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ABSTRACT: PurposeThe relative contributions of factors influencing lung injury immediately after birth are poorly understood. We hypothesized
that oxygen content and humidity of inspired air would influence markers of pulmonary inflammation in ventilated lambs.
MethodsLambs delivered at 140days gestation (term=150days) were assigned to one of five groups (n=5–6/group): unventilated controls, or ventilation with 21 or 100% O2 that was either heated and humidified or cold and dry. Lambs were ventilated gently for 3h: blood gases were recorded regularly.
Bronchoalveolar lavage and samples of tracheal mucosa and lung were collected post mortem.
ResultsArterial pH was lower [mean difference (95% CI): −0.07 (−0.13, −0.03)], while there was an increase in PaCO2 [mean difference (95% CI): 10.2 (2.4, 17.9)] and fold change in lung pro-inflammatory IL-1β cytokine mRNA [mean difference
(95% CI): 28.3 (0.3, 56.2)] or IL-8 [mean difference (95% CI): 27.8 (7.9, 47.7)] cytokine mRNA expression with 100% O2 relative to 21% O2. Cold dry inspired gas did not influence gas exchange or dynamic mechanics at 3h compared to heated humidified gas. Compared
to 100% inspired O2, cold dry inspired gas had less marked effect on fold change in lung pro-inflammatory IL-1β cytokine mRNA [mean difference
(95% CI): 27.2 (−0/8, 55.1)] or IL-8 [mean difference (95% CI): 14.5 (5.5, 34.4)] cytokine mRNA expression, although cilial
dysfunction/damage was evident on electron microscopy, especially when exposure to cold dry gas was combined with hyperoxia.
ConclusionsIn near-term neonatal lambs ventilated for 3h, hyperoxia was associated a more powerful stimulus for pulmonary dysfunction
and upregulation of inflammatory cytokines than cold dry gas.
Intensive Care Medicine 04/2012; 35(12):2157-2163. · 5.40 Impact Factor
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ABSTRACT: Bronchopulmonary dysplasia is associated with neutrophil infiltration into the lungs and oxidative injury. However, the pathological importance of neutrophil oxidants is still not clear. Nosocomial pneumonia is also implicated, but the evidence is limited, in part because of the difficulty of distinguishing genuine infection from bacterial colonization. Good biomarkers of neutrophil oxidant activity and lung infection are needed. We tested whether glutathione sulfonamide, a product of glutathione oxidation by myeloperoxidase-derived hypochlorous acid (HOCl) and a potential new neutrophil oxidant biomarker, is detectable in endotracheal aspirates from ventilated preterm infants. As infectious organisms stimulate neutrophils to generate HOCl, we determined whether levels of HOCl-specific biomarkers were increased in samples that were bacterial culture positive. Glutathione sulfonamide was detected in 66 of 87 endotracheal aspirate samples. Levels correlated with myeloperoxidase activity and another HOCl-specific marker, chlorotyrosine. Median levels of glutathione sulfonamide (4-fold) and other biomarkers (2-fold) were significantly higher in culture positive aspirates. Staphylococcus epidermidis, a frequent colonizer, was associated with glutathione sulfonamide levels no different from those in negative samples. Glutathione sulfonamide showed good sensitivity and specificity for detecting bacterial growth and has promise for detecting lung infection.
Pediatric Research 10/2010; 69(1):28-33. · 2.70 Impact Factor
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ABSTRACT: Chorioamnionitis frequently associates with preterm delivery and increased amniotic fluid IL-1, and causes fetal lung and systemic inflammation. However, chorioamnionitis is also associated with a paradoxical reduction in the incidence of surfactant deficiency-related respiratory distress syndrome in preterm infants.
To identify the role of IL-1 signaling in the mediation of pulmonary and systemic inflammation and lung maturation in a fetal sheep model of lipopolysaccharide (LPS) induced chorioamnionitis.
After confirming the efficacy of recombinant human IL-1 receptor antagonist (rhIL-1ra), fetal sheep were exposed to intraamniotic (IA) injections of Escherichia coli LPS with or without prior IA injections of rhIL-1ra. Preterm lambs were delivered at 82% of term gestation.
rhIL-1ra decreased IA LPS-induced lung inflammation assessed by decreased lung neutrophil and monocyte influx, inducible nitric oxide synthase expression, lung IL-6 and IL-1beta mRNA expression, and airway myeloperoxidase concentrations. rhIL-1ra inhibited IA LPS-induced fetal systemic inflammation assessed by decreased plasma IL-8, protein carbonyls, blood neutrophilia, and the expression of serum amyloid A3 mRNA in the liver. rhIL-1ra also partially blocked the lung maturational effects of IA LPS. Therefore blockade of IL-1 signaling in the amniotic compartment inhibited fetal lung and systemic inflammation and lung maturation in response to LPS-induced chorioamnionitis.
