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1.  Moderate tidal volumes and oxygen exposure during initiation of ventilation in preterm fetal sheep 
Pediatric research  2012;72(6):593-599.
Background
Preterm infants often receive mechanical ventilation and oxygen at birth. Exposure to large tidal volumes (VT) at birth causes lung inflammation and oxygen may amplify the injury. We hypothesized that normal VT ventilation at birth causes lung injury that is exacerbated by 95% oxygen.
Methods
The head and chest of anesthetized preterm fetal sheep (129±1d gestation) were surgically exteriorized while maintaining the placental circulation. Fetuses were randomized to four groups with either: 1) VT ventilation to 6 mL/kg or 2) CPAP of 5 cm H2O, and either: a) 95%O2/5%CO2 or b) 95%N2/5%CO2. Age-matched fetuses were controls. After a 15-minute intervention, the fetal lamb was returned to the uterus for 1 h 45 min.
Results
In ventilated lambs, VT was 6.2±0.4 mL/kg at 15 min. Ventilation increased pro-inflammatory cytokines compared to control and CPAP only lambs, with recruitment of primarily monocytes to bronchioalveolar lavage fluid. Early response protein 1 was activated around the bronchioles in VT ventilated animals. The 15-min oxygen exposure did not change inflammatory mediators or other markers of lung and oxidative stress.
Conclusions
A VT of 6–7 mL/kg at birth increased early markers of injury and lung inflammation. Brief exposure to 95% oxygen did not alter lung inflammation.
doi:10.1038/pr.2012.135
PMCID: PMC4073615  PMID: 23037872
2.  Modulation of lipopolysaccharide-induced chorioamnionitis by Ureaplasma parvum in sheep 
Objective
Ureaplasma colonization in the setting of polymicrobial flora is common in women with chorioamnionitis, and is a risk factor for preterm delivery and neonatal morbidity. We hypothesized that ureaplasma colonization of amniotic fluid will modulate chorioamnionitis induced by E. coli lipopolysaccharide (LPS).
Methods
Sheep received intra-amniotic (IA) injections of media (control) or live ureaplasma either 7 or 70d before delivery. Another group received IA LPS 2d before delivery. To test for interactions, U. parvum exposed animals were challenged with IA LPS, and delivered 2d later. All animals were delivered preterm at 125±1 day gestation.
Results
Both IA ureaplasmas and LPS induced leukocyte infiltration of chorioamnion. LPS greatly increased the expression of pro-inflammatory cytokines and myeloperoxidase in leukocytes, while ureaplasmas alone caused modest responses. Interestingly, 7d but not 70d ureaplasma exposure significantly downregulated LPS induced pro-inflammatory cytokines and myeloperoxidase expression in the chorioamnion.
Conclusion
Acute U. parvum exposure (7d) can suppress LPS induced chorioamnionitis.
doi:10.1016/j.ajog.2013.02.018
PMCID: PMC3637868  PMID: 23410690
Endotoxin tolerance; Preterm labor; Innate immunity; Fetal adaptation
3.  Ventilation-Induced Increases in EGFR Ligand mRNA Are Not Altered by Intra-Amniotic LPS or Ureaplasma in Preterm Lambs 
PLoS ONE  2014;9(4):e96087.
Chorioamnionitis and mechanical ventilation are associated with bronchopulmonary dysplasia (BPD) in preterm infants. Mechanical ventilation at birth activates both inflammatory and acute phase responses. These responses can be partially modulated by previous exposure to intra-amniotic (IA) LPS or Ureaplasma parvum (UP). Epidermal growth factor receptor (EGFR) ligands participate in lung development, and angiotensin converting enzyme (ACE) 1 and ACE2 contribute to lung inflammation. We asked whether brief mechanical ventilation at birth altered EGFR and ACE pathways and if antenatal exposure to IA LPS or UP could modulate these effects. Ewes were exposed to IA injections of UP, LPS or saline multiple days prior to preterm delivery at 85% gestation. Lambs were either immediately euthanized or mechanically ventilated for 2 to 3 hr. IA UP and LPS cause modest changes in the EGFR ligands amphiregulin (AREG), epiregulin (EREG), heparin binding epidermal growth factor (HB-EGF), and betacellulin (BTC) mRNA expression. Mechanical ventilation greatly increased mRNA expression of AREG, EREG, and HB-EGF, with no additional increases resulting from IA LPS or UP. With ventilation AREG and EREG mRNA localized to cells in terminal airspace. EGFR mRNA also increased with mechanical ventilation. IA UP and LPS decreased ACE1 mRNA and increased ACE2 mRNA, resulting in a 4 fold change in the ACE1/ACE2 ratio. Mechanical ventilation with large tidal volumes increased both ACE1 and ACE2 expression. The alterations seen in ACE with IA exposures and EGFR pathways with mechanical ventilation may contribute to the development of BPD in preterm infants.
doi:10.1371/journal.pone.0096087
PMCID: PMC4005755  PMID: 24788984
4.  Ureaplasma and BPD 
Seminars in perinatology  2013;37(2):94-101.
