Xiao-Du Ping

Exhaled breath condensate in intubated neonates- a window into the lung’s glutathione status

BackgroundAnalysis of exhaled breath condensates (EBC) is a non-invasive technique to evaluate biomarkers such as antioxidants in the pediatric population, but limited data exists of its use in intubated patients, particularly newborns. Currently, tracheal aspirate (TA) serves as the gold standard collection modality in critically ill newborns, but this method remains invasive. We tested the hypothesis that glutathione status would positively correlate between EBC and TA collections in intubated newborns in the Newborn Intensive Care Unit (NICU). We also hypothesized that these measurements would be associated with alveolar macrophage (AM) glutathione status in the newborn lung.MethodsReduced glutathione (rGSH), glutathione disulfide (GSSG), and total GSH (rGSH + (2 X GSSG)) were measured in sequential EBC and TA samples from 26 intubated newborns via high performance liquid chromatography (HPLC). Additionally, AM glutathione was evaluated via immunofluorescence. Pearson’s correlation coefficient and associated 95% confidence intervals were used to quantify the associations between raw and urea-corrected concentrations in EBC and TA samples and AM staining. Statistical significance was defined as p ≤ 0.05 using two-tailed tests. The sample size was projected to allow for a correlation coefficient of 0.5, with 0.8 power and alpha of 0.05.ResultsEBC was obtainable from intubated newborns without adverse clinical events. EBC samples demonstrated moderate to strong positive correlations with TA samples in terms of rGSH, GSSG and total GSH. Positive correlations between the two sampling sites were observed in both raw and urea-corrected concentrations of rGSH, GSSG and total GSH. AM glutathione staining moderately correlated with GSSG and total GSH status in both the TA and EBC.ConclusionsGSH status in EBC samples of intubated newborns significantly correlated with the GSH status of the TA sample and was reflective of cellular GSH status in this cohort of neonatal patients. Non-invasive EBC sampling of intubated newborns holds promise for monitoring antioxidant status such as GSH in the premature lung. Further studies are necessary to evaluate the potential relationships between EBC biomarkers in the intubated premature newborn and respiratory morbidities.

In Utero Ethanol Exposure Impairs Defenses Against Experimental Group B Streptococcus in the Term Guinea Pig Lung

BACKGROUND The effects of fetal alcohol exposure on the risks of neonatal lung injury and infection remain under investigation. The resident alveolar macrophage (AM) is the first line of immune defense against pulmonary infections. In utero ethanol (ETOH) exposure deranges the function of both premature and term guinea pig AM. We hypothesized that fetal ETOH exposure would increase the risk of pulmonary infection in vivo. METHODS We developed a novel in vivo model of group B Streptococcus (GBS) pneumonia using our established guinea pig model of fetal ETOH exposure. Timed-pregnant guinea pigs were pair fed +/-ETOH and some were supplemented with the glutathione (GSH) precursor S-adenosyl-methionine (SAM-e). Term pups were given GBS intratracheally while some were pretreated with inhaled GSH prior to the experimental GBS. Neonatal lung and whole blood were evaluated for GBS while isolated AM were evaluated using fluorescent microscopy for GBS phagocytosis. RESULTS Ethanol-exposed pups demonstrated increased lung infection and sepsis while AM phagocytosis of GBS was deficient compared with control. When SAM-e was added to the maternal diet containing ETOH, neonatal lung and systemic infection from GBS was attenuated and AM phagocytosis was improved. Inhaled GSH therapy prior to GBS similarly protected the ETOH-exposed pup from lung and systemic infection. CONCLUSIONS In utero ETOH exposure impaired the neonatal lungs defense against experimental GBS, while maintaining GSH availability protected the ETOH-exposed lung. This study suggested that fetal alcohol exposure deranges the neonatal lungs defense against bacterial infection, and support further investigations into the potential therapeutic role for exogenous GSH to augment neonatal AM function.

Delayed neonatal lung macrophage differentiation in a mouse model of in utero ethanol exposure