IL-1 plays a central role in the pathogenesis of chorioamnionitis-induced fetal inflammatory responses.
American Journal of Respiratory and Critical Care Medicine 03/2009; 179(10):955-61. · 11.08 Impact Factor
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ABSTRACT: Chorioamnionitis, a risk factor for bronchopulmonary dysplasia in preterm infants, causes an influx of inflammatory cells into the fetal lung. Using a fetal sheep model, we evaluated the time course of activation, functional maturity, and apoptosis of the leukocytes recruited to the fetal air spaces by lipopolysaccharide (LPS). Time-mated sheep were given intra-amniotic injections with 10 mg of Escherichia coli LPS or saline 2 or 7 days before preterm delivery at 124 days of gestation (term is 150 days). Both neutrophils and monocytes in bronchoalveolar lavage fluid (BALF) had activated NF-kappaB after 2- and 7-day LPS exposures. These neutrophils and monocytes expressed the activation factor CD11b and the maturation factor PU.1 at 2 days, and increased PU.1 expression was detected in macrophages at 7 days. Leukocyte oxidative burst activity was greatest at 7 days. BALF lipid peroxidation increased fivefold at 2 days, while protein carbonyls increased eightfold at 7 days. Nitrative stress was not detected in the BALF, but leukocytes in the lung expressed nitric oxide synthase (NOS)II (inducible NOS). BALF leukocytes expressed the antioxidant peroxiredoxin V. Lung glutathione peroxidase was also increased with LPS exposure. There was minimal apoptosis of airway and lung leukocytes assessed by caspase-3 activation. Intra-amniotic LPS recruits leukocytes to the fetal air space that have a persistent activation. These results have implications for the pathogenesis of lung inflammatory disorders in the preterm.
AJP Lung Cellular and Molecular Physiology 01/2009; 296(3):L384-93. · 3.66 Impact Factor
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ABSTRACT: Recent literature suggests hypothermia may protect against lung injury. We evaluated body temperature as a variable in lung inflammation due to oxygenation and mechanical ventilation following delivery of near-term lambs.
Twin fetuses were randomized prior to delivery at 140 d GA (term 150 d): unventilated controls, normothermic ventilated with room air, normothermic ventilated with 100% oxygen, low temperature ventilated (target 35 degrees C) with 100% oxygen, and high temperature (target 40 degrees C) with 100% oxygen. Lambs were intubated for gentle mechanical ventilation (tidal volume 7-8ml/kg). Temperature targeting was with radiant warmers and plastic wrap for normothermia, with heat lamps for hyperthermia, and with ice packs for hypothermia. Lambs were euthanized after 2h mechanical ventilation. Post-mortem, bronchoalveolar lavage fluid and lung tissue samples were evaluated for inflammatory responses by measuring inflammatory cell counts, protein, myeloperoxidase, protein carbonyl, and pro-inflammatory cytokine mRNA.
Target temperatures were achieved by 30min of age and tightly maintained for the 2h study. There were no differences in physiologic variables among groups except those directly resulting from study protocol-PaO2 from air vs. 100% oxygen and body temperature. Indicators of inflammation increased similarly in all ventilated groups compared to unventilated controls.
Moderate hyperthermia or hypothermia did not affect lung injury responses to the initiation of ventilation at birth in near-term lambs.