Ureaplasma is an organism with low virulence and is a commensal of the lower genito-urinary tract in females. From here, it can gain entry in the amniotic fluid to cause inflammation in the amniotic compartment during pregnancy. Ureaplasma spp. are the most common organisms isolated from women with chorioamnionitis. Ureaplasma spp. are associated with increased risk for preterm labor and morbidity in the preterm neonate. However, there is some controversy regarding the importance of Ureaplasma in the pathogenesis of bronchopulmonary dysplasia (BPD). This article will review the microbiology of Ureaplasma, host innate immune responses, and the pathology of lung injury in animal models of Ureaplasma chorioamnionitis. We will review epidemiological studies of Ureaplasma and BPD in preterm infants and efficacy of antibiotics in preventing preterm labor and BPD.
doi:10.1053/j.semperi.2013.01.005
PMCID: PMC3628630  PMID: 23582963
5.  The MANDATE model for evaluating interventions to reduce postpartum hemorrhage 
Objective
To create a comprehensive model of the comparative impact of various interventions on maternal, fetal, and neonatal (MFN) mortality.
Methods
The major conditions and sub-conditions contributing to MFN mortality in low-resource areas were identified, and the prevalence and case fatality rates documented. Available interventions were mapped to these conditions, and intervention coverage and efficacy were identified. Finally, a computer model developed by the Maternal and Neonatal Directed Assessment of Technology (MANDATE) initiative estimated the potential of current and new interventions to reduce mortality.
Results
For PPH, the sub-causes, prevalence, and MFN case fatality rates were calculated. Available interventions were mapped to these sub-causes. Most available interventions did not prevent or treat the overall condition of PPH, but rather sub-conditions associated with hemorrhage and thus prevented only a fraction of the associated deaths.
Conclusion
The majority of current interventions address sub-conditions that cause death, rather than the overall condition; thus, the potential number of lives saved is likely to be overestimated. Additionally, the location at which mother and infant receive care affects intervention effectiveness and, therefore, the potential to save lives. A comprehensive view of MFN conditions is needed to understand the impact of any potential intervention.
doi:10.1016/j.ijgo.2012.10.030
PMCID: PMC3628756  PMID: 23313144
Low-income countries; Maternal mortality; Model; Postpartum hemorrhage; Stillbirth
6.  A conference report on prenatal corticosteroid use in low- and middle-income countries 
Objective
To evaluate the evidence for prenatal corticosteroid use in low- and middle-income countries and to make recommendations regarding implementation and further research.
Methods
Studies and meta-analyses on prenatal corticosteroids relevant to low- and middle-income countries were identified and reviewed at the Maternal and Child Health Integrated Project (MCHIP) Antenatal Corticosteroid Conference held in Washington on October 19, 2010.
Results
There is strong evidence regarding the effectiveness of prenatal corticosteroid use in hospitals in high- and middle-income countries, usually in settings with high-level newborn care. For births occurring in hospitals in low-income countries without high-level neonatal care or for births outside hospitals, no studies have been conducted to evaluate prenatal corticosteroid use. The efficacy and safety of prenatal corticosteroid use in these settings must be evaluated.
Conclusions
The conference working group recommended expanding the use of prenatal corticosteroids in hospitals with high-level newborn care in low-income countries. For other low-income country settings, further research regarding efficacy and safety should precede the widespread introduction of prenatal corticosteroids.
doi:10.1016/j.ijgo.2011.07.017
PMCID: PMC3910250  PMID: 21930269
Low-resource countries; Prenatal corticosteroids; Preterm birth
7.  Effects of Intra-Amniotic Lipopolysaccharide and Maternal Betamethasone on Brain Inflammation in Fetal Sheep 
PLoS ONE  2013;8(12):e81644.
Rationale
Chorioamnionitis and antenatal glucocorticoids are common exposures for preterm infants and can affect the fetal brain, contributing to cognitive and motor deficits in preterm infants. The effects of antenatal glucocorticoids on the brain in the setting of chorioamnionitis are unknown. We hypothesized that antenatal glucocorticoids would modulate inflammation in the brain and prevent hippocampal and white matter injury after intra-amniotic lipopolysaccharide (LPS) exposure.
