The effect of human amnion epithelial cells on lung development and inflammation in preterm lambs exposed to antenatal inflammation

Lung inflammation and impaired alveolarization are hallmarks of bronchopulmonary dysplasia (BPD). We hypothesize that human amnion epithelial cells (hAECs) are anti-inflammatory and reduce lung injury in preterm lambs born after antenatal exposure to inflammation. Pregnant ewes received either intra-amniotic lipopolysaccharide (LPS, from E.coli 055:B5 4mg) or saline (Sal) on day 126 of gestation. Lambs were delivered by cesarean section at 128 d gestation (term ~150 d). Lambs received intravenous hAECs (LPS/hAECs: n = 7 30x10.sup.6 cells) or equivalent volumes of saline (LPS/Sal, n = 10 or Sal/Sal, n = 9) immediately after birth. Respiratory support was gradually de-escalated, aimed at early weaning from mechanical ventilation towards unassisted respiration. Lung tissue was collected 1 week after birth. Lung morphology was assessed and mRNA levels for inflammatory mediators were measured. Respiratory support required by LPS/hAEC lambs was not different to Sal/Sal or LPS/Sal lambs. Lung tissue:airspace ratio was lower in the LPS/Sal compared to Sal/Sal lambs (P<0.05), but not LPS/hAEC lambs. LPS/hAEC lambs tended to have increased septation in their lungs versus LPS/Sal (P = 0.08). Expression of inflammatory cytokines was highest in LPS/hAECs lambs. Postnatal administration of a single dose of hAECs stimulates a pulmonary immune response without changing ventilator requirements in preterm lambs born after intrauterine inflammation

Epidemiology of Neonatal Acute Respiratory Distress Syndrome:Prospective, Multicenter, International Cohort Study

OBJECTIVES: Age-specific definitions for acute respiratory distress syndrome (ARDS) are available, including a specific definition for neonates (the "Montreux definition"). The epidemiology of neonatal ARDS is unknown. The objective of this study was to describe the epidemiology, clinical course, treatment, and outcomes of neonatal ARDS. DESIGN: Prospective, international, observational, cohort study. SETTING: Fifteen academic neonatal ICUs. PATIENTS: Consecutive sample of neonates of any gestational age admitted to participating sites who met the neonatal ARDS Montreux definition criteria. MEASUREMENTS AND MAIN RESULTS: Neonatal ARDS was classified as direct or indirect, infectious or noninfectious, and perinatal (≤ 72 hr after birth) or late in onset. Primary outcomes were: 1) survival at 30 days from diagnosis, 2) inhospital survival, and 3) extracorporeal membrane oxygenation (ECMO)-free survival at 30 days from diagnosis. Secondary outcomes included respiratory complications and common neonatal extrapulmonary morbidities. A total of 239 neonates met criteria for the diagnosis of neonatal ARDS. The median prevalence was 1.5% of neonatal ICU admissions with male/female ratio of 1.5. Respiratory treatments were similar across gestational ages. Direct neonatal ARDS (51.5% of neonates) was more common in term neonates and the perinatal period. Indirect neonatal ARDS was often triggered by an infection and was more common in preterm neonates. Thirty-day, inhospital, and 30-day ECMO-free survival were 83.3%, 76.2%, and 79.5%, respectively. Direct neonatal ARDS was associated with better survival outcomes than indirect neonatal ARDS. Direct and noninfectious neonatal ARDS were associated with the poorest respiratory outcomes at 36 and 40 weeks' postmenstrual age. Gestational age was not associated with any primary outcome on multivariate analyses. CONCLUSIONS: Prevalence and survival of neonatal ARDS are similar to those of pediatric ARDS. The neonatal ARDS subtypes used in the current definition may be associated with distinct clinical outcomes and a different distribution for term and preterm neonates

Effects of intra-amniotic lipopolysaccharide and maternal betamethasone on brain inflammation in fetal sheep

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

Multipotent adult progenitor cells prevent functional impairment and improve development in inflammation driven detriment of preterm ovine lungs

