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1.  Intraamniotic Lipopolysaccharide Exposure Changes Cell Populations and Structure of the Ovine Fetal Thymus 
Reproductive Sciences  2013;20(8):946-956.
Chorioamnionitis induces preterm delivery and acute involution of the fetal thymus which is associated with postnatal inflammatory disorders. We studied the immune response, cell composition, and architecture of the fetal thymus following intraamniotic lipopolysaccharide (LPS) exposure.
Time-mated ewes received an intraamniotic injection of LPS 5, 12, or 24 hours or 2, 4, 8, or 15 days before delivery at 125 days gestational age (term = 150 days).
The LPS exposure resulted in decreased blood lymphocytes within 5 hours and decreased thymic corticomedullary ratio within 24 hours. Thymic interleukin 6 (IL6) and IL17 messenger RNA (mRNA) increased 5-fold 24 hours post-LPS exposure. Increased toll-like receptor 4 (TLR4) mRNA and nuclear factor κB positive cells at 24 hours after LPS delivery demonstrated acute thymic activation. Both TLR4 and IL1 mRNA increased by 5-fold and the number of Foxp3-positive cells (Foxp3+ cells) decreased 15 days after exposure.
Intraamniotic LPS exposure caused a proinflammatory response, involution, and a persistent depletion of thymic Foxp3+ cells indicating disturbance of the fetal immune homeostasis.
PMCID: PMC3702021  PMID: 23314960
fetal inflammatory response syndrome; prematurity; immune modulation; thymic involution
2.  Modulation of fetal inflammatory response upon exposure to LPS by chorioamnion, lung or gut in sheep 
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.
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.
Fetal systemic inflammatory responses occurred after chorioamnion, lung or gut exposures to LPS. The organ responses differed based on route of the fetal exposure.
PMCID: PMC2811227  PMID: 19801145
fetal inflammation; innate immunity; maturation; chorioamnionitis; antigen exposure
3.  Modulation of Lipopolysaccharide-Induced Chorioamnionitis in Fetal Sheep by Maternal Betamethasone 
Reproductive Sciences  2013;20(12):1447-1454.
We tested the hypothesis that the order of exposure to maternal betamethasone and intra-amniotic (IA) lipopolysaccharide (LPS) will differentially modulate inflammation in the chorioamnion.
Study Design:
Time-mated Merino ewes with singleton fetuses received saline alone, IA LPS alone, maternal betamethasone before LPS, or betamethasone after LPS. We assessed inflammatory markers in the chorioamnion and the amniotic fluid.
Inflammatory cell infiltration, expression of myeloperoxidase, serum amyloid A3 (acute phase reactant) in the chorioamnion, and levels of interleukin (IL)-8 in the amniotic fluid increased 7 days after LPS exposure. Betamethasone prior to LPS decreased infiltration of the inflammatory cells, CD3+ T cells, and decreased the levels of IL-1β and IL-8 in the amniotic fluid.
Betamethasone 7 days prior to LPS exposure suppressed LPS-induced inflammation. The markers of inflammation largely had returned to the baseline 14 days after LPS exposure.
PMCID: PMC3817667  PMID: 23653388
prematurity; preterm labor; innate immunity
4.  Comparison of Airway Responses in Sheep of Different Age in Precision-Cut Lung Slices (PCLS) 
PLoS ONE  2014;9(9):e97610.
Animal models should display important characteristics of the human disease. Sheep have been considered particularly useful to study allergic airway responses to common natural antigens causing human asthma. A rationale of this study was to establish a model of ovine precision-cut lung slices (PCLS) for the in vitro measurement of airway responses in newborn and adult animals. We hypothesized that differences in airway reactivity in sheep are present at different ages.
Lambs were delivered spontaneously at term (147d) and adult sheep lived till 18 months. Viability of PCLS was confirmed by the MTT-test. To study airway provocations cumulative concentration-response curves were performed with different allergic response mediators and biogenic amines. In addition, electric field stimulation, passive sensitization with house dust mite (HDM) and mast cells staining were evaluated.
PCLS from sheep were viable for at least three days. PCLS of newborn and adult sheep responded equally strong to methacholine and endothelin-1. The responses to serotonin, leukotriene D4 and U46619 differed with age. No airway contraction was evoked by histamine, except after cimetidine pretreatment. In response to EFS, airways in PCLS from adult and newborn sheep strongly contracted and these contractions were atropine sensitive. Passive sensitization with HDM evoked a weak early allergic response in PCLS from adult and newborn sheep, which notably was prolonged in airways from adult sheep. Only few mast cells were found in the lungs of non-sensitized sheep at both ages.
PCLS from sheep lungs represent a useful tool to study pharmacological airway responses for at least three days. Sheep seem well suited to study mechanisms of cholinergic airway contraction. The notable differences between newborn and adult sheep demonstrate the importance of age in such studies.