Resuscitation 12/2008; 80(1):133-7. · 3.60 Impact Factor
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ABSTRACT: Positive end-expiratory pressure (PEEP) protects the lung from injury during sustained ventilation, but its role in protecting the lung from injury during the initiation of ventilation in the delivery room is not established. We aimed to evaluate whether PEEP and/or tidal volume (VT) within the first 15-min of ventilation are protective against lung injury. Operatively delivered preterm lambs (133 +/- 1 d gestation) were randomly assigned to unventilated controls or to one of four 15 min ventilation interventions: 1) VT15 mL/kg, PEEP 0 cm H2O; 2) VT15 mL/kg, PEEP 5 cm H2O; 3) VT8 mL/kg, PEEP 0 cm H2O; and 4) VT8 mL/kg, PEEP 5 cm H2O. Each group was subsequently ventilated with VT 10 mL/kg, PEEP 5 cm H2O for 1 h 45 min. Lung function was assessed and measurements of lung injury were evaluated postmortem. After the 15 min ventilation maneuver, the VT15 groups were hypocarbic, had higher oxygenation, and required lower pressures than the VT8 groups; no consistent effect of PEEP was found. Markers of lung injury were significantly elevated in all ventilation groups compared with unventilated controls; no effect of PEEP was found. Ventilation resulted in localization of IL-6 to the small airways. Initial ventilation of preterm lambs with PEEP and/or VT of 8 mL/kg did not prevent an inflammatory injury to the lung.
Pediatric Research 06/2008; 64(5):517-22. · 2.70 Impact Factor
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ABSTRACT: Chorioamnionitis is a risk factor for the development of bronchopulmonary dysplasia. Endotoxin-induced oxidative stress to the fetus in the uniquely hypoxic intrauterine environment has not been reported. Using a model of chorioamnionitis, we measured markers of pulmonary and systemic oxidant exposures in fetal lambs at 124 d gestation (term = 150 d) exposed to 10 mg intra-amniotic endotoxin 2 d (n = 6) or 7 d (n = 6) before delivery, or saline as controls (n = 9). The 7 d endotoxin-exposed animals had 3-fold higher protein carbonyls (0.66 +/- 0.46 versus 0.23 +/- 0.14 nmol/mg protein) and 10-fold greater myeloperoxidase activity (2.38 +/- 1.87 versus 0.27 +/- 0.18 nM) in the bronchoalveolar lavage fluid (BALF), suggestive of neutrophil-derived oxidant activity. However, in the lung tissue, protein carbonyls, superoxide dismutase, and peroxiredoxin 1 were not different between groups. The expression of peroxiredoxin 1 was prominent, primarily in the peri-bronchiolar epithelium. Notably, evidence of oxidant exposure was minimal at 2 d when BALF inflammatory cells, lung IL-1beta, and IL-8 were highest. Intra-amniotic endotoxin induced systemic oxidative stress as plasma protein carbonyl was elevated at 7 d (0.14 +/- 0.04 nmol/mg protein; p = 0.005). Surfactant protein A and B mRNAs were highest at 2 d, suggesting that oxidative stress did not contribute to the lung maturation response. A modest lung oxidative stress in chorioamnionitis could contribute to bronchopulmonary dysplasia.
Pediatric Research 04/2008; 63(3):274-9. · 2.70 Impact Factor
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ABSTRACT: Hypochlorous acid (HOCl) is produced by the neutrophil enzyme, myeloperoxidase, and reacts with amines to generate chloramines. These oxidants react readily with thiols and methionine and can affect cell-regulatory pathways. In the present study, we have investigated the ability of HOCl, glycine chloramine (Gly-Cl) and taurine chloramine (Tau-Cl) to oxidize IkappaBalpha, the inhibitor of NF-kappaB (nuclear factor kappaB), and to prevent activation of the NF-kappaB pathway in Jurkat cells. Glycine chloramine (Gly-Cl) and HOCl were permeable to the cells as determined by oxidation of intracellular GSH and inactivation of glyceraldehyde-3-phosphate dehydrogenase, whereas Tau-Cl showed no detectable cell permeability. Both Gly-Cl (20-200 muM) and HOCl (50 microM) caused oxidation of IkappaBalpha methionine, measured by a shift in electrophoretic mobility, when added to the cells in Hanks buffer. In contrast, a high concentration of Tau-Cl (1 mM) in Hanks buffer had no effect. However, Tau-Cl in full medium did modify IkappaBalpha. This we attribute to chlorine exchange with other amines in the medium to form more permeable chloramines. Oxidation by Gly-Cl prevented IkappaBalpha degradation in cells treated with TNFalpha (tumour necrosis factor alpha) and inhibited nuclear translocation of NF-kappaB. IkappaBalpha modification was reversed by methionine sulphoxide reductase, with both A and B forms required for complete reduction. Oxidized IkappaBalpha persisted intracellularly for up to 6 h. Reversion occurred in the presence of cycloheximide, but was prevented if thioredoxin reductase was inhibited, suggesting that it was due to endogenous methionine sulphoxide reductase activity. These results show that cell-permeable chloramines, either directly or when formed in medium, could regulate NF-kappaB activation via reversible IkappaBalpha oxidation.