Background: Perinatal inflammation increases the risk for bronchopulmonary dysplasia in preterm neonates, but the underlying pathophysiological mechanisms remain largely unknown. Given their anti-inflammatory and regenerative capacity, multipotent adult progenitor cells (MAPC) are a promising cell-based therapy to prevent and/or treat the negative pulmonary consequences of perinatal inflammation in the preterm neonate. Therefore, the pathophysiology underlying adverse preterm lung outcomes following perinatal inflammation and pulmonary benefits of MAPC treatment at the interface of prenatal inflammatory and postnatal ventilation exposures were elucidated. Methods: Instrumented ovine fetuses were exposed to intra-amniotic lipopolysaccharide (LPS 5 mg) at 125 days gestation to induce adverse systemic and peripheral organ outcomes. MAPC (10 × 106 cells) or saline were administered intravenously two days post LPS exposure. Fetuses were delivered preterm five days post MAPC treatment and either killed humanely immediately or mechanically ventilated for 72 h. Results: Antenatal LPS exposure resulted in inflammation and decreased alveolar maturation in the preterm lung. Additionally, LPS-exposed ventilated lambs showed continued pulmonary inflammation and cell junction loss accompanied by pulmonary edema, ultimately resulting in higher oxygen demand. MAPC therapy modulated lung inflammation, prevented loss of epithelial and endothelial barriers and improved lung maturation in utero. These MAPC-driven improvements remained evident postnatally, and prevented concomitant pulmonary edema and functional loss. Conclusion: In conclusion, prenatal inflammation sensitizes the underdeveloped preterm lung to subsequent postnatal inflammation, resulting in injury, disturbed development and functional impairment. MAPC therapy partially prevents these changes and is therefore a promising approach for preterm infants to prevent adverse pulmonary outcomes

IL-1α Mediated Chorioamnionitis Induces Depletion of FoxP3+ Cells and Ileal Inflammation in the Ovine Fetal Gut

Endotoxin induced chorioamnionitis increases IL-1 and provokes an inflammatory response in the fetal ileum that interferes with intestinal maturation. In the present study, we tested in an ovine chorioamnionitis model whether IL-1 is a major cytokine driving the inflammatory response in the fetal ileum.Sheep bearing singleton fetuses received a single intraamniotic injection of recombinant ovine IL-1α at 7, 3 or 1 d before caesarian delivery at 125 days gestational age (term = 150 days).3 and 7 d after IL-1α administration, intestinal mRNA levels for IL-4, IL-10, IFN-γ and TNF-α were strongly elevated. Numbers of CD3+ and CD4+ T-lymphocytes and myeloidperoxidase+ cells were increased whereas FoxP3+ T-cells were detected at low frequency. This increased proinflammatory state was associated with ileal mucosal barrier loss as demonstrated by decreased levels of the intestinal fatty acid binding protein and disruption of the tight junctional protein ZO-1.Intraamniotic IL-1α causes an acute detrimental inflammatory response in the ileum, suggesting that induction of IL-1 is a critical element in the pathophysiological effects of endotoxin induced chorioamnionitis. A disturbed balance between T-effector and FoxP3+ cells may contribute to this process

The Role of the Multiple Banded Antigen of Ureaplasma parvum in Intra-Amniotic Infection: Major Virulence Factor or Decoy?

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

Ovine Fetal Thymus Response to Lipopolysaccharide-Induced Chorioamnionitis and Antenatal Corticosteroids

RATIONALE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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

The management of evolving bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is associated with increased mortality and significant long-term cardiorespiratory and neurodevelopmental sequelae. Treatment of evolving BPD in the neonatal intensive care unit (NICU) is challenging due to the complex interplay of contributing risk factors which include preterm birth per se, supplemental oxygen, positive pressure ventilation, patent ductus arterious, and pre- and postnatal infection. Management of evolving BPD requires a multimodal approach including adequate nutrition, careful fluid management, effective and safe pharmacotherapy, and respiratory support aiming at minimal lung injury. Among pharmacological interventions, caffeine has the best risk-benefit profile. Systemic postnatal corticosteroids should be reserved to ventilated infants at highest risk of BPD who cannot be weaned from the ventilator. Several ongoing randomised trials are evaluating optimal oxygen saturation targets in preterm infants. The most beneficial respiratory support strategy to minimise lung injury remains unclear and requires further investigation