PMCID: PMC4167544  PMID: 25229890
5.  Modulation of lipopolysaccharide-induced chorioamnionitis by Ureaplasma parvum in sheep 
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).
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.
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.
Acute U. parvum exposure (7d) can suppress LPS induced chorioamnionitis.
PMCID: PMC3637868  PMID: 23410690
Endotoxin tolerance; Preterm labor; Innate immunity; Fetal adaptation
6.  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.
PMCID: PMC3628630  PMID: 23582963
7.  Non-Invasive Serum Amyloid A (SAA) Measurement and Plasma Platelets for Accurate Prediction of Surgical Intervention in Severe Necrotizing Enterocolitis (NEC) 
PLoS ONE  2014;9(3):e90834.
To evaluate the value of biomarkers to detect severe NEC.
Summary Background Data
The time point of surgery in necrotizing enterocolitis (NEC) is critical. Therefore, there is a need for markers that detect severe NEC, because clinical signs of severe NEC often develop late. This study evaluated the value of biomarkers reflecting intestinal cell damage and inflammation to detect severe NEC.
29 neonates with NEC were included. Two definitions of moderate versus severe NEC were analyzed: medical NEC (n = 12) versus surgical or fatal NEC (n = 17); and Bell stage II NEC (n = 13) versus stage III NEC (n = 16). Urinary intestinal fatty acid binding protein (I-FABP), serum amyloid A (SAA), C3a and C5a, and fecal calprotectin were measured. C-reactive protein (CRP), white blood cell count (WBC) and platelet count data were measured in blood.
In both definitions of moderate versus severe NEC, urinary SAA levels were significantly higher in severe NEC. A cut-off value of 34.4 ng/ml was found in surgical NEC versus medical NEC (sensitivity, 83%; specificity, 83%; LR+, 4.88 (95% CI, 1.37–17.0); LR−, 0.20 (95% CI, 0.07–0.60)) at diagnosis of NEC and at one day prior to surgery in neonates who were operated later on. Combination of urinary SAA and platelet count increased the accuracy, with a sensitivity, 94%; specificity, 83%; LR+, 5.53 (95% CI, 1.57–20.0); and LR−, 0.07 (95% CI, 0.01–0.48).
Urinary SAA is an accurate marker in differentiating severe NEC from moderate NEC; particularly if combined with serum platelet count.
PMCID: PMC3946234  PMID: 24603723
8.  Preterm Birth and Childhood Wheezing Disorders: A Systematic Review and Meta-Analysis 
PLoS Medicine  2014;11(1):e1001596.
In a systematic review and meta-analysis, Jasper Been and colleagues investigate the association between preterm birth and the development of wheezing disorders in childhood.
Please see later in the article for the Editors' Summary
Accumulating evidence implicates early life factors in the aetiology of non-communicable diseases, including asthma/wheezing disorders. We undertook a systematic review investigating risks of asthma/wheezing disorders in children born preterm, including the increasing numbers who, as a result of advances in neonatal care, now survive very preterm birth.
Methods and Findings
Two reviewers independently searched seven online databases for contemporaneous (1 January 1995–23 September 2013) epidemiological studies investigating the association between preterm birth and asthma/wheezing disorders. Additional studies were identified through reference and citation searches, and contacting international experts. Quality appraisal was undertaken using the Effective Public Health Practice Project instrument. We pooled unadjusted and adjusted effect estimates using random-effects meta-analysis, investigated “dose–response” associations, and undertook subgroup, sensitivity, and meta-regression analyses to assess the robustness of associations.
We identified 42 eligible studies from six continents. Twelve were excluded for population overlap, leaving 30 unique studies involving 1,543,639 children. Preterm birth was associated with an increased risk of wheezing disorders in unadjusted (13.7% versus 8.3%; odds ratio [OR] 1.71, 95% CI 1.57–1.87; 26 studies including 1,500,916 children) and adjusted analyses (OR 1.46, 95% CI 1.29–1.65; 17 studies including 874,710 children). The risk was particularly high among children born very preterm (<32 wk gestation; unadjusted: OR 3.00, 95% CI 2.61–3.44; adjusted: OR 2.81, 95% CI 2.55–3.12). Findings were most pronounced for studies with low risk of bias and were consistent across sensitivity analyses. The estimated population-attributable risk of preterm birth for childhood wheezing disorders was ≥3.1%.
Key limitations related to the paucity of data from low- and middle-income countries, and risk of residual confounding.
There is compelling evidence that preterm birth—particularly very preterm birth—increases the risk of asthma. Given the projected global increases in children surviving preterm births, research now needs to focus on understanding underlying mechanisms, and then to translate these insights into the development of preventive interventions.