Methods
Time-mated ewes received saline (control), an intra-amniotic injection of 10 mg LPS at 106d GA or 113d GA, maternal intra-muscular betamethasone (0.5 mg/kg maternal weight) alone at 113d GA, betamethasone at 106d GA before LPS or betamethasone at 113d GA after LPS. Animals were delivered at 120d GA (term=150d). Brain structure volumes were measured on T2-weighted MRI images. The subcortical white matter (SCWM), periventricular white matter (PVWM) and hippocampus were analyzed for microglia, astrocytes, apoptosis, proliferation, myelin and pre-synaptic vesicles.
Results
LPS and/or betamethasone exposure at different time-points during gestation did not alter brain structure volumes on MRI. Betamethasone alone did not alter any of the measurements. Intra-amniotic LPS at 106d or 113d GA induced inflammation as indicated by increased microglial and astrocyte recruitment which was paralleled by increased apoptosis and hypomyelination in the SCWM and decreased synaptophysin density in the hippocampus. Betamethasone before the LPS exposure at 113d GA prevented microglial activation and the decrease in synaptophysin. Betamethasone after LPS exposure increased microglial infiltration and apoptosis.
Conclusion
Intra-uterine LPS exposure for 7d or 14d before delivery induced inflammation and injury in the fetal white matter and hippocampus. Antenatal glucocorticoids aggravated the inflammatory changes in the brain caused by pre-existing intra-amniotic inflammation. Antenatal glucocorticoids prior to LPS reduced the effects of intra-uterine inflammation on the brain. The timing of glucocorticoid administration in the setting of chorioamnionitis can alter outcomes for the fetal brain.
doi:10.1371/journal.pone.0081644
PMCID: PMC3866104  PMID: 24358119
8.  The New Bronchopulmonary Dysplasia 
Current Opinion in Pediatrics  2011;23(2):167-172.
Purpose of Review
BPD remains the most common severe complication of preterm birth. A number of recent animal models and clinical studies provide new information about pathophysiology and treatment.
Recent Findings
The epidemiology of BPD continues to demonstrate that birth weight or gestational age are most predictive of BPD. Correlations of BPD with chorioamnionitis are clouded by the complexity of the fetal exposures to inflammation. Excessive oxygen use in preterm infants can increase the risk of BPD, but low saturation targets may increase death. Numerous recent trials demonstrate that many preterm infants can be initially stabilized after delivery with CPAP and then be selectively treated with surfactant for RDS. The growth of the lungs of the infant with BPD through childhood remains poorly characterized.
Summary
Recent experiences in neonatology suggest that combining less invasive care strategies that avoid excessive oxygen and ventilation, decrease postnatal infections, and optimize nutrition may decrease the incidence and severity of BPD.
doi:10.1097/MOP.0b013e3283423e6b
PMCID: PMC3265791  PMID: 21169836
Ventilation; oxygen; prematurity; lung injury
9.  Lung Recruitment for Ventilation - Does it Work and Is it Safe? 
The Journal of pediatrics  2009;154(5):635-636.
doi:10.1016/j.jpeds.2009.01.059
PMCID: PMC2716052  PMID: 19364555
10.  Postnatal Corticosteroids for BPD 
Clinics in perinatology  2009;36(1):177-188.
Synopsis
Corticosteroids are used to improve lung function in infants who are progressing toward BPD. Corticosteroids facilitate extubation, but there is conflicting information about adverse effects on the developing brain. An approach to minimizing risk is to use low dose, short duration treatments in the highest risk ventilator dependent patients. Questions remain about which corticosteroid is the safest and how to dose that corticosteroid.
doi:10.1016/j.clp.2008.09.016
PMCID: PMC2663397  PMID: 19161874
Lung Injury; Alveoli; Premature; Neurodevelopment; Mechanical Ventilation
11.  Art and Science, Clinics in Perinatology 
Clinics in perinatology  2011;38(3):529-545.
doi:10.1016/j.clp.2011.06.013
PMCID: PMC3711408  PMID: 21890023
Prematurity; Respiratory distress syndrome; corticosteroids; neurodevelopmental outcomes
12.  Interleukin-1 in Lipopolysaccharide Induced Chorioamnionitis in the Fetal Sheep 
Reproductive Sciences  2011;18(11):1092-1102.