Biochemical Journal 06/2006; 396(1):71-8. · 4.90 Impact Factor
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ABSTRACT: Unresolved pulmonary inflammation in hyaline membrane disease (HMD) may be a precursor to the development of chronic lung disease of early infancy. We investigated whether nuclear factor kappaB (NF-kappaB), a transcription factor that regulates the inflammatory process, is activated in pulmonary leukocytes in tracheal aspirates from premature infants with HMD. A total of 172 samples were obtained from 59 infants, two thirds of whom showed NF-kappaB activation in lung neutrophils and macrophages on at least one occasion. Infants who had activated NF-kappaB showed elevated tumor necrosis factor-alpha concentrations in their tracheal aspirates. These infants also required a longer period of mechanical ventilation support. Almost half of the infants with HMD had antenatal exposure to chorioamnionitis on the basis of placental histopathologic examination. These infants had evidence of activated NF-kappaB and elevated cytokines and were more likely to have Ureaplasma urealyticum colonization in their airways. Together, these observations suggest that NF-kappaB activation in pulmonary leukocytes may be involved in the lung inflammatory process in infants with HMD.
Pediatric Research 06/2005; 57(5 Pt 1):616-23. · 2.70 Impact Factor
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ABSTRACT: In premature infants, inflammatory conditions in the lungs may result in the development of chronic lung disease. As neutrophil apoptosis is important for the resolution of inflammation and prevention of tissue injury, we set out to determine the extent of neutrophil apoptosis in tracheal aspirate samples from premature infants. Activation of the transcription factor nuclear factor (NF)-kappaB, which causes a delay in neutrophil apoptosis, was also investigated. We obtained 68 tracheal aspirate samples from 27 infants with median gestation and birthweight of 26 weeks and 860 g, respectively. Apoptosis was assessed by immunofluorescent detection of the active form of caspase-3, this assay being validated with peripheral blood neutrophils. Activation of NF-kappaB was monitored by the nuclear translocation of the p65 subunit, detected by immunofluorescence. Cleaved caspase-3 was detected in 11 of the 68 samples, and a median of 40% of the neutrophils showed activated caspase-3 (range 3-92%). A majority of the samples did not show evidence of apoptosis. Caspase activation was seen in cells with multilobed nuclear morphology, suggesting that early apoptosis was detectable. There was no significant difference in respiratory outcomes between infants with or without neutrophil apoptosis. Seventeen of the 68 samples (25%) had evidence of activated NF-kappaB, and a median of 20% (range 6-41%) of neutrophils showed activation. In all but one tracheal aspirate sample, there was a mutually exclusive relationship between activated caspase-3 and NF-kappaB activation, which supports in vitro observations that NF-kappaB activation delays neutrophil apoptosis.
Journal of Leukocyte Biology 04/2005; 77(3):432-7. · 4.99 Impact Factor
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ABSTRACT: We compared the Mycoplasma Duo kit (Sanofi Diagnostics Pasteur) with PCR for detection of Ureaplasma spp. in endotracheal aspirates from 60 premature neonates. The overall agreement between the two tests was 96%. The Mycoplasma Duo assay is a useful alternative to culture and PCR for detection of neonatal Ureaplasma infection.
Journal of Clinical Microbiology 02/2005; 43(1):509-10. · 4.15 Impact Factor
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ABSTRACT: We developed a bedside method for collecting exhaled breath condensate (EBC) from neonates who were ventilated or receiving nasal continuous positive airway pressure (CPAP) and analyzed their EBC for hydrogen peroxide levels. A sufficient volume for analysis could be collected over 25-40 min from neonates on the ventilator and nasal CPAP (medians 5.3 and 2.7 ml, respectively). There was no significant difference between hydrogen peroxide levels from neonates on a ventilator or CPAP (median 0.28 vs. 0.38 microM, p = 0.06) and these were no different from a background with the ventilator or CPAP system alone (median for each 0.31 microM). The dilution of breath condensate by humidified gases plus the existence of background hydrogen peroxide resulted in this collecting setup being insufficiently sensitive to use for the detection of exhaled hydrogen peroxide in infants who were ventilated or on nasal CPAP.
Biology of the Neonate 02/2003; 84(4):338-41. · 1.90 Impact Factor