Monitoring of lung volume recruitment and derecruitment using oscillatory mechanics during high-frequency oscillatory ventilation in the preterm lamb

Objectives: To determine whether the low-frequency forced oscillation technique (LFOT) can track changes in lung mechanics resulting from prophylactic intratracheal surfactant and a volume recruitment-derecruitment maneuver during high-frequency oscillatory ventilation (HFOV) and how this relates to the damping of the tracheal pressure waveform (P). Design: Interventional laboratory study. Setting: Rural animal research facility in Western Australia. Subjects: Sedated newborn preterm lambs. Interventions: Two separate studies were performed. Study 1 involved a volume recruitment- derecruitment maneuver during HFOV using stepwise changes (4 cm HO) in mean airway opening pressure (P, n = 5). Study 2 involved instillation of 4 mL/kg fetal lung fluid (n = 5) or exogenous surfactant (n = 8) at birth and subsequent intermittent mandatory ventilation for 40 mins. Measurements and Main Results: Arterial blood gases were recorded every 10 mins and ventilation was appropriately adjusted to achieve moderate hypercarbia (PaCO, 50-60 mm Hg). Lung mechanics were measured using LFOT 10 mins following each adjustment in P in parallel with measurements of P and tidal volume (study 1) or after 40 mins (study 2). Both the recruitment-derecruitment maneuver and prophylactic surfactant administration achieved similar improvements (decrease) in tissue impedance (Z). The coefficient of tissue resistance (G) decreased more than the coefficient of tissue elastance (H), consistent with improved homogeneity of the lung tissue compartment as lung volume was recruited. Minimum Z coincided with minimum tracheal pressure amplitude (ΔP) on the deflation limb of the volume recruitment-derecruitment maneuver during HFOV. There was a linear relationship between ΔP and H. Conclusions: In the preterm lamb, the LFOT can successfully detect changes in lung mechanics resulting from volume recruitment maneuvers during both HFOV and ventilation at conventional rates and may provide information on ventilation inhomogeneity. Minimization of Z is crucial to damping of P and may limit potential barotrauma to proximal alveolar units during establishment of HFOV

RESPIRATORY AND CHEST WALL MECHANICS IN VERY PRETERM INFANTS

: Data on static compliance of the chest wall (Ccw) in preterm infants are scarce. We characterised the static compliance of the lung (CL) and Ccw to determine their relative contribution to static compliance of the respiratory system (Crs) in very preterm infants at 36 weeks' postmenstrual age (PMA). We also aimed to investigate how these compliances were influenced by the presence of bronchopulmonary dysplasia (BPD) and impacted breathing variables. Airway opening pressure, esophageal pressure, and tidal volume (VT) were measured simultaneously during a short apnea evoked by the Hering-Breuer reflex. We computed tidal breathing variables, lung resistance (Rl) and dynamic lung compliance (CL,dyn), inspiratory capacity (IC), and Crs, CL and Ccw. Functional residual capacity was assessed by the multiple breath washout technique (FRCmbw). Breathing variables, compliances and lung volumes were adjusted for body weight. Twenty-three preterm infants born at 27.2 ± 2.0 weeks' gestational age (GA) were studied at 36.6 ± 0.6 weeks' PMA. Median (IQR) Crs/kg is 0.69 (0.6), CL 0.95 (1.0) and Ccw 3.0 (2.4). Infants with BPD (n=11) had lower Crs (p=0.013), CL (p=0.019), and Ccw (p=0.027) compared to infants without BPD. Ccw/CL ratio was equal between groups. FRCmbw/kg (p=0.044) and IC/kg (p=0.005) were decreased in infants with BPD. Infants with BPD have reduced static compliance of the respiratory system, the lung and chest wall. Decreased Crs, CL and Ccw in infants with BPD explains the lower FRC and IC seen in these infants