We tested the hypothesis that interleukin 1 (IL-1) mediates intra-amniotic lipopolysaccharide (LPS)-induced chorioamnionitis in preterm fetal sheep. Time-mated Merino ewes with singleton fetuses received IL-1α, LPS, or saline (control) by intra-amniotic injection 1 to 2 days before operative delivery at 124 ± 1 days gestational age (N = 5-9/group; term = 150 days). Recombinant human IL-1 receptor antagonist (rhIL-1ra) was given into the amniotic fluid 3 hours before intra-amniotic LPS or saline to block IL-1 signaling. Inflammation in the chorioamnion was determined by histology, cytokine messenger RNA (mRNA), protein expression, and by quantitation of activated inflammatory cells. Intra-amniotic IL-1 and LPS both induced chorioamnionitis. However, IL-1 blockade with IL-1ra did not decrease intra-amniotic LPS-induced increases in pro-inflammatory cytokine mRNAs, numbers of inflammatory cells, myeloperoxidase, or monocyte chemotactic protein-1-expressing cells in the chorioamnion. We conclude that IL-1 and LPS both can cause chorioamnionitis, but IL-1 is not an important mediator of LPS-induced chorioamnionitis in fetal sheep.
doi:10.1177/1933719111404609
PMCID: PMC3343143  PMID: 21493953
fetal inflammatory response syndrome; prematurity; innate immunity; IL-1 receptor antagonist
13.  Inflammation of the Fetal Ovine Skin Following in utero Exposure to Ureaplasma parvum 
Reproductive Sciences  2011;18(11):1128-1137.
There is increasing evidence linking in utero infection and inflammation to preterm birth. Many commensal urogenital tract microorganisms, including the Mycoplasmas and Ureaplasmas, are commonly detected in association with preterm birth. Using an ovine model of sterile fetal inflammation, we demonstrated previously that the fetal skin generates a robust inflammatory response following in utero exposure to lipopolysaccharides from Escherichia coli. The fetal skin’s response to colonization of the amniotic fluid by viable microorganisms remains unstudied. We hypothesised that in utero infection with Ureaplasma parvum serovar 3 would induce a proinflammatory response in the fetal skin. We found that (1) cultured fetal keratinocytes (the primary cellular constituent of the epidermis) respond to U. parvum exposure in vitro by increasing the expression of the chemotactant monocyte chemoattractant protein 1 (MCP-1) but not interleukin 1β (IL-1β), IL-6, IL-8, or tumor necrosis factor-α (TNF-α); (2) the fetal skin’s response to 7 days of U. parvum exposure is characterized by elevated expression of MCP-1, TNF-α, and IL-10; and (3) the magnitude of inflammatory cytokine/chemokine expression in the fetal skin is dependent on the duration of U parvum exposure. These novel findings provide further support for the role of the fetal skin in the development of fetal inflammation and the preterm birth that may follow.
doi:10.1177/1933719111408114
PMCID: PMC3343146  PMID: 22031190
preterm birth; uterine infection; inflammation
14.  Antenatal Inflammation Reduces Expression of Caveolin-1 and Influences Multiple Signaling Pathways in Preterm Fetal Lungs 
Bronchopulmonary dysplasia (BPD), associated with chorioamnionitis, results from the simultaneous effects of disrupted lung development, lung injury, and repair superimposed on the developing lung. Caveolins (Cavs) are implicated as major modulators of lung injury and remodeling by multiple signaling pathways, although Cavs have been minimally studied in the injured developing lung. We hypothesized that chorioamnionitis-associated antenatal lung inflammation would decrease the expression of Cav-1 in preterm fetal lungs. We tested whether changes occurred in the transcription factors Smad2/3, Smad1/5, Stat3, and Stat1, and we also studied the activation of acid-sphingomyelinase (a-SMase) with the generation of ceramide, along with changes in the expression of heme oxygenase–1 (HO-1) as indicators of possible Cav-1–mediated effects. Fetal sheep were exposed to 10 mg of intra-amniotic endotoxin or saline for 2, 7, or 2 + 7 days before preterm delivery at 124 days of gestation. The expression of Cav-1 and HO-1 and the phosphorylation of Smad and Stat were evaluated by real-time PCR, Western blotting, and/or immunohistochemistry. The activity of a-SMase and the concentrations of ceramide were measured. Intra-amniotic endotoxin decreased Cav-1 mRNA and protein expression in the lungs, with a maximum reduction of Cav-1 mRNA to 50% ± 7% of the control value (P < 0.05), and of Cav-1 protein expression to 20% ± 5% of the control value (P < 0.05). Decreased concentrations of Cav-1 were associated with the elevated phosphorylation of Smad2/3, Stat3, and Stat1, but not of Smad1/5. The expression of HO-1, a-SMase activity, and ceramide increased. Antenatal inflammation decreased the expression of Cav-1 in the preterm fetal lung. The decreased expression of Cav-1 was associated with the activation of the Smad2/3, Stat, and a-SMase/ceramide pathways, and with the increased expression of HO-1. The decreased concentrations of Cav-1 and changes in other signaling pathways may contribute to BPD.