Review Registration
PROSPERO CRD42013004965
Please see later in the article for the Editors' Summary
Editors' Summary
Most pregnancies last around 40 weeks, but worldwide, more than 11% of babies are born before 37 weeks of gestation (the period during which a baby develops in its mother's womb). Preterm birth is a major cause of infant death—more than 1 million babies die annually from preterm birth complications—and the number of preterm births is increasing globally. Multiple pregnancies, infections, and chronic (long-term) maternal conditions such as diabetes can all cause premature birth, but the cause of many preterm births is unknown. The most obvious immediate complication that is associated with preterm birth is respiratory distress syndrome. This breathing problem, which is more common in early preterm babies than in near-term babies, occurs because the lungs of premature babies are structurally immature and lack pulmonary surfactant, a unique mixture of lipids and proteins that coats the inner lining of the lungs and helps to prevent the collapse of the small air sacs in the lungs that absorb oxygen from the air. Consequently, preterm babies often need help with their breathing and oxygen supplementation.
Why Was This Study Done?
Improvements in the management of prematurity mean that more preterm babies survive today than in the past. However, accumulating evidence suggests that early life events are involved in the subsequent development of non-communicable diseases (non-infectious chronic diseases). Given the increasing burden of preterm birth, a better understanding of the long-term effects of preterm birth is essential. Here, the researchers investigate the risks of asthma and wheezing disorders in children who are born preterm by undertaking a systematic review (a study that uses predefined criteria to identify all the research on a given topic) and a meta-analysis (a statistical method for combining the results of several studies). Asthma is a chronic condition that is caused by inflammation of the airways. In people with asthma, the airways can react very strongly to allergens such as animal fur and to irritants such as cigarette smoke. Exercise, cold air, and infections can also trigger asthma attacks, which can sometimes be fatal. The symptoms of asthma include wheezing (a high-pitched whistling sound during breathing), coughing, chest tightness, and shortness of breath. Asthma cannot be cured, but drugs can relieve its symptoms and prevent acute asthma attacks.
What Did the Researchers Do and Find?
The researchers identified 30 studies undertaken between 1995 and the present (a time span chosen to allow for recent changes in the management of prematurity) that investigated the association between preterm birth and asthma/wheezing disorders in more than 1.5 million children. Across the studies, 13.7% of preterm babies developed asthma/wheezing disorders during childhood, compared to only 8.3% of babies born at term. Thus, the risk of preterm babies developing asthma or a wheezing disorder during childhood was 1.71 times higher than the risk of term babies developing these conditions (an unadjusted odds ratio [OR] of 1.71). In analyses that allowed for confounding factors—other factors that affect the risk of developing asthma/wheezing disorders such as maternal smoking—the risk of preterm babies developing asthma or a wheezing disorder during childhood was 1.46 times higher than that of babies born at term (an adjusted OR of 1.46). Notably, compared to children born at term, children born very early (before 32 weeks of gestation) had about three times the risk of developing asthma/wheezing disorders in unadjusted and adjusted analyses. Finally, the population-attributable risk of preterm birth for childhood wheezing disorders was more than 3.1%. That is, if no preterm births had occurred, there would have been more than a 3.1% reduction in childhood wheezing disorders.
What Do These Findings Mean?
These findings strongly suggest that preterm birth increases the risk of asthma and wheezing disorders during childhood and that the risk of asthma/wheezing disorders increases as the degree of prematurity increases. The accuracy of these findings may be affected, however, by residual confounding. That is, preterm children may share other, unknown characteristics that increase their risk of developing asthma/wheezing disorders. Moreover, the generalizability of these findings is limited by the lack of data from low- and middle-income countries. However, given the projected global increases in children surviving preterm births, these findings highlight the need to undertake research into the mechanisms underlying the association between preterm birth and asthma/wheezing disorders and the need to develop appropriate preventative and therapeutic measures.
Additional Information
Please access these websites via the online version of this summary at
The March of Dimes, a nonprofit organization for pregnancy and baby health, provides information on preterm birth (in English and Spanish)
Nemours, another nonprofit organization for child health, also provides information (in English and Spanish) on premature babies and on asthma (including personal stories)
The UK National Health Service Choices website provides information about premature labor and birth and a real story about having a preterm baby; it provides information about asthma in children (including real stories)
The MedlinePlus Encyclopedia has pages on preterm birth, asthma, asthma in children, and wheezing (in English and Spanish); MedlinePlus provides links to further information on premature birth, asthma, and asthma in children (in English and Spanish)
PMCID: PMC3904844  PMID: 24492409
9.  Effects of Intra-Amniotic Lipopolysaccharide and Maternal Betamethasone on Brain Inflammation in Fetal Sheep 
PLoS ONE  2013;8(12):e81644.
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.
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.
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.
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.
PMCID: PMC3866104  PMID: 24358119
10.  Mesenchymal Stem Cells Induce T-Cell Tolerance and Protect the Preterm Brain after Global Hypoxia-Ischemia 
PLoS ONE  2013;8(8):e73031.
