulse" that correlated with preterm labor (interleukin [IL]-1ß, tumor necrosis factor [TNF]-α, IL-6, IL-8, CCL2, prostaglandin E2, prostaglandin F2α, all P < .05). A similar inflammatory response was observed in amniocytes in vitro following mechanical stretch (IL1ß, IL6, and IL8 mRNA multiple time points, P < .05), in amnion of women with polyhydramnios (IL6 and TNF mRNA, P < .05) and in amnion (TNF-α) and myometrium of women with twins in early labor (IL6, IL8, CCL2, all P < .05). Genes differentially expressed in the nonhuman primate after balloon inflation and in women with polyhydramnios and twins are involved in tissue remodeling and muscle growth. CONCLUSION: Uterine overdistention by inflation of an intraamniotic balloon is associated with an inflammatory pulse that precedes and correlates with preterm labor. Our results indicate that inflammation is an early event after a mechanical stress on the uterus and leads to preterm labor when the stress is sufficiently great. Further, we find evidence of uterine tissue remodeling and muscle growth as a common, perhaps compensatory, response to uterine distension.

Comparative effect of lurasidone and blonanserin on cortical glutamate, dopamine, and acetylcholine efflux: role of relative serotonin (5-HT)2A and DA D2 antagonism and 5-HT1A partial agonism.

Atypical antipsychotic drugs (AAPDs) have been suggested to be more effective in improving cognitive impairment in schizophrenia than typical APDs, a conclusion supported by differences in receptor affinities and neurotransmitter efflux in the cortex and the hippocampus. More potent serotonin (5-HT)2A than dopamine (DA) D2 receptors antagonism, and direct or indirect 5-HT1A agonism, characterize almost all AAPDs. Blonanserin, an AAPD, has slightly greater affinity for D2 than 5-HT2A receptors. Using microdialysis and ultra performance liquid chromatography-mass spectrometry/mass spectrometry, we compared the abilities of the typical APD, haloperidol, three AAPDs, blonanserin, lurasidone, and olanzapine, and a selective 5-HT1A partial agonist, tandospirone, and all, except haloperidol, were found to ameliorate the cognitive deficits produced by the N-methyl-d-aspartate antagonist, phencyclidine, altering the efflux of neurotransmitters and metabolites in the rat cortex and nucleus accumbens. Blonanserin, lurasidone, olanzapine, and tandospirone, but not haloperidol, increased the efflux of cortical DA and its metabolites, homovanillic acid and 3,4-dihydroxyphenylacetic acid. Olanzapine and lurasidone increased the efflux of acetylcholine; lurasidone increased glutamate as well. None of the compounds significantly altered the efflux of 5-HT or its metabolite, 5-hydroxyindole acetic acid, or GABA, serine, and glycine. The ability to increase cortical DA efflux was the only shared effect of the compounds which ameliorates the deficit in cognition in rodents following phencyclidine.

Group B Streptococcus CovR regulation modulates host immune signalling pathways to promote vaginal colonization.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a frequent commensal organism of the vaginal tract of healthy women. However, GBS can transition to a pathogen in susceptible hosts, but host and microbial factors that contribute to this conversion are not well understood. GBS CovR/S (CsrR/S) is a two component regulatory system that regulates key virulence elements including adherence and toxin production. We performed global transcription profiling of human vaginal epithelial cells exposed to WT, CovR deficient, and toxin deficient strains, and observed that insufficient regulation by CovR and subsequent increased toxin production results in a drastic increase in host inflammatory responses, particularly in cytokine signalling pathways promoted by IL-8 and CXCL2. Additionally, we observed that CovR regulation impacts epithelial cell attachment and intracellular invasion. In our mouse model of GBS vaginal colonization, we further demonstrated that CovR regulation promotes vaginal persistence, as infection with a CovR deficient strainresulted in a heightened host immune response as measured by cytokine production and neutrophil activation. Using CXCr2 KO mice, we determined that this immune alteration occurs, at least in part, via signalling through the CXCL2 receptor. Taken together, we conclude that CovR is an important regulator of GBS vaginal colonization and loss of this regulatory function may contribute to the inflammatory havoc seen during the course of infection.