doi:10.1165/rcmb.2010-0519OC
PMCID: PMC3361364  PMID: 21562314
bronchopulmonary dysplasia; TGF-β; a-SMase; ceramide; chorioamnionitis
15.  Chronic fetal exposure to Ureaplasma parvum suppresses innate immune responses in sheep 
The chorioamnionitis associated with preterm delivery is often polymicrobial with ureaplasma being the most common isolate. To evaluate interactions between the different pro-inflammatory mediators, we hypothesized that ureaplasma exposure would increase fetal responsiveness to LPS. Fetal sheep were given intra-amniotic injections of media (control) or Ureaplasma parvum serovar 3 either 7d or 70d before preterm delivery. Another group received an intraamniotic injection of E.coli lipo-polysaccharide (LPS) 2d prior to delivery. To test for interactions, intraamniotic U. parvum exposed animals were challenged with intraamniotic LPS and delivered 2d later. All animals were delivered at 124±1d gestation (Term=150d). Compared to the 2d LPS exposure group, the U. parvum 70d+LPS group had: 1) decreased lung pro and anti-inflammatory cytokine expression 2) fewer CD3+ T-lymphocytes, CCL2+, myeloperoxidase+, and PU.1+ cells in the lung. Interestingly, exposure to U. parvum for 7d did not change responses to a subsequent intraamniotic LPS challenge, and exposure to intraamniotic U. parvum alone induced mild lung inflammation. Exposure to U. parvum increased pulmonary TGFβ1 expression but did not change mRNA expression of either the receptor TLR4 or some of the downstream mediators in the lung. Monocytes from fetal blood and lung isolated from U. parvum 70d+LPS but not U. parvum 7d+LPS animals had decreased in vitro responsiveness to LPS. These results are consistent with the novel finding of down-regulation of LPS responses by chronic but not acute fetal exposures to U. parvum. The findings increase our understanding of how chorioamnionitis exposed preterm infants may respond to lung injury and postnatal nosocomial infections.
doi:10.4049/jimmunol.1100779
PMCID: PMC3159703  PMID: 21784974
Prematurity; Chorioamnionitis; Fetal inflammatory response syndrome; endotoxin tolerance; lung inflammation
16.  The Role of the Multiple Banded Antigen of Ureaplasma parvum in Intra-Amniotic Infection: Major Virulence Factor or Decoy? 
PLoS ONE  2012;7(1):e29856.
The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigenic variation of the MBA is a potential mechanism by which ureaplasmas avoid immune recognition and cause chronic infections of the upper genital tract of pregnant women. We tested whether the MBA is involved in the pathogenesis of intra-amniotic infection and chorioamnionitis by injecting virulent or avirulent-derived ureaplasma clones (expressing single MBA variants) into the amniotic fluid of pregnant sheep. At 55 days of gestation pregnant ewes (n = 20) received intra-amniotic injections of virulent-derived or avirulent-derived U. parvum serovar 6 strains (2×104 CFU), or 10B medium (n = 5). Amniotic fluid was collected every two weeks post-infection and fetal tissues were collected at the time of surgical delivery of the fetus (140 days of gestation). Whilst chronic colonisation was established in the amniotic fluid of animals infected with avirulent-derived and virulent-derived ureaplasmas, the severity of chorioamnionitis and fetal inflammation was not different between these groups (p>0.05). MBA size variants (32–170 kDa) were generated in vivo in amniotic fluid samples from both the avirulent and virulent groups, whereas in vitro antibody selection experiments led to the emergence of MBA-negative escape variants in both strains. Anti-ureaplasma IgG antibodies were detected in the maternal serum of animals from the avirulent (40%) and virulent (55%) groups, and these antibodies correlated with increased IL-1β, IL-6 and IL-8 expression in chorioamnion tissue (p<0.05). We demonstrate that ureaplasmas are capable of MBA phase variation in vitro; however, ureaplasmas undergo MBA size variation in vivo, to potentially prevent eradication by the immune response. Size variation of the MBA did not correlate with the severity of chorioamnionitis. Nonetheless, the correlation between a maternal humoral response and the expression of chorioamnion cytokines is a novel finding. This host response may be important in the pathogenesis of inflammation-mediated adverse pregnancy outcomes.
doi:10.1371/journal.pone.0029856
PMCID: PMC3257234  PMID: 22253806
17.  Exposure to In Utero Lipopolysaccharide Induces Inflammation in the Fetal Ovine Skin 
Reproductive Sciences  2011;18(1):88-98.