Hypoxic-ischemic encephalopathy (HIE) in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC) in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI) was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 106 MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI), in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE.
PMCID: PMC3753351  PMID: 23991170
11.  Selective Exposure of the Fetal Lung and Skin/Amnion (but Not Gastro-Intestinal Tract) to LPS Elicits Acute Systemic Inflammation in Fetal Sheep 
PLoS ONE  2013;8(5):e63355.
Inflammation of the uterine environment (commonly as a result of microbial colonisation of the fetal membranes, amniotic fluid and fetus) is strongly associated with preterm labour and birth. Both preterm birth and fetal inflammation are independently associated with elevated risks of subsequent short- and long-term respiratory, gastro-intestinal and neurological complications. Despite numerous clinical and experimental studies to investigate localised and systemic fetal inflammation following exposure to microbial agonists, there is minimal data to describe which fetal organ(s) drive systemic fetal inflammation. We used lipopolysaccharide (LPS) from E.coli in an instrumented ovine model of fetal inflammation and conducted a series of experiments to assess the systemic pro-inflammatory capacity of the three major fetal surfaces exposed to inflammatory mediators in pregnancy (the lung, gastro-intestinal tract and skin/amnion). Exposure of the fetal lung and fetal skin/amnion (but not gastro-intestinal tract) caused a significant acute systemic inflammatory response characterised by altered leucocytosis, neutrophilia, elevated plasma MCP-1 levels and inflammation of the fetal liver and spleen. These novel findings reveal differential fetal organ responses to pro-inflammatory stimulation and shed light on the pathogenesis of fetal systemic inflammation after exposure to chorioamnionitis.
PMCID: PMC3656923  PMID: 23691033
12.  Fetal asphyctic preconditioning modulates the acute cytokine response thereby protecting against perinatal asphyxia in neonatal rats 
Perinatal asphyxia (PA) is a major cause of brain damage and neurodevelopmental impairment in infants. Recent investigations have shown that experimental sublethal fetal asphyxia (FA preconditioning) protects against a subsequent more severe asphyctic insult at birth. The molecular mechanisms of this protection have, however, not been elucidated. Evidence implicates that inflammatory cytokines play a protective role in the induction of ischemic tolerance in the adult brain. Accordingly, we hypothesize that FA preconditioning leads to changes in the fetal cytokine response, thereby protecting the newborn against a subsequent asphyctic insult.
In rats, FA preconditioning was induced at embryonic day 17 by clamping the uterine vasculature for 30 min. At term birth, global PA was induced by placing the uterine horns, containing the pups, in a saline bath for 19 min. We assessed, at different time points after FA and PA, mRNA and protein expression of several cytokines and related receptor mRNA levels in total hemispheres of fetal and neonatal brains. Additionally, we measured pSTAT3/STAT3 levels to investigate cellular responses to these cytokines.
Prenatally, FA induced acute downregulation in IL-1β, TNF-α and IL-10 mRNA levels. At 96 h post FA, IL-6 mRNA and IL-10 protein expression were increased in FA brains compared with controls. Two hours after birth, all proinflammatory cytokines and pSTAT3/STAT3 levels decreased in pups that experienced FA and/or PA. Interestingly, IL-10 and IL-6 mRNA levels increased after PA. When pups were FA preconditioned, however, IL-10 and IL-6 mRNA levels were comparable to those in controls.
FA leads to prenatal changes in the neuroinflammatory response. This modulation of the cytokine response probably results in the protective inflammatory phenotype seen when combining FA and PA and may have significant implications for preventing post-asphyctic perinatal encephalopathy.
PMCID: PMC3601975  PMID: 23351591
Asphyxia; Cytokines; Inflammation; Perinatal; Preconditioning
13.  Cerebral inflammation and mobilization of the peripheral immune system following global hypoxia-ischemia in preterm sheep 
Hypoxic-ischemic encephalopathy (HIE) is one of the most important causes of brain injury in preterm infants. Preterm HIE is predominantly caused by global hypoxia-ischemia (HI). In contrast, focal ischemia is most common in the adult brain and known to result in cerebral inflammation and activation of the peripheral immune system. These inflammatory responses are considered to play an important role in the adverse outcomes following brain ischemia. In this study, we hypothesize that cerebral and peripheral immune activation is also involved in preterm brain injury after global HI.
Preterm instrumented fetal sheep were exposed to 25 minutes of umbilical cord occlusion (UCO) (n = 8) at 0.7 gestation. Sham-treated animals (n = 8) were used as a control group. Brain sections were stained for ionized calcium binding adaptor molecule 1 (IBA-1) to investigate microglial proliferation and activation. The peripheral immune system was studied by assessment of circulating white blood cell counts, cellular changes of the spleen and influx of peripheral immune cells (MPO-positive neutrophils) into the brain. Pre-oligodendrocytes (preOLs) and myelin basic protein (MBP) were detected to determine white matter injury. Electro-encephalography (EEG) was recorded to assess functional impairment by interburst interval (IBI) length analysis.