We have previously demonstrated that fetal ethanol exposure deranges the function and viability of the neonatal alveolar macrophage. Although altered differentiation of the alveolar macrophage contributes to pulmonary disease states within the adult lung, the effects of fetal ethanol exposure on the normal differentiation of interstitial to alveolar macrophage in the newborn lung are unknown. In the current study, using a mouse model of fetal ethanol exposure, we hypothesized that altered terminal differentiation of the neonatal interstitial to alveolar macrophage contributes to the observed cellular dysfunction in the ethanol-exposed newborn mouse. Control alveolar macrophage differentiation was characterized by increased expression of CD32/CD11b (P < or = 0.05) and increased in vitro phagocytosis of Staphylococcus aureus (P < or = 0.05) compared with interstitial macrophage. After in utero ethanol exposure, both alveolar and interstitial macrophage lacked the acquisition of CD32/CD11b (P < or = 0.05) and displayed impaired in vitro phagocytosis (P < or = 0.05). Ethanol significantly increased transforming growth factor-beta(1) (TGF-beta(1)) in the bronchoalveolar lavage fluid (P < or = 0.05), as well as in both interstitial and alveolar macrophages (P < or = 0.05). Oxidant stress contributed to the ethanol-induced changes on the interstitial and alveolar cells, since maternal supplementation with the glutathione precursor S-adenosylmethionine during ethanol ingestion normalized CD32/CD11b (P < or = 0.05), phagocytosis (P < or = 0.05), and TGF-beta(1) in the bronchoalveolar lavage fluid and macrophages (P < or = 0.05). Contrary to our hypothesis, fetal ethanol exposure did not solely impair interstitial to alveolar macrophage differentiation. Rather, fetal ethanol exposure impaired both neonatal interstitial and alveolar macrophage phagocytic function and differentiation. Increased oxidant stress and elevated TGF-beta(1) contributed to the impaired differentiation of both interstitial and alveolar macrophage.

In Utero Nicotine Exposure Promotes M2 Activation in Neonatal Mice Alveolar Macrophages

Background:Maternal smoking in utero has been associated with adverse health outcomes including lower respiratory tract infections in infants and children, but the mechanisms underlying these associations continue to be investigated. We hypothesized that nicotine plays a significant role in mediating the effects of maternal tobacco smoke on the function of the neonatal alveolar macrophage (AM), the resident immune cell in the neonatal lung.Methods:Primary AMs were isolated at postnatal day 7 from a murine model of in utero nicotine exposure. The murine AM cell line MH-S was used for additional in vitro studies.Results:In utero nicotine increased interleukin-13 and transforming growth factor–β1 (TGFβ1) in the neonatal lung. Nicotine-exposed AMs demonstrated increased TGFβ1 and increased markers of alternative activation with diminished phagocytic function. However, AMs from mice deficient in the α7 nicotinic acetylcholine receptor (α7 nAChR) had less TGFβ1, reduced alternative activation, and improved phagocytic functioning despite similar in utero nicotine exposure.Conclusion:In utero nicotine exposure, mediated in part via the α7 nAChR, may increase the risk of lower respiratory tract infections in neonates by changing the resting state of AM toward alternative activation. These findings have important implications for immune responses in the nicotine-exposed neonatal lung.

Inflammatory mediator patterns in tracheal aspirate and their association with bronchopulmonary dysplasia in very low birth weight neonates

Objective:Alterations in inflammatory mediators are an important finding in neonates who develop bronchopulmonary dysplasia (BPD); however, there is a lack of research examining the relationship between multiple inflammatory mediators in premature neonates and the development of BPD. This study investigated whether the distribution of 12 inflammatory mediators detected in the tracheal aspirate (TA) of neonates within 24 h of birth could differentiate between neonates who did and who did not develop BPD.Study design:TA samples were collected from 27 very low birth weight neonates (BPD+=11), and the concentrations of 12 biomarkers associated with BPD were determined. Linear discriminant analysis (LDA) was used to classify neonates into two outcome groups.Result:LDA based on the 12 measured biomarkers displayed a significant level of discriminant function (P=0.007).Conclusion:Using linear discriminant analysis, predictive models of BPD can be generated. Our results suggest that multiple inflammatory mediators collected within 24 h of birth may be used to classify neonates into who will and who will not develop BPD.

Zinc Insufficiency Mediates Ethanol-Induced Alveolar Macrophage Dysfunction in the Pregnant Female Mouse

AIMS (a) Establish the minimum number of weeks of chronic ethanol ingestion needed to perturb zinc homeostasis, (b) Examine intracellular zinc status in the alveolar macrophages (AMs) when ethanol ingestion is combined with pregnancy, (c) Investigate whether in vitro zinc treatment reverses the effects of ethanol ingestion on the AM. METHODS C57BL/6 female mice were fed a liquid diet (±25% ethanol-derived calories) during preconception and pregnancy. The control group was pair-fed to the ethanol group. In the isolated AMs, we measured intracellular AM zinc levels, zinc transporter expression, alternative activation and phagocytic index. Zinc acetate was added to some cells prior to analysis. RESULTS Intracellular zinc levels in the AM decreased within 3 weeks of ethanol ingestion. After ethanol ingestion prior to and during pregnancy, zinc transporter expression and intracellular zinc levels were decreased in the AMs when compared with controls. Bacterial clearance was decreased because the AMs were alternatively activated. In vitro additions of zinc reversed these effects of ethanol. CONCLUSION Ethanol ingestion prior to and during pregnancy perturbed AM zinc balance resulting in impaired bacterial clearance, but these effects were ameliorated by in vitro zinc treatments.