Inflammation is a defensive process by which the body responds to both localized and systemic tissue damage by the induction of innate and adaptive immunity. Literature from human and animal studies links inappropriate in utero inflammation to preterm parturition and fetal injury. The pathways by which such inflammation may cause labor, however, are not fully understood. Any proinflammatory agonist in the amniotic fluid will contact the fetal skin, in its entirety, but a potential role of the fetal skin in the pathways to labor have not previously been explored. We hypothesized that the fetal skin would respond robustly to the presence of intra-amniotic lipopolysaccharide (LPS) in our ovine model of in utero inflammation. In vitro and in utero exposure of fetal ovine keratinocytes or fetal skin to Escherichia coli LPS reliably induced significant increases in interleukin 1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), and IL-8 expression. We demonstrate that, in utero, this expression requires direct exposure with LPS suggesting that the inflammation is triggered directly in the skin itself, rather than as a secondary response to a systemic stimuli and that inflammation involves Toll-like receptor (TLR) regulation and neutrophil chemotaxis in concordance with an acute inflammatory reaction. We show that this response involves multiple inflammatory mediators, TLR regulation, and localized inflammatory cell influx characteristic of an acute inflammatory reaction. These novel data strongly suggests that the fetal skin acts as an important mediator of the fetal inflammatory response and as such may contribute to preterm birth.
doi:10.1177/1933719110380470
PMCID: PMC3343071  PMID: 20923949
skin; inflammation; preterm birth; uterine infection
18.  Pulmonary and Systemic Expression of Monocyte Chemotactic Proteins in Preterm Sheep Fetuses Exposed to LPS Induced Chorioamnionitis 
Pediatric research  2010;68(3):210-215.
Monocyte chemoattractant proteins (MCP-1 and MCP-2) mediate monocyte and T-lymphocyte chemotaxis, and IL-1 contributes to the pathogenesis of chorioamnionitis-induced lung inflammation and fetal inflammatory responses. We tested the hypothesis that IL-1 mediates the systemic and pulmonary induction of MCP-1 and MCP-2 in response to lipopolysaccharide (LPS) induced chorioamnionitis. MCP-1 mRNA, MCP-2 mRNA and MCP-1 protein expression were measured in two models: 1) intra-amniotic LPS and 2) intra-amniotic recombinant sheep IL-1α given at varying intervals prior to preterm delivery at 124d gestational age. Intra-amniotic LPS or IL-1α induced MCP-1 mRNA and protein and MCP-2 mRNA in fetal lung many fold at 1-2d. LPS induced intense MCP-1 expression in sub-epithelial mesenchymal cells and interstitial inflammatory cells in the lung. Inhibition of IL-1 signaling with recombinant human IL-1 receptor antagonist (rhIL-1ra) did not attenuate LPS induced increase in MCP-1 or MCP-2 expression. MCP-1 and MCP-2 were not induced in liver or chorioamnion, but MCP-1 increased in cord plasma. LPS or IL-1 can induce robust expression of MCP-1 or MCP-2 in the fetal lung. LPS induction of MCP-1 is not IL-1 dependent in fetal sheep. MCP-1 and MCP-2 may be significant contributors to fetal inflammation.
doi:10.1203/PDR.0b013e3181e9c556
PMCID: PMC3123719  PMID: 20703142
19.  The Severity of Chorioamnionitis in Pregnant Sheep Is Associated with In Vivo Variation of the Surface-Exposed Multiple-Banded Antigen/Gene of Ureaplasma parvum1 
Biology of Reproduction  2010;83(3):415-426.
Ureaplasma species are the bacteria most frequently isolated from human amniotic fluid in asymptomatic pregnancies and placental infections. Ureaplasma parvum serovars 3 and 6 are the most prevalent serovars isolated from men and women. We hypothesized that the effects on the fetus and chorioamnion of chronic ureaplasma infection in amniotic fluid are dependent on the serovar, dose, and variation of the ureaplasma multiple-banded antigen (MBA) and mba gene. We injected high- or low-dose U. parvum serovar 3, serovar 6, or vehicle intra-amniotically into pregnant ewes at 55 days of gestation (term = 150 days) and examined the chorioamnion, amniotic fluid, and fetal lung tissue of animals delivered by cesarean section at 125 days of gestation. Variation of the multiple banded antigen/mba generated by serovar 3 and serovar 6 ureaplasmas in vivo were compared by PCR assay and Western blot. Ureaplasma inoculums demonstrated only one (serovar 3) or two (serovar 6) MBA variants in vitro, but numerous antigenic variants were generated in vivo: serovar 6 passage 1 amniotic fluid cultures contained more MBA size variants than serovar 3 (P = 0.005), and ureaplasma titers were inversely related to the number of variants (P = 0.025). The severity of chorioamnionitis varied between animals. Low numbers of mba size variants (five or fewer) within amniotic fluid were associated with severe inflammation, whereas the chorioamnion from animals with nine or more mba variants showed little or no inflammation. These differences in chorioamnion inflammation may explain why not all women with in utero Ureaplasma spp. experience adverse pregnancy outcomes.