Global HI resulted in profound activation and proliferation of microglia in the hippocampus, periventricular and subcortical white matter. In addition, non-preferential mobilization of white blood cells into the circulation was observed within 1 day after global HI and a significant influx of neutrophils into the brain was detected 7 days after the global HI insult. Furthermore, global HI resulted in marked involution of the spleen, which could not be explained by increased splenic apoptosis. In concordance with cerebral inflammation, global HI induced severe brain atrophy, region-specific preOL vulnerability, hypomyelination and persistent suppressed brain function.
Our data provided evidence that global HI in preterm ovine fetuses resulted in profound cerebral inflammation and mobilization of the peripheral innate immune system. These inflammatory responses were paralleled by marked injury and functional loss of the preterm brain. Further understanding of the interplay between preterm brain inflammation and activation of the peripheral immune system following global HI will contribute to the development of future therapeutic interventions in preterm HIE.
PMCID: PMC3614445  PMID: 23347579
Hypoxic-ischemic encephalopathy; Inflammation; Microglia; Neutrophils; Oligodendrocytes; Preterm; Spleen
14.  Synergistic Effect of Caffeine and Glucocorticoids on Expression of Surfactant Protein B (SP-B) mRNA 
PLoS ONE  2012;7(12):e51575.
Administration of glucocorticoids and caffeine is a common therapeutic intervention in the neonatal period, but possible interactions between these substances are still unclear. The present study investigated the effect of caffeine and different glucocorticoids on expression of surfactant protein (SP)-B, crucial for the physiological function of pulmonary surfactant. We measured expression levels of SP-B, various SP-B transcription factors including erythroblastic leukemia viral oncogene homolog 4 (ErbB4) and thyroid transcription factor-1 (TTF-1), as well as the glucocorticoid receptor (GR) after administering different doses of glucocorticoids, caffeine, cAMP, or the phosphodiesterase-4 inhibitor rolipram in the human airway epithelial cell line NCI-H441. Administration of dexamethasone (1 µM) or caffeine (5 mM) stimulated SP-B mRNA expression with a maximal of 38.8±11.1-fold and 5.2±1.4-fold increase, respectively. Synergistic induction was achieved after co-administration of dexamethasone (1 mM) in combination with caffeine (10 mM) (206±59.7-fold increase, p<0.0001) or cAMP (1 mM) (213±111-fold increase, p = 0.0108). SP-B mRNA was synergistically induced also by administration of caffeine with hydrocortisone (87.9±39.0), prednisolone (154±66.8), and betamethasone (123±6.4). Rolipram also induced SP-B mRNA (64.9±21.0-fold increase). We detected a higher expression of ErbB4 and GR mRNA (7.0- and 1.7-fold increase, respectively), whereas TTF-1, Jun B, c-Jun, SP1, SP3, and HNF-3α mRNA expression was predominantly unchanged. In accordance with mRNA data, mature SP-B was induced significantly by dexamethasone with caffeine (13.8±9.0-fold increase, p = 0.0134). We found a synergistic upregulation of SP-B mRNA expression induced by co-administration of various glucocorticoids and caffeine, achieved by accumulation of intracellular cAMP. This effect was mediated by a caffeine-dependent phosphodiesterase inhibition and by upregulation of both ErbB4 and the GR. These results suggested that caffeine is able to induce the expression of SP-transcription factors and affects the signaling pathways of glucocorticoids, amplifying their effects. Co-administration of caffeine and corticosteroids may therefore be of benefit in surfactant homeostasis.
PMCID: PMC3522739  PMID: 23272120
15.  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.
PMCID: PMC3343143  PMID: 21493953
fetal inflammatory response syndrome; prematurity; innate immunity; IL-1 receptor antagonist
16.  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.
PMCID: PMC3361364  PMID: 21562314
bronchopulmonary dysplasia; TGF-β; a-SMase; ceramide; chorioamnionitis
17.  New Surfactant with SP-B and C Analogs Gives Survival Benefit after Inactivation in Preterm Lambs 
PLoS ONE  2012;7(10):e47631.
Respiratory distress syndrome in preterm babies is caused by a pulmonary surfactant deficiency, but also by its inactivation due to various conditions, including plasma protein leakage. Surfactant replacement therapy is well established, but clinical observations and in vitro experiments suggested that its efficacy may be impaired by inactivation. A new synthetic surfactant (CHF 5633), containing synthetic surfactant protein B and C analogs, has shown comparable effects on oxygenation in ventilated preterm rabbits versus Poractant alfa, but superior resistance against inactivation in vitro. We hypothesized that CHF 5633 is also resistant to inactivation by serum albumin in vivo.