Impaired defenses of neonatal mouse alveolar macrophage with cftr deletion are modulated by glutathione and TGFβ1

Our understanding of the intrinsic effects of cystic fibrosis (CF) transmembrane conductance regulator (cftr) deletion on resident neonatal alveolar macrophage (AM) remains limited. We previously demonstrated that diminished glutathione (GSH) or excessive AM transforming growth factor beta one (TGFβ1) contributes to AM dysfunction in a variety of disease states. In this study, using a gut‐corrected cftr neonatal knockout (KO) mouse model and a siRNA‐manipulated macrophage‐like cell line (THP‐1 cell), we hypothesized (1) that cftr mutation alone increases neonatal AM oxidant stress and cellular TGFβ1 signaling via altered GSH, thereby impairing cellular function, and (2) that exogenous GSH attenuates AM alterations and dysfunction in the KO AM. In neonatal KO mice, the baseline bronchoalveolar lavage fluid demonstrated a near doubling in mixed disulfides (P ≤ 0.05) and oxidized GSSG (P ≤ 0.05) without concurrent inflammation compared to WT littermates. KO AM demonstrated diminished AM thiols (P ≤ 0.05), increased AM mitochondrial ROS (P ≤ 0.05), increased AM TGFβ1 (P ≤ 0.05) with increased TGFβ1 signaling (P ≤ 0.05), and impaired phagocytosis (P ≤ 0.05). KO AM mitochondrial ROS was modulated by exogenous GSH (P ≤ 0.05). Conversely, TGFβ1 was reduced (P ≤ 0.05) and impaired phagocytosis was rescued (P ≤ 0.05) by exogenous GSH in the KO AM. These results suggest that an altered neonatal AM phenotype may contribute to the initiation of lung inflammation/infection in the CF lung. Modulation of the AM in the neonatal CF lung may potentially alter progression of disease.

407 ETHANOL CONSUMPTION INCREASES SYSTEMIC OXIDATIVE STRESS IN THE GRAVID GUINEA PIG

Background Maternal alcohol abuse remains a significant problem for our society and ˜ 35% of women consume alcohol during pregnancy. Alcohol-induced injury is augmented in females compared to males. Although in utero alcohol exposure is known to increase oxidative stress in the developing fetus, the effects of alcohol use on the gravid female have not been thoroughly explored. Previous studies from our laboratory have demonstrated that in utero ethanol (ETOH) exposure increased fetal oxidative stress in part via a decrease in the availability of the antioxidant glutathione (GSH). We hypothesize that alcohol used during pregnancy increases oxidative stress and alters cytokines in the gravid female. Objective To 1) evaluate the effects of ETOH consumption on maternal markers of oxidative stress and proinflammatory cytokines and 2) evaluate the effects of GSH supplementation during ETOH ingestion in the gravid female. Methods Using a guinea pig model of fetal ETOH exposure, timed-pregnant guinea pigs were randomly assigned on ˜ d32 (term d70) to ± ETOH in drinking water up to 4% (25% calories + 8 mg/100 mL saccharin) by d40 gestation. Where appropriate the GSH donors SAM or NAC were added to the water containing the ETOH. Cesarean section was performed on d55 of gestation and maternal blood obtained. Malonyldialdehyde (MDA) a marker of oxidative stress, and the proinflammatory cytokines TNF-α and IL-6 were measured with commercially available ELISAs with values normalized to plasma protein. Values are mean (pg/μg protein) ± SEM. Results MDA was significantly increased with ETOH consumption (control 0.0142 ± 0.001, ETOH 0.0285 ± 0.00329, p < .05, n = 3). The level of MDA was normalized with the addition of SAM or NAC during ETOH ingestion (NAC 0.0156 ± 0.00263 vs. SAM 0.0166 ± 0.00201, p < .05 respectively, n = 3). In preliminary studies, plasma TNF-α was nearly doubled and IL-6 increased by 60% in the ethanol dam. The addition of SAM or NAC blunted this increase of proinflammatory cytokines. Conclusions ETOH consumption during pregnancy increases systemic oxidative stress in the female dam. The addition of GSH donors during ETOH ingestion protects the dam from ETOH-induced oxidant stress. We speculate that alcohol ingestion during pregnancy increases the risk of oxidant and proinflammatory mediated injury in the female. Funded by NIH RO1 AA013972-02 (to T.W.G.).