After intra-amniotic inoculation of pregnant sheep with Ureaplasma parvum serovar 3 and serovar 6, infections persisted in utero for 70 days, causing high titer chorioamnion infections, while some chorioamnions showed no histological evidence of infection.
doi:10.1095/biolreprod.109.083121
PMCID: PMC2924804  PMID: 20519696
chorioamnionitis; immunology; multiple-banded antigen gene; pregnancy; ureaplasma species
20.  Long term consequences of oxygen therapy in the neonatal period 
Summary
Preterm and term infants are frequently exposed to high concentrations of oxygen for prolonged periods. In experimental models, high and prolonged oxygen exposures cause delayed alveolar septation and a bronchopulmonary dysplasia phenotype. Often, however, the oxygen exposure is tolerated in that the infants recover without severe lung or systemic injury. Multiple exposures change oxygen sensitivity in adult and newborn animals. Examples are antenatal corticosteroids, inflammatory mediators or preconditioning with oxygen, which will increase tolerance to oxygen injury. Intrauterine growth restriction or postnatal nutritional deficits will increase oxygen injury. Different infants probably have quite variable sensitivities to oxygen injury, but there are no biomarkers available to predict the risk of oxygen injury.
doi:10.1016/j.siny.2010.03.007
PMCID: PMC2910185  PMID: 20452844
Bronchopulmonary dysplasia; Injury; Lung; Prematurity
21.  Body Temperature Effects on Lung Injury in Ventilated Preterm Lambs 
Resuscitation  2010;81(6):749-754.
Aims:
Mechanical ventilation causes lung injury in premature infants. Hypothermia may protect against and hyperthermia may augment lung injury. We tested the effects of hypo- and hyperthermia on ventilation induced acute lung injury in preterm lambs.
Methods:
Twin sheep fetuses at 128 d GA (term 150 d) were surgically delivered and randomized to unventilated control (UVC), normothermia (38-39 °C) without lung injury (NTNI), or to 1 of 3 injurious ventilation groups: hypothermic (33-34 °C, LT), normothermic (38-39 °C, NT) or hyperthermic (40-41 °C, HT). NT, LT and HT groups had 15 min of injurious ventilation (PEEP 0 cmH2O, VT escalation to 15 mL/kg) following delivery and prior to surfactant. The animals were then gently ventilated (PEEP 5 cmH2O, VT 7.5 mL/kg) for 2 h 45 min. NTNI lambs received surfactant at birth prior to gentle ventilation. The lambs were then euthanized, and bronchoalveolar lavage (BAL) fluid and lung tissue were used to evaluate lung injury, inflammatory cell counts, inflammatory markers and cytokine mRNA.
Results:
Target temperatures were achieved by 15 min of age and maintained for 3 h. All ventilated groups had increased BAL protein, lung inflammation and increased cytokine mRNA. HT animals developed acidosis, premature death, pneumothoraces, impaired lung function and increased inflammatory mRNA expression. LT animals remained clinically stable without pneumothoraces or death, had improved ventilatory efficiency and trended toward lower inflammatory mRNA expression than NT animals.
Conclusion:
Hyperthermia exacerbated ventilator induced lung injury, while hypothermia may protect against lung injury in the preterm lamb.
doi:10.1016/j.resuscitation.2009.12.007
PMCID: PMC2871967  PMID: 20299144
22.  Thymic changes after chorioamnionitis induced by intraamniotic LPS in fetal sheep 
Background
Treg mediates homeostasis of the immune system and differentiate under the control of the transcription factor FoxP3 in the fetal thymus.
Objective
We asked if fetal inflammation caused by chorioamnionitis would modulate thymus development.
Methods
Fetal sheep were exposed to an intraamniotic injection (IA) of 10 mg LPS 5h, 1d, 2d or 5d before delivery at 123d gestation days. Cord blood lymphocytes, plasma cortisol and thymus weight were measured. Glucocorticoid receptor-, activated caspase-3-, Ki67-, PCNA-, NF-κB- and FoxP3-positive cells were immunohistochemically evaluated in thymus.
Results
IA LPS decreased the number of circulating lymphocytes by 40% after 1d. Thymus-to-body weight ratios were reduced in all LPS groups by a maximum of 40% at 5d. LPS modestly increased plasma cortisol concentration, increased NF-κB immunostaining in fetal thymus and reduced the number of FoxP3-positive cells by 60% at 1d.