Methodology/Principal Findings
Nineteen preterm lambs of 127 days gestational age (term = 150 days) received CHF 5633 or Poractant alfa and were ventilated for 48 hours. Ninety minutes after birth, the animals received albumin with CHF 5633 or Poractant alfa. Animals received additional surfactant if PaO2 dropped below 100 mmHg. A pressure volume curve was done post mortem and markers of pulmonary inflammation, surfactant content and biophysiology, and lung histology were assessed. CHF 5633 treatment resulted in improved arterial pH, oxygenation and ventilation efficiency index. The survival rate was significantly higher after CHF 5633 treatment (5/7) than after Poractant alfa (1/8) after 48 hours of ventilation. Biophysical examination of the surfactant recovered from bronchoalveolar lavages revealed that films formed by CHF 5633-treated animals reached low surface tensions in a wider range of compression rates than films from Poractant alfa-treated animals.
For the first time a synthetic surfactant containing both surfactant protein B and C analogs showed significant benefit over animal derived surfactant in an in vivo model of surfactant inactivation in premature lambs.
PMCID: PMC3473048  PMID: 23091635
18.  Neurally Mediated Airway Constriction in Human and Other Species: A Comparative Study Using Precision-Cut Lung Slices (PCLS) 
PLoS ONE  2012;7(10):e47344.
The peripheral airway innervation of the lower respiratory tract of mammals is not completely functionally characterized. Recently, we have shown in rats that precision-cut lung slices (PCLS) respond to electric field stimulation (EFS) and provide a useful model to study neural airway responses in distal airways. Since airway responses are known to exhibit considerable species differences, here we examined the neural responses of PCLS prepared from mice, rats, guinea pigs, sheep, marmosets and humans. Peripheral neurons were activated either by EFS or by capsaicin. Bronchoconstriction in response to identical EFS conditions varied between species in magnitude. Frequency response curves did reveal further species-dependent differences of nerve activation in PCLS. Atropine antagonized the EFS-induced bronchoconstriction in human, guinea pig, sheep, rat and marmoset PCLS, showing cholinergic responses. Capsaicin (10 µM) caused bronchoconstriction in human (4 from 7) and guinea pig lungs only, indicating excitatory non-adrenergic non-cholinergic responses (eNANC). However, this effect was notably smaller in human responder (30±7.1%) than in guinea pig (79±5.1%) PCLS. The transient receptor potential (TRP) channel blockers SKF96365 and ruthenium red antagonized airway contractions after exposure to EFS or capsaicin in guinea pigs. In conclusion, the different species show distinct patterns of nerve-mediated bronchoconstriction. In the most common experimental animals, i.e. in mice and rats, these responses differ considerably from those in humans. On the other hand, guinea pig and marmoset monkey mimic human responses well and may thus serve as clinically relevant models to study neural airway responses.
PMCID: PMC3467211  PMID: 23056631
19.  A Clinical Prediction Rule for Histological Chorioamnionitis in Preterm Newborns 
PLoS ONE  2012;7(10):e46217.
Histological chorioamnionitis (HC) is an intrauterine inflammatory process highly associated with preterm birth and adverse neonatal outcome. HC is often clinically silent and diagnosed postnatally by placental histology. Earlier identification could facilitate treatment individualisation to improve outcome in preterm newborns.
Develop a clinical prediction rule at birth for HC and HC with fetal involvement (HCF) in preterm newborns.
Clinical data and placental pathology were obtained from singleton preterm newborns (gestational age ≤32.0 weeks) born at Erasmus UMC Rotterdam from 2001 to 2003 (derivation cohort; n = 216) or Máxima MC Veldhoven from 2009 to 2010 (validation cohort; n = 206). HC and HCF prediction rules were developed with preference for high sensitivity using clinical variables available at birth.
HC and HCF were present in 39% and 24% in the derivation cohort and in 44% and 22% in the validation cohort, respectively. HC was predicted with 87% accuracy, yielding an area under ROC curve of 0.95 (95%CI = 0.92–0.98), a positive predictive value of 80% (95%CI = 74–84%), and a negative predictive value of 93% (95%CI = 88–96%). Corresponding figures for HCF were: accuracy 83%, area under ROC curve 0.92 (95%CI = 0.88–0.96), positive predictive value 59% (95%CI = 52–62%), and negative predictive value 97% (95%CI = 93–99%). External validation expectedly resulted in some loss of test performance, preferentially affecting positive predictive rather than negative predictive values.
Using a clinical prediction rule composed of clinical variables available at birth, HC and HCF could be predicted with good test characteristics in preterm newborns. Further studies should evaluate the clinical value of these rules to guide early treatment individualisation.
PMCID: PMC3465298  PMID: 23071549
20.  IFN-γ and TNF-α Synergize to Inhibit CTGF Expression in Human Lung Endothelial Cells 
PLoS ONE  2012;7(9):e45430.
Connective tissue growth factor (CTGF/CCN2) is an angiogenetic and profibrotic factor, acting downstream of TGF-β, involved in both airway- and vascular remodeling. While the T-helper 1 (Th1) cytokine interferon-gamma (IFN-γ) is well characterized as immune-modulatory and anti-fibrotic cytokine, the role of IFN-γ in lung endothelial cells (LEC) is less defined. Tumour necrosis factor alpha (TNF-α) is another mediator that drives vascular remodeling in inflammation by influencing CTGF expression. In the present study we investigated the influence of IFN-γ and TNF-α on CTGF expression in human LEC (HPMEC-ST1.6R) and the effect of CTGF knock down on human LEC. IFN-γ and TNF-α down-regulated CTGF in human LEC at the promoter-, transcriptional- and translational-level in a dose- and time-dependent manner. The inhibitory effect of IFN-γ on CTGF-expression could be almost completely compensated by the Jak inhibitor AG-490, showing the involvement of the Jak-Stat signaling pathway. Besides the inhibitory effect of IFN-γ and TNF-α alone on CTGF expression and LEC proliferation, these cytokines had an additive inhibitory effect on proliferation as well as on CTGF expression when administered together. To study the functional role of CTGF in LEC, endogenous CTGF expression was down-regulated by a lentiviral system. CTGF silencing in LEC by transduction of CTGF shRNA reduced cell proliferation, but did not influence the anti-proliferative effect of IFN-γ and TNF-α. In conclusion, our data demonstrated that CTGF was negatively regulated by IFN-γ in LEC in a Jak/Stat signaling pathway-dependent manner. In addition, an additive effect of IFN-γ and TNF-α on inhibition of CTGF expression and cell proliferation could be found. The inverse correlation between IFN-γ and CTGF expression in LEC could mean that screwing the Th2 response to a Th1 response with an additional IFN-γ production might be beneficial to avoid airway remodeling in asthma.
PMCID: PMC3447888  PMID: 23029004
21.  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.
PMCID: PMC3159703  PMID: 21784974
Prematurity; Chorioamnionitis; Fetal inflammatory response syndrome; endotoxin tolerance; lung inflammation
22.  Ovine Fetal Thymus Response to Lipopolysaccharide-Induced Chorioamnionitis and Antenatal Corticosteroids 
PLoS ONE  2012;7(5):e38257.
Chorioamnionitis is associated with preterm delivery and involution of the fetal thymus. Women at risk of preterm delivery receive antenatal corticosteroids which accelerate fetal lung maturation and improve neonatal outcome. However, the effects of antenatal corticosteroids on the fetal thymus in the settings of chorioamnionitis are largely unknown. We hypothesized that intra-amniotic exposure to lipopolysaccharide (LPS) causes involution of the fetal thymus resulting in persistent effects on thymic structure and cell populations. We also hypothesized that antenatal corticosteroids may modulate the effects of LPS on thymic development.
Time-mated ewes with singleton fetuses received an intra-amniotic injection of LPS 7 or 14 days before preterm delivery at 120 days gestational age (term = 150 days). LPS and corticosteroid treatment groups received intra-amniotic LPS either preceding or following maternal intra-muscular betamethasone. Gestation matched controls received intra-amniotic and maternal intra-muscular saline. The fetal intra-thoracic thymus was evaluated.
Intra-amniotic LPS decreased the cortico-medullary (C/M) ratio of the thymus and increased Toll-like receptor (TLR) 4 mRNA and CD3 expression indicating involution and activation of the fetal thymus. Increased TLR4 and CD3 expression persisted for 14 days but Foxp3 expression decreased suggesting a change in regulatory T-cells. Sonic hedgehog and bone morphogenetic protein 4 mRNA, which are negative regulators of T-cell development, decreased in response to intra-amniotic LPS. Betamethasone treatment before LPS exposure attenuated some of the LPS-induced thymic responses but increased cleaved caspase-3 expression and decreased the C/M ratio. Betamethasone treatment after LPS exposure did not prevent the LPS-induced thymic changes.
Intra-amniotic exposure to LPS activated the fetal thymus which was accompanied by structural changes. Treatment with antenatal corticosteroids before LPS partially attenuated the LPS-induced effects but increased apoptosis in the fetal thymus. Corticosteroid administration after the inflammatory stimulus did not inhibit the LPS effects on the fetal thymus.
PMCID: PMC3365024  PMID: 22693607
23.  Poractant alfa (Curosurf®) increases phagocytosis of apoptotic neutrophils by alveolar macrophages in vivo 
Respiratory Research  2012;13(1):17.