Conclusion
Intraamniotic exposure to LPS induced thymic changes and influenced thymic FoxP3 expression.
doi:10.1016/j.ajog.2010.02.035
PMCID: PMC2868266  PMID: 20452494
preterm; fetal inflammatory syndrome; immune development; T lymphoyctes; FoxP3; Treg
23.  Airway Injury from Initiating Ventilation in Preterm Sheep 
Pediatric research  2010;67(1):60-65.
Premature infants exposed to ventilation are at risk of developing bronchopulmonary dysplasia (BPD) and persistent lung disease in childhood. We report where injury occurred within the lung following brief ventilation at birth. Preterm sheep (129d gestation) were ventilated with an escalating VT to 15mL/kg by 15 min to injure the lungs, with the placental circulation intact (Fetal) or after delivery (Newborn). Fetal lambs were returned to the uterus for 2h 45min, while Newborn lambs were maintained with gentle ventilatory support for the same period. The control group was not ventilated. Bronchoalveolar lavage fluid (BALF) and lung tissue were analysed. In both Fetal and Newborn lambs, ventilation caused bronchial epithelial disruption in medium-sized airways. Egr-1, MCP-1, IL-6, and IL-1β mRNA increased in lung tissue from Fetal and Newborn lambs. Egr-1, MCP-1 and IL-6 mRNA were induced in mesenchymal cells surrounding small airways, whereas IL-1β mRNA localized to the epithelium of medium/small airways. Ventilation caused loss of HSP70 mRNA from the bronchial epithelium, but induced mRNA in smooth muscle surrounding large airways. HSP70 protein decreased in lung tissue and increased in BALF with ventilation. Initiation of ventilation induced a stress response and inflammatory cytokines in small and medium-sized airways.
doi:10.1203/PDR.0b013e3181c1b09e
PMCID: PMC2795027  PMID: 19816239
24.  Modulation of fetal inflammatory response upon exposure to LPS by chorioamnion, lung or gut in sheep 
Objectives
We hypothesized that fetal LPS exposures to the chorioamnion, lung or gut would induce distinct systemic inflammatory responses.
Study Design
Groups of 5–7 time-mated ewes were used to surgically isolate the fetal respiratory and the gastrointestinal systems from the amniotic compartment. Outcomes were assessed at 124d gestational age, 2d and 7 d after LPS (10 mg, E.coli 055:B5) or saline infusions into the fetal airways or amniotic fluid.
Results
LPS induced systemic inflammatory changes in all groups in the blood, lung, liver, and thymic lymphocytes. Changes in lymphocytes in the posterior mediastinal lymph node draining lung and gut, occurred only after direct contact of LPS with the fetal lung or gut.
Conclusions
Fetal systemic inflammatory responses occurred after chorioamnion, lung or gut exposures to LPS. The organ responses differed based on route of the fetal exposure.
doi:10.1016/j.ajog.2009.07.058
PMCID: PMC2811227  PMID: 19801145
fetal inflammation; innate immunity; maturation; chorioamnionitis; antigen exposure
25.  Betamethasone Dose and Formulation for Induced Lung Maturation in Fetal Sheep 
Objectives
We hypothesized that maternal treatments with betamethasone acetate (Beta-Ac) induce fetal lung maturation comparably to the betamethasone phosphate (Beta-PO4) + Beta-Ac (Celestone®) used clinically.
Study Design
Ewes with singleton pregnancies were treated with single doses of 0.25 mg/kg or 0.5 mg/kg Beta-Ac, 4 doses of 0.25 mg/kg Beta-PO4, a single dose of 0.5 mg/kg Beta-Ac + 0.25 mg/kg Beta-PO4, 2 doses of 0.25 mg/kg Beta-Ac + 0.25 mg Beta-PO4 or vehicle beginning 48 h before preterm delivery. Fetal lung maturation was evaluated.
Results
All treatments induced lung maturation relative to vehicle controls. The relatively insoluble Beta-Ac resulted in low maternal blood Beta and no detectable fetal blood Beta in 2 of 3 fetuses, but induced fetal lung maturation comparable to the 2 dose Beta-Ac + Beta-PO4 or 4 doses of Beta-PO4.
Conclusions
A single maternal dose of Beta-Ac effectively induces fetal lung maturation in sheep with minimal fetal exposure.
doi:10.1016/j.ajog.2009.07.014
PMCID: PMC2789907  PMID: 19800603
Corticosteroid; Fetal Therapy; Preterm; Respiratory Distress Syndrome; Sheep 7

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