Clearance of apoptotic neutrophils in the lung is an essential process to limit inflammation, since they could become a pro-inflammatory stimulus themselves. The clearance is partially mediated by alveolar macrophages, which phagocytose these apoptotic cells. The phagocytosis of apoptotic immune cells by monocytes in vitro has been shown to be augmented by several constituents of pulmonary surfactant, e.g. phospholipids and hydrophobic surfactant proteins. In this study, we assessed the influence of exogenous poractant alfa (Curosurf®) instillation on the in vivo phagocytosis of apoptotic neutrophils by alveolar macrophages.
Poractant alfa (200 mg/kg) was instilled intratracheally in the lungs of three months old adult male C57/Black 6 mice, followed by apoptotic neutrophil instillation. Bronchoalveloar lavage was performed and alveolar macrophages and neutrophils were counted. Phagocytosis of apoptotic neutrophils was quantified by determining the number of apoptotic neutrophils per alveolar macrophages.
Exogenous surfactant increased the number of alveolar macrophages engulfing apoptotic neutrophils 2.6 fold. The phagocytosis of apoptotic neutrophils was increased in the presence of exogenous surfactant by a 4.7 fold increase in phagocytosed apoptotic neutrophils per alveolar macrophage.
We conclude that the anti-inflammatory properties of surfactant therapy may be mediated in part by increased numbers of alveolar macrophages and increased phagocytosis of apoptotic neutrophils by alveolar macrophages.
PMCID: PMC3310829  PMID: 22405518
Inflammation; Resolution; Anti inflammation; Drug therapy; Surfactant
24.  Systemic hydrocortisone to prevent bronchopulmonary dysplasia in preterm infants (the SToP-BPD study); a multicenter randomized placebo controlled trial 
BMC Pediatrics  2011;11:102.
Randomized controlled trials have shown that treatment of chronically ventilated preterm infants after the first week of life with dexamethasone reduces the incidence of the combined outcome death or bronchopulmonary dysplasia (BPD). However, there are concerns that dexamethasone may increase the risk of adverse neurodevelopmental outcome. Hydrocortisone has been suggested as an alternative therapy. So far no randomized controlled trial has investigated its efficacy when administered after the first week of life to ventilated preterm infants.
The SToP-BPD trial is a randomized double blind placebo controlled multicenter study including 400 very low birth weight infants (gestational age < 30 weeks and/or birth weight < 1250 grams), who are ventilator dependent at a postnatal age of 7 - 14 days. Hydrocortisone (cumulative dose 72.5 mg/kg) or placebo is administered during a 22 day tapering schedule. Primary outcome measure is the combined outcome mortality or BPD at 36 weeks postmenstrual age. Secondary outcomes are short term effects on the pulmonary condition, adverse effects during hospitalization, and long-term neurodevelopmental sequelae assessed at 2 years corrected gestational age. Analysis will be on an intention to treat basis.
This trial will determine the efficacy and safety of postnatal hydrocortisone administration at a moderately early postnatal onset compared to placebo for the reduction of the combined outcome mortality and BPD at 36 weeks postmenstrual age in ventilator dependent preterm infants.
Trial registration number
Netherlands Trial Register (NTR): NTR2768
PMCID: PMC3245429  PMID: 22070744
25.  Increased Number of Cerebellar Granule Cells and Astrocytes in the Internal Granule Layer in Sheep Following Prenatal Intra-amniotic Injection of Lipopolysaccharide 
Cerebellum (London, England)  2011;11(1):132-144.
Chorioamnionitis is an important problem in perinatology today, leading to brain injury and neurological handicaps. However, there are almost no data available regarding chorioamnionitis and a specific damage of the cerebellum. Therefore, this study aimed at determining if chorioamnionitis causes cerebellar morphological alterations. Chorioamnionitis was induced in sheep by the intra-amniotic injection of lipopolysaccharide (LPS) at a gestational age (GA) of 110 days. At a GA of 140 days, we assessed the mean total and layer-specific volume and the mean total granule cell (GCs) and Purkinje cell (PC) number in the cerebelli of LPS-exposed and control animals using high-precision design-based stereology. Astrogliosis was assessed in the gray and white matter (WM) using a glial fibrillary acidic protein staining combined with gray value image analysis. The present study showed an unchanged volume of the total cerebellum as well as the molecular layer, outer and inner granular cell layers (OGL and IGL, respectively), and WM. Interestingly, compared with controls, the LPS-exposed brains showed a statistically significant increase (+20.4%) in the mean total number of GCs, whereas the number of PCs did not show any difference between the two groups. In addition, LPS-exposed animals showed signs of astrogliosis specifically affecting the IGL. Intra-amniotic injection of LPS causes morphological changes in the cerebellum of fetal sheep still detectable at full-term birth. In this study, changes were restricted to the inner granule layer. These cerebellar changes might correspond to some of the motor or non-motor deficits seen in neonates from compromised pregnancies.
PMCID: PMC3311858  PMID: 21773814
Chorioamnionitis; LPS; Intra-amniotic; Fetal; Cerebellum; Granule cell loss; Astrocytes; Stereology

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